Viral Infections and Treatment (Infectious Disease and Therapy, 30)

Viral Infections and Treatment

edited by

Helga Rubsamen-Waigmann Karl Deres Guy Hewlett Reinhold Welker Bayer Healthcare Wuppefial, Germany

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INFECTIOUS DISEASE A N D THERAPY Series Editor

Burke A. Cunha Winthrop- UniversityHospital Mineola, and State University of New York School of Medicine Stony Brook, New York

I.Parasitic Infections in the Compromised Host, edited by Peter D. Walzer and Robert M. Genta 2. Nucleic Acid and Monoclonal Antibody Probes: Applications in Diagnostic Methodology, edited by Bala Swaminathan and Gyan Prakash 3. Opportunistic Infections in Patients with the Acquired Immunodeficiency Syndrome, edited by Gifford Leoung and John Mills 4. Acyclovir Therapy for Herpesvirus Infections, edited by David A. Baker 5. The New Generation of Quinolones, edited by Clifford Siporin, Carl L. Heifetz, and John M. Domagala 6. MethiciI Iin-Resistant Staphylococcus aureus: CIinical Mlanagement and Laboratory Aspects, edited by Mary T. Cafferkey 7. Hepatitis B Vaccines in Clinical Practice, edited by Ronald W. Ellis 8. The New Macrolides, halides, and Streptogramins: Pharmacology and Clinical Applications, edited by Harold C. Neu, Lowelil S. Young, and Stephen H. Zinner 9. Antimicrobial Therapy in the Elderly Patient, edited by Thomas T. Yoshikawa and Dean C. Norman 10. Viral Infections of the Gastrointestinal Tract: Second Edition, Revised and Expanded, edited by Albert Z. Kapikian 11. Development and Clinical Uses of Haemophilus b Conjugate Vaccines, edited by Ronald W. Ellis and Dan M. Granoff 12. Pseudomonas aeruginosa Infections and Treatment, edited by Aldona L. Baltch and Raymond P. Smith 13. Herpesvirus Infections, edited by Ronald Glaser and James F. Jones 14. Chronic Fatigue Syndrome, edited by Stephen E. Straus 15. lmmunotherapy of Infections, edited by K. Noel Masihi 16. Diagnosis and Management of Bone Infections, edited by Luis E. Jauregui 17. Drug Transport in Antimicrobial and Anticancer Chemotherapy, edited by Nafsika H. Georgopapadakou

18. New Macrolides, Azalides, and Streptogramins in Clinical Practice, edited by Harold C. Neu, Lowell S. Young, Stephen H. Zinner, and Jacques F. Acar 19. Novel Therapeutic Strategies in the Treatment of Sepsis, edited by David C. Morrison and John L. Ryan 20. Catheter-Related Infections, edited by Harald Seifert, Bernd Jansen, and Barry M. Farr 21. Expanding Indications for the New Macrolides, Azalides, and Streptogramins, edited by Stephen H. Zinner, Lowell S. Young, Jacques F. Acar, and Harold C. Neu 22. Infectious Diseases in Critical Care Medicine, edited by Burke A. Cunha 23. New Considerations for Macrolides, Azalides, Streptogramins, and Ketolides, edited by Stephen H. Zinner, Lowell S. Young, Jacques F. Acar, and Carmen Ortiz-Neu 24. Tickborne Infectious Diseases: Diagnosis and Management, edited by Burke A. Cunha 25. Protease Inhibitors in AIDS Therapy, edited by Richard C. Ogderi and Charles W. Flexner 26. Laboratory Diagnosis of Bacterial Infections, edited by Nevi0 Cimolai 27. Chemokine Receptors and AIDS, edited by Thomas R. O’Brien 28. Antimicrobial Pharmacodynamics in Theory and Clinical Practice, edited by Charles H. Nightingale, Take0 Murakawa, and Paul G. Ambrose 29. Pediatric Anaerobic Infections: Diagnosis and Management, ‘Third Edition, Revised and Expanded, ltzhak Brook 30. Viral Infections and Treatment, edited by Helga Riibsamen-Waigmann, Karl Deres, Guy Hewleit, and Reinhold Welker 31. Community-Acquired Respiratory Infections, edited by Charles H. Nightingale, Paul G. Ambrose, and Thomas M. File, Jr.

Additional Volumes in Production

Preface

The obliteration of diseases that impinge on our health is a regal yardstick of civilization’s success, and those who accomplish that task will be among the true navigators of a brave new world. Michael B. A. Oldstone, Viruses, Plagues and History, New York: Oxford University Press, 1998 Viruses are regarded by some as thieves, parasites, and murderers. Others regard them as the ultimate examples of informational nanotechnology. This book is concerned with the dark side of viruses and the diseases they cause. However, it should not be forgotten that it is because viruses are so technologically advanced that we have been able to make so much progress in our understanding of genetics, cell biology, biochemistry, and molecular biology. Paradoxically, the knowledge given us by the study of viruses has also led to the discovery of many antiviral compounds. Viral infections have long been regarded as untreatable. In the middle of the last century, the science of virology was in its infancy and viral chemotherapy was but a gleam in the eye of leading microbiologists like Robert Doerr, who fervently believed in the existence of chemotherapeutic agents that would be effective against a large number of virusiii

iv

Preface

related diseases [1]. However, the realization that viruses usurped the metabolic machinery of the cell raised the intellectual barrier that a ‘‘virotoxic’’ agent would also be toxic toward the normal cell. Thus, therapeutic nihilism became an accepted philosophy within the majority of medical circles. Successful control of some viral diseases came about only through the advent of prophylactic vaccination for now nearly extinct viruses such as smallpox and polio. Unfortunately, not all viral diseases lend themselves to vaccination. For example, researchers have tried for decades to develop vaccines against herpes simplex viruses but without much success. At the turn of the new millennium, a glimmer of understanding emerged as to why previous attempts had failed: herpesviruses have evolved strategies to interact with the mechanisms that alarm the immune system and inactivate or divert the signals. Similarly, major efforts to develop a vaccine against human immunodeficiency virus have thus far failed to reach their target, partly because of the enormous genetic drift of the virus and partly because of the lack of knowledge about the immune reactions that have to be triggered in order to create an effective vaccine against a virus that attacks and modulates the immune system itself. Therefore, although our understanding of immune mechanisms in the fight against viruses is increasing, some viruses may never be controlled by vaccines but will almost certainly require the development of low-molecular-weight antiviral compounds or novel immunomodulating principles before they succumb. A major conceptual breakthrough in chemotherapy occurred in the late 1940s when Hitchings and Elion realized that the rate of nucleic acid synthesis in infected tissues is much higher than that of the normal host tissue. This led to the idea of antimetabolite chemotherapy for cancer and parasitic diseases that ultimately led to antiviral agents such as iododeoxyuridine, deoxycytidine, hydroxyethoxymethylguanine, and azidothymidine. The latter two are better known as acyclovir and AZT, respectively. Although a thiosemicarbazone derivative was the first lowmolecular-weight antiviral compound used in humans—as a prophylactic treatment for smallpox contacts in the early 1960s [2]—herpesvirus disease was the first viral infection for which a truly efficient treatment was developed, in the form of acyclovir. When AIDS was recognized as a viral disease in the early 1980s, treatment at first seemed unlikely if not impossible. However, only four years after the virus had been discovered, the first drug, azidothymidine, had been introduced onto the market. We now have several antiviral drugs in our arsenal, and not all are antimetabolites: two novel drugs against influenza have been developed that interact with, and inhibit, the viral neuraminidase,

Preface

v

whereas the viral protease and reverse transcriptase of HIV are principal targets for current treatments with non-nucleosidic inhibitors. Some of the nucleoside analogs developed for HIV therapy have been found to be effective against hepatitis B as well. Ribavirin, in combination with interferon, has also been successfully introduced for the treatment of hepatitis C and can be regarded as an effective immunomodulatory principle. Daunting as it seemed for years, in the year 2003 effective drug therapy does exist for a variety of viral diseases, just as Doerr and colleagues predicted 60 years ago. Concomitant with this, we have also witnessed the development of a new era of diagnostic tools. Polymerase chain reactions combined with appropriate methods for sequence detection now allow not only the diagnosis of a viral disease but also the determination of a viral subtype. In addition, methods that allow the quantification of the virus in body fluids or tissues in order to determine viral loads are now standard in clinical trials, and drugs are being approved on the basis of showing efficacy in reducing this particular parameter. Thus, the demonstration of efficacy of an antiviral drug no longer depends entirely on clinical endpoints once a relationship has been established between the reduction in viral load and resolution of clinical signs has been established. All these advances in understanding and treating viral diseases are relatively recent developments and are proceeding at a rapid pace. Thus there is often a gap in knowledge about the diagnostic and therapeutic options currently available for clinical management. It is our hope that this book will fill that gap, and we have tried to present in a practical and cohesive manner all the latest developments and their use. The introductory chapter gives an overview of the most important human viral pathogens and their transmission as well as emerging viral pathogens. At the time of writing, severe acute respiratory syndrome (SARS) dominates the media; it is an example of the unpredictability of emerging viral diseases and illustrates the potential for explosive dissemination of viruses throughout our global community. Not only does the novelty of the SARS-associated virus make it difficult to assess the long-term significance of the outbreak, but also the SARS epidemic itself exposes the desperate need for rapid diagnosis and reporting of unusual outbreaks and for international cooperation in investigating such occurrences. The main structure of this book consists of specific sections on infections of the respiratory tract, the skin and mucosa, and the liver as well as special sections on human immunodeficiency virus and on

vi

Preface

herpesvirus infections of the immunocompromised host. We have included chapters on what are currently regarded as the most clinically relevant viruses—influenza viruses, respiratory syncytial virus, rhinovirus, the herpes simplex viruses, varicella zoster virus, papillomaviruses, hepatitis viruses, human immunodeficiency virus, human cytomegalovirus, Epstein-Barr virus, and the recently identified human herpesviruses 6, 7, and 8. Each chapter describes the respective diagnostic measures guiding therapy, approved therapeutic agents, their mode of action, and their clinical applications. This book is intended not only for infectious-disease physicians and virologists who want to update their knowledge on transmission, diagnosis, and treatment of viral diseases but also for the nonspecialist who wishes to obtain a greater understanding of the clinically important viral pathogens. It is clear that there is still a less than complete understanding in the community of the burden of mortality and morbidity caused by viruses. HIV continues to devastate the world population and now ranks equal to tuberculosis as a global killer. The spread of the virus is still exponential in many areas of the world, and a constant 35,000–40,000 people are being newly infected in both the United States and Europe each year. Persistent viral infections develop unusual patterns of disease and unusual severity under conditions of immunosuppression by HIV, be it herpesvirus, cytomegalovirus, or papillomaviruses. Similarly, many of the viruses discussed in this book cause considerable, even life-threatening, problems in immunocompromised states after transplantation or in neonates. Therein lies the reason for our putting this book together, and it is our profound hope that it will serve the reader well. We hope that it will be a constant companion for physician, student, and layperson alike. We are grateful to everyone who has accompanied us on the sometimes tortuous path of producing this volume, especially the contributors and the editorial staff of Marcel Dekker, Inc. Helga Ru¨bsamen-Waigmann Karl Deres Guy Hewlett Reinhold Welker References 1.

Doerr R. The chemotherapy of viral diseases. In: Doerr R, Hallauer C, eds. Handbook of Virus Research: 1st Suppl. (in German). Vienna: SpringerVerlag, 1944:271–348.

Preface 2.

vii

Bauer DJ, St. Vincent L, Kempe CH, Downie AW. Prophylactic treatment of smallpox contacts with N-methylisatin b-thiosemicarbazone. Lancet 1963; ii(7306):494–496.

Contents

Preface Contributors 1.

Emerging and Reemerging Viral Pathogens Ulrich Desselberger

iii xi 1

Infections of the Respiratory Tract 2.

Influenza: The Virus, the Disease, and Its Control Thorsten Wolff and Rene´ Snacken

39

3.

Respiratory Syncytial Virus Philip R. Wyde and Pedro A. Piedra

91

4.

Rhinovirus Ronald B. Turner and Frederick G. Hayden

139

5.

Herpes Simplex Virus Kimberly A. Yeung-Yue, Gisela Torres, Mathijs H. Brentjens, Patricia C. Lee, and Stephen K. Tyring

165

6.

Varicella-Zoster Virus Jashin Joaquin Wu, Kimberly A. Yeung-Yue, Mathijs H. Brentjens, and Stephen K. Tyring

193

7.

Human Papillomaviruses Guy Hewlett, Philip S. Shepherd, and Jenny C. Luxton

227

ix

x

Contents

Infections of the Liver 8.

Hepatitis A Virus Verena Gauss-Mu¨ller and Reinhart Zachoval

259

9.

Hepatitis B Virus Guido Gerken and Christoph Jochum

277

Hepatitis C Virus Miriam Kerstin Huber, Ulrike Sarrazin, and Stefan Zeuzem

295

10.

Human Immunodeficiency Virus 11.

12.

13.

HIV Infection: Epidemiology, Pathogenesis, and Principles of Antiretroviral Therapy Reinhold Welker and Helga Ru¨bsamen-Waigmann

369

Diagnosis and Management of HIV Infection Using Immunoassays and Molecular Technologies Rainer Ziermann, Charlene E. Bush-Donovan, and David A. Hendricks

433

Nucleoside/Nucleotide Inhibitors of HIV Reverse Transcriptase Erik De Clercq

485

14.

Non-Nucleoside Inhibitors of HIV Reverse Transcriptase Matthias Go¨tte and Mark A. Wainberg

505

15.

HIV Protease Inhibitors Richard Ogden

523

16.

Emerging Therapies for HIV Infection Julie M. Strizki

555

Systemic Herpesvirus Infections and the Immune-Compromised Host 17.

Human Cytomegalovirus: Diagnosis, Pathophysiology, and Treatment Hermann Einsele and Gerhard Jahn

587

18.

Epstein-Barr Virus: Pathogenesis and Treatment Nancy Raab-Traub and Shannon C. Kenney

623

19.

The Human Herpesviruses HHV-6, HHV-7, and HHV-8 Dharam V. Ablashi and Gerhard R. F. Krueger

659

Index

707

Contributors

Dharam V. Ablashi, D.V.M., M.S., Dip.Bact.* Adjunct Professor of Microbiology and Director, Herpesvirus Program, Georgetown University Medical School, Washington, D.C., and Advanced Biotechnologies Inc., Columbia, Maryland, U.S.A. Mathijs H. Brentjens, M.D. Department of Dermatology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Charlene E. Bush-Donovan, Ph.D. Director of Research, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A. Erik De Clercq, M.D., Ph.D. Professor, Rega Institute for Medical Research, Catholic University of Leuven, Leuven, Belgium Ulrich Desselberger, M.D., F.R.C.Path., F.R.C.P.(Glasgow, London){ Consultant Virologist and Director, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, England

* Retired { Current affiliation: Virologie Mole´culaire et Structurale, UMR 2472, CNRS, Gif-sur-Yvette, France.

xi

xii

Contributors

Hermann Einsele, M.D. Division of Hematology/Oncology, Department of Internal Medicine, Universita¨tsklinikum Tu¨bingen, Tu¨bingen, Germany Verena Gauss-Mu¨ller, Ph.D. Professor, Institute of Medical Molecular Biology, University of Lu¨beck, Lu¨beck, Germany Guido Gerken, M.D. Director, Gastroenterology and Hepatology Clinic, Center for Internal Medicine, University of Essen, Essen, Germany Matthias Go¨tte, Ph.D. Assistant Professor, Departments of Medicine and Microbiology and Immunology, McGill University, and Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada Frederick G. Hayden, M.D. Stuart S. Richardson Professor of Clinical Virology and Professor, Departments of Internal Medicine and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. David A. Hendricks, Ph.D. Senior Research Fellow and Director, Medical and Scientific Affairs, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A. Guy Hewlett, Ph.D. Principal Scientist, Department of Virology, Bayer HealthCare, Wuppertal, Germany Miriam Kerstin Huber, M.D. Department of Gastroenterology, Johann-Wolfgang Goethe University Clinic, Frankfurt/Main, Germany Gerhard Jahn, M.D. Professor, Institute of Medical Universita¨tskinikum Tu¨bingen, Tu¨bingen, Germany

Virology,

Christoph Jochum, M.D. Department of Gastroenterology and Hepatology, Center of Internal Medicine, University of Essen, Essen, Germany Shannon C. Kenney, M.D. Professor, Lineberger Comprehensive Cancer Center, Department of Medicine and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

Contributors

xiii

Gerhard R. F. Krueger, M.D., Ph.D. Professor, Division of Allergy and Clinical Immunology, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Patricia C. Lee, M.D. Associate Professor, Department of Microbiology/Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Jenny C. Luxton, Ph.D. Department of Immunobiology, Guy’s, King’s and St. Thomas’ Medical and Dental Schools, London, England Richard Ogden, Ph.D. Senior Director, Scientific Development, Agouron Pharmaceuticals, Inc., A Pfizer Company, La Jolla, California, U.S.A. Pedro A. Piedra, M.D. Associate Professor, Department of Molecular Virology and Microbiology and Department of Pediatrics, Baylor College of Medicine, Houston, Texas, U.S.A. Nancy Raab-Traub, Ph.D. Professor, Lineberger Comprehensive Cancer Center, Department of Medicine, and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Helga Ru¨bsamen-Waigmann, Ph.D. Vice President, Division of Antiviral Research, Anti-Infective Research, Bayer HealthCare, Wuppertal, and Professor, Department of Biochemistry and Virology, University of Frankfurt, Frankfurt, Germany Ulrike Sarrazin, M.D. Clinic for Internal Medicine II, Saarland University Hospital, Homberg, Germany Philip S. Shepherd, M.B., C.H.B. M.Sc., M.R.C.P., F.R.C.P. Senior Lecturer and Honorary Consultant, Department of Immunobiology, Guy’s, King’s and St. Thomas’ Medical and Dental Schools, London, England Rene´ Snacken, M.D., M.P.H. Head, Influenza Branch, Department of Epidemiology, Scientific Institute of Public Health, Brussels, Belgium Julie M. Strizki, Ph.D. Principal Scientist, Antiviral Therapeutics, Schering–Plough Research Institute, Kenilworth, New Jersey, U.S.A.

xiv

Contributors

Gisela Torres, M.D. Clinical Research Fellow, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Ronald B. Turner, M.D. Professor, Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. Stephen K. Tyring, M.D., Ph.D. Professor, Department of Dermatology and Department of Microbiology/Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Mark A. Wainberg, Ph.D. Professor, Department of Microbiology, McGill University, and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada Reinhold Welker, M.D. Senior Scientist, Department of Virology, Bayer HealthCare, Wuppertal, Germany Thorsten Wolff, M.D. Germany

Group Leader, Robert Koch-Institut, Berlin,

Jashin Joaquin Wu, M.D. Department of Internal Medicine, Baylor College of Medicine, Houston, Texas, U.S.A. Philip R. Wyde, Ph.D. Professor, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A. Kimberly A. Yeung-Yue, M.D. Dermatology Clinical Research Fellow, Center for Clinical Studies, University of Texas Medical Branch, Galveston, Texas, U.S.A. Reinhart Zachoval, M.D. Professor, Department of Internal Medicine II, Grosshadern Medical Center, Munich, Germany Stefan Zeuzem, M.D. Professor, Department of Internal Medicine II, Saarland University Hospital, Homburg, Germany Rainer Ziermann, Ph.D. Principal Staff Scientist, Medical and Scientific Affairs, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A.

1 Emerging and Reemerging Viral Pathogens Ulrich Desselberger Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, England*

1

INTRODUCTION

Over the last 25–30 years numerous viral and other microbial pathogens have been discovered to be etiological agents of human disease. This chapter presents a review on the viral pathogens found to cause novel or reemerging human disease. Animals were found or suspected to be the source of human infection in a number of cases. Prospective, laboratorybased, epidemiological surveillance is of paramount importance for early detection and management of emerging or reemerging infectious diseases. In 1990 the Institute of Medicine of the United States, in Washington, D.C., convened a committee under the cochairmanship of Joshua Lederberg and Robert Shope that conducted an extensive study on emerging microbial threats to health and the conditions under which they occurred. In the ensuing report [1] a large number of microbes (bacteria, * Current affiliation: Virologie Mole´culaire et Structurale, UMR 2472, CNRS, Gif-sur-Yvette, France.

