PerspectiveVirology

A Molecular Whodunit

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Science  07 Sep 2001:
Vol. 293, Issue 5536, pp. 1773-1775
DOI: 10.1126/science.1065206

T wo influenza [HN1] outbreaks in the 20th century challenge current beliefs about patterns of influenza virulence. The “Spanish flu” pandemic of 1918 [HN2], rather than sparing young healthy adults, killed millions in the prime of life. It wiped out entire villages at opposite ends of the Earth and depressed world population growth for 10 years. In 1997, a lethal avian influenza virus was transmitted directly to humans from chickens in Hong Kong [HN3]. Six of 18 clinically diagnosed human cases were fatal, and, again, many of the victims were young adults. Both of these outbreaks suggest the emergence of highly virulent influenza variants. Unfortunately, until the basis of influenza virulence is understood, the human population will be defenseless against similar outbreaks in the future. In this issue of Science, Hatta and colleagues (page 1840) (1) [HN4] and Gibbs and co-workers (page 1842) (2) [HN5] offer new insights into the virulence [HN6] of these influenza strains.

The virulence of a virus is defined by its comparative capacity to produce disease in a host (3). The 1918 Spanish flu virus was extremely virulent: It killed 10 times as many persons in the United States as did the 1957 Asian flu and about 20 times as many as the 1968 Hong Kong flu [HN7]. Both the Asian and 1968 Hong Kong viruses were reassortants [HN8], that is, their genes were acquired from flu viruses infecting different host species. Genes encoding the hemagglutinin (HA) and polymerase 1 (PB1) proteins [HN9] of these two flu viruses—and the enzyme neuraminidase (NA) [HN10] of the Asian strain—were acquired from a Eurasian avian influenza virus; the remaining genes were all acquired from the human influenza virus circulating at the time.

The origin of the 1918 Spanish influenza virus, however, is still a work in progress. Taubenberger's group is analyzing short fragments of RNA from the tissues of 1918 victims: preserved specimens from soldiers and lung tissue from an Inuit woman buried in the Alaskan permafrost (4) [HN11]. Sequence and phylogenetic analysis of the HA, NA, and nonstructural (NS) gene segments of these samples suggests that an avian influenza virus was transmitted to humans and pigs, developing separate lineages sometime before 1918. The available data do not suggest that the 1918 virus is a reassortant, rather, it seems to be more akin to the “bird flu” that emerged in Hong Kong in 1997.

As many as 10% of poultry workers in Hong Kong were serologically reactive to the 1997 “bird flu” virus (subtype H5N1) (5). Late in that year, the deaths of 6 of 18 clinically diagnosed persons suggested that a variant that was highly virulent in humans had emerged. When the viruses isolated from humans were inoculated into mice, they differed in virulence: One group of viruses replicated in the lungs, spread to the brain, and was lethal, whereas the other replicated only in the lungs and did not cause death. Because there was a general correspondence between lethality in humans and in mice, the mouse offered an experimental system for dissection of the genetic basis of the virulence of these viruses.

Genetic manipulation of segmented negative-sense RNA viruses such as influenza virus was extremely difficult until 1989, when Palese [HN12] and collaborators developed an appropriate reverse genetics [HN13] method (6). Only in the past 2 years has it become possible to recover all eight gene segments of infectious influenza viruses from bacterial plasmids (1). Because these plasmid-only systems can be used in any laboratory, influenza viruses can now be “made to order.” Taking advantage of plasmid-based reverse genetics [HN14], Hatta et al. (4) compared a pair of the mouse-lethal and mouse-nonlethal H5N1 influenza virus strains from Hong Kong. They show that a glutamic acid-to-lysine substitution at residue 627 of the PB2 polymerase protein, together with an HA glycoprotein that can be readily cleaved, determined the extreme virulence of the H5N1 Hong Kong flu virus. Interestingly, although mice are not men, the PB2 of all human influenza viruses (subtypes H1, H2, and H3) so far analyzed has a lysine at position 627, whereas only 3 of the 17 H5N1 human isolates from Hong Kong in 1997 had a lysine at this position. The H5N1 viruses isolated from humans were probably evolving rapidly because of their recent introduction to a new host.

