The ecology of influenza viruses: a WHO Memorandum*

Bulletin of the World Health Organization, 59 (6): 869-873 (1981) The ecology of influenza viruses: a WHO Memorandum* InfluenzaA viruses continue to be isolatedfrom man, pigs, horses, anda wide range of avian species, especially ducks. The recent isolation ofan influenza A virusfrom seals has added an additional mammal to the list of natural hosts for these viruses. In contrast, influenza B viruses have been isolated onlyfrom man. The haemagglutinin ofa virus isolatedfrom gulls in the United States ofAmerica could not be identified with reference antisera and may constitute a new haemagglutinin subtype. Studies in swine suggest that HINI viruses related to A/New Jersey/8/76 continue to circulate in some countries ofthe world, and that the H3N2 andHINI viruses can be trans- mitted from man to pigs. The isolation from pigs of an HINI virus that is antigenically similar to avian HINI isolates and the isolation of "classical" swine influenza virusfrom a piglet in Czechoslovakia are ofparticular interest and raise the question of the source of the viruses. Preliminary studies on equine influenza viruses (H3N8) (Heq2Neq2) from horses in Europe suggest that significant antigenic drift has occurred in viruses of this subtype since the prototype was identified in 1963. The role ofinfluenza virusesfrom lower animals and birds in the genesis ofnew human strains is unknown, and the genes responsiblefor host range and virulence have notyet been identified. The availability oftechniquesfor analysis ofall theRNAs and the geneproducts will permit further studies on these important questions. Over the past 5 - 8 years, large numbers of influ- enza A viruses have been isolated from lower animals, especially birds, but the role of these viruses in the genesis of human influenza strains and of influenza viruses that cause disease in domestic animals is not yet known. This Memorandum summarizes current knowledge on the ecology of influenza viruses in the various animal species. RECENT DEVELOPMENTS IN COMPARATIVE ANALYSIS OF INFLUENZA VIRUSES Influenza A viruses contain 8 single-stranded seg- ments of RNA; these code for 9 different proteins, 2 of which are not incorporated into virus particles. Each of these gene products is potentially important in determining the biological properties of the virus and methods have been developed for comparative analysis of both the RNAs and the proteins. Genetic studies using these methods have provided confir- mation for the revised system of nomenclature of This Memorandum was drafted by the signatories listed on page 873 on the occasion of a meeting held in Munich in November 1980. A French translation will appear in a future issue of the Bulletin. Requests for reprints should be addressed to Chief, Virus Diseases, World Health Organization, 1211 Geneva 27, Switzer- land. influenza A viruses which was introduced by the World Health Organization in 1980 (1) and was based on antigenic analysis. Nucleotide and amino acid sequencing Sequence data have been described for the genes coding for 11 of the 12 haemagglutinin antigen sub- types (Hl-H9, HIl, and H12) and the 9 neur- aminidase antigen subtypes (NI - N9). Up to 350 nucleotides from the 3' end of the viral RNA were sequenced using dideoxy chain termination and the predicted amino acid sequence of the N-terminal region of HAl was obtained. Analysis of the haemag- glutinin showed that the signal peptide from each of the 11 subtypes showed little or no homology, except for the occurrence of hydrophobic amino acids in this region. Thereafter, apart from cysteine residues and a number of other residues that were totally conserved in all subtypes, there was little homology between the subtypes in the N-terminal region of the heavy poly- peptide chain (HAI) of the haemagglutinin molecule.a During antigenic drift in the haemag- glutinin of H2N2 and HO-H INI subtypes, there were many nucleotide changes that did not lead to amino a The influenza A haemagglutinin molecule is comprised of two polypeptide chains, a heavy-chain, HAI, and a light-chain, HA2, linked by disulfide bonds. 