1

2

Desselberger

TABLE 1 Major Etiologic Agents of Viral Infectious Diseases in Humans Identified Since 1972a Year 1972

Agent

1973

Small round structure viruses (SRSVs; caliciviruses); Norwalk virus Rotaviruses

1975 1975

Astroviruses Parvovirus B19

1977 1977

Ebola virus Hantaan virus

1980

Human T-cell lymphotropic virus-1 (HTLV-1)

1982 1983 1984 1985 1988

HTLV-2 Human immunodeficiency viruses (HIV-1, HIV-2) Puumulavirus SRSV: sapporovirus Human herpesvirus-6 (HHV-6)

1989

Hepatitis C virus (HCV)

1990 1990

Human herpesvirus-7 (HHV-7) Hepatitis E virus (HEV)

1991 1991 1992

Hepatitis F virus (HFV) Aichi virus Dobravavirus

1993

Sin nombre virus

1993 1994

Hepatitis G virus (HGV) Sabia virus

Human disease Diarrhea (outbreaks)

Major cause of infantile diarrhea worldwide Diarrhea (outbreaks) Aplastic crisis in chronic hemolytic anemia; fifth disease; fetal infection Ebola hemorrhagic fever Hemorrhagic fever with renal syndrome (HFRS) Adult T-cell leukemia/ lymphoma; tropical spastic paraparesis (TSP)/HTLV-1 associated myelopathy (HAM) Hairy T-cell leukemia Acquired immunodeficiency syndrome (AIDS) Nephropathia epidemica Diarrhea (outbreaks) Exanthema subitum (roseola infantum); pneumonitis, excephalitis in bone marrow transplant recipients and AIDS patients Parenterally transmitted non-A, non-B hepatitis Exanthema subitum, others? Enterically transmitted non-A, non-B hepatitis Severe non-A, non-B hepatitis Diarrhea Hemorrhagic fever with renal syndrome Hantavirus pulmonary syndrome (‘‘Four corners disease’’) Non A-C hepatitis? Brazilian hemorrhagic fever and necrotizing hepatitis

Emerging and Reemerging Viral Pathogens

3

TABLE 1 Continued Year

Agent

Human disease

1994

1995

Human herpesvirus-8 (HHV-8) or Kaposi’s sarcoma-associated herpesvirus (KSHV) Borna disease virus

1995 1996

Hendravirus Prion (BSE?)b

1996 1996 1997 1997 1997 1998 1999 1999 2000 2001

Whitewater Arroyo virus Influenza A virus (H7N7) Influenza A virus (H5N1) Transfusion-transmitted virus (TTV) Enterovirus 71 (EV71) Nipahvirus Influenza A virus (H9N2) West Nile encephalitis virus Cantalago virus Metapneumovirus

Kaposi’s sarcoma; body cavitybased lymphoma; Castleman’s disease Neuropsychiatric disorders (disputed for human) Meningitis, encephalitis New variant Creutzfeldt-Jakob disease (nv-CJD) Hemorrhagic fever Conjunctivitis (England) Influenza (Hong Kong) No firm association yet

2001

SEN virus (TTV-related)

Epidemic encephalitis Meningitis, encephalitis Influenza (Hong Kong) Encephalitis (New York) Vesicular rash (Brazil) Respiratory tract infection (children) No firm association yet

a

Date of discovery assigned on the basis of the year of isolation or identification of etiological agent. b Prions are proteinaceous infectious particles lacking nucleic acid (224), not viruses. Source: Refs. 8,11,12 (updated).

rickettsiae, chlamydiae, viruses, fungi, parasites) were identified that were either already known as human pathogens but appeared to be associated with changed disease patterns and/or increased case or infection rates, or were recognized as new human pathogens. Factors involved in their emergence or reemergence were population increase and increasing urbanization, industrial and economic development including man-made perturbation of the environment, global travel and mass movements (refugees), and civil unrest and wars but also microbial genomic change and adaptation [1–12]. It was further recognized that gradually improved surveillance using clinical, pathological (laboratory), epidemiological, and public health approaches has led to rapid identification of newly emerging or reemerging infectious agents. Advances in the science of virology have allowed the introduction of a

4

Desselberger

number of molecular diagnostic techniques that have greatly enhanced the discovery of previously unknown viruses pathogenic for humans [13]. Numerous journal publications and books [e.g., 2,6,14,15] and even a dedicated journal, Emerging Infectious Diseases, have described the emergence of new pathogens in the 1990s. The major viral pathogens identified as causes of human disease since 1972 are listed in Table 1. (This time point is arbitrary, because shortly before 1972 Lassa virus, Marburg virus, and others had emerged.) In the following pages, selected aspects of diagnosis, epidemiology, and treatment and prevention of infection with these new or reemerged viruses are briefly described.

2 2.1

SPECIFIC PATHOGENS Small Round Structured Viruses (Caliciviridae)

Norwalk virus (NV) was identified as the first small round structured virus (SRSV) when it caused outbreaks of acute gastroenteritis in 1972 [16]. In 1990 the NV genome, a single-stranded RNA of positive sense and approximately 7.5 kilobases (kb) in size was cloned [17], and subsequently its genome and those of other emerging related viruses were sequenced [e.g., 18–21], allowing their classification as members of the Caliciviridae family. Human gastroenteric caliciviruses occur in two genera [Norwalk-like viruses (genogroups I and II) and Sapporo-like viruses] [22]. These genera have recently been designated as Norovirus and Sapovirus, respectively [22a]. Several genogroups of SRSV have been found that in part cocirculate and even coinfect [23,24]. Calicivirus recombinants have been observed to occur naturally [25]. The virus structure, that of a nonenveloped viral particle, has been established by cryoelectron microscopy and image reconstruction [26]. Expression of the NV capsid protein from baculovirus recombinants in insect cells [27] and its use as an antigen in enzyme-linked immunosorbent assays (ELISAs), have allowed assessment of the agerelated seroprevalence of specific antibody to the virus [28–30], which starts to infect children early in life, often asymptomatically, and is causing much more widespread human infection than previously thought [31]. However, recently human caliciviruses were also recognized as a major cause of apparent gastroenteritis in children by using the now widely available reverse transcription-polymerase chain reaction (RT-PCR) for diagnostic purposes and outbreak investigation (screening) [32–34]. The evidence of close genetic relationships between porcine enteric caliciviruses and Sapporo-like viruses and between

Emerging and Reemerging Viral Pathogens

5

bovine caliciviruses and Norwalk-like viruses suggests that animals are potential reservoirs for human infections [35, 35a–c].

2.2

Rotaviruses (Reoviridae)

Rotaviruses were discovered as the cause of infantile human gastroenteritis in 1973 [36,37] after similar viruses had already been recognized as the cause of acute gastroenteritis in a variety of young animals (mice, monkeys, calves) in the 1960s. Since their discovery, human rotaviruses have been found to be the main etiological agent of gastroenteritis in infants and young children worldwide [38]. At least seven groups (A–G) and, within group A, various types exist (classified as G and P types). Group A rotaviruses cocirculate at any one time [38,39]. Whereas G1P[8], G2P[4], G3P[8], and G4P[8] represent the majority of viruses cocirculating in temperate climates, G5 and particularly G9 viruses have been found in recent years, first in Asia, Africa, and South America, but G9 also in Europe and North America [40–44]. After many years of research, a live-attenuated, tetravalent (TV), rhesus rotavirus (RRV)–based, human reassortant vaccine was developed [45–47] that was licensed in the United States in August 1998. Guidelines for its application appeared in March 1999 [48]. In the period between 1 September 1998 and 7 July 1999, when 1.8 million doses of the vaccine were administered in the United States, gut intussusceptions were observed in 15 infants, of whom 13 developed the condition after the first dose of the three-dose RRV-TV vaccine course, and 12 developed the symptoms within one week of any dose (see Refs. [49] and [50] for further details). Although the number of reported cases was within the expected range by chance during the week following the receipt of any dose, the well-known incompleteness and delays of reporting through the Vaccine Adverse Event Reporting System (VAERS) led the U.S. Centers for Disease Control (CDC) and the American Academy of Pediatrics (AAP) to recommend postponing administering RRV-TV to infants between July and November 1999. A study with evidence supporting an association between vaccination with RRV-TV and intussusception has been published [51], but the true vaccine-attributable risk is still under investigation. The original CDC Advisory Committee on Immunization Practices (ACIP) guidelines [48] have been revoked, and the vaccine was taken off the market in October 1999 [52]. Alternative concepts for developing candidate rotavirus vaccines are under active investigation [53].

6

2.3

Desselberger

Astroviruses (Astroviridae)

Human astroviruses were first detected in 1975 by electron microscopy (EM) in stool specimens of infants and children with diarrhea [54] and named after the pathognomonic star-shaped appearance of some of the particles in electron micrographs [55]. These viruses are now classified within a separate family, the Astroviridae [56]. They are nonenveloped particles and possess a genome of single-stranded RNA of positive polarity and 6.8 kb size with three open reading frames (ORFs) of which the second encodes the single capsid protein [57]. So far eight different human astrovirus types have been recognized [58,59], and Noel et al. [60] have shown a very good correlation of serotypes and genotypes for seven astrovirus types, using ELISAs and RT-PCR, respectively. Astroviruses have also been recognized as the cause of major outbreaks of gastroenteritis [e.g., 61,62]. In hospitalized children in Australia, astroviruses were found to be the second most frequent viral cause of diarrhea, after rotaviruses [63]. 2.4

Parvovirus B19 (Parvoviridae)

Parvovirus B19 was discovered as an agent infecting humans in 1975 [64]. The viral particles measure only 18–26 nm in diameter, are nonenveloped, and have an icosahedral symmetry. Their genome consists of linear, single-stranded DNA of both polarities and is approximately 5 kb in size. For their replication, parvoviruses have an almost absolute requirement of rapidly dividing cell populations such as those found in bone marrow and embryonic tissues [65]. This explains parvoviruses as the cause of aplastic crises in chronic hemolytic anemia (sickle cell anemia) patients [66] and of intrauterine infections followed sometimes by hydrops and early abortion as a clinical outcome but mostly remaining an inapparent infection [67,68]. Parvovirus B19 is also the etiological agent of the childhood disease erythema infectiosum (fifth disease) [69]. 2.5

Ebola Virus (Filoviridae)

The Ebola virus was found to be the cause of a hemorrhagic fever with high mortality in Central Africa in the mid-1970s [70,71] and reemerged there in the mid-1990s [72,73]. Early recognition of Ebola virus as the causative agent of the outbreaks in Kikwit hospital in Zaire in 1995 was due to a broad international collaboration of physicians, virologists, immunologists, molecular biologists, pathologists, epidemiologists, and public health doctors. Ebola virus (like Marburg virus) is a member of the

Emerging and Reemerging Viral Pathogens

7

Filoviridae family and has now been thoroughly characterized at the molecular level (for review, see Ref. [73]). Recently reverse genetics systems for Ebola virus have been devised by two groups [74,75]. An intensive search for a true animal reservoir of these viruses is ongoing [76,77]. 2.6

Sabia Virus, Whitewater Arroyo Virus (Arenaviridae)

Other emerging hemorrhagic fever viruses were observed in different parts of the world. Examples include Sabia virus, associated also with severe hepatitis [78,79], and Whitewater Arroyo virus [80,81]. Both of these viruses are members of the Arenaviridae family, of which the best known member is Lassa virus, detected as the cause of severe hemorrhagic fever in 1970 [82]. 2.7

Bunyaviruses (Bunyaviridae)

Viruses of the Bunyaviridae family were discovered several times as novel etiologic agents of human diseases. In 1977 Hantaan virus was found to be the cause of hemorrhagic fever with renal syndrome (HFRS) [83], which by then had been known as Korean hemorrhagic fever for more than 20 years [84]. Nephropathia epidemica, a mild form of HFRS occurring in Scandinavia, was found in 1984 to be caused by a bunyavirus, Puumala virus [85,86]. Another hantavirus, Dobrava virus, was shown to be the cause of severe HFRS in the Balkans [87]. Rodents (mice, rats, etc.) were recognized as the main, asymptomatic reservoir of bunyaviruses [84,88,89]. In 1993 another member of the Bunyaviridae family, sin nombre virus, was identified as the cause of the hantavirus pulmonary syndrome (HPS or ‘‘four corners disease’’) [89]. It was shown that climatic changes in the Southwestern United States (due to el Nin˜o) had led to an increase in food for rodents and a subsequent increase in their numbers, making it more likely for humans in close contact with the countryside (hiking, summer houses, etc.) to become infected [89,90]. For sin nombre virus, coevolution of virus and host species was extensively documented, each rodent species acting as the primary reservoir for only a single specific hantavirus [90,91]. Since then hantaviruses have been found throughout wide areas of the United States and also in Central and South America. The data suggest that hantaviruses and their rodent hosts have coevolved over 20–30 million years [90]. Bunyaviruses are widespread in animal reservoirs [90,92,93]. Transmission in animals seems to occur mainly horizontally by infectious excretions, and transmission to humans by inhalation of contaminated dust. Although bunyaviruses contain three segments of negative-stranded RNA as their genome,

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reassortment has so far not been found to be a major factor in the emergence of new strains (in contrast to influenza viruses, where reassortment events have repeatedly led to the emergence of pandemic strains; see below). A reverse genetics system was devised by Bridgen and Elliott [94] that is starting to help unravel the molecular biology of the virus. 2.8

Human T-Cell Leukemia Viruses (Retroviridae, Oncovirinae)

Two types of human T-cell leukemia viruses (HTLVs) have been recognized as the cause of human disease: type 1 (HTLV-1) in 1980 as being closely associated with T-cell lymphoma-leukemia (the first human tumor firmly associated with a viral infection [95]) and type 2 (HTLV-2) in 1982 as being associated with atypical hairy T-cell leukemia [96]. In addition, HTLV-1 was shown to cause tropical spastic paraparesis (TSP) [97], also termed HTLV-1-associated myelopathy (HAM) [98]. These HTLVs are members of a large subfamily (Oncovirinae) of the Retroviridae family, which have for some time been known to be associated with a large number of tumors in animals (Rous sarcoma virus, mouse mammary tumor virus, etc.). HTLV-1 infections were discovered in Japan but are now found worldwide. Like HIV, HTLV-1 is transmitted sexually (mainly male to female), through infected blood, or vertically from mother to child. Although not of recognized animal origin, HTLV-1 has been transmitted to rats and rabbits, with some animals developing features of the human disease [99]. 2.9

Human Immunodeficiency Virus (Retroviridae, Lentivirinae)

A virus isolated in 1983 from a homosexual patient was first termed lymphadenopathy-associated virus (LAV) [100]. Soon afterward the unequivocal association of LAV infection with the acquired immunodeficiency syndrome (AIDS) was demonstrated, and the virus was renamed human immunodeficiency virus (HIV). At least two types (HIV-1, HIV-2) and within them a large variety of subtypes/clades (HIV-1: clades A–J in group M, groups N and O; HIV-2: clades A–E) exist and cocirculate. The origin of HIV as a human pathogen has for a long time been an enigma. However, the findings that HIV-2 isolates are closely related to simian immunodeficiency virus (SIV) isolates from sooty mangabeys (SIVsmm) [101] and that HIV-1 isolates are closely related to SIVs obtained from chimpanzees (SIVcpz) [102] strongly suggested that chimpanzees and sooty mangabeys are the animal reservoirs for a zoonosis in humans.

Emerging and Reemerging Viral Pathogens

9

There is evidence for SIV–host coevolution [103]. The striking diversity within and between clades is achieved by the accumulation of point mutations and by frequent recombinatory events in regions of the world where HIVs of different clades cocirculate in sufficient numbers and frequencies, thus increasing the chance of coinfection [104–106]. Even intergroup recombination has recently been found to occur in nature [107]. This chapter is clearly not the place to review the replication, pathogenesis, diagnosis, and treatment of HIV in detail [103,108–110]. However, it should be noted that highly active antiretroviral therapy (HAART), which has benefited many HIV-infected patients since 1996, has exerted a strong selective pressure on HIV, leading to the emergence of drug-resistant HIV mutants (in both the reverse transcriptase and protease genes). Although genotypic resistance assays were helpful in the formulation of antiretroviral regimes [111], increasingly (up to 27% of), new infections occur with HIV variants that are already resistant against one or several of the licensed drugs [112–115; D. Pillay, personal communication]. Thus, there is an accelerated evolution and emergence of drug-resistant viruses. 2.10

New Herpesviruses (Herpesviridae)

Since 1988, three new human herpesviruses (HHV-6, HHV-7, and HHV8) have been discovered. HHV-6, initially termed human B-lymphotropic virus (HBLV), was discovered in patients with lymphoproliferative disorders [116] but was later found to be the cause of the infancy and childhood disease exanthema subitum/roseola infantum [117,118]. Most cases of exanthema subitum are caused by the variant HHV-6B [119]. The primary infection with this ubiquitous virus mainly occurs within the first three years of life [120], not infrequently associated with encephalitis [121,122]. Reactivation of HHV-6 in allogeneic bone marrow transplant patients was found to be associated with fever, skin rashes, pneumonitis, encephalitis, bone marrow suppression, and graft-versus-host disease [123–125]. In 1990, a new human herpesvirus was isolated from a healthy carrier and termed HHV-7 [126]. As with HHV-6, infection with HHV-7 seems to occur ubiquitously and to cause infection, mainly in children [127–129]. At present, the extent of the involvement of HHV-7 in human disease is not clear, although the virus has been isolated from patients with exanthema subitum [118,130] and pityriasis rosea [131]. In 1994, cDNA sequences with homology to herpesvirus sequences were classified as those of a new type of human herpesvirus, called

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HHV-8. Subsequently, HHV-8 was found to be firmly associated with the occurrence of Kaposi’s sarcoma (angioplastic sarcoma) and therefore also termed Kaposi’s sarcoma associated herpesvirus (KSHV) [132]. In 1996, a cell line (BCBL-1) propagating KSHV/HHV-8 was described [133]. Phylogenetically, KSHV is a g2-herpesvirus [134] related to the EpsteinBarr virus (EBV), a g1-herpesvirus. Knowledge of the epidemiology and transmission of the virus is still rudimentary. KSHV occurs in several variants, based on sequence differences at the left and right ends of the viral genome [135,136]. Several subtypes have been distinguished, subtype B being found mainly in Africa and subtypes A and C also found in Africa but mostly in Europe [137,138]. Seroprevalence data show differences among geographical regions, also depending on the antigen used in serological assays. In general, there is an age-related increase of antibody prevalence. Transmission is mainly sexual, due to the occurrence of the virus in seminal fluid [139]. However, horizontal transmission among children has also been observed [140,141]. Parenteral transmission seems possible, but this observation needs to be supported by further studies [141]. A causative role of KSHV in a rare non-Hodgkin’s lymphoma, body cavity based lymphoma, and another a typical lymphoproliferative disorder, Castleman’s disease (a multicentric B-cell lymphoma), is also likely [138]. 2.11 2.11.1

New Hepatitis Viruses Hepatitis C Virus (Flaviviridae)

The study of hepatitis virology has moved tremendously fast since 1989 when, entirely by the use of molecular techniques, hepatitis C virus (HCV) was discovered as the main cause of transfusion-transmitted, nonhepatitis A, non-hepatitis B (non-A, non-B) viral infections [142]. The virus belongs to the Flaviviridae family but has now been classified as a separate genus (Hepacivirus) [143]. A first diagnostic test was developed in 1989 [144], but reliable second and third generation tests became generally available in 1991 and were then immediately made obligatory for the screening of every blood donation. That measure virtually closed a previously predominant transmission pathway (blood, blood products), and needle sharing among intravenous drug users remained the main transmission route. Transmission by sexual contacts and vertical transmission are relatively rare events compared to the frequency of these transmission pathways being used by other bloodborne viruses (HBV, HIV). The infection resolves in only 20% of the cases; in 80% a chronic hepatitis results, along with an increased risk for the development of hepatocellular carcinoma (HCC). Chronic HCV infection is very

Emerging and Reemerging Viral Pathogens

11

difficult to manage. HCV-infected individuals with chronic liver disease are the most frequent subpopulation of patients becoming candidates for liver transplants. The seroprevalence of HCV infection in blood donors worldwide is between 0.02% and 1.2%, with higher rates in Japan, Spain, Hungary, Italy, and Saudi Arabia [145]. Exceptionally high donor infection rates of almost 20% have been recorded in Egypt [146]. Hepatitis C viruses are highly heterogeneous, and at least six different HCV types with 11 subtypes are recognized at present [147]. Treatment is by a-interferon [148,149], more recently in combination with other antiviral agents (ribavirin, lamivudine), but is of limited success; there is a high relapse rate after cessation of treatment. Different HCV types vary in responsiveness to interferon treatment, with type 1 strains being more resistant than type 2 and 3 strains [149]. 2.11.2

Hepatitis E Virus (Caliciviridae)

Hepatitis E virus (HEV) infection became known as a separate entity in the late 1980s and was termed epidemic non-A, non-B or enterically transmitted non-A, non-B (ENANB) hepatitis. It followed a transmission pathway and caused disease similar to hepatitis A but was not reactive with HAV-specific serological assays. In 1990 Reyes et al. [150] succeeded in cloning and sequencing part of the genome of this virus. The complete sequence of a number of isolates has now been determined [151–156], and the ENANB virus has been renamed hepatitis E virus (HEV). Its genome is similar to that of caliciviruses; however, the order of genes in HEV is not identical, and therefore HEV may be placed into a separate family at some stage [143]. The genomes of several HEV strains from Asia and Mexico have been entirely sequenced, and partial sequences are available for some other strains [157]. In all parts of the genome the Mexican strain was most different from the sequences studied [152]. Although moderate genetic heterogeneity has been identified among HEV strains, evidence for serological heterogeneity is limited [158]. The course of infection in experimentally infected primates is similar to that in humans [159,160]. The incubation period is 3–8 weeks, followed by an increase of liver enzyme concentrations in the blood. Peak viremia and shedding of HEV in feces occur during the incubation period and the very early acute phase of disease. In most cases the infection resolves completely. However, the severity of HEV infections is on average somewhat greater than the severity of HAV infections. Mortality of hepatitis E has varied in different reports and has been as high as 1%, compared to 0.2% of hepatitis A [161]. More important is the severity of hepatitis E in

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pregnant women, the mortality in pregnancy increasing with each trimester and possibly reaching 20% [161–163]. The reason for the excessive mortality of hepatitis E in pregnancy is at present unknown. The diagnosis is based on HEV-specific IgM and IgG ELISAs, using recombinant expressed capsid antigen [158,162,164]. The age-specific clinical attack rate with its peak among young adults is striking. In endemic areas the age-related prevalence of HEV antibodies reaches only 40% [165–167]. There is at present no vaccine, but protection of monkeys against experimentally induced hepatitis E by vaccination with recombinant HEV proteins has yielded encouraging results.

2.11.3

Hepatitis F Virus (Paramyxoviridae?)

In 1991, Phillips et al. [168] described 10 cases of a syncytial giant cell hepatitis observed sporadically in the United States between 1979 and 1988 and associated with a severe clinical course. The virological studies suggested a paramyxovirus as putative cause because paramyxoviruslike nucleocapsids were found by electron microscopy in patients’ livers (8/10). Chimpanzees inoculated with infected liver homogenate raised an antibody response that was cross-reactive with measles virus and parainfluenza virus type 4; however, the animals did not develop a hepatitis. Although this putative viral infection figured as hepatitis F at the time, the work by Phillips et al. [168] has not been pursued further.

