Isolation of an influenza A virus from seals

Summary Influenza A virus of serotype Hav1 Neq1 (H7N7 by the 1980 revised influenza typing system proposed by WHO experts) was repeatedly isolated from lung and brain tissues taken from harbor seals (Phoca vitulina) found suffering from pneumonia on Cape Cod Peninsula (U.S.A.) in the winter of 1979–1980. The seal isolates, although of a serotype identical to some fowl plague virus strains, were harmless to chickens and turkeys in transmission experiments. An earlier human infection by a Hav1 Neq1 influenza virus and the serologic relatedness of this avian serotype with the equine 1 serotype are cited in support of the view that influenza viruses with these antigenic characteristics seem to have a facility to pass from birds to mammals.     

Duck influenza lacking evidence of disease signs and immune response.

Influenza viruses A/duck/Hokkaido/5/77 (Hav7N2), A/budgerigar/Hokkaido/1/77 (Hav4Nav1), A/Kumamoto/22/76 (H3N2), A/Aichi/2/68 (H3N2), and A/New Jersey/8/76 (Hsw1N1) were experimentally inoculated into Pekin ducks. Of these, the influenza viruses of duck and budgerigar origin replicated in the intestinal tract of the ducks. The infected ducks shed the virus in the feces to high titers, but did not show clinical signs of disease and scarcely produced detectable serum antibodies. Using immunofluorescent staining, we demonstrated that the target cells of the duck virus in ducks were the simple columnar epithelial cells which form crypts in the large intestines, especially in the colon. After primary infection, the birds resisted reinfection with the duck virus at least for 28 days, but from 46 days onward they were susceptible to reinfection. These infections were quickly restricted by a brisk secondary immune response, reflected in the rapid appearance of high titers of antibody after reinoculation. In contrat to the avian influenza viruses, the remaining three influenza viruses of human origin did not replicate in the intestinal tract but did cause a serum antibody response.     

Isolation and characterization of influenza A viruses from wild ducks in northern Japan: appearance of HSW1 antigens in the Japanese duck population.

Twenty-six influenza A viruses were isolated from cloacal and tracheal samples of 235 resident and 396 migratory ducks in Miyagi prefecture, Japan, in 1977--78. Of these, twelve were antigenically related to the avian-origin HSW1 virus, A/duck/Alberta/35/76 (HSW1N1), but their neuraminidase antigens were characterized as Nav2-3, Nav4 or N2. These antigenic configuration have not previously been reported. In addition, one strain in which the neuraminidase antigen was identified as Nav4, was demonstrated to be a mixture of two haemagglutinins, HSW1 and Hav7. Two distinct strains were separated from the mixture and characterized as HSW1Nav4 and Hav7Nav4. The antigenic identification of an additional 13 influenza A viruses revealed the presence of six haemagglutinin subtypes (Hav1, Hav3, Hav4, Hav6, Hav7, and Hav8) and five neuaraminidase subtypes (Nav1, Nav2-3, Nav4, Neq2, and N2) in various combinations. The results suggest that the avian influenza A viruses among feral ducks may be isolated in various combinations of haemagglutinins and neuraminidase subtypes in Japan, and that feral ducks may be the site of genetic recombination occurring as a result of dual infection with different subtypes of influenza A virus.     

Replication of avian influenza A viruses in mammals.

The recent appearance of an avian influenza A virus in seals suggests that viruses are transmitted from birds to mammals in nature. To examine this possibility, avian viruses of different antigenic subtypes were evaluated for their ability to replicate in three mammals-pigs, ferrets, and cats. In each of these mammals, avian strains replicated to high titers in the respiratory tract (10(5) to 10(7) 50% egg infective doses per ml of nasal wash), with peak titers at 2 to 4 days post-inoculation, similar to the pattern of human and other mammalian viruses in these animals. Most avian strains were recovered for 5 to 9 days post-inoculation. One avian H1N1 virus initially replicated poorly in pigs, but was adapted to this host and even transmitted to other pigs. Replication of the avian viruses occurred in the respiratory tracts of mammals, whereas, in birds, they replicate in the intestinal tract as well. The infected mammals had no significant disease signs and produced low levels of humoral antibodies; however, challenge experiments in ferrets indicated that they were immune. These studies suggest that influenza A viruses currently circulating in avian species represent a source of viruses capable of infecting mammals, thereby contributing to the influenza A antigenic pool from which new pandemic strains may originate.     

Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza A viruses.

Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza. A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6. The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 degrees C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals.     

Isolation and serological characterization of influenza A viruses from birds that were dead on arrival at Tokyo airport

Summary Twenty-two strains of influenza A virus were isolated from caged birds which had been imported into Japan from India and Thailand and had died during transportation to Tokyo.Serological tests divided these strains into two groups. Viruses in the first group contained Hav7 hemagglutinin and were related antigenically to A/duck/ Ukraine/1/63 [Hav7 Neq2]; viruses in the second group contained Hav4 hemagglutinin and were related to A/duck/Czech/56 (Hav4 Nav1]. All strains contained Neq2 neuraminidase that was closely related to that of A/equine/Miami/1/63 [Heq2 Neq2] and A/duck/Ukraine/1/63 [Hav7 Neq2]. It was concluded that the strains in the first group were Hav7 Neq2 and those in the second group were Hav4 Neq2; both groups of viruses showed antigenic drift from the prototype strains.With 2 Figures     

Genetic reassortment of influenza A viruses in the intestinal tract of ducks

Genetic reassortment between influenza A viruses was shown to occur in the intestinal tract of ducks during mixed infection; this phenomenon was examined both in naturally and laboratory-infected ducks. Studies on cloacal samples from Canadian feral ducks demonstrated that 7% of these samples contained two or more antigenically distinguishable influenza viruses, indicating that mixed infections occur rather frequently in nature. The RNAs of multiple viruses isolated from one cloacal sample were separated by polyacrylamide gel electrophoresis; the RNA migration patterns showed that this sample contained a mixture of viruses, including antigenically identical viruses with heterogeneous RNAs. To determine if genetic reassortment may be responsible for the RNA heterogeneity, laboratory ducks were infected with two antigenically distinct avian influenza viruses (Hsw1N1 and Hav1Nav2). Antigenic recombinants (Hav1N1) were readily isolated, without selective antibody pressure, from the feces of these mixedly infected ducks. Comparisons between the RNA migration patterns of the parental viruses and multiple isolates from the feces showed that antigenically identical isolates (as the Hav1N1 recombinants) possessed different gene constellations. These findings show that genetic reassortment between viruses occurs readily in the duck's intestinal tract. Other parameters of virus infection in ducks were also examined. Juvenile ducks, inoculated with influenza virus, shed virus in their feces for 30 days, produced low levels of circulating antibodies (HI titer of 1:20), and were resistant to challenge with homologous virus. These studies illustrate the mechanism by which genetically diverse influenza A viruses may evolve in nature.     

Characterisation of influenza A viruses isolated from turkeys in England during March-May 1979

During the early spring of 1979 turkeys on at least twelve sites in England became infected with influenza A viruses. On five of these sites no virus was isolated but birds were shown to have antibodies to Havl (four sites) and Hav2 antigenic subtypes of influenza A viruses. The eight viruses isolated were typed: A/turkey/England/192-328/79 (Havl Nav2/3), A/turkey/England/192-329/79 (Hav1 N2), A/turkey/England/199/79 (Hav1 Neq1), A/turkey/ England/214/79 (Hav1 Neq1), A/turkey/England/250/79 (Hsw1 N1), A/turkey/England/262/79 (Hav1 Nav2/3), A/turkey/England/272/79 (Havl Neq1), A/turkey/England/384/79 (Hav2 Nav4). Pathogenicity index tests in 6-week-old chickens agreed with the clinical signs seen in turkeys in the field. Three of the isolates: 199, 214 and 272 were of extremely high virulence, 384 showed intermediate virulence, while the other isolates were of low virulence.     

Studies on the origin of pandemic influenza: III. Evidence implicating duck and equine influenza viruses as possible progenitors of the Hong Kong strain of human influenza

Two strains of influenza virus isolated from horses and ducks in 1963, A/equine/Miami/1/63 (Heq2 Neq2) and A/duck/Ukraine/1/63 (Hav7 Neq2) were found to possess hemagglutinin subunits which cross-reacted in hemagglutination-inhibition and immunodiffusion tests with those of the Hong Kong strain of human influenza A/Hong Kong/1/68 (H3 N2).Peptide maps of the heavy polypeptide chains from the hemagglutinin subunits of these three strains showed a number of differences, but maps of the light chains were almost identical, indicating that the light polypeptide chains from the hemagglutinin subunits of these animal, avian and human viruses had practically the same amino acid sequence.One explanation of these results is that the three viruses arose, by genetic recombination, from a common ancestor.     