4128 869- WHO MEMORANDUM acid sequence changes and only a very few that did. This indicates strong pressure to conserve the primary sequence of the HA protein. Analysis of the neuraminidase showed that the pre- dicted first 12 amino acids of the N terminus were totally conserved through all 9 subtypes, and the next six were conserved through most subtypes. There- after, the predicted sequences were totally different, with the exception of certain short sequences that appeared at different positions in some of the subtypes. In contrast, during antigenic drift, there was marked conservation of the amino acid sequence in the neuraminidases. Base sequence homology studies Genetic studies using hybridization techniques have shown that: (a) H5 (Hav5) should be considered as a unique subtype, unrelated to the HI (HO, HI, Hswl) group; (b) the N9 (Nav6) subtype was distinct from the N3 (Nav2, Nav3) group; (c) segment 8 (non- structural protein, NS) could be used to divide avian strains into 2 groups. Within each group, sequence homology was 85 - 100%; between the two groups, it was about 40%. The mammalian strains exhibited about 85% homology with one of these avian groups. One peculiar property of the NS gene is that the mRNA functions as a template for two different pro- teins read in different reading frames (the second protein from a spliced mRNA). Also, there is an open reading frame for a third protein on the vRNA of segment 8; (d) repressor recombinants were isolated from a ts-mutant with a ts defect in segment 8. After rescue of this ts-mutant with PR8, ts+-isolates were obtained, in which segment 2 had been replaced but segment 8, which had the ts defect, had not. The product of the segment 2 gene of PR8 suppressed the ts-genotype of the NS protein phenotypically; (e) reassortment between two influenza strains that were non-neurotropic for mice yielded recombinants that were neurotropic for mice. The implications of these findings for the ecology of influenza are obvious, since by "recombination" the gene products of a new constellation can give a different host range and/or organ tropism and, in this way, new patho- genic properties may be attained. Oligopeptide mapping Oligopeptide mapping showed much more conser- vation in the light chain of the haemagglutinin molecule (HA2) than in the heavy chain of the molecule. Analysis with monoclonal antibodies Analysis of the haemagglutinin molecule of influ- enzaA viruses with monoclonal antibodies has shown that there are 3 - 4 non-overlapping antigenic deter- minants on the molecule. Selection of antigenic vari- ants with monoclonal antibodies and examination of the amino acid sequence has permitted identification of the regions of the HAl polypeptide that are associ- ated with the antigenic determinants. Some of these probably constitute the antigenic determinants them- selves, while others may result from changes within the molecule and refolding of the antigenic area. Monoclonal antibodies to the haemagglutinin have practical use in specialized laboratories and can be used to differentiate closely related strains. Five different monoclonal antibodies to the nucleoprotein (NP) of A/WSN/33 [HiNl] (HON1) have been used to show antigenic variation in this protein. The nucleoproteins of human strains isolated between 1933 and 1979 could be divided into six groups, which did not correlate with any particular HA or NA subtype. The influenza A viruses from swine and avian sources were grouped with the A/WSN/33 strain, while the equine strains were in another group. These monoclonal antibodies are proving useful for studies on the origin of the NP gene in influenza A viruses. Studies on antigenic variation of the haemag- glutinin of HI NI human strains with monoclonal antibodies revealed that most natural variants exhibited changes at an epitope recognized by one antibody. Laboratory studies confirmed that selec- tion of mutants by neutralization in the presence of monoclonal antibodies yielded variants that no longer reacted with one particular monoclonal antibody and also exhibited significant antigenic drift with ferret sera. This suggests that significant antigenic drift can occur by point mutation in the haemagglutinin of this strain. INFLUENZA IN MAN Until 1977, only one influenza A virus was known to circulate in man at any one time, but there are now two co-circulating influenza A subtypes, i.e., HINi and H3N2. Two variants of the HINI subtype are currently circulating in different areas, A/USSR/ 90/77-like viruses and A/Brazil/11/79-like viruses. Among the H3N2 viruses, A/Texas/l/77- and A/Bangkok/1/79-like viruses are being isolated in different parts of the world. In addition, influenza B outbreaks in 1979-80 involved viruses similar to B/Singapore/222/79. The co-circulation of different influenza A sub- types in man offers the possibility of genetic reassort- ment. Following reports of mixed infections with HINI and H3N2 strains and the isolation in 1978 - 79 of recombinant HINI strains possessing H3N2 poly- 