2.11.4

Hepatitis G Virus (Flaviviridae)

Hepatitis G virus (HGV), also a member of the Flaviviridae family, was discovered in 1993 from cloned cDNA fragments in blood donations. Subsequently, sequences of this virus were found in 1–3% of all blood donations in different parts of the world; it seemed to replicate in liver cells and was thought to be associated with hepatitis. However, a close association of infection by this virus with liver disease has so far not been secured [169,170], and replication in the liver has not been confirmed (P. Simmonds, personal communication). Therefore testing of blood donations for the presence of HGV sequences is at present not mandatory. Recently, two reports appeared demonstrating significantly higher survival rates in HIV-infected patients coinfected with the HGV (GB virus C) (170a, 170b). The HIV load was significantly lower in the coinfected patients (170b). The mechanisms underlying this remarkable effect remain to be explored.

Emerging and Reemerging Viral Pathogens

2.12

13

Transfusion-Transmitted Virus (Circoviridae)

In 1997 transfusion-transmitted virus (TTV) was discovered as the cause of some cases of hepatitis transmitted by infected blood donations [171]. The prevalence of TTV antibodies in various populations was found to be very variable [172]. TTV is the first human-transmitted member of the Circoviridae family [143] (P. Simmonds, personal communication). The virus was found as a coinfection in HCV-infected patients but the two viruses reacted differently upon treatment with interferon [173]. In an American study, one-third of healthy blood donors were found to be infected with TTV. A connection with disease is still being debated [174]. Significant numbers of chickens, pigs, cows, and sheep were found to be infected [175]. Erker et al. [176] found sequence diversities of up to 30% among more than 10 full-length genomic sequences of TTV isolates. This very substantial amount of variation suggests that there are at least three types of the virus. Ball et al. [177] followed up several chronically HCVinfected patients longitudinally and found in some a stable form of TTV over several years; however, in others there were fluctuating levels of at least seven distinct variants of the virus over a 5-year period. The natural history of TTV is rapidly expanding; the high prevalence of TTV worldwide with apparently no significant associated disease is astonishing [174]. Another virus of this family, the recently discovered SEN virus, was found to be common in people at high risk for bloodborne viral infections in Taiwan but not to be significantly associated with hepatitis [177a].

2.13

Influenza Viruses (Orthomyxoviridae)

Type A and B influenza viruses regularly cause outbreaks of severe respiratory disease in large segments of the world’s population during winter and spring. Several influenza A virus pandemics have been described (caused by subtype H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, and H1N1 in 1977). Influenza viruses have a wide animal reservoir, and, by the mechanism of reassortment, animal type A influenza viruses have contributed genes, for instance those coding for hemagglutinin H3, to viruses that became human pandemic viruses. It has been shown that human H1N1 and also H3N2 influenza A viruses can infect pigs and, vice versa, that related pig viruses can infect humans. By contrast, avian influenza viruses, representing by far the greatest diversity and biggest reservoir of influenza A viruses, are thought to circulate only within their original host or closely related species. Contribution of avian genes into viruses able to replicate in humans until recently was thought to be possible only by reassortment, pigs being the likely host, because they

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were shown to be able to replicate avian influenza viruses to a certain extent (‘‘mixing vessel’’ theory of Scholtissek et al. [178]). Recently, however, different events were recorded. During 1997 at least 18 people in Hong Kong came down with influenza-like symptoms, some of them with severe generalized disease, which was found to be caused by influenza A viruses of the H5N1 subtype. Six out of 18 patients with confirmed H5N1 influenza died. Six out of 18 patients with confirmed H5N1 influenza died. Viral isolates were very closely related in all their eight RNA segments to viruses isolated from chickens on several farms in and around Hong Kong in the spring of 1997. From the molecular data it could be excluded that reassortants between animal and human strains had been formed. This was the likely reason for the inability of these viruses to spread from human to human and thus widely within the human population. In fact, human-to-human transmission was never convincingly recorded. The recent isolation of H5N1 influenza A viruses from humans was seen as the possible advent of a new pandemic strain. The first virus was isolated in May 1997 in Hong Kong from a 3-year-old boy who subsequently died of Reye’s syndrome after treatment with aspirin. A very close surveillance ensued from this case, and in the following 8 months a total of 16 more confirmed and two suspected human cases of infection with influenza A virus of subtype H5N1 were observed. Twelve of the 16 patients became ill in December 1997, and rapid diagnosis was the result of intensified surveillance in hospital and healthcare centers. Seven patients were under the age of 5 years, three between 5 and 14, and six over 14 years. Whereas in patients under 5 the infection was generally mild, in the older patients there was a high rate of complications such as gastroenteritis and renal and liver dysfunction [179]. There was an H5N1 epidemic in chickens between March and May 1997 in Hong Kong and southern China. Extensive epidemiological surveillance has so far not revealed significant spread in humans. Most important, a human-to-human infection has not been definitely proven. A close virological investigation, including partial sequencing of the whole genome, has shown that in at least the first cases the H5N1 isolate was a true avian isolate, i.e., all eight segments were of avian origin [180,181]. This finding greatly decreases, but does not exclude, the likelihood that these viruses may spread widely in humans. The World Health Organization (WHO) and various nations have worked out plans to cope with the sudden emergence of a new pandemic influenza virus strain. In the United Kingdom, both the Department of Health and the Public Health Laboratory Service have such plans in place. With the emergence of the first isolate in May 1997 in Hong Kong,

Emerging and Reemerging Viral Pathogens

15

stage 1 of the plan was activated, entailing constant review of the situation and signifying increased surveillance of both humans and animals. By the end of 1997, the Hong Kong government took the bold step of killing the chicken population of Hong Kong (approximately 1.5 million) [182,183]. WHO sent a fact-finding mission to southern China whose participants found relatively intensive surveillance practices in place. No human cases or seroepidemiological evidence of wider spread of this virus in animals or humans has been found so far. The finding of 16 cases of human infection with H5N1 strains that are ‘‘pure’’ avian viruses is in itself a highly unusual event, and these isolates will be scrutinized very intensely for factors that might have changed their host tropism and allowed the emergence of pathogenicity for humans. The human isolates were found to remain pathogenic for chickens [180,181]. It is possible that an unusual sequence around the trypsin cleavage site of the H5N1 viruses is in part responsible for the wider host spectrum [180,181,184,184a]. A mutation in the PB2 gene was found to be correlated with pathogenicity in mice [184a]. In March 1999 two isolates of influenza A virus of subtype H9N2 were obtained from two children with influenza in Hong Kong [185]. Genetically all the genes except for hemagglutinin (HA) and neuraminadase (NA) were very similar to those of the H5N1 viruses, suggesting that these genes may be important for efficient transmission from birds to humans [184,186]. Thus, the animal influenza virus reservoir is a permanent threat for transmission of infectious agents to humans. An influenza A virus of subtype H7N7 was isolated in 1998 from an inflamed eye of a woman in Oxfordshire who was keeping ducks on a pond. No such virus was isolated from the woman’s ducks, but molecular analysis showed the human isolate to be of animal origin [187] and to be closely related to an H7N7 virus isolated from a turkey in Ireland in 1995 [188]. There is the sword of Damocles hanging over the population of Hong Kong, southern China, and the whole world that avian influenza viruses carrying H4–H14, which have so far not circulated in humans, might reassort somewhere with influenza A viruses that replicate well in humans, leading to a new pandemic strain that could spread rapidly throughout the world (similarly to the ‘‘Asian’’ and ‘‘Hong Kong’’ influenza viruses in 1957 and 1968, respectively). Such an event could indeed occur relatively easily in southeast Asia, where humans and domestic animals live in very close proximity, often under the same roof [184,189]. Thus, although at present there is no acute cause for alarm, a high degree of vigilance is clearly indicated.

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New Paramyxoviruses (Paramyxoviridae)

In September 1994 an outbreak of severe respiratory disease affected 18 horses, their trainer, and a stablehand in Queensland, Australia. Fourteen horses and one human died. A novel virus was isolated from those affected and named equine morbillivirus (EMV) or Hendra virus [190]. In the following year several other humans became infected with this new virus, most of them developing meningitis and encephalitis [191]. Serological evidence showed that a paramyxovirus related to EMV was present in Pteropus, a species of fruit bat [190,192]. In the autumn of 1998 through the spring of 1999 an outbreak of encephalitis in pig farmers and slaughterhouse workers in Malaysia and Singapore occurred, with more than 250 cases and over 100 deaths. There were also sick animals (pigs, dogs, and cats). A virus was grown from patient material that formed syncytia in Vero cells and yielded positive immunofluorescence with a Hendra virus–specific antiserum. The virus was termed Nipah virus and found to be homologous to 89% at the nucleic acid level with Hendra virus. However, it is clearly different from other paramyxovirus genera, so a new genus is proposed [193,193a]. Recently a cytopathic infectious agent was isolated from the kidneys of an apparently healthy tree shrew (Tupaia belangen) that had been captured in the area of Bangkok. The virus turned out to be a paramyxovirus termed tupaia paramyxovirus (TPMV), and partial sequences of its genome (more than 4000 nucleotides) showed that this virus had the highest homologies with Hendra virus [194]. This supports the hypothesis that new human morbilliviruses are likely to be derived from animal reservoirs. 2.15

Human Metapneumovirus (hMPV)

Metapneumovirus is a second genus of the subfamily Pneumovirinae of the Paramyxoviridae family. Until recently it was considered to infect only birds, e.g., the turkey rhinotracheitis virus (TRTV). In June 2001 a report was published of novel viral isolates obtained from acute respiratory tract infections in children in the Netherlands during the winter [195]. The genomes of these viruses termed hMPV had 60–80% sequence homology with genes of TRTV (not all genes of hMPV have been identified yet). Preliminary serological data suggest that by the age of 5 years, >70% of children had been infected with this virus; practically 100% of adults are seropositive [195]. Preliminary data from England of clinical pediatric respiratory samples that had been negative for other known viruses yielded 10% positivity for incidence of infections with this virus and the virus detected in the respiratory tract of immunocompro-

Emerging and Reemerging Viral Pathogens

17

mised adults (P. Cane, personal communication), and the virus has now also been found in Australia [195a] and Canada [195b]. 2.16

Enterovirus 71 and Aichivirus (Picornaviridae)

Enterovirus 71 (EV71), one of the major causative agents of hand, foot, and mouth disease (HFMD), is also sometimes associated with severe central nervous system disease. HFMD epidemics were recorded in Malaysia and Japan in 1997 and in Taiwan in 1998. They resulted in sudden death among young children, often from encephalitis, and were mainly due to the A-2 B genotypes of this virus [196]. Central nervous system complications were observed in previous HFMD outbreaks [197– 199]. By contrast, in large HFMD epidemics in Japan in 1973 and 1978, there was hardly any CNS involvement [200]. In 1989, an outbreak of gastroenteritis occurred in the Aichi prefecture, Japan, for which a new enterovirus, termed Aichi virus, was found to be the causative agent [201,202]. This virus was recently defined as a new genus (Aichivirus) of the Picornaviridae family [143,203]. The virus is also found to cause sporadic cases of diarrhea, mainly in travelers in Southeast Asia [202,204]. 2.17

West Nile Fever Virus (Flaviviridae)

West Nile (WN) virus has long been known for its wide host spectrum (including mammalian, avian, amphibian, and insect species) and was found to infect humans in many parts of Africa, eastern Europe, and Asia [205]. It emerged in the western hemisphere in 1999 when it infected 59 mainly elderly people in the New York City area; those infected developed fever, headache, rashes, myositis, polyneuropathy, meningitis, or meningo-encephalitis, and seven died [206,207]. The virus was also isolated from sick crows and other birds and mosquitoes [208] and found to be closely related genetically to a Middle Eastern virus isolate [206]. Despite extensive efforts to eliminate the virus by vector control, it reappeared in 2000 in New York City and New Jersey [209] and spread into the bird population of the east coast of the United States. Molecular analyses of a number of WN virus isolates support the model of migrating birds as hosts that spread the virus to local mosquito populations along their migrating routes; the spread into North America may also have been due to an infected traveler [205]. Physicians in the eastern United States have now been asked to consider WN virus infection in their differential diagnosis in hospitalized patients with encephalitis (particularly when occurring in conjunction with muscle weakness) and in adults with viral meningitis [207]. By 2002, WN virus

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had spread in animals and humans throughout the midwestern United States and California [207a].

2.18

Cantalago Virus (Orthopoxviridae)

A virus isolated from vesicular lesions of cattle and milkers in Cantalago County, Rio de Janeiro State, Brazil, turned out to be an orthopoxvirus and was genetically most closely related to the vaccinia virus strain (VVIOC) used for over 20 years in Brazil. The data suggest that Cantalago virus derived from VV-IOC after long-term persistence in an as yet unidentified animal reservoir [210].

2.19

Borna Disease Virus (Bornaviridae)

Borna disease virus (BDV) has for a long time been recognized as the causative agent of chronic neurodegenerative disease in horses, sheep, cattle, and other vertebrates. It also replicates in experimentally infected rats. The virus is the prototype of the Bornaviridae family in the order Mononegavirales [211]. After infection in the periphery (olfactory epithelium), the virus is transported intra-axonally toward the CNS, which it infects, with a preference for the limbic system [212]. The disease, a nonpurulent encephalomyelitis, develops by a T-cell-dependent immune mechanism and is characterized clinically by movement abnormalities and by hyperactive and abnormal behavior followed by apathy, somnolence, and depression. In the neonatal rat a chronic persistent infection without immune response and without clinical signs develops (persistent tolerant infection of the newborn). These different features of disease in animals have led to the development of animal models for neuropsychiatric disorders [213]. The virus came to wide attention around 1995 when antibody to the virus was found in the sera of patients with mood disorders [214]. The advent of RT-PCR led to reports of human BDV isolates being obtained from blood and tissues of psychiatric patients [215,216], but these data require broader confirmation [217,218]. A recent review [217] concluded that ‘‘a critical evaluation indicates that no laboratory has to date been able to present solid evidence that BDV is infecting humans.’’ The validity of testing for BDV antibody in human sera has recently been questioned [219], but highavidity antibodies against BDV-specific peptides have been found in human sera [219a].

Emerging and Reemerging Viral Pathogens

TABLE 2

19

Zoonotic Origin of Emerging Viral Pathogens in Human Disease

Virus (genus or type species)

Human disease

Animal source

Probabilitya

Calicivirus Rotavirus Ebola virus Sin nombre virus HIV HEV Influenza virus

Diarrhea Diarrhea Hemorrhagic fever HFRS, HPS

Swine, cattle Swine, cattle Monkeys Rodents

L L P C

AIDS Hepatitis Influenza

Hendravirus

Meningoencephalitis

Monkeys Swine Pigs Horses Birds Fruit bat

L/C P reassortants L reassortants L C L

Nipahvirus

Encephalitis

Metapneumovirus

Respiratory tract infection Encephalitis Vesicular rash Encephalomyelitis

Tree shrew Pigs Dogs Birds

L L P ??P

Birds Cattle Horses

C C ??P

Cattle

P

West Nile virus Cantalagovirus Borna disease virus BSE agentb a b

nvCJD

P, possible; L, likely; C, confirmed. See Table 1, footnote b.

2.20

Transmissible Bovine Spongiform Encephalopathy and New Variant Creutzfeldt-Jakob Disease (nv-CJD)

During the mid-1980s, a rapid spread of bovine spongiform encephalopathy (BSE) was observed in British cattle herds. The likely origin of this infection was the scrapie agent, one of the prion agents [220], originating from meat and bone meal used for cattle feed in 1981–1982 when a previously established processing step of extraction with solvents and hot steam was omitted. It was then calculated that an incubation period of 4–5 years had to be taken into account, and it was hypothesized that 5 years would have to pass after a ruminant protein ban was set in force in July 1988 before the epidemic would peak. The peak of the epidemic occurred in 1994. During the epidemic, BSE cases within a herd remained constant at approximately 4%, but over the years more herds became

SRSVs (NLVs, SLVs) Rotavirus Astrovirus Parvovirus B19 Ebola virus Hantavirus HTLV-1 HIV HHV6/7 HCV HEV SNV HHV-8 Hendra virus Nipahvirus Influenza virus WNV Metapneumovirus Alchi virus X

X

X

X

X

X X

Genetic changes/ evolution of viruses

X X X X

X

X

X

Globalization: industry, trade, travel

X

X X

Changed behavior: IVDU, etc

X?

X

X

X X

Changes in environment: deforestation, urbanization, industrialization

X?

X?

X

X?

X X X

Contaminated food or water

X

Civil unrest, wars, refugee camps

X?

X?

X

Anti-microbial resistance

X

X X

X

X

Immunosuppression

X X X X X X X X X X X

X X

X X X

Improved surveillance: clinical, diagnostic, epidemiological

Factors Contributing to Emergence of Viral Pathogens in Humans (Other Than Animal Reservoir)

Emerging viruses

TABLE 3

20 Desselberger

Emerging and Reemerging Viral Pathogens

21

infected. The reason for this finding was likely the continuation of cattleto-cattle recycling for food purposes, which may have gone on for some time after the initiation of the ruminant protein ban. In early 1996 several cases of a rapidly progressive form of Creutzfeldt-Jacob disease (CJD) in humans were described that also seemed to differ pathologically from previously identified forms [220,221]. Some molecular data point to the possibility that the causative agent of this so-called new variant CJD (nvCJD) may be the BSE agent [222,223]. However, the epidemiology of nvCJD has so far not confirmed this hypothesis [224,225]. By 21 March 2002, there were 109 confirmed cases of nvCJD counted and seven awaiting confirmation [226]. The predictions of how many cases may become apparent over the next 3–5 years vary grossly [227]. One major unknown in this calculation is the uncertainty about the variability of the incubation period. There has been enormous progress in recognizing the nature of the transmissible agent and the molecular genetics of the prion–host relationship but major riddles remain [224,227a].

3

ANIMAL RESERVOIRS FOR EMERGING VIRUSES

Long before 1972, animal reservoirs for human viral infection were a well-known fact (e.g., rabiesvirus, herpesvirus B, influenza viruses, bunyaviruses, flaviviruses). It is remarkable that many of the new emerging viruses do or may originate from an animal reservoir. In Table 2 an attempt is made to assess the significance of animal reservoirs for human infection by emerging viruses. Although in detail some of the considered possible links may not stand up to scrutiny, the concept is compelling and should lead to close surveillance of animal as well as human populations for emerging diseases and their causes.

4

SIGNIFICANCE OF OTHER FACTORS INVOLVED IN VIRAL EMERGENCE AND REEMERGENCE

Besides animal reservoirs, numerous other factors mentioned in the Introduction contribute to the emergence and or reemergence of viral (and other) infections. Table 3 is an attempt to allocate certain factors to the viral infections reviewed in the preceding sections. The evolution of viruses, forces driving globalization, changes in the environment, food contamination, and immunosuppression are recognized as major factors. It is very clear that improved tools of surveillance have significantly helped to recognize emerging infections early.

22

Desselberger

The issue of emerging, antiviral drug-resistant virus mutants is already of major concern for the highly active antiretroviral therapy (HAART) of HIV infection [112–115]; is well described for herpes simplex viruses, cytomegalovirus, influenza viruses, and others; and is likely to become a very significant factor in virus evolution as increasing numbers of antiviral drugs are being developed and applied. In fighting emerging infections, the balance of treatment with antiviral agents, immunization procedures, and exposure prophylaxis will have to be constantly reviewed and redefined. More detailed considerations on this topic will be found in other parts of this book. 5

EMERGING NONVIRAL PATHOGENS

Besides viruses, many other microorganisms (bacteria, fungi, parasites) have emerged as human pathogens. These have been reviewed elsewhere [e.g., refs. 1,6,11,15]. 6

CONCLUSIONS

Increases in population sizes, global travel, and changes in the ecology all contribute to the assumption that more new infectious diseases will arise in the future. It is likely that there are more viruses around causing hepatitis than are recognized at present, and the number of human retroviruses (including endogenous retroviruses) is likely to be underrecognized. Clinical attentiveness; good laboratory facilities, including the application of molecular identification techniques; and comprehensive epidemiological surveillance systems for infections in both humans and animals, which often form a reservoir for human infections, have to be combined for early recognition of emerging infections. Lack of facilities can have adverse consequences and can lead to misdiagnoses. Finally, economic enablement in the public health sector will be necessary to allow early recognition and comprehensive management of emerging infections. Given the record of emerging and reemerging microorganisms as the cause of infectious diseases over the last three decades there is every prospect of this continuing for some time, and high vigilance in detecting them is of paramount importance. Acknowledgments The author gratefully acknowledges the support of Lynne Bastow and Narguesse Stevens, who typed and processed the manuscript, and the

Emerging and Reemerging Viral Pathogens

23

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2 Influenza: The Virus, the Disease, and Its Control Thorsten Wolff Robert Koch-Institut, Berlin, Germany

Rene´ Snacken Scientific Institute of Public Health, Brussels, Belgium

1

INTRODUCTION

Influenza is a highly contagious acute respiratory disease that has global significance because it affects all age groups and can recur in any individual. The etiologic agent of the disease, influenza virus, was first isolated in 1933 [180] and has served since then as a paradigm of an important viral pathogen. Thus, fundamental principles such as antigenic drift and shift have been recognized with influenza viruses. Molecular analyses have revealed that the unique potential of influenza viruses to cause epidemics annually and pandemics occasionally is based on an amazing variability of its segmented negative-strand RNA genome. This is reflected in the existence of several antigenically distinct subtypes, a wide host range that comprises a variety of mammalian and avian species, and the propensity to escape from selective conditions such as neutralizing antibodies or antiviral drugs by rapid mutation. Thus, influenza viruses have in the past equally concerned basic life 39

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scientists, clinicians, public health authorities, and pharmaceutical companies. Undoubtedly this will continue in the future, because influenza viruses have a large natural host reservoir in feral birds that provide an ineradicable pool for viral genes that may emerge in future novel pandemic virus strains. Here we discuss current knowledge and recent developments in the different fields of molecular biology, antiviral therapy, and immunoprophylaxis that together promise improved understanding, control, and management of influenza in the future.