The virulence of H5N1 in humans is likely to involve more genomic changes than the PB2 point mutation. Classical reassortant experiments indicate that the virulence of influenza virus is a polygenic trait involving HA and a constellation of other gene segments that can vary with the virus strain and host (see the figure) (7). Furthermore, although mice are a good model of human H5N1 infection (because the viruses replicate in mice without adaptation), the viruses are not transmitted from mouse to mouse. The wholesale slaughter of poultry in Hong Kong in 1997 eliminated the source of H5N1 from live poultry markets and interrupted any mutation and reassortment that would have permitted human-to-human spread. The Hatta et al. work is a big step forward because it defines the molecular basis of the virulence of two examples of the Hong Kong 1997 H5N1 virus in mice (1). But just as important is the fact that it provides a proof-of-principle that reverse genetics has finally come of age in influenza research.

The devil is in the details.

The proteins of the influenza virus and their importance in virulence. The glycoprotein hemagglutinin (HA) is the principal antigen on the surface of the flu virus and is cleaved by proteases in host epithelial cells. The enzyme neuraminidase (NA), also on the flu virus surface, cleaves sialic acid residues from the host cell receptor for the virus, freeing virus particles and enabling them to spread throughout the body. A point mutation in the internal protein PB2 (a polymerase) is associated with the virulence of the 1997 Hong Kong flu virus (1). The NS1 protein is an interferon antagonist and blocks the host's ability to make interferon. For a virus to be highly virulent with pandemic potential, it must circumvent the host immune response. To do this, the virus must express new epitopes on its proteins (HA, NA, NP, PA, and PB1) that will not be recognized by the host's T and B cells.

We do not yet know the basis of the virulence of the 1918 Spanish flu virus. In their report, Gibbs et al. (2) propose that a recombinant HA was responsible for the virulence of this virus. Their proposal is definitely a stretch for influenza virologists because homologous recombination (portions of a single gene segment from two different flu virus strains) is a rare event among RNA viruses, and many influenza virologists are not convinced that it even occurs. However, unorthodox proposals like this one can make everyone stop and reconsider the evidence. The authors contend that the Spanish influenza virus HA was a recombinant whose globular domain (HA1), which contains antigenic and host cell receptor binding sites, was acquired from a swine influenza virus and whose stalk region (HA2) was derived from the human virus. Their proposal that the 1918 swine lineage diverged from the human lineage before 1918 is consistent with the results of earlier phylogenetic analyses of the nucleoprotein (NP) and matrix (M) flu virus genes, which placed the divergence of the swine and human lineages several years before the pandemic (8, 9). There is evidence that a much milder strain of the 1918 H1N1 virus was circulating before the pandemic began. Military medical records (long kept secret) reveal that there were a large number of deaths from respiratory infection in military camps in France in 1916 (10). The heliotrope cyanosis (bluish-purple discoloration of skin and mucous membranes) described in these records from 1916 is similar to that seen in 1918 flu victims. Therefore, the Spanish flu virus of 1918 or its precursor viruses are likely to have preceded the arrival of American troops in Europe, although the origin and route of the viruses are unknown. The widely distributed coincidental outbreaks of the Spanish flu in different parts of the world seem to correspond with the return of soldiers from Europe to their home countries at the end of World War I.

Definitive genetic analysis of the 1918 human influenza virus is difficult. The primary sequences of swine and avian influenza viruses before 1930 are unknown, and available samples of these viruses after 1930 have acquired mutations because of their passage in chicken eggs and mice. The transfer of viruses from pigs to humans or vice versa and the infection of either host with both pig and human viruses before 1918 would provide possible conditions for reassortment or recombination. Swine H1N1 virus [HN15] is frequently transmitted to humans and occasionally causes human deaths (11). Influenza viruses are subject to different selective pressures in pigs and in humans—for example, HA undergoes antigenic drift (the accumulation of single amino acid changes) more slowly in pigs. Thus, although the highly pathogenic 1918 virus may have come from the pig lineage, the evidence is not conclusive. These questions may be resolved if archival samples containing swine influenza viruses can be found.