870 ECOLOGY OF INFLUENZA VIRUSES merase and nucleoprotein genes, a prospective study of the relative prevalence of "recombinant HINI" and "true HINI" viruses was undertaken. This revealed that, although recombinant HINI strains were widely distributed in Asia, Europe, and North and South America, true H1N1 viruses continued to circulate in 1979 and were isolated during outbreaks and sporadic cases in the USA in 1980. Thus, displacement of true HINI viruses by recombinant HINI viruses has not been demonstrated. INFLUENZA IN BIRDS A large number of antigenically distinct influenza A viruses have been isolated from wild (feral) and domestic avian species in most parts of the world where studies have been carried out. Among feral species, ducks are the major source of viruses but influenzaA viruses have also been isolated from other aquatic birds. These viruses are found less frequently in passerine birds, so it is of interest that an avirulent H7N7 (HavINeqI) influenza virus has been isolated from starlings in Israel. The influenza viruses from feral birds are not usually associated with any disease signs. The antigenic spectrum of influenza A viruses isolated from wild birds throughout the world is very broad. Many different antigenic subtypes have been isolated from water birds in the same area and most of the known antigenic subtypes of influenza A viruses are found in feral ducks throughout the world. An influenza A virus isolated from gulls in North America has been found to possess a haemagglutinin that is unrelated to any of the reference subtypes and may constitute a new influenza A haemagglutinin subtype. Influenza A viruses of many different antigenic subtypes [H4N8 (Hav4Neq2), H6N2 (Hav6N2), HION7 (Hav2Neql), H7N3 (HavINav2), H7N2 (HavlN2), HINI (HswiNi), H7N7 (HaviNeql), H1ON4 (Hav2Nav4)] have been associated with disease in domestic turkeys in the United Kingdom and United States of America. In particular, a highly virulent H7N7 (HavINeql) virus has been isolated from turkeys in the United Kingdom, but it is not known where the virus originated or how it was main- tained in the turkeys. It has been suggested that wild avian species may have been involved in the introduction of the viruses to domestic flocks. A very broad spectrum of antigenic subtypes of influenza A viruses have been isolated from domestic ducks, especially in Hong Kong, but have not been associated with overt disease. In contrast, influenza viruses from domestic ducks in North America have been associated with respiratory disease. INFLUENZA IN SWINE Studies carried out in 1979- 80 in various parts of the world demonstrated the presence of influenza A viruses of the HINI and H3N2 subtypes in pigs. The predominant viruses were related to A/New Jersey/ 8/76 [H INI] but others related to the currently circulating H3N2 strains of man were also isolated. There have been limited reports of the isolation of A/USSR/90/77-like viruses from pigs, and this, together with serological evidence, suggests that the HI N I viruses currently circulating in man may also be transmitted to pigs. It is of interest that a virus related to "classical" swine influenza virus (A/swine/Iowa/ 15/30) has been isolated from a piglet in Czechoslovakia, particularly since other studies in the recent past have provided no evidence for the virus in that country, and it is not clear how it is maintained in nature. There have also been reports of HINI influenza A viruses, isolated from pigs in Belgium, that were antigenically similar to H INI (HswiNI) viruses isolated from avian sources. In haemag- glutination inhibition tests, these Belgian isolates were not inhibited with antiserum to A/New Jersey/ 8/76, but were inhibited by antisera to avian HINI (HswINI) strains (A/duck/Bavaria/1/77 and A/ duck/Alberta/35/76). Pigs experimentally infected with A/swine/Belgium/2/79 showed a moderate rise in antibody titre to the homologous virus but not to A/New Jersey/8/76. Molecular analysis of H3N2 virus isolates from pigs in Hong Kong in 1976 showed that the haemag- glutinin and neuraminidase antigens were most closely related to strains that previously circulated in the human population. Oligonucleotide mapping revealed that two swine H3N2 isolates from 1976 were slightly different from each other and from 1968 reference strains, although common oligonucleotides were evident. Preliminary results of oligonucleotide mapping and RNA hybridization on individual genes of the swine isolates indicated that most genes were of H3N2 origin. The findings substantiate the view that early H3N2 strains persisted