2

THE BURDEN OF INFLUENZA

Influenza epidemics and pandemics impose a considerable socioeconomic burden on individuals and society, which is due to morbidity and mortality and direct medical costs as well as to indirect economic losses related to work absenteeism and decrease in productivity [62,108,187]. Whereas uncomplicated influenza is a self-limited disease, severe respiratory or systemic complications may develop that require in-patient medical attention (see Sec. 6). A recent analysis reported an average of 49 pneumonia and influenza-associated hospitalizations per 100,000 persons among all age groups during the years 1970–1995 in the United States (average number of annual influenza-associated hospitalizations: 114,000) [178]. The relative risk for hospitalization is highest among the very young (birth to 1 year) and the elderly (>65 years) [18]. Influenza and pneumonia together were ranked the seventh most frequent cause of all deaths in the United States in 1999 [2]. However, precise quantification of the impact of influenza on mortality is difficult, because the infection is not routinely confirmed by laboratory diagnostics and because impact estimates are not transposable from country to country. Influenzaassociated mortality is usually expressed as the number of deaths during seasonal virus circulation that exceeds a projected baseline level of expected deaths that occur in the absence of influenza. The available U.S. data indicate that between 1972 and 1992 influenza was responsible on average for 21,300 fatalities per year, with great variation among seasons (range: 0–46,200 deaths) [177]. In interpandemic years, more than 90% of all influenza victims were older than 64 years, indicating a distinct need for improvement of immunoprophylaxis in this age group [18]. In total, interpandemic influenza years accounted for many more deaths than pandemic-associated fatalities. Immunization and the use of antiviral agents for prophylaxis and treatment can only reduce the impact of the disease; vaccine and antiviral agents are not exclusive but complementary for controlling the disease.

Influenza

3

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INFLUENZA VIRUSES—STRUCTURAL AND MOLECULAR PROPERTIES

Influenza viruses are divided into types A, B, and C on the basis of antigenic differences of their matrix- and nucleoproteins. Influenza viruses are systematically specified by indication of the particular virus type (A, B, or C), the species, the geographic site from which the virus was isolated, a strain number, and the year of isolation, as exemplified in A/Swine/Iowa/15/30. For human strains, indication of the host species is omitted as in A/HK/1/68. The influenza A viruses are epidemiologically most relevant and are considered as the prototype of the Orthomyxoviridae. These viruses not only infect and replicate in humans but have also been isolated from pigs, horses, minks, and marine mammals as well as from domestic and wild aquatic birds. Only type A influenza viruses are further categorized into subtypes that reflect antigenic differences in their two major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Currently, we know of 15 different HA subtypes (numbered H1–H15) and nine NA subtypes (N1– N9), which have all been found in viruses isolated from feral birds. Human influenza A virus strains that have caused epidemics and pandemics in the last century belonged to the groups H1N1, H2H2, and H3N2. However, future epidemic strains may carry other HA subtypes as suggested by the recent sporadic occurrences of human infections by H5N1 and H9N2 viruses [26,102]. Influenza B viruses can cause similar severe disease symptoms; however, the host spectrum of influenza B viruses is restricted mainly to humans, which lowers their capacity for genetic alterations. This chapter focuses on type A and B influenza viruses, because influenza C viruses are less prone to antigenic changes and are clinically less relevant. In the laboratory, influenza viruses can be propagated in embryonated chicken eggs or a variety of standard tissue culture cell lines such as Madin-Darby canine kidney (MDCK). Virus titers are usually quantified either by plaque assays on tissue culture cells or by hemagglutination of erythrocytes [6]. Accordingly, virus titers are given either in plaque-forming units (PFUs) or in hemagglutination units (HAUs). Moreover, hemagglutination of erythrocytes by influenza viruses can be inhibited by HA-specific antibodies in immune sera. Thus, titers of antisera can be expressed as their activity in hemagglutination inhibition (HI) [221]. Influenza A and B viruses are characterized by an outer membrane envelope and a genome that consists of eight single-stranded RNA segments of negative polarity (complementary to mRNA sense). The

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FIGURE 1 Structure of influenza viruses. (A) Electron micrograph of influenza B viruses (magnification 6140,000). (Courtesy of Hans Gelderblom, RKI Berlin.) (B) Schematic drawing of a virion particle and of viral structural proteins.

diameter of spherical virion particles (Fig. 1) is in the range of 80–120 nm. The genomic viral RNAs (vRNAs) are replicated in the nucleus of the infected cell via synthesis of positive-strand copy RNA (cRNA) intermediates that serve as template for the production of new vRNA molecules [99,107]. The viral RNA segments are between 2.4 and 0.9 kilobase (kb) in size, adding up to a total of about 13.6 kb (type A) and 14.6 kb (type B), respectively (Table 1). The coding capacities of these viruses comprise 11 known proteins each, which is relatively few in comparison to large DNA viruses such as herpes- or poxviruses, which encode more than 300 viral gene products. The vRNA segments carry short stretches of conserved nucleotides (nts) at their 50 (type A: 13 nts; type B: 11 nts) and 30 ends (type A: 12 nts; type B: 9 nts), respectively. These sequences are in part complementary, and thus the ends of viral RNAs can engage in base-pairing interactions resulting in a partially double-stranded promoter structure that is recognized by the viral RNA polymerase. More than half of the genomic information is dedicated to the four replicative proteins PA, PB1, PB2, and NP [86]. The former three proteins assemble into a trimeric RNA-dependent RNA polymerase that provides the enzymatic functions for replication and transcription. The nucleoprotein NP and the polymerase encapsidate the genomic RNA, forming a viral ribonucleoprotein (vRNP). Within the virion, the vRNPs are embedded into a layer of the viral M1 matrix protein that is associated with a few copies of the viral NS2/NEP protein, a putative viral RNA export factor. The viral envelope contains three viral transmembrane proteins: the trimeric hemagglutinin (HA), the tetra-

Influenza

TABLE 1

43 Influenza A and B Viral Genomesa A/PR/8/34 Encoded protein (AA)

B/Lee/40

vRNA (nts)

Encoded protein (AA)

Segment

vRNA (nts)

1

2341

PB2, 759

2396

PB2, 769

2

2341

PB1, 757

2368

PB1, 752

PB1-F1, 87

3

2233

PA, 716

2304

PA, 726

4

1778

HA, 566

1882

HA, 584

5

1565

NP, 498

1841

NP, 560

6

1413

NA, 454

1557

7

1027

M1, 252 M2, 97

1191

NA, 466 NB, 100 M1, 248

8

890

NS1, 230

1096

BM2, 109 NS1, 248

NS2/NEP, 121

NS2/NEP, 122

Functions(s) Subunit of viral RNA polymerase; capbinding Catalytic subunit of viral RNA polymerase Mitochondrial localization; induction of apoptosis Subunit of viral RNA polymerase Surface glycoprotein; receptor binding, membrane fusion Nucleoprotein; encapsidation of viral genomic and antigenomic RNA Neuraminidase Putative ion channel Matrix protein Ion channel; acidification of virions, protecting HA conformation Structural protein Post-transcriptional regulator: inhibition of pre-mRNA splicing, polyadenylation and PKR activation Nuclear export factor

a The lengths of the eight viral RNA segments and the encoded polypeptides of the influenza A/PR8/34 and B/Lee/40 viruses are given in nucleotides (nts) and amino acids (AA), respectively. The functions of the gene products are indicated in the rightmost column.

meric neuraminidase (NA), and the M2 ion channel (type A) or NB protein (type B). Influenza viruses express one major nonstructural polypeptide in infected cells that is designated NS1. The NS1 protein has several regulatory functions at the post-transcriptional level, as it inhibits host

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cell RNA processing and transport [25,143,214]. Furthermore, the NS1 protein antagonizes activation of unspecific cellular defense mechanisms by its ability to bind to double-stranded (ds) RNA that can be generated during replication of the negative-sense vRNA through the plus-sense cRNA intermediates [70,106]. This property prevents both the activation of the dsRNA-activated protein kinase (PKR), which downregulates cellular translation, and activation of the transcriptional regulators NF-Kb, ATF-2/c-Jun, and IRF-3, which induce expression of type I interferons (IFNs) [55]. IFNs are secreted from infected cells and induce in neighboring cells the expression of gene products that establish an antiviral status [183]. Influenza viruses apparently counteract activation of this defense mechanism through NS1-mediated masking of dsRNA. Consequently, an influenza virus with a genetic knockout of the NS1 gene was completely avirulent in animal studies and replicated only in interferon-deficient hosts to considerable titers [56]. There are two viral proteins that are uniquely found in either type A or type B influenza viruses. A very recent discovery is the expression by influenza A viruses of the 87 amino acid PB1-F2 protein that is transported into mitochondria and has the ability to induce apoptosis [24]. The capability for PB1-F2 expression is not conserved in all virus strains and has been shown to be dispensable for efficient viral replication in tissue culture cells. It remains to be determined if and how the PB1-F2 protein contributes to viral virulence and/or pathogenesis. Only influenza B viruses express the BM2 polypeptide that is incorporated into the virion, but its function has not been recognized yet [141]. The main target tissue of human influenza viruses is the epithelial cell layer that lines the respiratory tract. The viruses initiate infection by binding of the HA to sialic acids attached to cellular surface sialoglycoproteins or sialoglycolipids followed by internalization of the virus through receptor-mediated endocytosis [114] (Fig. 2). Two events that are crucial for the establishment of the infection occur in the low-pH environment (pH 5–6) of the endosome. First, the ion channel activity of the M2 protein facilitates influx of protons into the interior of the virion, which destabilizes the tight association of the viral vRNPs with the M1 protein [83,154]. It is the acidification through the M2 ion channel that is blocked by amantadine and rimantadine, the first discovered class of compounds with anti-influenza virus activity (see Sec. 7). Second, the HA undergoes a major structural rearrangement leading to the exposure of a short a-helical hydrophobic domain that initiates the fusion of viral and endosomal membranes [179].

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FIGURE 2 Replication cycle of influenza viruses in an infected cell. See text for a description of the steps involved. The virus and the cellular structures are not drawn to scale.

After fusion, the vRNPs are released into the cytoplasm and are transported to the nucleus, where they serve as templates for transcription of viral mRNA transcripts. Synthesis of viral mRNAs is primed by capped oligonucleotides of 10–13 bases that the polymerase acquires by nucleolytic cleavage from the 50 ends of cellular mRNAs [156]. Polyadenylation at the 30 end occurs through a ‘‘stuttering’’ mechanism at an oligo-U signal in the vRNA template [109]. Mature viral transcripts are exported to the cytoplasm, where they are translated. Influenza viruses efficiently downregulate cellular protein synthesis, whereas translation of viral transcripts is maintained at high levels [57]. Replication of the negative-strand RNA genome through synthesis of positive-strand cRNA intermediates is a primer-independent process and continues for several hours [174]. Newly replicated vRNPs are exported to the cytoplasm in the late stage of infection, which is presumably mediated through the activity of the M1-NS2/NEP complex [140]. Recent analysis suggests that this step requires activation of the intracellular Raf/MEK/ERK signaling cascade [155]. Subsequently, the

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vRNPs assemble with other viral structural proteins at the plasma membrane into new virions that are released to the exterior by budding. The activity of the viral neuraminidase in removing sialic acids from cellular and viral glycoproteins is critical to prevent retention and aggregation of virions at the plasma membrane [151]. Therefore, the recently developed neuraminidase inhibitor substances inhibit the release of progeny viruses from the cell. A prerequisite for the initiation of a new replication cycle is proteolytic cleavage activation of the viral HA by secreted proteases. For most human influenza virus strains, cleavage of the HA precursor occurs at a single arginine residue, which generates the HA1 and HA2 subunits [97]. This is an essential step, because only cleaved HA molecules can undergo the pH-dependent structural rearrangement in the endosome that leads to membrane fusion and release of vRNPs (reviewed in Ref. 179). Only a few cellular proteases, including plasmin and tryptase clara, have been identified that function in HA cleavage activation in humans or animals [94]. In viral infections of tissue culture cells, trypsin is added as an adequate enzyme. Depending on the cell type and the strain, influenza virus infections normally result in lysis and death of the cell. Influenza viruses became amenable to targeted genetic alteration more than a decade ago [44]. However, efficient procedures that allow rapid and systematic reverse genetic analysis through de novo generation of recombinant influenza A viruses from transfected cloned cDNA were established only recently [50,132]. In essence, tissue culture cells are cotransfected with four plasmids expressing the viral replicative proteins and eight plasmids, each of which encodes a complete viral gene segment in an RNA polymerase I expression cassette. The viral RNAs are transcribed within the cell and packaged into ribonucleoprotein by the viral NP and polymerase. This, in turn, allows expression of all viral gene products and the assembly of new infectious virus particles. It is expected that this methodical breakthrough will greatly stimulate and accelerate basic research on virus pathogenicity and host cell tropism. Moreover, these systems should also have great potential in the development of novel tailormade influenza vaccines and expression vectors.

4 4.1

EPIDEMIOLOGY, EVOLUTION, AND HOST RESERVOIRS OF INFLUENZA VIRUSES Epidemiology

Influenza A and B viral infections are a significant cause of morbidity and mortality, because they affect all age groups and, in contrast to many

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other pathogens, can occur repeatedly in all persons. In epidemic years attack rates of influenza can reach 10–20% of the total population [35,61]. In this section we discuss epidemiological and molecular data that are relevant for the evolution, spread, and survival of these viruses in the population. Human epidemics in the last century were caused by three different HA subtypes of influenza A viruses that circulated during different decades. Thus, the epidemiology of these viruses is characterized by the predominance of one or two subtypes that can last for decades. The introduction of a new subtype that succeeds in establishing an independent evolutionary lineage is usually associated with a pandemic and often results in the extinction of the previous predominant strain. The H1N1 viruses probably appeared shortly before the 1918 pandemic and circulated until 1956 [190]. In 1957, the H1N1 subtypes were replaced by the H2N2 Asian pandemic viruses that had acquired the HA, NA, and PB1 segments from avian strains while maintaining the five other segments [90,173]. In 1968, these strains were replaced by viruses of the H3N2 subtype, which contained novel HA and PB1 genes derived from duck strains [46,90]. The year 1977 witnessed the reappearance of H1N1 viruses that were almost identical to strains that circulated shortly before the 1957 pandemic and thus appear to have been maintained in a frozen state for 20 years. H1N1 and H3N2 strains have cocirculated since that time. In countries with a temperate climate, influenza epidemics are observed almost exclusively in the winter months (November to April and May through September in the northern and southern hemispheres, respectively). In tropical areas, the influenza activity is less linked to the season and the virus can be isolated during the whole year. Globally, influenza epidemics move from north to south across the globe, crossing the equator twice annually [85]. Influenza activity is divided into five levels—no activity, sporadic cases, local outbreak, regional activity, and widespread activity—according to the number of isolated viruses and the number of observed cases of flulike syndromes in comparison with the baseline used by the international surveillance network [49]. The main epidemiological factors related to the host are age, prior immunity, and indoor crowding. Health status does not affect the attack rate but is the main factor that underlies the high morbidity and mortality related to influenza. In the elderly, cardiopulmonary complication rates increase with age up to 70% in persons over 70 years of age [14] (see also Sec. 6). Attack rates are higher in young people, whereas complications and mortality are higher in the elderly, although their incidence rates are lower. Healthy babies are also affected, with an influenza-related hospitalization rate of 104/10,000 in children less than

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6 months of age and 50/10,000 in children between 6 and 12 months [133]. These rates are comparable to those observed in adults with high risk conditions [134]. Herd immunity determines the severity of the incidence rates in the community: The higher the levels of circulating neutralizing antibodies, the lower the infection rate [104]. Crowding greatly influences the attack rate, and the most common factor is the institutionalization of individuals who are in poor condition. Nosocomial outbreaks of influenza are common, even in pediatric wards, with high variable attack rates of up to 47% of hospitalized persons [219]. Influenza outbreaks with very high attack rates were also observed in confined healthy populations: 42% in a U.S. Navy ship despite an optimal vaccination rate [43], 49% in a ski school hotel [110], and 72% in an aircraft [130]. Surveillance is of particular importance for the early detection of influenza activity, the identification of circulating strains, and the estimate of the impact of the outbreak. National surveillance networks often merge both virological and epidemiological data, simultaneously including the activity of the respiratory syncytial virus (RSV). This latter virus is essentially known for causing bronchiolitis in infants, but it is also responsible for respiratory infections that can mimic influenza with a comparable morbidity and mortality [45]. Information provided by surveillance networks* from the general population and hospitals gives additional arguments for clinical diagnosis. Public health authorities are also informed about the scope and the impact of the outbreak, and the World Health Organization (WHO) collects useful data for deciding the composition of vaccines for the next season. Timeliness of the latter information is essential, because the vaccine composition has to be declared by WHO in February in the northern hemisphere and in September in the southern hemisphere, leaving 6 months for the pharmaceutical companies to prepare the appropriate trivalent vaccine. 4.2

Evolution

A characteristic feature that distinguishes influenza viruses from other human respiratory viral pathogens is their enormous spectrum of genetic diversity and changeability. For instance, the HA protein sequences of different subtypes differ by up to 60% although they probably have very similar spatial structures [139]. Numerous serological and nucleic acid sequencing studies have shown that several mechanisms account for the * http://www.cdc.gov/nip/flu/News.htm#Bulletin in the United States and http:// www.eiss.org in Europe.

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geno- and phenotypical flexibility of influenza viruses. Antigenic drift is the most important principle during interpandemic periods by which influenza viruses escape increased immunity in the population toward an epidemic strain. This involves the appearance of influenza A or B virus variants with point mutations that can evade neutralization or clearance mediated by HA- and NA-specific antibodies generated after infection or immunization with their predecessor. Thus, individuals who have experienced infection with a given virus subtype can be reinfected by a drift variant. The ‘‘original antigenic sin’’ that gives a preferential response against prior infecting strains and may inhibit a specific response to the newly infecting virus might explain some poor reactivity, especially in the elderly. Structural analyses have shown that drift variants differ by only a few amino acids, which are confined to five epitopes (A–E) within the globular head domain of the HA [212] and at least two epitopes on the NA [168]. A successful drift variant usually replaces the prevalent strain of the previous seasons and circulates on average for 2–5 years. Therefore, the composition of the influenza vaccines that contain components of recent influenza A/H3N2, A/H1N1, and B viruses is annually adjusted to ensure the best possible level of protection against circulating virus strains (see Sec. 8). On a molecular level, antigenic drift is facilitated by the relatively high error rate of the viral RNA polymerase, which appears to lack a proofreading function. This results in a mutation rate of about 1 6 10
5 base pairs per site per replication, which is in a range similar to those of other RNA viruses [184]. Thus, a given wild-type virus population usually contains at low frequency variants that may gain a replicative advantage under selective conditions such as the presence of neutralizing antibodies or antiviral substances. For the HA and NA genes, mutational rates of 6.7 6 10
3 and 2.6 6 10
3 substitutions per site per year, respectively, have been determined [48,218]. However, the genes of the internal viral proteins such as NP, M1, or NS1 that are believed not to underlie any immune surveillance also evolve, albeit at somehow slower rates [20,87,175]. A second important mechanism that contributes to genetic diversity among influenza viruses is the reassortment of viral gene segments. This is based on the capability of cells to produce progeny viruses that contain RNA segments from both parents after infection by two different virus strains [147]. It is important that when genes of the viral surface glycoproteins are involved, reassortment can lead to the emergence of viruses with novel subtypes of HA and/or NA proteins that are antigenically unrelated to strains that circulated previously among humans. Such a fundamental change in viral antigenicity that occurs in

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unpredictable intervals is termed antigenic shift. Shift variants such as the pandemic H2N2 and H3N2 viruses of the years 1957 and 1968 encounter a naive population that largely lacks immunological protection and is therefore highly susceptible to widespread infection. Thus, every human influenza virus strain that contains novel HA and/or NA subtypes must be considered a potentially dangerous pathogen. Apart from immunogenicity, reassortment can have further consequences that are unpredictable. For instance, upon crossing two virulent avian strains, both pathogenic and nonpathogenic reassortant viruses were recovered [167]. Thus, not only the presence of a specific segment (such as an HA containing a multibasic cleavage site) may be important for viral virulence, but also the constellation of reassorted genes. A third mechanism is the transfer in toto of a non-human strain into humans. This infection by an animal virus without prior reassortment has been rarely observed [27,91,165] but has to be considered as a pandemic threat because a reassortment with circulating strains could also occur in a human host. Although human-to-human transmission was not demonstrated in the A/H5N1 bird flu infections in Hong Kong in 1997, mass slaughtering of domestic poultry prevented a possible reassortment with human influenza strains. Finally, there are few reports on viruses carrying genes with insertions of cellular or viral RNAs [13,92]. Thus, in contrast to some positive-strand RNA viruses such as polio virus, true recombination between RNA strands appears to play in general only a minor role in the evolution of influenza virus. However, recent phylogenetic analysis suggests that the HA of influenza viruses that circulated during the devastating Spanish influenza in 1918 may have originated from recombination between human and swine genes [59]. It has been proposed that the fusion of human and swine HA sequences granted the 1918 virus unique immunogenicity or tissue specificity that may have contributed to its extreme virulence. 4.3

Host Range of Influenza Viruses

As indicated above, influenza A viruses are not restricted to humans but have been isolated from a broad range of hosts including pigs, horses, minks, seals, whales, and a variety of avian species [217]. For influenza B and C viruses, humans appear to be the major host, although recently type B viruses were also isolated from diseased seals [144] and influenza C viruses have been found in pigs. All influenza A viruses are believed to have originated from the avian reservoir [88]. This hypothesis is supported by the findings that all 15 HA subtypes and nine NA subtypes currently known exist in avian influenza viruses that circulate in feral

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51

and domestic birds. Furthermore, phylogenetic analyses suggest that avian viruses are close to evolutionary stasis, because their proteins are highly conserved. In their natural hosts the viruses multiply predominantly in the cells lining the intestinal tract, from which they can be excreted in large quantities. From this reservoir, the viruses can enter, directly or after adaptation, other host populations in which they may establish independent evolutionary lineages. However, crossing of the interspecies barrier followed by continued circulation in the new hosts has been rarely observed. Avian influenza viruses replicate only poorly in humans [9]. One important factor in this species restriction appears to be the strong preference of avian influenza viruses for a2,3-linked sialic acid receptor determinants [117]. Such glycoconjugates are abundant in avian target cells but seem to be largely absent from epithelial cells in the human respiratory tract [7,32,89]. Instead, these cells carry a2,6-linked neuraminic acids that are recognized by human influenza viruses [7,33,164]. The basis for preferential binding to one or the other type of receptor is not a matter of a specific HA serotype but rather depends on the presence of distinct amino acids in the receptor binding site of the HA [117]. Indeed, reassortment of avian HA genes into virulent mammalian viruses was associated with alterations of the avian consensus sequence at amino acid positions 190, 225, and 226, resulting in increased binding to a2,6-linked sialic acids [115]. However, the recent occurrence of severe human infections by avian H5N1 viruses whose HA did not carry such adaptations suggests that such interspecies transmissions can be successful in spite of inappropriate receptor specificity [116]. Thus, additional factors may also define the host range of influenza virus strains. For instance, several internal viral genes including the NP, matrix, NS, and polymerase segments have been suggested to restrict the replication of avian viruses in monkeys and humans, although the precise mechanisms are not known yet [182,194,200]. How can the emergence of avian–human reassortant viruses be envisioned when humans are less susceptible to infection with avian strains and human viruses do not spread in birds? Although unlikely, it cannot be excluded that such reassortants are directly generated in either host. However, several findings suggest that reassortment of genes from avian and human viruses may more favorably occur in swine as an intermediate host that facilitates the adaptation of avian virus genes to a mammalian environment [170]. Pigs are relatively susceptible to infection with both avian and human virus strains [93] and contain both a2,3-and a2,6-linked sialic acid receptor determinants in their trachea cells [89]. Furthermore, in rural areas pigs are abundant hosts

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that can come into close contact with humans and birds, which would be a prerequisite for the acquisition of progenitor strains. In fact, H3N2 influenza viruses containing segments of avian and human strains have been shown to circulate in pigs [23] and have also been isolated from diseased children [27]. This demonstrates that avian–human reassortant viruses can be transmitted from the porcine population to humans and strengthens the suspicion that pigs may play an important role in the generation of novel virulent strains [172]. 5

TRANSMISSION AND PATHOGENESIS OF HUMAN INFLUENZA

Influenza viruses are most likely perpetuated by continuous human-tohuman transmissions, because there is no evidence for persistent infections in immunocompetent individuals. The viruses are usually disseminated in small droplets (23–40 kg. In principle, NIs should not be used without confirmation of the presence of influenza virus, because they are ineffective against other pathogens that may induce similar symptoms. For this main reason, presumptive treatment must remain the exception. Summarized indications for treatment with neuraminidase inhibitors (Tamiflu from 1 year of age and Relenza from 7 years of age): Proven cases of uncomplicated influenza A and B infection Unconfirmed secondary cases, if postexposure prophylaxis was not used. The use of neuraminidase inhibitors could be envisaged in Primary influenzal pneumonia and other influenza-associated complications, excluding superinfections. Presumptive treatment of an infected person with very suggestive symptoms during epidemic activity if no microbiological confirmation is possible. Presumptive treatment of an infected person with suggestive symptoms in case of antigenic mismatch during an influenza outbreak, regardless of the vaccination status. Immunosuppressed patients who did not receive prior chemoprophylaxis. in this case, diagnostic criteria will be less demanding. In any case, salicylates have to be avoided as supportive treatment in children, and paracetamol should be preferred.