Unfortunately, the proposed recombination events through which the Spanish flu virus may have arisen bring us no closer to understanding its virulence. To be highly virulent, a virus must possess new B and T cell epitopes on its HA, NA, NP, PA, and PB1 proteins that have not been seen previously by the host lymphocyte population. In this way, the flu virus is able to rapidly invade host epithelial cells before the immune system has a chance to become mobilized. In addition, extreme virulence requires that the interaction of virus with host lymphocytes must trigger a devastating cytokine and apoptotic response resulting in severe inflammation and the death of large numbers of cells (12).

The parts played by PB2 and other proteins of the 1918 flu virus in the overwhelming immune responses that killed healthy young soldiers within a single day remain to be understood. The NS1 protein turns out to be a potent type I interferon antagonist (13). Whether NS1 was a crucial player in the virulence of the 1918 virus remains an open question. In preliminary studies, a highly laboratory-adapted reassortant strain of the A/WSN/33 (H1N1) virus containing the 1918 NS gene sequence was not virulent in mice (4).

These questions cannot be resolved until the entire primary sequence of the 1918 Spanish influenza genome is known. The sequencing and assembly of the shorter gene segments (HA, NA, and NS) from short RNA fragments is a major achievement. The sequencing and assembly of the larger PB1 and PB2 genes remains a huge challenge. Although it appears likely that the entire genome sequence will be obtained, the possibility of error will increase with the length of the genes, and multiple genomes must be analyzed to ensure an authentic sequence. Additional helpful clues might be obtained from the sequence of the causative agent of the mild influenza outbreak in early 1918 and from the genomes of other ancient avian or mammalian viruses that may be found in the permafrost. Efforts are under way to obtain frozen penguin and gull droppings from ancient nesting sites in the Antarctic permafrost.

Recent advances in reverse genetics, such as those described by Hatta et al., now permit complete manipulation of all genes of the influenza virus. This progress offers many advantages. It is now possible to make human vaccines more quickly and efficiently by creating tailor-made rapid-growth, high-yield reassortants [HN16]. Specific changes can be inserted into future live attenuated vaccine strains, and all functional domains of flu virus proteins, and their interactions with host cells, can be defined. Creation of the global influenza laboratory proposed by Layne and colleagues (see the editorial on page 1729) [HN17] will provide advance warning of a new pandemic influenza virus that, together with reverse genetics and human genomics, will resolve the molecular basis of influenza virulence. That information in turn will allow the selection of vaccine strains with greater certainty and may in the future allow us to identify influenza viruses that are potential human pandemic strains.

Manipulation of influenza viruses by reverse genetics is also cause for caution. When the complete sequence of the 1918 virus is obtained, it may be possible to create the virus anew. Such a study should be attempted only if its benefits warrant the risk and if high-level biosafety laboratories are used. Of more immediate concern is the ability to make H5N1 “bird flu”-like viruses that can be transmitted among mammals. Although any influenza virus can theoretically arise in the natural environment, scientists will possess the knowledge and the tools to assemble viruses that are tailored for virulence in the desired host. Safety issues concerning the manipulation of influenza viruses by reverse genetics were explored at a National Institute of Allergy and Infectious Diseases (USA) conference in July this year. Discussions centered on using local biosafety committees to examine the specific planned work and to make risk assessments and safety recommendations. The need to re-examine the current biosafety guidelines in light of technical advances was also debated.