in pigs at least until 1976, undergoing genetic mutation during transmission. INFLUENZA IN HORSES Evidence of outbreaks of influenza in horses caused by H7N7 (HeqINeqI) and H3N8 (Heq2Neq2) viruses has been reported from several countries. H7N7 (HeqINeqI) infection has occurred in Czecho- slovakia and the USA, and outbreaks associated with H3N8 (Heq2Neq2) viruses have been reported from Sweden and the USA. The strain isolated from 871 WHO MEMORANDUM Sweden showed significant antigenic drift from earlier strains and constitutes a threat to horses that may necessitate a change in vaccine composition. Studies are recommended to determine the extent of antigenic drift among recent H3N8 (Heq2Neq2) isolates. INFLUENZA IN OTHER ANIMALS Seals Collaborative studies between laboratories in Canada and the USA resulted in the isolation of an influenza A virus that was antigenically similar to A/fowl plague virus/Dutch/27 [H7N7J (HavlNeql) from harbor seals (Phoca vitulina) that had died of primary viral pneumonia on Cape Cod peninsula, USA, in the winter of 1979- 80. High concentrations of virus were obtained from the lungs and low concentrations from the brain. Antigenic analysis of the haemagglutinin and neuraminidase showed that the surface antigens of the seal isolates were anti- genically indistinguishable from recent isolates from birds. Genetic analysis of the RNAs by competitive hybridization showed that the genes coding for the internal proteins of the virus were closely related to various avian influenza isolates from several different species (gulls, turkeys, and ducks). Biologically, the virus behaved more like a mammalian, than an avian, strain; it replicated poorly in avian species and could be recovered only from the respiratory tract, while in mammals such as ferrets, cats, and pigs, it replicated readily. The virus caused no disease signs in any of the species inoculated experimentally. Preliminary studies detected no evidence of sero- conversion among persons who worked with either the dead seals or with the virus. It is not yet known whether the influenza virus isolated from seals was responsible for the high mortality in this species, but the studies suggest that the virus originated from avian species. Limited serological studies in seals from the Boston area showed evidence of antibodies to the virus, but antibodies have not been detected in a limited number of sera from seals in other areas of the world. Whales Genetic and antigenic studies on the influenza A virus isolated from whales in the Pacific Ocean in 1978, showed that the virus was antigenically similar to HlNl (HswlNl) viruses from avian sources. Hybridization studies have shown that the virus is intermediate between avian and mammalian strains of influenza A viruses and further studies are indicated. INTERSPECIES TRANSMISSION OF INFLUENZA VIRUSES There is abundant virological and serological evi- dence that human H3N2 viruses are frequently trans- mitted to swine, but usually do not produce overt signs of disease. They may, however, persist in swine for prolonged periods after they have ceased to circu- late in the human population, as originally reported by workers in Hong Kong. Evidence for the trans- mission of swine HlNl (HswlNI) viruses from pigs to human subjects in the USA was documented in 1976 and 1977, and in 1980 there was one case of transmission of HlNI (HswlN1) virus from pigs to man at an agricultural animal display in the USA. Experimental infection ofcattle with H3N2 influenza viruses Subclinical infection was demonstrated in cows experimentally infected with an H3N2 isolate from cattle in Czechoslovakia in 1978. Seroconversion was very low with a peak at 21 days and a sharp decline. This is in keeping with observations in the field where similar infections may frequently escape attention. Experimental infection of domestic chickens with H7N7 strains White Leghorn chickens inoculated with H7N7 (HeqlNeql) viruses showed signs of acute infection. Virus was recovered from tracheal swabs for 3 days and in one instance, from a cloacal swab. Serocon- version was low; egg yolk antibody was detected. Experimental infection of mammals with avian influenza viruses Avian influenza A viruses of different antigenic subtypes have been examined for their ability to replicate in pigs and ferrets. Four avian HlNl (HswlNl) isolates replicated readily during initial passage and viruses were recovered for 6-7 days postinfection. Studies on other antigenic subtypes [H3N2 (Hav7N2), H6N2 (Hav6N2), H2N3 (H2Nav2), H2N2, H2N1, H1ON7 (Hav2Neq1), and H7N7 (HavlNeql)] have