8 8.1

IMMUNIZATION Inactivated Vaccine

Current influenza vaccines are made from purified hemagglutinin of inactivated egg-grown viruses. This split or subunit surface protein is derived from the three A/H3N2, A/H1N1, and B circulating strains annually recommended by WHO. Randomized controlled trials have shown 70–90% efficacy [125], i.e., protection against laboratory-con-

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firmed influenza infection, when circulating and vaccination strains match well. As expected, postvaccination antibody titers decrease with age [161] but nevertheless induce protecting antibodies in 50% of vaccinated elderly persons [64]. Despite an important mismatch, a very low efficacy (12%) by protecting HI antibodies might be observed in old persons [39]. Twenty cohort studies on vaccine effectiveness were evaluated by metaanalysis that showed prevention rates in the elderly of 56% for respiratory illness, 53% for pneumonia, 50% for hospitalization, and 68% for death [65]. Even if vaccine efficacy is low for protecting elderly individuals against infection, the vaccine remains highly effective in preventing outcomes associated to influenza in this particular high-risk group. In addition, economic studies have shown that immunization can reduce work absenteeism by 43% [135] and physician visits by 42% [19] in healthy adults. Influenza vaccination was ranked first among 587 lifesaving interventions with the best cost per life gained [192]. The costeffectiveness ratio, in comparison with other interventions in the elderly, was found to be the best one, and the vaccine was even considered costsaving for the elderly [47]. Even though safe and effective, inactivated vaccine has important limitations: Included strains have to be chosen, and possibly changed, by WHO each year on a timely basis. Mismatch between vaccine and circulating strains can occur. During the period 1982–1991, 12% of vaccine compositions were inadequate [146]. In contrast with other vaccines, influenza vaccine offers partial protection. Vaccine efficacy and vaccine effectiveness are often confounded [28]. Compliance of vaccination is limited, especially in health care workers. Production in eggs increases vaccine production time, and it is a real race against time from the moment the appropriate composition of the vaccine is declared by WHO. 8.2

Persons Recommended for Vaccination

The main objective of influenza vaccination is to reduce the impact of the disease, and targeted vaccination is essentially oriented to persons who are at high risk for complications. Influenza immunization policies vary greatly from country to country [138], reflecting uncertainties concerning

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the risks of influenza and benefits of vaccination. For instance, advanced age (>65 years) without any high-risk condition was not recognized by some countries as additional risk, whereas publications have shown that even healthy elderly individuals should be vaccinated [211]. Moreover, American health authorities recently extended their recommendations by lowering the age for vaccination from 65 to 50 [18]. The main reason is that one-third of this age group (50–64 years old) has at least one highrisk medical condition, and the argument for extending immunization policy is that an age-based strategy is more efficient than strategies based on individuals’ medical conditions. Selection of persons to be vaccinated relies on the reduction of complications in high-risk groups, the reduction of morbidity related to influenza in particular individuals, and breaking the circulation of the virus by immunizing persons who can transmit the diseases to high-risk groups. In this way, it is debated whether all children have to be vaccinated for enhancing herd immunity as suggested by the Japanese experience, where vaccination of schoolchildren was shown to protect and decrease the mortality in the elderly [159]. Individual protection and societal benefits unfortunately often compete for the support of policymakers, and particular attention should be drawn if the use of attenuated vaccine were generalized in the child population. Health care workers (HCWs) are also important groups of people who can transmit the disease to high-risk persons, and publications on the benefits of HCW vaccination for the protection of high-risk persons are convincing [21,157]. That is why current U.S. recommendations about which persons should absolutely be vaccinated have included HCWs, and these seem to be applicable to all countries [18]. Summarized indications for influenza vaccination: 1. Persons of 65 and more, even healthy ones 2. Institutionalized persons (those in nursing homes and health care facilities) 3. Persons of the age of 6 months and older with at least one underlying chronic condition: a. Lung disease, essentially asthma and COPD b. Cardiovascular diseases such as cardiac failure, valvulopathy, and pulmonary hypertension c. Hepatic disorders such as cirrhosis or chronic viral hepatitis d. Renal diseases, particularly in dialysis or transplant patients

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e.

Metabolic diseases such as diabetes types I and II, cystic fibrosis, or hemoglobinopathies f. Immune disorders, in all situations threatening immunocompetence: therapy with corticoids, therapy for malignant tumors, HIV infection, organ transplants, pregnancy in which the second or third trimester will occur during the winter g. Children aged 6 months to 18 years who are receiving longterm aspirin therapy (risk of Reye’s syndrome) 4.

Persons who can transmit the disease to high-risk groups a.

Medical and administrative staff of health care facilities, nursing homes, or outpatient care settings b. Households of high-risk persons 5.

6. 7.

Travelers who are at high risk and who travel to the southern hemisphere between April and September or to the northern hemisphere between October and March. Normally these persons should have been vaccinated in the autumn, and a second immunization should be given with a vaccine or the last available composition. Persons between 50 and 64 years, with particular attention to smokers, excessive alcohol drinkers, and obese persons. Anyone else who wishes to be protected against the infection.

The only contraindication is allergy to egg protein, and the vaccine should not be given, for psychological reasons, to persons with an upper respiratory infection. Side effects are essentially local (redness, pain) or rarely general (low grade fever) and in both cases are self-limiting. 8.3

Live Attenuated Vaccines

Live cold-adapted influenza vaccine was first developed in the 1960s [111], and this reassortant attenuated vaccine is currently submitted to the Food and Drug Administration. The vaccine reassortant strain contains six genes from attenuated master strains and two genes coding for hemagglutinin and neuraminidase of contemporary wild viruses. Safe and highly immunogenic, this attenuated intranasally administered vaccine has a 92% efficacy rate in preventing laboratory-confirmed influenza infection [11]. In 1997, when the A/Sydney/5/97(H3N2) strain was not included in its composition, the vaccine was nevertheless 86% efficacious against the wild strain [10]. Effectiveness of the vaccine is also appreciable in that it reduces febrile otitis media by 30% and illness

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necessitating antibiotics by 29% [11]. Rhinorrhea, especially after the first dose, is the main side effect.

9

SUMMARY OF APPROACH FOR CONTROLLING AND MANAGING INFLUENZA IN INDIVIDUALS

Control and management of influenza are intended to decrease the incidence rate, the spread of the virus, the intensity of symptoms, and the severity of associated complications. Vaccination is the most important preventive intervention among persons at high risk or persons who are likely to transmit the disease to high-risk persons. Dosage varies according to age and prior vaccination. Recommendations are as follows: In previously unvaccinated children aged from 6 months to 35 months: two half-doses (0.25 mL) separated by 4 weeks In previously unvaccinated children aged from 36 months to 8 years: two doses (0.50 mL) separated by 4 weeks In previously vaccinated children over 36 months of age, whatever the prior vaccination status: one dose (0.50 mL) The intramuscular route of administration has to be used, in the anterolateral part of the thigh in children less than 2 years of age and in the shoulder in both children over the age of 2 years and adults. Vaccination must be given between mid-October and the end of November in the northern hemisphere. For chemoprophylaxis, neuraminidase inhibitors (NIs) are preferred in place of M2 blockers. Long-term prophylaxis (4 weeks) with oseltamivir can be initiated in persons aged 13 years or older when the risk of exposure to influenza virus is high, with a dosage of 75 mg/day. In this case, combined chemoprophylaxis and vaccination provide additional protection. Contact chemoprophylaxis with oseltamivir may also be given in a family setting where an index case is discovered. The same dosage is used for 7 days. Treatment of uncomplicated cases of confirmed influenza can be initiated with Relenza, 5 mg twice daily in persons aged 7 years or more, or Tamiflu, 75 mg twice daily in adults. As mentioned above (Sec. 7.2.5), dosage of Tamiflu in children aged 1 year and older depends on the weight. Both drugs have to be taken for 5 days. Effectiveness cannot be expected if the treatment is not started within 2 days after the onset of illness. As for chemoprophylaxis, both NIs might be given as treatment in vaccinated persons.

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10 10.1

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PANDEMICS OF INFLUENZA Three Major Pandemics of the Twentieth Century

Three major pandemics occurred during the 20th century, namely the Spanish flu in 1918, the Asian flu in 1957, and the Hong Kong flu in 1968. Possible mechanisms of the occurrence of new strains and disappearance of old strains have been described above. The death toll of each pandemic was impressive, with 20–40 million worldwide during the Spanish flu and 100,000 deaths in the United States for both pandemics in 1957 and 1968 [58]. A large proportion of deaths occurred in young adults, suggesting a putative acquisition of protection against the illness in younger persons [176]. This age pattern and the variable severity of the pandemic virus have to be taken into account for preparing contingency plans including scenarios of variable intensity. Lessons could also be learned from the Spanish flu to help plan for the next influenza pandemic. Issues observed in 1918, such as authoritative measures for wearing masks or closing schools, which were often unpopular and rejected, could arise again. The main objectives of a national plan are to reduce panic-related problems, to ensure a reliable communication strategy, and to ensure equity in access to prevention and treatment measures. 10.2

Preparedness Plans

The World Health Organization issued guidelines in 1998 for helping national and regional authorities prepare a preparedness plan that can be used in the case of an influenza pandemic [210]. The latter is defined as The emergence of an influenza A strain with a different hemagglutinin subtype than strains that have been circulating for many years A high proportion of susceptible people in the community, i.e., no or low antibody titers to the novel hemagglutinin High person-to-person transmissibility, with accompanying human disease To assess the risk before proposing ways for managing and controlling a pandemic, it was necessary to rank the different threat levels and the successive phases of a pandemic. This makes it possible to define a strategy according to the importance of the risk. If the risk is to be assessed by international institutions and teams, then each country is responsible for the management process. It is strongly recommended that a National Pandemic Committee be established in each country or

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that it be improved if it already exists. To prevent inadequate measures in the case of false alarms, preparedness levels have been defined that must exist before an influenza pandemic is declared: Phase 0, preparedness level 1 corresponds to the detection during the interpandemic period of a novel human strain without evidence of outbreak. In phase 0, preparedness level 2, two or more human infections are observed. Human transmission is confirmed by WHO in preparedness level 3. In phase I, outbreaks occur in at least one country. In phase II, the outbreak is extended to multiple countries. In phase III, outbreak activity has stopped in initially affected regions and epidemics occur elsewhere. After the end of the first world wave, additional waves are expected in phase IV. In phase V, WHO will declare the pandemic to be over. When there is a pandemic or a pandemic alert, WHO will collaborate closely with the four collaborating centers in London (UK), Atlanta (USA), Tokyo (Japan), and Melbourne (Australia), essentially for preparing diagnostic agents, characterizing the strain, and preparing a new vaccine. The most delicate actions to be taken if a pandemic occurs will be organizing antiviral agent stockpiles for the first wave and obtaining a specific vaccine before the next wave. Inequity in distribution and social disruption will be the main problems to be addressed by the authorities.

11

CONCLUSIONS

Interpandemic influenza outbreaks pose an important challenge for both individuals and society. Management and control of the disease rely essentially on surveillance, immunization, and treatment. The reduction of the impact that can be obtained depends on the awareness of the illness by both clinical staff and decision-makers. Strategies for enhancing herd immunity by vaccinating children have to be considered, because immunization of groups at high risk will not be sufficient for controlling the disease. Likewise, the appropriate use of new antiviral agents in both chemoprophylaxis and treatment will help to reduce the impact. Meanwhile, national preparedness for an influenza pandemic, which is certain to occur, is of major importance.

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Acknowledgments We thank J. Manuguerra (Institute Pasteur, Paris, France) and S. Pleschka (University of Giessen, Germany) for critical comments on the manuscript and D. Heuer (Max Planck Institute of Infection Biology, Berlin, Germany) for help in the preparation of figures.

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antibodies in the serum of healthy volunteers. Clin Pharmacokinet 36:51– 58. Webster, R. G., Y. Kawaoka, W. J. Bean, C. W. Beard, and M. Brugh. 1985. Chemotherapy and vaccination: a possible strategy for the control of highly virulent influenza virus. J Virol 55:173–176. Welliver, R., A. S. Monto, O. Carewicz, E. Schatteman, M. Hassman, J. Hedrick, H. C. Jackson, L. Huson, P. Ward, and J. S. Oxford. 2001. Effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial. JAMA 285:748–754. WHO. 1999 posting date. Influenza pandemic preparedness plan. The role of WHO and guidelines for national and regional planning. World Health Organization. http://www.who.int/emc-documents/influenza/whocdscs redc99lc.html. Wijma, G., and G. J. Ligthart. 1996. Influenza vaccination for all elderly. Gerontology 42:270–273. Wiley, D. C., I. A. Wilson, and J. J. Skehel. 1981. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289:373–378. Wingfield, W. L., D. Pollack, and R. R. Grunert. 1969. Therapeutic efficacy of amantadine HCl and rimantadine HCl in naturally occurring influenza A2 respiratory illness in man. N Engl J Med 281:579–584. Wolff, T., R. E. O’Neill, and P. Palese. 1998. NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells. J Virol 72:7170–7180. Woods, J. M., R. C. Bethell, J. A. Coates, N. Healy, S. A. Hiscox, B. A. Pearson, D. M. Ryan, J. Ticehurst, J. Tilling, S. M. Walcott, et al. 1993. 4Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro. Antimicrob Agents Chemother 37:1473–1479. Wright, P. F., J. D. Bryant, and D. T. Karzon. 1980. Comparison of influenza B/Hong Kong virus infections among infants, children, and young adults. J Infect Dis 141:430–435. Wright, P. F., and R. G. Webster. 2001. Orthomyxoviruses. In: B. N Fields (ed.), Virology. 4th ed. Lippincott-Raven, Philadelphia, pp. 1533–1579. Xu, X., N. J. Cox, C. A. Bender, H. L. Regnery, and M. W. Shaw. 1996. Genetic variation in neuraminidase genes of influenza A (H3N2) viruses. Virology 224:175–183. Yassi, A., M. McGill, D. Holton, and L. Nicolle. 1993. Morbidity, cost and role of health care workers transmission in an influenza outbreak in a tertiary care hospital. Can J Infect Dis 4:52–56. Zambon, M., and F. G. Hayden. 2001. Position statement: global neuraminidase inhibitor susceptibility network. Antiviral Res 49:147–156.

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3 Respiratory Syncytial Virus Philip R. Wyde and Pedro A. Piedra Baylor College of Medicine, Houston, Texas, U.S.A.

1 1.1

INTRODUCTION Human and Economic Impact of Respiratory Syncytial Virus Infections

Respiratory syncytial virus (RSV) is ubiquitous, causing epidemics annually worldwide [1]. Community attack rates are usually high (30– 50%), and almost all children have an RSV infection by 3 years of age [2]. The majority of these infections are subclinical or mild. However, if virus descends to the lower respiratory tract, serious manifestations and even death can occur. Respiratory syncytial virus infections impact a number of populations especially hard [3]. In the very young (those less than 2 years of age), RSV is the leading producer of bronchiolitis, pneumonia, and lower respiratory tract infection (LRTI). This virus can also cause significant problems in children with underlying chronic health conditions, for example, those with nephritic syndrome, chronic heart disease (CHD), or bronchopulmonary dysplasia (BPD). Children with cystic fibrosis (CF) are also at high risk. In this populations, RSV infections result in reduced lung function and a greater rate of hospitalization (43%) than any other viral infection [4]. Adults with chronic pulmonary and/or cardiac 91

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disease are also at high risk, and in the elderly the impact of RSV appears to be second only to that of nonpandemic influenza [5]. However, most at risk are immunocompromised individuals [6], especially those receiving chemotherapy for leukemia or undergoing bone marrow transplantation. In these populations, once pneumonia develops, the overall mortality can range from 50–100% without treatment; even with treatment, unless therapy begins before infection of the lower respiratory tract takes place the prognosis is very poor. In toto, the human and economic impact of RSV is staggering. Presently there are approximately 100,000 hospital admissions and 4,500 deaths in the United States annually due to RSV infections [7]. These infections may be responsible for 40–50% of hospitalizations for bronchiolitis and 25% of pediatric hospitalizations for pneumonia [8]. Worse, many RSV infections may have long-lasting consequences because a significant number of infected children may develop hyperactive airway disease and diminished pulmonary function later in life [9]. Here, too, the numbers are astounding. Between 40% and 50% of infants hospitalized with RSV bronchiolitis have recurrent episodes of wheezing during early childhood, and many go on to develop asthma or other long-term airway morbidity. There may also be a link between RSV infection in infancy and the development of chronic obstructive pulmonary disease (COPD) in adult life [10]. Indeed, it has been reported that even children who have mild RSV disease can have recurrent wheezing up to 6 or even 10 years after the acute episode [11,12]. However, a causal relationship between RSV infection early in life and the development of asthma later in life has not been proven. Adding to the problem, RSV epidemics often occur in clusters (usually during late fall and winter in the northern hemisphere), and this clustering of cases can cause major problems in both primary and secondary care centers [13]. In terms of dollars, the average hospitalization charge for an RSV infection (weighted in 1998 US dollars) was $7,140 for infants and $6,910 among all children younger than 5 years old [14]. Overall, it is estimated that in the United States alone, RSVrelated illnesses cost between $300 million [7] and $400 million annually [15]. Amazingly, these statistics may be an underestimation. Bronchiolitis-associated hospitalizations among U.S. children is rising [16], and recent data suggest that annual bronchiolitis hospitalizations associated with RSV infection among infants may be greater than previous estimates for RSV bronchiolitis and pneumonia hospitalization combined [17].