The human population is most vulnerable to influenza viruses that have new antigenic properties. It now takes about 6 months to prepare an appropriate vaccine. Although advances in reverse genetics will shorten this time, several months will still be needed to prepare a vaccine. During the period between detection of a pandemic strain and the availability of a vaccine, antiviral drugs will be essential (see the Perspective on page 1776). It is gravely disquieting that no action has yet been taken to create strategic stockpiles of such drugs.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

This issue of Science has a related Enhanced Perspective by G. Laver and E. Garman titled “The origin and control of pandemic influenza.”

Dictionaries and Glossaries

The On-line Medical Dictionary is provided by CancerWeb.

The InteliHealth Web site makes available the Merriam-Webster Medical Dictionary.

The Academic Press Dictionary of Science and Technology is made available by the publisher Harcourt.

Web Collections, References, and Resource Lists

P. Gannon's Cell & Molecular Biology Online is a collection of annotated links to Internet resources.

The Google Web Directory offers collection of virology Internet resources.

The library of the Karolinska Institutet, Stockholm, provides links to biomedical information resources on the Internet. A section on virus diseases is included.

MEDLINEplus, a resource maintained by the U.S. National Library of Medicine, provides reference information and links to Internet biomedical resources.

BioWurld, hosted by the European Bioinformatics Institute, is a searchable index of resources in the fields of bioinformatics and molecular biology.

Science's Functional Genomics Web site provides links to news, educational, and scientific resources in genomics and postgenomics.

The CMS Molecular Biology Resource is a compendium of electronic and Internet-accessible tools and resources for molecular biology, biotechnology, molecular evolution, biochemistry, and biomolecular modeling.

The WWW Virtual Library of Microbiology and Virology is maintained by S. Sutton of the Microbiology Network.

All the Virology on the WWW is a resource collection of virology information maintained by D. Sanders.

The Influenza Sequence Database is maintained by the Theoretical Biology and Biophysics Group at Los Alamos National Laboratory.

The National Center for Infectious Diseases (NCID) of the U.S. Centers for Disease Control and Prevention (CDC) offers an influenza information Web page. The CDC's National Vaccine Program Office (NVPO) offers a presentation on pandemic influenza.

The Communicable Disease Surveillance and Response (CSR) division of the World Health Organization (WHO) provides an influenza information page. FluNet is WHO's geographical information system to monitor influenza activity. The WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, provides an introduction to influenza and Internet links.

The Division of Microbiology and Infectious Diseases of the U.S. National Institute of Allergy and Infectious Diseases (NIAID) offers an information page on influenza.

InfluenzaNews.com is provided by the International Influenza Education Panel. An introduction to influenza virus strains, a collection of Internet links, and a glossary are provided.

Online Texts and Lecture Notes

J. Kimball presents Kimball's Biology Pages, an online biology textbook and glossary. Presentations on viruses and on influenza are included.

A student project on the influenza virus was prepared for a Brown University course on the development of vaccines to infectious diseases taught by A. De Groot and P. Knopf. A student presentation on antigenic variation with a section on influenza is also available.

Medical Microbiology is an online textbook edited by S. Baron, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston. A chapter by R. Couch on the orthomyxoviruses and a chapter by W. R. Fleischmann on viral genetics are included.

Microbiology and Immunology On-line from the Department of Microbiology and Immunology, University of South Carolina School of Medicine, offers lecture notes on virology. A presentation by M. Hunt on the influenza virus is included.

The University of Florida Continuing Medical Education Web site makes available a tutorial on influenza by P. Small and B. Bender.

The Department of Microbiology and Immunology, University of Leicester, UK, makes available lecture notes and other resources for a virology course; an overview of orthomyxoviruses is included. The Infection & Immunity Web page provides a microbiology glossary and a virology glossary.

E. Rybicki, Department of Molecular and Cell Biology, University of Cape Town, South Africa, presents an introduction to molecular virology.

M. Hewlett, Department of Molecular and Cellular Biology, University of Arizona, offers lecture notes for a virology course.