indicated that several avian strains can infect mammals, but cause no disease signs and differ in the length of time they replicate. ANTIGENIC CLASSIFICATION AND NOMENCLATURE OF INFLUENZA VIRUSES Antigenic and RNA analyses are in complete agree- ment with the revised nomenclature for influenza 872 ECOLOGY OF INFLUENZA VIRUSES 873 viruses (1) and all investigators are encouraged to use this system of nomenclature. It will be necessary over the next few years to establish a set of reference strains within each subtype of influenza viruses from animal and avian sources, corresponding to those for human strains, to reflect antigenic drift in the subtypes. For example, in the H3N2 subtype, the reference strains are A/Hong Kong/1/68, A/England/42/72, A/Port Chalmers/l/73, etc. CONCLUSIONS Influenza A viruses have been isolated from man, pigs, horses, and a wide range of avian species, especi- ally ducks. Influenza B viruses have been isolated only from man. The isolation of an influenza A virus from seals adds an additional mammal to the list of natural hosts for influenza viruses. Most of the known haemagglutinin and neuramini- dase subtypes of influenza A viruses have been iso- lated from wild avian sources throughout the world where they provide a large gene pool but cause no overt disease in their natural host. The haemag- glutinin of a virus isolated from gulls in the USA could not be identified with reference antisera and may constitute a new haemagglutinin subtype. There is increasing evidence that some avian influenza viruses can replicate in mammalian species. Studies in swine suggest that HlNl viruses related to A/New Jersey/8/76 continue to circulate in some countries of the world and that H3N2 and HlNl viruses can be transmitted from man to pigs. The iso- lation from pigs of an HIN I virus that is antigenically similar to avian HlNl isolates and the isolation of "classical" swine influenza virus from a piglet in Czechoslovakia are of particular interest and raise the question of the source of the viruses. Preliminary studies on equine influenza viruses H3N8 (Heq2Neq2) from horses in Europe suggest that significant antigenic drift may have occurred in these viruses and further studies are needed to deter- mine whether a change in vaccine composition is necessary. The role of influenza viruses from lower animals and birds in the genesis of new human strains is not resolved, and it is not yet known which genes are responsible for host range and virulence. The avail- ability of techniques for analysis of all the RNAs and the gene products will permit further studies on these important questions, especially after the appearance of the next "new" pandemic strain in man. * * * D. J. Alexander, MAFF Agricultural Development and Advisory Service, Central Veterinary Labora- tory, Weybridge, Surrey, England F. Assaad, Virus Diseases, World Health Organiz- ation, Geneva, Switzerland P. A. Bachmann, Institute of Microbiology and Infectious Diseases of Animals, Department of Veterinary Medicine, Ludwig-Maximillians Uni- versity, Munich, Federal Republic of Germany K. B6gel, Veterinary Public Health, World Health Organization, Geneva, Switzerland H. Chu, Department of Clinical Veterinary Medicine, Cambridge University, Cambridge, England B. C. Easterday, Department of Veterinary Science, University of Wisconsin, Madison, WI, USA C. M. Hannoun, Pasteur Institute, Paris, France V. S. Hinshaw, St Jude Children's Research Hos- pital, Division of Virology, Memphis, TN, USA M. M. Kaplan, Pugwash Conferences on Science and W.orld Affairs, Geneva, Switzerland W. G. Laver, Department of Microbiology, John Curtin Medical School, Australian National Uni- versity, Canberra, Australia K. Ottis, Institute of Microbiology and Infectious Diseases ofAnimals, Ludwig-Maximillians Univer- sity, Munich, Federal Republic of Germany J. J. Romvary, Veterinary Medical Research Insti- tute, Hungarian Academy of Sciences, Budapest, Hungary C. Scholtissek, Institute of Virology, Justus-Liebig University, Giessen, Federal Republic of Germany K. F. Shortridge, Department of Microbiology, Uni- versity of Hong Kong, Hong Kong A. N. Slepuskin, The D. I. Ivanovskij Institute of Virology, Moscow, USSR B. Tumova, Institute of Hygiene and Epidemiology, Centre of Epidemiology and Microbiology, Prague, Czechoslovakia R. G. Webster, Division of Virology, St Jude Chil- dren's Research Hospital, Memphis, TN, USA REFERENCE 1. A revision of the system of nomenclature for influenza viruses: a WHO Memorandum. Bulletin of the World Health Organization, 58: 585 - 591 (1980).