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Epidemiology

There are two major subgroups of RSV, designated A and B. Strains are placed into one of these groups primarily on the basis of their reactivity with panels of monoclonal antibodies raised against epitopes on RSV fusion (F) and attachment (G) envelope proteins [18,19]. During an epidemic, a single strain may circulate, or strains belonging to both subtypes can be present [20]. Reinfections with RSV are common [21], possibly due to the antigenic variability that occurs among RSV strains. However, the actual mechanisms involved remain unclear. Regardless, significant illness and appreciable morbidity can occur during reinfections, and it does not seem to matter which subtypes the individuals were initially infected with. Although most reinfections do not lead to serious disease, they may be an important source of virus that can lead to infection of more vulnerable populations (e.g., infants or immunoincompetent individuals). Spread of RSV appears to take place primarily through saliva drops and close contact and not by aerosol. Infected persons with symptoms are highly contagious [22]. Moreover, the virus can remain infectious for 6 hr on solid surfaces. Thus, disinfection and hygienic practices are very important in preventing transmission, with hand washing being of paramount importance. Other steps include (1) avoidance of close contact with infants by persons who have symptoms of respiratory tract infections, (2) reducing contact of very young infants (50% have been reported between different group A and B strains for this protein, and even within the same antigenic group, 20% differences in G protein sequences have been determined [33–35]. Indeed, G proteins from different RSV strains have been reported to vary markedly in length and to have differences in molecular weight ranging from 84 to 90 kDa (mature form of the protein). Highly varied glycosylation adds to this heterogeneity [33]. The role of this glycosolation is not clear, but it has been speculated that it may protect epitopes on the G protein from being recognized as a foreign antigen [36]. Regardless, although the G protein is responsible for promoting attachment and entry into host cells and can induce virusneutralizing antibodies, it has not been the target of many RSV vaccines. The significant variability just discussed is partially responsible for this. However, a second factor is that several studies have suggested that the G protein is capable of inducing eosinophilia and other adverse immune responses [37]. (This subject is discussed in more detail below.) The third coat protein, SH, is small (only 64 amino acids) and hydrophobic. Although it has been shown to facilitate virus-host cell fusion [26], it is not essential to this process or to infection [38]. However, SH deletion mutants have been shown to be attenuated in mice compared to wild-type RSV strains and may therefore be of some interest in developing attenuated live virus vaccines [38]. The large polymerase protein (L) and phosphoprotein (P) that are part of the polymerase complex and viral nucleocapsid are not well characterized. To date, neither protein has been a major target for either vaccines or chemotherapeutics. Neither have the two matrix proteins, M and M2, or the two nonstructural genes, NS1 and NS2. 2.2

Replication

Respiratory syncytial virus replicates primarily in the ciliated epithelial cells lining the respiratory tract. Infection is initiated by the binding of the linear heparin-binding domain of the virion’s G protein to receptors on the host cell plasma membrane [39]. What exactly the receptor is is not known, but it is thought to be a sulfated peptidoglycan [40]. The virus then penetrates and enters the host cell, where it uncoats, releasing the matrix protein and the nucleocapsid [26]. Transcription of vRNA to positive-stranded messenger RNA (mRNA) then occurs, followed by translation of the viral mRNA into early viral proteins. There is then a switch to the production of positive sense RNA that serves as a template for the synthesis of progeny genomes. Viral proteins accumulate in the

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FIGURE 2 Diagramatic representation of respiratory syncytial virus. Shown are the lipid bilayer (light gray) with the three coat proteins (G, F and SH) of RSV penetrating. Inside the virion are the matrix protein (depicted in black), the matrix 2 protein (black dots), and negative-strand RNA, which is surrounded with nucleocapsid protein (NP). Associated with the NP are the two other proteins associated with the transcriptase complex, the large polymerase (L) and phospho (P) proteins. The two nonstructural proteins (NS1 and NS2) of RSV are found only in the host cell and not in RSV virions. (Modeled after the general representation of a paramyxovirus virion presented in Ref. 244.)

host cell cytoplasm and associate with viral genetic material and polymerase to form the nucleocapsid. This complex is incorporated into the virion, which then buds through the apical membrane, where the virions acquire their lipid envelopes [41].

3

PATHOGENESIS

Respiratory syncytial virus infections usually begin in the nasopharynx, from where they may spread to the lower airways (Fig. 3). In

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FIGURE 3 Comparison of the general kinetics of respiratory syncytial virus shedding and relationship to clinical illness in infants and adults.

immunocompetent individuals, infection of cells other than the respiratory epithelium is unusual, and the virus is usually restricted to the respiratory tract [42,43]. The major exception to this is that the virus often spreads to the middle ear, so the development of otitis media is common in this population [44]. In immunoincompetent persons, extrapulmonary dissemination can occur, and virus may migrate to the kidneys, liver, central nervous system, and heart with some regularity [6,26]. There is a high frequency of virus isolation from the nasopharynx in the early phase of infection, especially in children [45]. After 14 days, virus recovery from this region is less likely. However, in immunosuppressed individuals, excretion of virus may continue for 28 days or longer. In general, the peak viral infection occurs about 5–7 days after onset of illness [3]. Many things can influence clinical outcome, including poor general condition and poor nutritional status [46,47]. Gender, ethnic group, and

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levels of maternal antibody [48]; age at which the first RSV infection takes place [49]; an underlying pulmonary or cardiac condition [3]; reduced lung function; or an immunodeficiency condition can also contribute to disease severity. In addition to causing bronchiolitis and pneumonia, RSV has been implicated in the pathogenesis of childhood asthma and reactive airway disease [50,51]. Indeed, although other viruses have been associated with wheezing [52,53], RSV appears to be the single most important agent in children 99.9% F glycoprotein. It has been shown to be safe and immunogenic in healthy young, seropositive children [126], in seropositive children with BPD [127], in seropositive children with CF [128], in ambulatory adults over age 60 [129], and in the elderly [130]. As with PFP-1, no significant adverse reactions were associated with PFP-2 during testing. Moreover, no enhanced RSV disease was seen in any of the trials in vaccinated children who subsequently became infected with RSV. Unfortunately, neither PFP preparation has been studied in young infants, particularly those with underlying risk factors. A third generation of PFP, PFP-3, is also being developed. This material is currently in phase II clinical trials. A systematic overview of the PFP trials was recently performed to assess whether these vaccine preparations are efficacious in preventing RSV-induced LRTI [131]. It was concluded that they do reduce the overall incidence of all RSV infections but that the clinically important outcome of RSV LRTI is not reduced. It was recommended that, because of concerns about the pooling of data that was done in different clinical trials, these vaccines be tested in large field trials. BBG2Na is a synthetic polypeptide produced in Escherichia coli. It is composed of residues 130–230 of RSV-A G protein (G2Na) fused to BB, an albumin-binding domain of streptococcal protein G. (This fusion potentiates the immunogenicity of the polypeptide.) Within this domain are 12 amino acids that are present in both A and B RSV subtypes. BBG2Na has been shown to be immunogenic and protective in mice following administration by different inoculation routes including intramuscular [132] and intranasal [133]. Equivalent protection was seen against both subgroup A and B RSV strains [134]. In other animal studies, BBG2Na administered intranasally with cholera toxin B or zwittergent 3–14 generated both mucosal and systemic antibody responses that protected the test animals against RSV challenge and did not induce lung immunopathology upon subsequent RSV challenge [135]. This finding is important because early studies showed that BBG2Na induced a predominant type 2 T-cell response upon immunization. In recent testing in adults, BBG2Na appeared to be both safe and immunogenic [136]. Less favorable results may have occurred in other trials since testing of this product has been discontinued. Synthetic RSV peptides have also been tested as potential vaccine candidates. Mucosal delivery of one of these, when combined with enterotoxin-based adjuvants, elicited CD8þ T-cell responses in mice [137]. However, this response appeared to be both protective and immunopathogenic. All of these peptide vaccine candidates are still experimental and in preclinical study.

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Vaccines based on RSV subunits or synthetic peptides may be particularly useful in older children, adults, and the elderly. These are populations that have already experienced RSV infections and are thus immunologically primed. For this reason, a peptide or subunit vaccine may be able to induce protective secondary immune responses in these populations that it does not induce in infants. These vaccines may also be useful to boost levels of protective antibodies in pregnant women. (These women would be immunologically primed and thus should respond relatively well to subunit or peptide vaccines.) In addition to standard testing, subunit RSV vaccines have been tested in combination with recombinant virus preparations [138]. Other investigators have been investigating the effects of various adjuvants on the immunogenicity of subunit vaccines (see, e.g., Ref. 139). One caution should be emphasized. Increased pulmonary cellularity has been seen in cotton rats infected with RSV after immunization with one of the chimeric FG preparations [140]. 5.2.4

DNA Vaccines

Several RSV DNA vaccines aimed at inducing expression of proteins with protective epitopes have been developed [141–144]. Such vaccines have the potential to be highly immunogenic and capable of inducing strong protective humoral and CMI responses. However, a major concern with DNA vaccines is their safety, especially in the long term and in infants who have relatively high endogenous DNA synthesis and replication. For this reason, DNA vaccines may be more suitable in an older population, because there is less cellular replication taking place in these individuals.

6 6.1

ANTIVIRAL AGENTS Passive Antibodies

At the present time, two agents, RSV immunoglobulin (RSVIG) (RespiGamTM, MedImmune, Inc., Gaithersburg, MD) and palivizumab (SynergisTM, MedImmune, Inc., Gaithersburg, MD), are currently licensed for prophylactic use against RSV. The former material contains polyclonal antibody to RSV and is prepared from serum that has been shown by screening tests to have high Nt antibody titers to this virus. For this reason RSVIG is more efficient against RSV than standard Ig preparations. RSVIG is generally administered intravenously once a month during the RSV season and has been shown to prevent or reduce

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RSV disease severity in infants and young children who are exposed to this virus after its administration [145–148]. RSVIG was licensed in the United States in 1996 [167]. However, it is not recommended for use in infants with cyanotic CHD, because it was associated with an excess of adverse events in this population during testing. In addition, it is generally not used outside the United States because of the difficulty in administrating it (i.e., i.v.) and because of the large fluid volumes (15 mL/kg) and high protein load (750 mg/kg) involved in its use. Because passively administered antibodies can interfere with vaccine efficacy, administration of live virus vaccines (e.g., MMR and varicella) is often postponed after use of RSVIG. Another problem associated with RSVIG is its high cost [149,150]. Palivizumab was developed because it was desirable to find a material that did not require intravenous infusion, administration of relatively large volumes, multiple visits, and a hospital setting for proper administration [146]. This humanized monoclonal IgG1 antibody has virus-Nt activity against antigenic site A on the F protein of RSV and thus potentially can prevent infection of both A and B subtypes of RSV [151]. Although it contains a low amount (5%) of murine amino acid sequences, no adverse effects have been seen with this product. Palivizumab has been shown to prevent or reduce RSV disease severity in preterm infants and infants with chronic lung disease who are exposed to this virus after its administration [90,151–153]. Currently palivizumab is usually given intramuscularly at a dose of 15 mg/kg prior to the beginning of the RSV season, with subsequent doses given monthly throughout the RSV season. Specific prophylaxis usually starts when circulation of RSV in the community is verified. The Food and Drug Administration approved palivizumab for clinical use in 1999, and guidelines have been put forth by the American Academy of Pediatrics Committee on Infectious Diseases [154]. However, it is approved only for prophylactic use because it has not been shown to prevent RSV replication following therapeutic administration. Interestingly, unlike RSVIG, it has not been shown to reduce development of otitis media [155]. Regardless, palivizumab is the first monoclonal antibody ever licensed for use against any infectious disease. Like RSVIG, palivizumab is expensive, and this has caused heated debate about its cost-effectiveness [156,157]. The debate is in flux because of differences in analysis, the increasing cost of an average hospital stay, and the ethics of withholding a treatment that has been shown to be effective and safe [10]. Because of the expense, it has been suggested that patients selected for immunoprophylaxis with palivizumab be selected carefully [158]. The American Academy of Pediatrics [154] has issued

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guidelines recommending that the decision to use palivizumab should be made by a specialist and that it be used in children at greatest risk of complication to RSV (e.g., babies with chronic lung disease severe BPD and very preterm babies discharged from the neonatal unit shortly before or during the RSV season). Palivizumab is currently not recommended for use in some high-risk groups such as children with congenital heart malformations, cystic fibrosis, malignancies, immune deficiencies, or neuromuscular diseases, because it has not been studied in these groups.

6.2 6.2.1

Chemotherapeutic Agents Antiviral Targets

Most antiviral agents are designed to prevent virus replication by interfering with one or more of the replicative steps essential for virus replication. In the case of RSV this would be virus attachment to and penetration of the host cell, transcription of negative-stranded vRNA into mRNAs and more vRNA, translation of mRNAs into functional (e.g., P and L polymerase enzymes), structural (e.g., M, SH, F, and G), and nonstructural (e.g., NS1 and NS2) proteins, and/or viral assembly and/ or egress (budding) of the virus out of the host cell. Interference at any of these steps can reduce or truncate infection and result in decreased infection and disease. These agents can come in many forms, some of which are discussed below. In the case of RSV, some host cell enzymes that have been targeted are inosine monophosphate dehydrogenase (IMPDH), S-adenosylhomocysteine hydrolase (SAH), L-aspartic acid transcarbamoylase, ornithine monophosphate decarboxylase (OMP decarboxylase), and cytosine triphosphate synthetase (CTP synthetase; see Ref. 159 for a detailed review). Selectivity apparently comes about in this situation because in most nonrapidly dividing host cells these enzymes are functioning at a significantly lower level than occurs in virus-infected cells. Regardless, these compounds generally have lower therapeutic indices (i.e., generally are less selective toward virus-infected cells) and can be cytotoxic to the host. Many nucleoside analogs, the next subject in this chapter, fall in this category. The only one that will be discussed in detail is ribavirin. That is because this nucleoside analog is currently the only one that is licensed for use against RSV, and it is, of course, a prototype for this class of antiviral agents. Its successes and problems are probably true, to a greater or lesser extent, of many nucleoside analogs, particularly those designed to inhibit negativestranded RNA viruses.

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Ribavirin

Ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) (Virazole, ICN Pharmaceuticals) is a synthetic nucleoside analog of guanosine. It was approved in 1986 to treat RSV infections when administered as an aerosol [160] following demonstration that it could inhibit RSV replication in tissue culture [161], protect cotton rats from pulmonary RSV infection [162], and in clinical trials provide medical benefit (i.e., significantly improve overall clinical scores, decrease lower respiratory tract–associated symptoms, and improve the arterial saturated air (SaO2) in treated infected infants [163,164], even in those with underlying CHD or BPD) [165]. Mechanism studies have shown that ribavirin must be phosphorylated to manifest its antiviral activity. Initial phosphorylation to its monophosphate form occurs as the drug is transported into the host cell. It is then serially phosphorylated to a triphosphate, which is believed to be the active form of the drug that inhibits IMPDH in host cells. Inhibition of this enzyme is thought to lead to a depletion of intracellular pools of guanosine triphosphate, which in turn interferes with vRNA synthesis [166]. Support for this mechanism is provided by the fact that the addition of guanosine can reverse the inhibitory activity of ribavirin in cell culture [167]. Ribavirin triphosphate may also interfere with virusspecific RNA polymerase initiation and elongation steps required for the synthesis of essential viral proteins, because it is known to do this during influenza virus replication [168,169]. The half-life of ribavirin in lung tissues is approximately 2 hr. In contrast, its T1/2 in serum is 300 hr. The major catabolite of ribavirin is triazole carboxamide, which is inactive and is excreted in the urine [170]. It is important that ribavirin treatment be started as soon as possible after the onset of symptoms because RSV pulmonary titers peak shortly after the onset of symptoms [23]. This point deserves emphasis, because it may be responsible for much of the variable findings and controversy associated with this compound. It is also important to emphasize that although it is highly desirable to deliver ribavirin early in infection, it is also desirable that the RSV diagnosis be verified, e.g., by antigen detection. Ribavirin alone or combined with RSVIG is recommended for use with transplant patients with mild or moderate pneumonia (infiltration with or without mild or moderate hypoxia) [171]. However, transplant patients with serious pneumonia requiring ventilator treatment presently have a mortality rate of 100%, irrespective of antiviral therapy, and therefore such therapy is not recommended for these patients [172,173]. It

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is highly recommended that transplant patients exhibiting symptoms of respiratory infection be tested frequently for RSV and if this virus is present, that they be treated before it descends to the lower respiratory tract. Despite its long use and licensing, ribavirin’s favor has been declining steadily. There are a number of reasons for this. First, there are potential safety problems, not only to patients receiving the drug but also to health care workers working in the vicinity of where ribavirin is being aerosolized [174]. The primary concern is that ribavirin is known to have teratogenic, carcinogenic, and mutagenic side-effects in animal models [175,176]. Thus, it is important that pregnant women (e.g., female nurses and doctors) not be exposed to the drug. It is recommended that all individuals concerned be informed about possible exposure risks during ribavirin aerosolizations and that adequate ventilation be ensured during the procedure. Another factor contributing to the declining acceptance of ribavirin has been the failure of many more recent studies to demonstrate any significant beneficial clinical effect of treatment with ribavirin. Indeed, many of the early studies have now been criticized with respect to their methodology and their use of subjective endpoints such as clinical score rather than major endpoints such as mortality, SaO2 , and mechanical ventilation. Furthermore, aerosolized water was used in some of these studies as the placebo, and this may have induced bronchospasms in the placebo groups [177]. These problems and the high cost of ribavirin ($3,300 per case [178]) led the American Academy of Pediatrics in 1996 to alter their recommendations for the use of ribavirin from ‘‘should be used’’ to ‘‘may be considered.’’ In addition, their new guidelines indicate that treatment with ribavirin should be limited to use in high-risk children (i.e., those with CHD, BPD, or premature infants or those aged 417 to >2500. It was not effective in cotton rats when administered parenterally, but it was when given topically [210]. RFI-641 has also been shown to have antiviral activity in African green monkeys [211]. In the latter model, prophylactic administration of RFI-641 significantly reduced viral titers in nasal washes (1.7 log10 measured at the peak of virus infection). The fact that this compound can significantly inhibit RSV in animals following topical administration is exciting. These are really the first studies to provide evidence that

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molecules that can inhibit virus attachment and/or penetration into host cells can be practically administered and still be active. Development of this compound is ongoing [211]. 6.2.7

Miscellaneous Compounds and Materials

Human eosinophil–derived neurotoxin (EDN) has been shown to mediate the ribonucleolytic destruction of extracellular RSV [212]. Cetirizine (2-[2-[4-(4-chlorophenyl)phenyl-methyl]-1-piperazinyl] ethoxyacetic acid) is an antihistamine that has been reported in a patent application to inhibit RSV replication [213]. RD3-0028 [1,4-dihydro-2,3bonzodithiin] is an interesting compound that inhibits RSV infection both in tissue culture and in mice. In tissue culture, this compound inhibited laboratory and clinical isolates of both RSV A and B subgroups. The EC50 obtained was *4 mM, and its median toxic concentration to cells was 271 mM [214]. NMSO3 is a sulfated sialyl lipid (molecular weight 1,478.8) that appears to inhibit RSV replication by several mechanisms: (1) binding directly to the F protein and inhibiting fusion, (2) inhibiting penetration (seen by shifting temperature during the period of contact between the virus and cells), and (3) inhibiting syncytia formation [215]. A peptide comprising amino acids 77–95 of Rho A (a member of the Ras superfamily of small GTP-binding proteins) has been shown to block RSV syncytia formation and RSV entry into host cells [216]. In tissue culture assays, an EC50 of 0.54 mg peptide/mL was obtained. When administered intranasally to mice at a dose of 500 mg/animal, a 200-fold reduction in pulmonary RSV titer was seen if the Rho A was administered at the same time as virus challenge, and a 20-fold reduction was observed in this model if the Rho A was given 4 days postinfection (1 day prior to killing) [217]. Interestingly, mice given the Rho A simultaneously with virus had no apparent illness or weight loss, whereas those treated 4 days after infection exhibited the same degree of illness and weight loss as infected control animals given placebo. In a related study, mice were treated with lovastatin, a drug that inhibits prenylation pathways in cells by directly inhibiting hydroxymethylglutaryl coenzyme A reductase [218]. Giving this material to mice up to 24 hr after RSV inoculation caused a significant reduction in mean pulmonary virus titer, weight loss, and illness compared to control animals that did not get this treatment. Because lovastatin also reduces syncytia formation in cell culture and eliminates RSV replication in HEp2 cells, it is more likely that this compound inhibited RhoA membrane localization and virus fusion. Because lovastatin is already approved for use, it has been recommended that this compound should be considered

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for evaluation as a preventive antiviral therapy for selected groups of patients at high risk for severe RSV disease such as the institutionalized elderly and bone marrow or lung transplant recipients [218]. A number of plant extracts have been shown to inhibit RSV replication. Some examples reported recently include an extract from Eleutherococcus senticosus roots [219], iridoid glycosides isolated from Barleria prionitis [220], ferulic and isoferulic acid extracted from Cimicifuga beracleifolja rhizomes [221], and a 10,425 Da protein purified from the edible mushroom Rozites caperata [222]. Several extracts from the medicinal plant Barleria prionitis found throughout Africa, India, Sri Lanka, and tropical Asia [220] and extracts of three species of medicinal plants obtained from Argentina (i.e., Polygonum punctatum, Lithraea molleoides, and Myrcianthes cisplatenis) [223] have also been reported to inhibit RSV. None of these plant extracts has been evaluated in vivo. Other miscellaneous compounds that have been reported to have RSV-inhibiting activity are pyridobenzazoles [224], a synthetic derivative of 1,5-dideoxy-1,5-imino-D-glucitol [225], pyridobenzazol [226], benz[de]anthracen-7-one [227], and 2,20 ,4’-methylidynetriphenol [228]. The latter is of particular interest because it has been reported by Viropharma Inc. to have a selective index of >150,000 against RSV. 6.2.8

Interferons

In mice, interferon gamma (IFN-g) clearly appears to have the potential to limit RSV replication and inflammatory responses, because animals to which the gene for IFN-g has been transferred are protected against infection with this virus [229]. Moreover, knockout mice that cannot produce this cytokine innately or following inoculation with antibodies specific for IFN-g develop more extensive inflammation of the airways and disease than control mice after RSV infection [230]. However, the IFN-defective mice also develop less obstruction of their airways, suggesting that this cytokine may have a pathogenic as well as protective role in these models. Also of interest is the fact that IFN-g production in mice does not appear to reduce pulmonary eosinophilia. The latter can occur following experimental infection with RSV despite abundant IFN-g production by local T-cells [231]. In humans, IFN-g is often detected following RSV infection and has been detected in the lower respiratory tracts of infected babies [232]. It has also been shown that significant levels of IFN-g are produced following infection of human leukocyte cultures with RSV [233]. A protective effect for IFN-g is indicated by the fact that children with severe RSV LRTI that requires mechanical ventilation is associated with low IFN-g production [234]. In addition, peripheral blood mononuclear

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cells obtained from infants with severe RSV disease express less IFN-g than peripheral blood mononuclear cells obtained from other test groups [233]. However, there are also data suggesting that IFN-g may contribute to some RSV-induced disease. For example, higher levels of this cytokine have been reported in children with virus-induced wheezing than in nonwheezers [230]. Thus, the roles of IFN-a and IFN-b in RSV disease remain unclear. The effects of exogenously administered IFN-a on the course of RSV infection have been studied following administration intramuscularly [235–237], intranasally, or as a small-particle aerosol [238]. No clinical efficacy or cytotoxicity was seen in these studies, and IFN therapy for RSV infections has not been pursued. 6.2.9

Bronchodilators and Corticosteroids

Although not true antiviral agents, bronchodilators and corticosteroids have commonly been used to try to mitigate the inflammatory effects that take place in the lower respiratory tract of the host in response to RSV infection and that appear to play an important role in the bronchiolitis and other respiratory problems initiated by this virus. However, highly variable results have been obtained following the use of these agents, and their effectiveness in ameliorating RSV disease is unclear and a subject of much debate (see reviews in Refs. 239–241). 6.2.10

Oxygen

There seems to be a consensus that oxygen is useful for the treatment of acute bronchiolitis by functioning to maintain oxygen saturation (SaO2) levels [10]. 6.2.11

Vitamin A

Because vitamin A concentrations in infants with RSV disease are often inversely proportional to disease severity, it was thought that this vitamin might be a useful adjunctive therapy for treating severe RSV infections [242–243]. However, although proven safe, oral administration of vitamin A has not proved to be effective in decreasing morbidity in children with acute RSV infection [242]. 7

CONCLUSIONS

An ever-increasing appreciation of the prevalence of RSV and its medical impact in multiple populations has led to a burgeoning interest in this virus as a target for prophylactic and therapeutic agents. The success of Synagis as a prophylactic agent and the significant deficiencies

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associated with ribavirin as a therapeutic agent have further heightened interest in this area. Live attenuated, recombinant, subunit, peptide, and DNA RSV vaccines are being developed and evaluated. However, licensing of any of these vaccines may take years. Moreover, if any is approved, it will not likely be approved for, or efficacious in, all target populations (especially immunoincompetent individuals and very young infants). Use of a combination of separate strategies, e.g., maternal immunization followed by immunization of the infant with a subunit vaccine, may be necessary to increase the utility and protection of any approved vaccine(s) [82]. Regardless, even following the licensing of one or more RSV vaccines, there will still likely be a need for effective chemotherapeutic or biological antiviral agents to be used against infections caused by this virus. This is certainly true in the absence of an approved RSV vaccine. Similarly, when new safe and efficacious RSV antiviral agents are approved, they may not be effective alone and may have to be given in combination or coadministered with an antiinflammatory agent in order to more effectively alleviate disease symptoms. Clearly it is desirable to have available both safe and effective vaccines and antiviral agents to reduce the medical impact of RSV. Based on current efforts, there is good reason to be optimistic that this state will come about in the not too distant future.