General Reports and Articles

Genetic Engineering of Viruses and Viral Vectors is a 1996 report of a National Academy of Sciences colloquium, made available online by the National Academy Press. These symposium papers were also published in the 15 October 1996 issue of the Proceedings of the National Academy of Sciences.

The July-September 1998 issue of Emerging Infectious Diseases had an article by R. Webster titled “Influenza: An emerging disease.”

The 29 September 1997 issue of the New Yorker had an article by M. Gladwell about the search for the 1918 influenza virus titled “The dead zone.”

Numbered Hypernotes

1. Influenza and the influenza virus. The Johns Hopkins Healthcare Epidemiology and Infection Control Web site offers a presentation on influenza. GlaxoSmithKline's Worldwide Vaccines Web site includes an information page on influenza. The National Foundation for Infectious Diseases offers information about influenza and the influenza virus. J. Love's Science Explained offers a presentation on influenza viruses. Wong's Virology Web site provides a presentation on influenza viruses. The National Influenza Centre of Switzerland provides an introduction to influenza and a collection of Internet links. The Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York, Stony Brook, makes available lecture notes by J. Hearing on influenza and influenza viruses for a medical microbiology course. M. Taylor, Department of Biology, Indiana University, offers lecture notes on the influenza virus for a virology course. R. Siegel, Department of Microbiology and Immunology, Stanford University School of Medicine, makes available a student presentation by R. Shadman about the Orthomyxoviridae on his Human Virology Web page. The Division of Medical Virology, University of Cape Town, South Africa, makes available a presentation on influenza in a collection of medical virology lecture notes.

2. The 1918 influenza pandemic. R. Siegel makes available a student presentation by M. Billings about the Influenza Pandemic of 1918 on his Human Virology Web page. Influenza 1918 is presented by PBS Online. The UK National Institute for Medical Research makes available the 1998 Mill Hill essay by R. Daniels titled “In search of an enigma: The ‘Spanish Lady’.” Discovery.com offers a presentation about the 1918 pandemic titled “The flu: The hunt for a killer in disguise.” The Spartacus Educational Web site provides an introduction to the 1918 influenza pandemic. The P. L. Duffy Resource Centre, Trinity College, Western Australia, provides a collection of links to Internet resources on the 1918 influenza epidemic. The November-December 1998 issue of the Pennsylvania Gazette had an article by E. Lynch titled “The flu of 1918.” The 1918 Project is a presentation about an expedition to Longyearbyen, Norway, to recover tissue samples from Spanish flu victims buried in the permafrost.

3. The 1997 Hong Kong avian influenza. The Population and Public Health Branch of Health Canada provides a December 1997 information page about the Hong Kong avian flu. The CDC's NCID provides information about the 1997 influenza A(H5N1) in Hong Kong. The 13 December 1997 issue of Science News had an article by N. Seppa titled “Chicken flu virus raises concerns.” WHO issued a 6 January 1998 press release titled “Investigation of origins of H5N1 influenza virus stepped up in Hong Kong”; links to related articles are included. Time magazine makes available a 23 February 1998 article by E. Larson about the 1997 Hong Kong flu outbreak titled “The flu hunters.” NIAID Council News had a February 1998 article titled “NIAID in front lines of Hong Kong flu crisis.” A section on the 1997 avian flu outbreak in Hong Kong is included in a presentation by N. Cox on bird flu and influenza prepared for a lecture series on emerging infections of international public health importance at the School of Public Health and Community Medicine, University of Washington. The 12 September 1997 issue of Science had a News and Comment article by J. Cohen titled “The flu pandemic that might have been.” The 16 January 1998 issue had a report by K. Subbarao et al. titled “Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness” and a Research News article by G. Vogel titled “Sequence offers clues to deadly flu.” The March-April 1999 issue of the CDC's Emerging Infectious Diseases had an article by R. Snacken et al. titled “The next influenza pandemic: Lessons from Hong Kong, 1997.”