Detection of the determinants of influenza virus H3 hemagglutinin by the technic of radioimmunologic analysis

The interrelations between H3/73 hemagglutinin of human influenza virus and the other 16 mammalian and avian hemagglutinin subtypes (a total of 50 strains) were studied by the method of radioimmunologic analysis (RIA). The antigenic relations of H3, Hav7 and Heq2 were confirmed, certain common determinants were also found in H3/73 hemagglutinin and avian viral Hav6 and Hav9 hemagglutinins. No interrelations were revealed with previously circulating human influenza viruses H0, H1, H2 as well as with swine influenza virus and avian viruses Hav1-Hav5, Hav8. It has been shown that the H3/73 determinant in some avian viruses evolves similarly to drift-variants of human influenza virus. The method can be recommended for fine analysis of influenza virus antigenic structure as it allows detecting small antigenic quantities.     

Hemagglutinin molecules of Hong Kong, equine-2, and duck/Ukraine influenza viruses lack N-terminal aspartic acid.

We have found that hemagglutinin molecules from equine-2 (Heq2Neq2) and duck/Ukraine (Hav7Neq2) influenza viruses do not possess N-terminal aspartic acid and that the N-terminus of HA1 from these viruses seems to be blocked. In this respect, these hemagglutinin molecules are similar to those of Hong Kong influenza virus (H3N2) and its variants and unlike those of every other influenza virus examined (a total of 12 strains, including those of the Asian H2N2 series) which have been found to possess N-terminal aspartic acid (or asparagine) on the hemagglutinin polypeptides.     

Effects of streptovirudin on influenza viruses type A and B: inhibition of the lipid-linked oligosaccharide synthesis of fowl plague virus

Antibiotics of the streptovirudin complex (SV) inhibited the growth of influenza A and B viruses such as influenza A/fowl plague virus (FPV), strain Weybridge (Hav1 Neq1), influenza A/England 42/72 (H3N2), influenza A/Port Chalmers 1/73 (H3N2), influenza B/Leningrad 235/74, influenza B/Tokyo 7/66, and influenza B/Jamagata in chick embryo cell (CEC) cultures, in permanent canine kidney cells (MDCK), and in suspended fragments of chick embryo chorioallantoic membranes (CAM). As revealed by spectrophotometric turbidity measurements, SV completely inhibited the FPV-induced cytopathic effect (CPE). A 99.99% reduction of infectious virus yield was obtained in one-step growth cycle experiments and in the plaque reduction test. The haemagglutination inhibition titres of influenza viruses in suspended CAM fragment cultures in the presence of SV drugs were also substantially reduced. The incorporation assays indicated that SV exhibited no effect on virus-induced RNA synthesis, but influenced virus maturation by inhibition of lipid-linked oligosaccharide synthesis. A partial protection from infection was found in influenza virus A/England infected mice.     

Isolation of influenza a viruses from commercial ducks on a farm in Norfolk between August 1979 and March 1980

Investigation of respiratory disease and high mortality which occurred on a commercial duck fattening farm between August 1979 and March 1980 resulted in the isolation of 10 influenza A viruses. The viruses were characterised as Hav6 N2 (three isolates), Hav4 Navl (four isolates), Hav4 Nl (two isolates) and Hav7 Neq2 (one isolate) subtypes by haemagglutination-inhibition and neuraminidase-inhibition tests. A Newcastle disease virus isolate was also obtained from the ducks. All isolates had low intravenous pathogenicity indices in 6-week-old chickens.     

Contact infection of mink with influenza A viruses of avian and mammalian origin

Summary Avian influenza A virus Hav 7 N 2 was transmitted to mink by contact. Other avian influenza A viruses, Hav 4 Nav 1 and Hav 6 Nav 5, were not transmitted, and human, swine and equine influenza A viruses were transmitted to mink by a similar contact.     

Genetic diversity among avian influenza viruses

The RNAs of a series of avian influenza viruses of the subtype Hav7Neg2 were examined to determine if their antigenic similarity reflected an overall conservation of their RNA sequences. Genetic analysis by gel electrophoresis showed a marked variability in all of the RNA segments of the isolates. Analysis by competitive hybridization indicated that with some genome segments this variability represented major genetic differences. These differences were present even among concurrent isolates from one geographical area. In contrast, similar analysis of human H3N2 influenza virus isolates showed that viruses isolated 9 years apart were much more similar than the cocirculating avian viruses. The genetic diversity in avian influenza viruses may result from the cocirculation of many different influenza A viruses in ducks and their ability to recombine in nature.     