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4 Rhinovirus Ronald B. Turner and Frederick G. Hayden University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.

1

INTRODUCTION

The isolation of a rhinovirus in cell culture in 1956 was promptly followed by the recognition that these viruses were a major cause of the common cold. Rhinoviruses are now recognized as the most frequent cause of viral upper respiratory tract infections [1–3]. Epidemiological studies based on isolation of virus in cell culture indicate that both adults and children experience a rhinovirus infection every 1–2 years [4,5]. The relative insensitivity of cell culture isolation suggests that these attack rates are underestimates, but systematic epidemiological surveys using more sensitive polymerase chain reaction methods have not been done. The risk of rhinovirus infection is highest in young infants and gradually declines with increasing age. These infections are frequently brought into the home by young children, and there is a slight increase in the incidence of infection in young adult parents. The rhinoviruses cause infection yearround but are associated with seasonal epidemics in the fall and spring. The onset of common cold symptoms associated with rhinovirus infection typically occurs 1–2 days after infection. The time to peak symptoms is generally 2–4 days after infection [6]. Nasal obstruction, 139

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rhinorrhea, and sneezing are present early in the course of the illness, but sore or ‘‘scratchy’’ throat is frequently reported as the most bothersome symptom on the first day of symptoms [1,6–8]. The sore throat usually resolved quickly, and by the second and third day of illness the nasal symptoms predominate. Cough is associated with approximately 30% of colds and typically does not become the most bothersome symptom until later in the illness when the nasal symptoms decrease in severity [6,7]. The usual cold lasts about a week, although 25% last 2 weeks [7]. Virus shedding persists after the resolution of symptoms, and virus may be cultured from 10–20% of subjects for 2–3 weeks after infection [9]. The common cold is generally associated with little morbidity, although the complications of these illnesses have a substantial medical impact (see Table 1). The most important complications of common colds are otitis media in children and exacerbations of reactive airways disease. Bacterial sinusitis is also a recognized complication of viral upper respiratory infection, although recent evidence indicates that sinus involvement is a part of the common cold syndrome and it is difficult to differentiate this viral sinusitis from bacterial superinfection. In spite

TABLE 1 Illnesses Associated with Rhinovirus Infection

Illness Common cold Otitis media

Sinusitis Exacerbation of asthma Exacerbation of cystic fibrosis Exacerbation of chronic obstructive pulmonary disease

Population at risk All ages Primary children, although ~70% of colds are complicated by abnormal middle ear pressure in all age groups All ages All ages

Proportion of illnesses associated with rhinovirus 50% 30%

All ages

6–50% 60–70% in children, 19% in adults 16%

Adult

23%

Source: Refs. 9a, 9b, 9c, 9d, 9e, 9f, 62.

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of the medical significance of the common cold and its complications, attempts to develop effective treatments have been relatively limited and unsuccessful. Another important consequence of the common cold is the inappropriate use of antibiotics for these illnesses and the associated contribution to the problem of increasing antibiotic resistance in pathogenic respiratory bacteria. In the United States in 1998, there were an estimated 84 million office visits for acute respiratory illnesses (ARIs), including 25 million primary care office visits for colds [10]. Antibiotics were prescribed for over one-half of all ARIs and 30% of colds, despite lack of evidence to indicate clinical benefit [11,12]. Effective prevention or treatment of rhinovirus infection would potentially have a beneficial impact on this problem. 2 2.1

PATHOGENESIS Transmission

The pathogenesis of the common cold presents several potential targets for interrupting rhinovirus transmission, infection, and rhinovirusassociated illness. During a rhinovirus cold, the virus is present in nasal secretions at titers of 102–103 TCID50/mL of nasal lavage fluid [13,14]. This virus is readily transmitted to the hands of the infected individual and to objects in the environment. Virus can be recovered from the hands of approximately 50% of infected individuals and approximately 10% of objects in the environment of these individuals. Once in the environment, rhinovirus can survive hours to days [13–15]. Rhinovirus may be transmitted either by direct contact of a susceptible individual with virus on the hands of the infected person or on objects in the environment, followed by self-inoculation into the nose or eye, or by large-particle aerosols generated by the infected individual [15,16]. The role of direct contact in the transmission of rhinovirus infection suggests the possibility that transmission could be prevented by the use of virucidal agents directed at removing rhinovirus from the hands. 2.2

Replication

Rhinovirus replication is initiated by attachment to a receptor on the cell surface. The cellular receptor for most rhinovirus serotypes (major receptor group) is intercellular adhesion molecule-1 (ICAM-1) [17–19]. The rhinovirus capsid has an icosahedral symmetry formed of 60 identical protomers consisting of four proteins designated VP1–VP4. Crystallographic techniques have identified a depression in the capsid

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surface, the so-called ‘‘canyon,’’ surrounding each of the 12 fivefold axes of symmetry [20]. This canyon is the binding site for the ICAM-1 receptor molecule [21]. At the bottom of the viral canyon is a hydrophobic pocket. The current working hypothesis for the mechanism of viral uncoating suggests that this pocket contains a lipid moiety or ‘‘pocket factor’’ that is expelled when ICAM-1 binding occurs (reviewed in Ref. 22). The loss of the lipid moiety destabilizes the viral capsid and results in a change in conformation that allows release of the viral RNA. The hydrophobic pocket is also the site of binding for the capsid-binding agents that are currently under investigation as antiviral agents for treatment of rhinovirus infections [23]. Binding of these agents in the capsid pocket appears to inhibit attachment to ICAM-1 and stabilize the conformation of the viral capsid, thus inhibiting viral uncoating [24–27]. The relative importance of these mechanisms of action appears to vary depending on the viral serotype. A second aspect of viral replication that has been targeted by antiviral agents is the post-translational modification of viral proteins. The rhinovirus genome encodes a single large polyprotein that is cleaved to produce the individual structural and enzymatic proteins of the virus (reviewed in Ref. 28). Most of these cleavage reactions are catalyzed by a protease designated the 3C protease. This protease has a structure similar to that of trypsin, a serine protease, but the active site of the protease is a cysteine sulfhydryl [29]. The active site sequences are highly conserved in the different rhinovirus serotypes, and inhibitors of 3C protease have potent anti-rhinovirus activity for a broad spectrum of rhinovirus serotypes [30]. As discussed below, one of these agents is currently under study as treatments for rhinovirus colds. 2.3

Pathogenesis of Symptoms

The role of viral replication in the initiation of rhinovirus illness is clear, but efforts to treat rhinovirus colds by inhibition of virus replication have been generally disappointing. Recent research efforts have been directed at understanding the pathogenesis of rhinovirus-induced illness with the expectation that this effort might lead to more effective treatments for common cold symptoms. Only a small proportion of nasal epithelial cells are infected during a rhinovirus cold, and there is little evidence of direct viral damage to the nasal epithelium [31–35]. The observation that a polymorphonuclear leukocyte response was present during symptomatic rhinovirus infection but was absent in infected volunteers who were asymptomatic suggested that the host response to the virus might contribute to the symptom complex [36]. Subsequent studies identified

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interleukin-8 as one potential mediator of this host response [37,38]. Increased concentrations of IL-8 are present in the nasal secretions of subjects with symptomatic rhinovirus infection [38–40], and in experimental rhinovirus infection there is a modest correlation between the severity of common cold symptoms and the concentrations of IL-8 in the nasal secretions [40]. The observation that intranasal challenge of normal subjects with IL-8 produces symptoms that in some respects mimic the common cold also provides support for the hypothesis that IL-8 may contribute to common cold symptoms [41]. Other proinflammatory mediators have also been associated with rhinovirus cold symptoms. The kinins, bradykinin and lysylbradykinin, have been found in the nasal secretions of volunteers with rhinovirus colds, both experimentally induced and naturally acquired [36,42]. The concentration and time course of the production of kinins were roughly correlated with the severity and time course of symptoms in these subjects. Subjects who were infected with rhinovirus but who did not develop symptoms did not have an increase in nasal secretion kinin concentration. Intranasal challenge of uninfected volunteers with increasing concentrations of bradykinin resulted in symptoms of nasal obstruction, rhinorrhea, and sore throat [43]. The role of kinins in the pathogenesis of common cold symptoms is less clear, however, in light of the failure of a bradykinin antagonist to moderate common cold symptoms [44]. Similarly, in a more recent study, steroid therapy significantly reduced the concentration of kinins in nasal washes but had no effect on symptoms [45]. The interleukins IL-1 and IL-6 have also been reported in the nasal secretions of symptomatic subjects with experimental rhinovirus colds [46,47]. As with IL-8 and the kinins, the concentration of these proteins increases and then decreases as symptoms wax and wane. The concentration of Il-6 in nasal secretions appears to be directly correlated with the severity of the common cold symptoms [47]. In spite of these data demonstrating an association between common cold symptoms and various inflammatory mediators, the role of these mediators in pathogenesis will not be clear until specific inhibitors are available for use in human studies. If these proinflammatory mediators are involved in the symptomatic response to rhinovirus infection, then treatments directed at inhibiting the elaboration or action of these mediators might be beneficial. It remains to be determined, however, whether the nonspecific proinflammatory response to rhinovirus infection is an important contributor to the adaptive host responses that are required for natural recovery from, and development of immunity to, the rhinovirus infection.

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DIAGNOSIS OF RHINOVIRUS INFECTIONS

There are no rapid diagnostic methods currently available for the detection of rhinovirus infection that will be useful for guiding appropriate antiviral therapy. In the absence of effective antiviral treatments, a specific diagnosis of rhinovirus infection is not useful for the management of the common cold. Thus efforts to develop point-ofcare diagnostics for rhinovirus have been limited. Initial efforts to develop an immunoassay by using antibodies directed at the rhinovirus 3C protease have recently been described [48]. This assay, which uses thin-film biosensor technology that has been successfully adapted for rapid point-of-care diagnosis of other human pathogens was capable of detecting a broad spectrum of different rhinovirus serotypes although it remains to be determined whether the assay will be sufficiently sensitive for clinical use. The only generally available method for the laboratory diagnosis of rhinovirus infection is isolation of the virus in cell culture. Polymerase chain reaction (PCR) has been used in the research setting and appears to be the most sensitive method for detection of rhinovirus infection (reviewed in Ref. 49). Under ideal conditions, cell culture will detect approximately 75% of the infections documented by PCR. Neither of these currently available diagnostic methods is useful for guiding clinical decisions in patients with the common cold syndrome. In the absence of clinically useful laboratory methods, the diagnosis of rhinovirus infection relies on clinical criteria. Recent evidence suggests that 60–80% of patients with a common cold syndrome (afebrile, prominent nasal symptoms, and minimal systemic symptoms) that occurs between late August and early November will have a rhinovirus infection. The sensitivity of clinical diagnosis of rhinovirus infection is not known. 4 4.1

GENERAL ISSUES IN USE OF ANTI-RHINOVIRAL AGENTS Assessment of Antiviral Action

The virologic course of experimentally induced and naturally occurring rhinovirus colds has usually been characterized by determining the yields of infectious virus in sequentially collected nasal secretion samples. During the first several days of infection the titers of infectious virus peak at relatively low levels [approximately 103–104 50% tissue culture infectious doses (TCID50) per milliliter] and generally correlate with symptom severity (reviewed in Ref. 50). Recent studies indicate that viral RNA levels in nasal secretions are higher but also correlate broadly

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with symptom scores. However, clear relationships between pharmacokinetic (PK) properties and pharmacodynamic (PD) effects in terms of antiviral or clinical measures have not been defined for the investigational anti-rhinoviral agents currently available or previously studied. For example, it is uncertain what magnitudes of antiviral effect with respect to reductions in upper respiratory tract viral titers are needed to prevent or treat rhinovirus illness. One limitation to defining PK–PD relationships is the absence of a practical animal model of human rhinovirus infection. These viruses can successfully infect non-human primates, specifically chimpanzees and gibbons, but experimentally induced infections in susceptible human volunteers have provided the most informative in vivo model for assessing candidate anti-rhinoviral agents. For example, experimental prophylaxis studies with intranasally administered interferons have shown that dose-related protection can be achieved against rhinovirus infection and illness, such that high doses can prevent both infection, defined by virus recovery and/or virus-specific serologic responses, and common cold illness (reviewed in Ref. 51). Somewhat lower doses allow laboratory-documented infection but protect against illness, whereas even lower doses do not prevent illness. Substantial evidence indicates that the specific mechanism of antiviral action and pharmacokinetic properties of candidate antirhinovirus compounds are important factors in determining route and frequency of administration as well as probable clinical utility. Agents that interact directly with intact virions (e.g., receptor decoys, capsidbinding agents), and possibly those that bind to specific host cell receptors (e.g., interferons, anti-receptor antibodies), need to achieve adequate concentrations in the extracellular fluids lining the respiratory epithelium, whereas agents that inhibit an intracellular event in viral replication (e.g., viral protease or transcriptase action) need to reach adequate intracellular concentrations in the mucosa. These characteristics; the anatomy and physiology of the upper respiratory tract, including the effects of mucociliary clearance; and the pathogenesis of infection heavily influence the potential antiviral and clinical activities of antiviral agents for prevention and treatment of rhinovirus infections. 4.2

Prophylaxis Versus Treatment

In general, prevention of illness is easier to achieve than treatment of established rhinovirus colds. Several investigational agents (for example, intranasal interferons or pirodavir) are effective for chemoprophylaxis but not treatment of experimental or natural colds (reviewed in Ref. 51).

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Although these agents exert significant but incomplete antiviral effects when used for treatment, no symptom benefit has been recognized in treating colds. This lack of clinical efficacy is probably due to the pattern of viral replication during rhinovirus colds, with early peaking and rapid declines in nasal viral titers, and to the fact that host inflammatory responses and neurogenic reflexes play important roles in the symptom pathogenesis (reviewed in Ref. 52). In addition, the magnitude of the observed antiviral effects with these agents was likely insufficient. More recent studies with orally administered pleconaril establish that treatment of rhinovirus colds with selective inhibitors can reduce symptom duration and severity [53]. These findings indicate that ongoing, that is, continuing, viral replication is important in symptom pathogenesis and offer the possibility that prompt termination of replication might provide even greater clinical effects. Although symptom pathogenesis is incompletely understood, combinations of antiviral and host response–modifying agents would likely provide the greatest therapeutic benefit. Several current challenges are to identify key host inflammatory responses and selective, welltolerated inhibitors of these responses. For example, studies of systemic or intranasally applied corticosteroids have found evidence for both lack of consistent symptom benefit and upregulation of rhinovirus replication [45,54]. One treatment trial in young children with rhinovirus colds found that intranasal fluticasone not only failed to provide clinical benefit but also appeared to increase the likelihood of developing acute otitis media compared to placebo [55]. Such experiences indicate that host immune modulators need to be carefully assessed with regard to adverse effects on the virologic course of infection and will likely need to be used in combination with antiviral therapy. 4.3

Intranasal Administration

Available data indicate that delivery of antiviral agent to the nasal mucosa alone would be adequate for chemoprophylaxis of most rhinovirus colds. In particular, intranasal deposition of small quantities of infectious virus is sufficient to initiate infection in almost all persons lacking serotype-specific humoral immunity. Most infections appear to start after virus reaches the posterior nasopharynx, which is rich in ICAM-1-expressing cells overlying the adenoidal tissue [56]. Furthermore, studies of intranasal interferon established that intranasal delivery of antiviral agents to the nasopharyngeal mucosa by coarse sprays or drops is protective against both experimentally induced and naturally occurring rhinovirus infection and illness (reviewed in Ref. 51).

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The method used for intranasal administration may have implications for clinical effectiveness. Several studies employing radiolabeled tracers found broader, more uniform distribution but more rapid clearance after administration by nasal drops in the supine position than by nasal sprays in the sitting position [57,58]. Although drops appear more active under experimental infection conditions, in which the virus inoculum is also given as drops, a definitive study comparing the efficacy of antiviral sprays and drops has not been reported for natural rhinovirus colds. Although some rhinovirus infections may result from deposition of the virus in the lower respiratory tract, the clinical observations to date indicate that intranasal drug delivery is sufficient for prevention of most rhinovirus colds. Rapid mucociliary clearance of topically applied agents that do not interact with specific cellular receptors (e.g., unlike interferons, antiICAM-1 antibodies) or reach inhibitory intracellular levels would likely necessitate frequent intranasal dosing. For example, pirodavir is a capsid-binding agent that presumably interacts with extracellular virus to inhibit early replication events. Intranasal sprays of pirodavir were protective against experimental rhinovirus when given six times daily but not when given three times daily [59]. A topically applied agent such as soluble ICAM-1 (tremacamra) that serves as a reversible receptor decoy would also probably require frequent intranasal dosing. In contrast, a topically applied agent that inhibits an intracellular replication event (e.g., the 3C protease inhibitor ruprintrivir or AG7088) might be effective on a relatively infrequent dosing basis if taken up and retained by cells in an antivirally active form. Although mucociliary clearance is reduced, often dramatically, during established colds, it is unclear what implications this pathophysiological event has for the distribution, retention, and activity of topically administered antiviral agents. In addition, the increased respiratory secretions due to vascular leak, goblet cells, and glandular activity during colds would likely diminish the local antiviral effects of topically applied agents. 4.4

Systemic Administration

Once symptoms have developed, broader antiviral distribution within the respiratory tract appears to be desirable for treatment of established illness. The extent of viral replication, as reflected by peak viral titers in nasal secretions and by in situ hybridization studies of nasal mucosal biopsies [31], appears limited in rhinovirus colds. However, rhinovirus colds cause radiologically documented, self-limited sinusitis in most cold sufferers [60] and are frequently associated with otitis media in children

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[61,62], as well as otological abnormalities indicative of Eustachian tube dysfunction in adults [63]. In situ hybridization studies indicate that virus replication can occur in the tracheobronchial epithelium [64] and in the maxillary sinus mucosa [65], and rhinoviral RNA is frequently detected in the middle ear fluid of children with acute otitis media. Such observations suggest that replication commonly occurs at sites beyond the nasopharyngealmucosa and that inhibition of virus replication at such sites would be important to providing therapeutic benefit and reducing the risk of complications. For example, drug delivery to the tracheobronchial tree might be needed to reduce the likelihood of exacerbations of asthma or chronic obstructive pulmonary disease. This would require an inhaled route of delivery for agents that lack oral bioavailability. Conversely, an orally administered agent that distributes widely within the respiratory tract has a greater likelihood of inhibiting viral replication at extranasal sites (i.e., sinus, Eustachian tube, middle ear, tracheobronchial tree) and reducing associated complications. However, adequate drug distribution to the respiratory tract from the blood compartment following oral administration was a problem for several earlier capsid-binding agents, which achieved reasonable concentrations in the blood but ineffective concentrations in nasal secretions [66]. Furthermore, systemic administration might increase the risk of unacceptable drug-related toxicities.