4. M. Hatta, P. Gao, P. Halfmann, and Y. Kawaoka are at the Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin.

5. M. J. Gibbs, J. S. Armstrong, and A. J. Gibbs are in the Division of Botany and Zoology, Australian National University, Canberra. The 11 May 2001 issue of Science had a News of the Week article about Gibbs' research by J. Pickrell titled “Killer flu with a human-pig pedigree?”

6. Viral virulence. M. Taylor provides lecture notes on virulence for a virology course.

7. The 1957 and 1968 influenza epidemics. The CDC's National Vaccine Program Office provides information about the 1957 Asian flu and the 1968 Hong Kong flu pandemics. Kimball's Biology Pages includes a section on the 1957 and 1968 influenza pandemics in the presentation on influenza. A section about the 1957 Asian flu is included in an article by H. Collins titled “The man who changed your life: Maurice Hilleman” provided by the New Jersey Association for Biomedical Research.

8. Reassortant and reassortment are defined in the Academic Press Dictionary of Science and Technology. The On-line Medical Dictionary defines reassortant viruses.

9. Hemagglutinin and other influenza virus proteins. On-line Medical Dictionary provides a definition of haemagglutinin glycoproteins, influenza virus. A class project on influenza, prepared for a microbiology course taught by S. Grove, Department of Biology, Goshen College, IN, provides an introduction to influenza virus proteins. A list of influenza virus proteins is included in a student project on influenza prepared for a course on viruses taught by G. Linquester, Department of Biology, Rhodes College, Memphis, TN. M. Hewlett provides a diagram of RNA segments and proteins of the Orthomyxoviridae genome in lecture notes on negative-strand RNA viruses for a virology course. The Viruses: From Structure to Biology Web site, made available by Washington University School of Medicine, provides an introduction to hemagglutinin in the influenza virus. “The Hemagglutinin Protein of the Influenza Virus” is a student presentation by P. Curry prepared for a biochemistry course taught by K. Moreman, Department of Biochemistry and Molecular Biology, University of Georgia. W. Gallagher, Department of Chemistry, University of Wisconsin, Eau Claire, makes available a student project by H. Rose on hemagglutinin prepared for course on biophysical chemistry.

10. Neuraminidase enzyme. Viruses: From Structure to Biology includes a presentation on neuraminidase. The Department of Biology, Lancaster University, UK, makes available a student presentation on neuraminidase, prepared for a course on proteins and biotechnology. “Influenza A subtype 2 neuraminidase: A protein critical to virus survival” is a student report by A. Dixon prepared for K. Moreman's biochemistry course.

11. Genetic analysis of the 1918 virus by Taubenberger's group. Influenza 1918 from PBS Online includes an interview with J. Taubenberger. J. Taubenberger is in the Division of Molecular Pathology, Armed Forces Institute of Pathology (AFIP). The AFIP provides a press release about the research of A. Reid and J. Taubenberger. The July 1999 issue of ASM News from the American Society for Microbiology had an article by J. Taubenberger titled “Seeking the 1918 Spanish influenza virus.” The 21 March 1997 issue of Science had a report by J. Taubenberger et al. titled “Initial genetic characterization of the 1918 ‘Spanish’ influenza virus” and a Research News article by E. Pennisi about the research. The 22 March 1997 issue of Science News had an article by S. Sternberg titled “A doughboy's lungs yield 1918 flu virus.” The 27 February 2001 issue of the Proceedings of the National Academy of Sciences had an article by C. Basler et al. titled “Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes” and a commentary by J. Lederberg titled “H1N1-influenza as Lazarus: Genomic resurrection from the tomb of an unknown.” The 6 June 2000 issue had an article by A. Reid et al. titled “Characterization of the 1918 ‘Spanish’ influenza virus neuraminidase gene.” The 16 February 1999 issue had an article by A. Reid, T. Fanning, J. Hulton, and J. Taubenberger titled “Origin and evolution of the 1918 Spanish influenza virus haemagglutinin gene” and a commentary on the research by R. Webster titled “1918 Spanish influenza: The secrets remain elusive.” Nature provides a 4 March 1999 Science Update about this research by E. Lawrence titled “Spanish 'flu keeps its secrets.” BBC Online offers a presentation about the research of Taubenberger and others on the 1918 virus. InfluenzaNews.com makes available an article by J. Oxford titled “Recovery of Spanish 1918 influenza virus genes from formalin-fixed and frozen respiratory tissues.”