4.5

Combination Therapies

The combined use of anti-rhinoviral agents may offer enhanced antiviral action, reduced toxicity, and reduced likelihood of the emergence of resistance. However, the number of active agents available is very limited, and combined use of antiviral agents has received little study. Additive or synergistic antiviral activity has been found with combinations of certain anti-rhinoviral agents (e.g., interferon-alfa and enviroxime), but one clinical trial found no greater protection from the combination than with interferon alone [67]. More effort has focused on using combinations of antiviral agents and host response modifiers to exert greater therapeutic benefits. Intranasal interferon-alfa 2b combined with an oral nonsteroidal antiinflammatory drug (naproxen) and a topical anticholinergic drug (ipratropium) exerted greater therapeutic activity than individual agents in experimental rhinovirus infections [68], and further studies of this combination antiviral–antimediator approach are under way for treating naturally occurring colds. Progress in this approach will depend on an

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improved understanding of the pathogenesis of rhinovirus illness and the identification of selective inhibitors of key host inflammatory responses. 5

ANTI-RHINOVIRUS AGENTS OF INVESTIGATIVE INTEREST

A substantial number of agents have been identified that have antirhinoviral activity in vitro (reviewed in Ref. 51), and candidate agents from several different mechanistic classes have progressed to clinical testing (Table 2). However, most have been abandoned because of lack of efficacy, limited antiviral spectrum, poor tolerance or pharmacokinetic properties, or high cost of production. Several investigational agents that have been recently studied or remain under active clinical development are discussed below. 5.1

Pleconaril

Pleconaril (3-[3,5-dimethyl-4-[[3-(3-methyl-5-isoxazolyl)propyl]oxy]phenyl]-5-(trifluoromethyl)-1,2,4-oxadiazole; VP-63843) is a novel, orally bioavailable, small-molecule inhibitor of picornaviruses that interacts with the virus capsid. It has progressed the furthest in clinical development of anti-rhinovirus agents to date. 5.1.1

Spectrum of Activity

Pleconaril has broad activity in vitro against rhinoviruses and enteroviruses, including polioviruses, coxsackieviruses, echoviruses, and TABLE 2 Antiviral Targets for Rhinovirus and Representative Agents That Have Been Studied in Clinical Trials Antiviral mechanism/ viral target

Antiviral class

Alteration of cell susceptibility Attachment

Interferons

Uncoating/capsid function Nucleic acid synthesis

Capsid binders

Post-translational protein modification

Receptor blockers

Transcriptase complex inhibitors 3C Protease inhibitors

Representative agents Recombinant IFN-a 2a/b, leukocyte IFN-a Anti-ICAM-1 antibody, soluble ICAM-1 Pleconaril, pirodavir Enviroxime Ruprintrivir (AG7088)

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human enterovirus. In cell culture, concentrations of 40.18 mM inhibit 90% of enterovirus clinical isolates [69]. The median EC50 value for rhinovirus clinical isolates is 0.04 mg/mL, but the range (1.0 mg/mL) is broad [70]. Approximately 10% of the numbered rhinovirus serotypes and 10% of clinical isolates are not inhibited by pleconaril concentrations of 1.0 mg/mL or more. Plasma concentrations in adults after a single 400 mg dose of pleconaril exceed the IC90 values for approximately 95% of enterovirus and 90% of rhinovirus serotypes. Oral pleconaril is active in experimental murine models of enteroviral central nervous system infection and in experimental human coxsackie A21 virus infection of the respiratory tract [71]. 5.1.2

Mechanism of Action

The capsid of picornaviruses plays essential roles in binding of the virus to and entry into the host cell. The crystallographic structure of picornaviruses shows a relatively conserved hydrophobic pocket within the capsid protein VP1 for both enteroviruses and rhinoviruses. Pleconaril was developed to bind into this hydrophobic pocket, and pleconaril binding induces conformational changes in the viral capsid that lead to altered attachment and/or viral uncoating. For major receptor group rhinoviruses, which use ICAM-1 as their cellular receptor, pleconaril alters the conformation of the canyon floor and inhibits the receptor binding interaction. For these and other picornaviruses, pleconaril filling the pocket stabilizes the capsid and inhibits the intracellular uncoating and release of viral RNA. This is indicated in part by increased thermostability of the virions. 5.1.3

Pharmacology

Pleconaril is orally absorbed with an estimated bioavailability that approaches 70% when it is administered with food. Pharmacokinetic studies using both a hard gelatin capsule and oral solution show firstorder absorption and dose-proportional blood concentrations [72–74]. Coadministration of pleconaril with food results in two- to four-fold enhanced bioavailability. Following multiple doses of 400 mg given with food, peak plasma concentrations occur at 2–3 hr and average 2.2 mg/mL on day 1 and 3.4 mg/mL on day 7 in healthy adults [75]. The elimination half-life of pleconaril is best characterized by a two-compartment model with a shorter initial phase (T1/2 of 2–3 hr) and a prolonged terminal elimination phase (T1/2 of approximately 180 hr). Studies in children suggest decreased maximal concentrations and smaller area under the curve compared to adults [73]. In neonates, oral bioavailability of a liquid formulation is variable with normalized Cmax averaging 29 ng/mL/mg

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dose [76]. Animal studies have shown that pleconaril distributes to the liver, nasal epithelium, and brain within 2 hr of the administration of oral solution; concentrations of drug in the central nervous system and nasal secretions exceed those of plasma. Pleconaril has a large volume of distribution consistent with significant tissue distribution [77]. It undergoes slow but extensive metabolism, with over 30 metabolites that apparently lack antiviral activity. The major route appears to be reductive cleavage of the trifluoromethyl oxadiazole ring. Pleconaril and its metabolites are excreted primarily in the feces, with approximately 80% of an orally administered dose excreted in this way within 48 hr of administration; less than 1% of pleconaril is excreted unchanged in the urine. 5.1.4

Adverse Effects

Pleconaril appears to be safe and generally well tolerated in adults and children. In placebo-controlled studies of the use of pleconaril for rhinoviral illness in adults, the only significant differences in adverse events between pleconaril and placebo recipients have been in gastrointestinal complaints (nausea, emesis) and menstrual irregularities. In a review of 32 patients treated with compassionate release pleconaril for severe enterovirus disease, 13 experienced no adverse effects, and the most common adverse events among the remaining patients were nausea and vomiting. No important effects on hepatic levels of CYP450 isoenzymes in rat or dog microsomes were observed following in vivo dosing (100 mg/kg) for 26 weeks [78]. One human study found modest (18 yr >18 yr

720 EL.U 25 U 1440 EL.U 50U

0.5 mL 0.5 mL 1.0 mL 1.0 mL

a

Schedule (months) 0, 0, 0, 0,

6–12 6–18 6–12 6

EL.U ¼ ELISA unit.

injection [60–64]. The protective efficacy of both vaccines was evaluated in double-blind, controlled, randomized clinical trials conducted in Thailand among 40,000 children and in a semiclosed New York community with a high incidence of HAV infections and found to be 94–100% [8–10]. Surveillance data were collected to monitor long-term protective efficacy in these vaccinated individuals and to determine the possible need for a booster injection. In the longest follow-up study available, no hepatitis A cases were detected among children followed for 7 years after vaccination [65]. Protective levels of anti-HAV are estimated to last for at least 20 years [66–68]. Reduced immunogenicity is observed when the hepatitis A vaccine is administered simultaneously with serum immunoglobulin [69,70]. Reduced anti-HAV antibody levels after vaccination were also found in risk groups, such as HIV-infected men and patients with chronic liver disease of viral or nonviral origin [71,72]. Other factors, such as age, smoking, and obesity, that might affect the immunogenicity of hepatitis A vaccines have not been elucidated so far. Other vaccines (e.g., for hepatitis B, DTP, yellow fever, rabies, Japanese encephalitis, poliovirus) can be administered simultaneously with hepatitis A vaccine without affecting either vaccine’s immunogenicity or increasing the rate of adverse events [73,74].

8.2

Side Effects and Adverse Events

Pre- and postlicensure studies have demonstrated the safety of both vaccines. Among adults, the most frequently reported side effect is a local reaction at the injection site (soreness, 56%) and systemic reactions such as headache (14%) and malaise (7%). Serious adverse events are in the range of background incidence rates and cannot be attributed to the vaccine.

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271

Testing for Anti-HAV Before and After Vaccination

The decision to determine the immune status by serological testing prior to vaccination should be based on the expected prevalence of immunity and the cost of serological testing. In most industrialized countries screening of individuals >40 years might be cost-effective. Postvaccination testing is not indicated due to the high immunogenicity of the vaccine in adults and children. Anti-HAV concentrations after vaccination are usually 10–100-fold lower than those after natural infection. Highly sensitive assays [lower limit of detection 20 mlU/mL (milliinternational units per milliliter) minimal protective antibody level] can sometimes detect low antibody liters, whereas the routine anti-HAV (IgG and IgM) test (lower limit of detection 100 mlU/mL) is usually insufficient. Anti-HAV tests are standardized by a WHO reference immunoglobulin, and the results are expressed in milli-international units per milliliter.

8.4

Postexposure Prophylaxis

Nonimmune persons who have recently been exposed to HAV should receive a single intramuscular dose of Ig (0.02 mL/kg) as soon as possible but not later than 10–14 days after exposure. Candidates for Ig administration are people who have had close personal contact with individuals acutely infected with HAV, unvaccinated staff or attendees of day care centers or homes where cases of hepatitis A have been recognized, classroom contacts of an index case, or those in comparable situations. The hepatitis A vaccine has been successfully used to control community-wide outbreaks because it prevents secondary infections in those in contact with infected people [75]. Active immunization can easily be combined with IG administration.

9

PERSPECTIVES

Hepatitis A virus infection of the liver causes significant morbidity and even mortality. Great success has been achieved by taking preventive measures with the killed hepatitis A vaccine, which provides long-lasting protection from infection by all known viral variants. Therefore, the future goal in the control of HAV infections will be vaccination of people at risk. Therapeutic intervention will be restricted to patients refractory to vaccination.

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9 Hepatitis B Virus Guido Gerken and Christoph Jochum University of Essen, Essen, Germany

1

DIAGNOSIS OF INFECTION

The hepatitis B virus (HBV) is an enveloped partially double-stranded circular DNA virus with a diameter of 42 nm (Dane particle) (Fig. 1). It belongs to the Hepadna family of viruses. HBV can infect only humans and chimpanzees. Its genome consists of approximately 3200 base pairs. It codifies various genes in an open reading frame (Fig. 2) [1,2]. The preC/C gene codes for polypeptides that make up the nucleocapsid containing the HB core antigen (HBcAg) and the HBe antigen (HBeAg), which is detectable in the blood after post-translational processing. The pre-S1/pre-S2/S gene codes for the lipoproteins that make up the viral envelope containing the HBs antigen (HBsAg). The P gene codes for the viral polymerase. This protein has several functions such as RNA pregenome encapsidation, priming of DNA synthesis, reverse transcription, and plus-strand polymerization [3]. The true function of the X gene is still unclear. It seems to be involved in regulatory functions and plays a role in hepatocarcinogenesis in chronic HBV infection. The HBV causes either acute or chronic inflammation of the liver. The incubation period from inoculation to disease ranges from 4 weeks to 6 months. About 50% of the cases do not develop jaundice. Of the 277

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FIGURE 1

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The structure of hepatitis B virus.

acute infections, 1–2% lead to acute liver failure. Between these two distinctively different courses of hepatitis B, a wide spectrum of symptoms exists. The prodrome usually consists of malaise, fatigue, nausea, elevated body temperature, and in some cases arthralgia and rash. Clinical hepatitis manifests as jaundice and elevated serum aminotransaminase values, pruritus, and nausea. Fulminant hepatitis shows symptoms of liver failure such as coagulopathy, encephalopathy, and rising bilirubin level. In most of the cases aminotransaminase levels decrease to normal within the following weeks. Eighty to ninety percent of the adults have normal aminotransaminase values and achieve clearance of the virus from the blood after 6 months. In 10–20% of the adults the virus persists in the blood. In these cases the hepatitis B advances to a chronic disease. In newborns and young children the rate of chronicity is remarkably higher. A perinatal infection becomes chronic in more than 90% of the cases. Chronic HBV infection is often completely asymptomatic and may not be detected until symptoms of cirrhosis appear. Chronic hepatitis B could appear with chronic fatigue and diffuse upper abdominal pain. In

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FIGURE 2 The structure of the HBV genome. For simplification, the circular genome is shown as a straight line.

some cases of chronic hepatitis B, extrahepatic manifestations such as vasculitis [4], porphyria cutanea tarda [5], essential mixed cryoglobulinemia [6], and other autoimmune phenomena [7,8] may occur.

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The diagnosis of HBV infection is made by antibody and antigen tests and viral DNA detection. During HBV infection antibodies against HBcAg, HBeAg, and HBsAg are detectable: anti-HBc, anti-HBe, and antiHBs. Viral proteins such as HBeAg and HBsAg are detectable in the blood. Qualitative and quantitative DNA tests are available to prove viral load. The presence of anti-HBc of the IgM type and HBsAg confirm the diagnosis of acute HBV infection. The HBeAg, if detectable, is a marker for active viral replication, but the lack of HBeAg does not exclude viral replication. Some viral strains have lost the HBeAg by a mutation in the precore region [9]. These precore mutant viruses are particularly common in the Mediterranean region and in Asia. Therefore the ‘‘gold standard’’ for confirmation of viral replication is qualitative DNA detection. Virus DNA in blood or tissue may be detected by using the technique of molecular hybridization or polymerase chain reaction (PCR) assay. Molecular hybridization allows quantification and is able to detect as little as 2 pg of HBV DNA/mL serum. With the PCR technique, as few as 200 copies per milliliter are detectable [10]. Seroconversion is indicated by the occurrence of anti-HBe. Loss of HBsAg is associated with the ‘‘healing’’ of the disease. The combination of the different markers is characteristic for different stages of the acute disease (Fig. 3). Chronic HBV infection is defined by persistence of HBsAg for more than 6 months. The HBsAg-positive patient with normal aminotransaminase levels, low DNA levels, and positive anti-HBe detection is considered an asymptomatic carrier and does not need therapy in most cases. A therapeutic decision has to be made according to the individual constellation of HBV markers (Table 1). 2

VACCINATION

Today highly effective passive and active vaccines against HBV are available. HBV-specific immunoglobulin (HBIg) contains high anti-HBs titers. It has been shown to be effective for passive immunization against HBV infection if given prophylactically or within a few hours after infection. There is an indication for vaccination of persons who had sexual contact with an infected individual, neonates born to an HBsAgpositive mother, and persons who have had parenteral exposure (needle injury, etc.) to HBsAg-positive blood or body fluids [11–15]. Passive immunization can be effective for up to 6 months. Persistent protection with a consequent reduction of the community acquired infection rate requires active immunization.

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FIGURE 3 Serum markers during an acute hepatitis B infection.

The first active HBV vaccine was introduced in the early 1970s [16]; it was plasma-derived. Since the early 1990s the vaccine has been produced in the western countries by recombinant DNA technology. In both types of vaccine the essential immunogen is HBsAg. They confer a long-lasting protection against HBV infection [17]. The vaccines actually in use do not contain the highly immunogenic pre-S1 and pre-S2 determinants [18]. Incorporation of the pre-S1 and pre-S2 antigen determinants in a new vaccine can improve the seroconversion rate after vaccination [19]. Some data also suggest that the pre-S1 and pre-S2 vaccine is an effective immune therapy in chronic HBV infection [20], but these results are controversial. This vaccine has not yet been approved. In Germany and in over 80 other countries, immunization against HBV is generally recommended for children and adolescents [21]. Universal immunization of newborns against HBV in epidemic countries such as Taiwan has resulted in a remarkable decrease in the HBV carrier rate and in the incidence of hepatocellular carcinoma (HCC) in children [22]. In 1995 the World Health Organization (WHO) therefore recommended the general vaccination of all children worldwide [23]. In addition to this general recommendation, persons who are at special risk for HBV infection should be vaccinated in any case (Table 2).

HBeAg negative (HBV pre-core variants)

HBeAg-positive (wild-type HBV)

Treatment

IFN plus antivirals

Favor nucleotide/nucleoside analogs:

Favor nucleotide/nucleoside analogs with:

Favor IFN with:

Treatment in Chronic Hepatitis B

HBV-DNA-positive patients

TABLE 1

Long-term treatment usually required Favor therapies with low resistance risk (e.g., ADV) Inclusion into studies

High ALT Low HBV-DNA No contraindications Motivated patients Low ALT High HBV-DNA Patient’s preference

Options

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TABLE 2

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Individuals Who Are at Risk for HBV Infection

Illicit injection drug users Health care workers Chronically transfused persons Hemodialysis patients Homosexually active men Heterosexuals with multiple partners or other sexually transmitted disease Police officers, emergency medical technicians, firefighters Household and sexual contacts of infected persons Clients and staff of institutions for the developmentally disabled Newborns of infected mothers

The currently recommended schedule for active vaccination consists of three doses given at 0, 6, and 24 weeks. A successful vaccination should result in an anti-HBs titer of 10 IU/mL or higher, which is achieved in 95% of young immunocompetent individuals. Adults younger than 40 years of age show a better response rate to vaccination than older individuals [24]. Persons with severe immunosuppression may not respond, even after a second course of vaccination. It remains unclear how long after vaccination the protection will continue. Anti-HBs may disappear within 10 years after successful immunization in about 40% of vaccinated adults. Immunity against clinical disease, however, may persist for years and even remain lifelong after the loss of measurable anti-HBs titer. Nevertheless, booster immunization is recommended in immunosuppressed individuals with anti-HBs levels below 10 IU/mL. 3

VIRAL REPLICATION STRATEGY AND TARGETS FOR THERAPY

The replication cycle of HBV is very complex and not completely understood. Viral replication takes place in the cytoplasm [1,25,26]. HBV is not cytopathic and does not kill the host cell, which is an outstanding feature of HBV infection. Only after long-term infection do secondary cytopathic effects occur. The initial steps of adhesion to the cell and cell invasion are not yet understood. The cellular receptor is still unknown. After invading the cytoplasm, the viral core particle is uncoated and is translocated to the nucleus of the host cell. Here the synthesis of the (þ)-strand HBV DNA is completed and DNA repair takes place. The HBV DNA is converted to a covalently closed circular DNA (cccDNA or

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supercoiled DNA). The cccDNA remains episomal and serves as a template for the cellular polymerase II, which produces pregenomic RNA and messenger RNAs [2]. The pregenomic RNA transcript serves both as a template for the reverse transcription of the first (
)-strand HBV DNA and as a messenger RNA for the viral polymerase and the nucleocapsid. The smaller pre-S and pre-C transcripts are translated to viral surface and core proteins. The synthesis of the first (
)-strand HBV DNA is followed by the production of a shorter (þ)-strand DNA to become a partially double-stranded DNA. This generated DNA can serve as viral nucleic acid in mature virions budding out from the host cells to infect new target cells. In addition, this new synthesized DNA can build new cccDNA, resulting in more than one cccDNA copy per nucleus [27]. This replication cycle offers a number of targets for specific antiviral treatment strategies. The transcription could be influenced by cytokines and antisense constructs. The viral polymerase with its reverse transcriptase activity is the main target of existing drugs, especially the nucleoside analogs. Another target is the protein synthesis and viral assembly, where cytokines and other non-nucleosidic inhibitors have been shown to be effective (27a). Figure 4 summarizes the replication cycle of the HBV and indicates the targets for therapy. The best target for antiviral therapy in HBV infection seems to be the inhibition of cccDNA formation in the nucleus. However, most of the antiviral agents that have been investigated so far show no or little effect against cccDNA. This accounts for the rapid reappearance of HBV after termination of antiviral therapy. In theory, complete viral clearance could be achieved if potent antiviral therapy, which completely inhibits viral synthesis, were administered as long as the duration of treatment outlasted the pool of existing cccDNA. However, the half-life of cccDNA depends on the loss of hepatocytes [28]. The half-life of infected hepatocytes is 10–100 days [29], so complete clearance requires an effective treatment of 1–10 years. However, costs, side effects, and resistant viral mutants preclude such a long therapy. Therefore the development of new therapeutic strategies should be focused on the blockade of cccDNA synthesis and/or cccDNA eradication.

4

TREATMENT AND MONITORING OF TREATMENT SUCCESS

Acute hepatitis B does not require specific therapy in most cases. In cases of progression toward liver failure, lamivudine, a nucleoside analog, could be used. Severe liver failure requires urgent liver transplantation.

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FIGURE 4 HBV replication cycle and possible targets of an antiviral therapy. The black arrows indicate actions that are of predominantly cellular origin. The dashed arrows indicate actions of viral proteins. Potential targets of antiviral therapy are shown in gray.

In the prevention of liver cirrhosis and hepatocarcinoma, chronic HBV infection is getting increasing attention. Currently there are two therapeutic strategies available for treating chronic HBV infection: (1) the use of immunomodulatory drugs such as interferon and (2) the use of nucleoside analogs. The second group serves to block the reverse transcriptase activity of the viral polymerase. Both therapeutic options are of limited success and/or have serious side effects. Therefore the indication for therapy and drug regimen should be carefully defined. An asymptomatic carrier (see Table 1) does not need specific therapy, but periodic controls are required. In the case of superinfection with the hepatitis D virus no therapy is possible. All other cases of chronic hepatitis B should be treated. Immunomodulatory agents are mostly cytokines or derivatives, which are important in the regulation of natural defense against viral

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infection. The potential mechanism of action is the restitution of the host immune system, which is incompetent to eliminate the virus. Interferon-a was the first drug approved for the treatment of chronic HBV infection. Although the exact mechanism of action of interferon-a is not yet understood, it has been shown to inhibit the replication of HBV and to establish disease remission under prolonged therapy. Interferon is administered subcutaneously (6–10 Mio IU [1 million international units ¼ 1 Mio unit] three times a week) for 4–6 months. In general, interferon therapy is effective in only 20–30% of the patients [30]. The best candidates for successful interferon therapy are patients with high aminotransaminase levels and low viral load. Interferon has a variety of side effects, which exclude some patients from therapy. In addition, the relatively low success rate makes interferon not an ideal antiviral agent. Table 3 gives a summary of the side effects of interferon-a and the contraindications to interferon treatment. A second hopeful immunomodulatory drug is thymosin. Thymosin is a thymic extract that mediates a variety of immunological effects including augmentation of suppressor T-cell activity and the stimulation of IgG production. Thymosin is able to promote disease remission and cessation of HBV replication in patients with HBeAgpositive chronic HBV without severe side effects [31–33]. Its synthetic derivative thymosin-a1 was effective and safe in therapy of chronic HBV infection in several multicenter phase III studies in the People’s Republic

TABLE 3

Side Effects of and Contraindications to Interferon-a

Side effects of interferon-a

Contraindications to interferon-a

Flu-like illness Bone marrow suppression Myalgia Headache Depression Irritability Sleep disturbance Weight loss Alopecia Skin rash Fatigue Arthralgia Hyperthrosis

Thrombopenia,