12. P. Palese is the Department of Microbiology, Mount Sinai School of Medicine, New York. The 1996 report Genetic Engineering of Viruses and Viral Vectors includes a chapter by P. Palese et al. titled “Negative-strand RNA viruses: Genetic engineering and applications.”

13. Reverse genetics. The On-line Medical Dictionary defines reverse genetics. Reverse genetics is defined in the genetics glossary provided by the Biology Teaching Organisation, University of Edinburgh, UK. The Human Molecular Genetics On-Line Teaching Site, made available by the Department of Biological Sciences, Brunel University, London, provides an introduction to reverse genetics in the section on molecular techniques. C. Kennedy, Department of Plant Pathology University of Arizona, offer lecture notes on reverse genetics for a microbial genetics course.

14. Pasmid-based reverse genetics. The Kawaoka Laboratory Web site provides a research presentation titled “Reverse genetics - Generation of influenza viruses entirely from cloned cDNA.” The 3 August 1999 issue of the Proceedings of the National Academy of Sciences had an article by G. Neumann et al. titled “Generation of influenza A viruses entirely from cloned cDNAs” and a commentary by A. Pekosz, B. He, and R. Lamb titled “Reverse genetics of negative-strand RNA viruses: Closing the circle.” The 23 May 2000 issue had an article by E. Hoffmann et al. titled “A DNA transfection system for generation of influenza A virus from eight plasmids.” The January 2000 issue of the Journal of Virology had an article by G. Neumann, T. Watanabe, and Y. Kawaoka titled “Plasmid-driven formation of influenza virus-like particles.”

15. The swine influenza virus H1N1. The e-AnimalHealth Web site offers a presentation about swine influenza. MARK, an information resource on swine husbandry from the Department of Animal Science at North Carolina State University, makes available a symposium paper by C. Woodlief on swine influenza. Influenza as a Zoonotic Disease, a tutorial by C. Olsen, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, includes a section zoonotic swine influenza.

16. Developing new types of vaccines. The June 2000 issue of Influenza, the bulletin of the European Scientific Working Group on Influenza, had an article titled “Reverse genetics for the control of influenza.” An overview of human virus vaccines by R. Chanock is available on the NIH Clinical Center Round Table Web site. Eurekah.com makes available a chapter on recombinant viral vaccines by R. Spaete with a section on influenza vaccines from a book on new vaccine technologies. The 13 November 1999 issue of BMJ had an article by M. Liu titled “Vaccines in the 21st century.” Access Excellence provides a science update by S. Henahan titled “DNA provides better flu vaccine.” NIAID provides a 2 March 2000 press release about the Jordan Report: Accelerated Development of Vaccines; a section on influenza vaccines begins on page 49 of the report, which is available in Adobe Acrobat format. The July 1999 issue of Scientific American had an article by D. Weiner and R. Kennedy titled “Genetic vaccines.”

17. The editorial in this issue titled “A global lab against influenza” is by S. Layne, T. Beugelsdijk, K. Patel, J. Taubenberger, N. Cox, I. Gust, A. Hay, M. Tashiro, and D. Lavanchy. S. Layne is in the Department of Epidemiology, School of Public Health, University of California, Los Angeles. The summer 2000 issue of the UCLA Magazine had an article about Layne's proposal by G. Taubes titled “The hot zone.” The National Academy Press makes available online a 2001 book Firepower in the Lab: Automation in the Fight Against Infectious Diseases and Bioterrorism edited by S. Layne et al.

18. R. G. Webster is in the Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN.

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