Novel influenza A viruses isolated from Canadian feral ducks: including strains antigenically related to swine influenza (Hsw1N1) viruses.

Twelve influenza A viruses, antigenically related to the Ho, H1 and Hsw1 subtypes, were isolated from cloacal samples of feral ducks in Canada. Antigenic comparisons showed that these viruses were most closely related to the recent HSW1N1 isolates from man and pigs, whereas in vivo pathogenicity tests revealed differences between the Hsw1N1 viruses from the ducks and those from humans and pigs. Antigenic characterization of 94 additional influenza A viruses from the ducks showed four haemagglutinin subtypes (Hav1, Hav4, Hav5 and Hav7), an unclassified haemagglutinin, and six neuraminidase subtypes (N1, N2, Neq2, Nav1, Nav2 and Nav5) in various combinations, some of which are novel and have not previously been reported. Three of these duck influenza viruses possessed a haemagglutinin antigenically related to that of classical fowl plaque virus. A much higher percentage of virus isolations were from juvenile ducks (18.5%) than from adults (5%). All of the ducks, from which viruses were isolated, appeared healthy at the time of sampling. Serological studies on a limited number of humans and domestic birds living in close proximity to the Canadian ducks revealed no evidence of interspecies transmission. Our findings suggest that these birds serve as a substantial reservoir of antigenically diverse influenza viruses, including isolates antigenically related to the current human and animal influenza viruses. This reservoir in nature may be perpetuated by a cycle involving annual infection of juvenile birds followed by transmission to the remaining susceptible birds until the next congregation during the breeding season.     

The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses.

Comparative studies on naturally occurring avian influenza viruses have been carried out in order to investigate the determinant(s) for pathogenicity for chickens. At least one virus isolate from each of the nine different hemagglutinin (HA) subtypes was included. The polypeptides of these viruses were studied by analyzing infected cell extracts on SDS-polyacrylamide gels. Both viral glycoproteins, HA and neuraminidase, showed remarkable variation in their electrophoretic mobility even among serologically closely related viruses. Pulse-chase experiments revealed that most avian influenza virus strains had an HA which was not susceptible to proteolytic cleavage in MDCK, turkey (TEC), and chicken embryo cells (CEC). Only viruses belonging to the subtype Hav5 and some strains of the subtype Hav1 possessed a cleaved HA in these cells. Only the virus strains with cleaved HA were produced in infectious form in MDCK, CEC, TEC, as well as in duck embryo cells (DEC) and quail embryo cells (QEC). The other virus strains produced plaques in these cells only in the presence of trypsin. There was a strict correlation between the cleavability of the HA, the potential of the virus to be produced in infectious form in a wide range of host cells, and their pathogenicity for chickens. No evidence was obtained for an involvement of the neuraminidase in determining pathogenicity. For the nonpathogenic viruses it could be shown that they can replicate and produce infectious progeny in some organs of the chicken. The results obtained permit the conclusion that in naturally occurring avian influenza viruses the structure of the hemagglutinin, that is its susceptibility to proteolytic cleavage in a broad spectrum of host cells, is the determining factor for pathogenicity.     

Growth of avian and human influenza viruses in organ cultures of duck and chicken colons

Summary Colons from ducks and chicken 1, 7, 14 and 28 days old maintained near-normal morphology up to 48 and 96 hours respectively in a system using NCTC 135 medium (1 part)+Dulbecco's modified Eagle's medium (9 parts), at 37° C and 95 per cent 0₂/5 per cent CO₂. In the colon of 1 and 28 day-old ducks, duck influenza virus (Hav7N2) and budgerigar influenza virus (Hav4Nav1) grew to peak titer by hour 72, whereas human influenza virus (H3N2) did not grow. In the colon of 1 day-old chicken, the three viruses grew in the order of first duck virus, then budgerigar virus and then human virus, but in the colon of 28 day-old chicken, the growth of human virus was much less. Specific fluorescence was demonstrated in the mucosal epithelium of the colon of ducks and chicken, and intensity of fluorescence correlated with virus yield. The fact that the avian and not the human influenza viruses showed good growth in the duck clons coincided with the fact that influenza viruses possessing avian hemagglutinin subtypes have frequently been isolated in nature from duck intestines.With 3 Figures     

Type A influenza virus strains isolated from free living ducks in Czechoslovakia during 1978-1981

Eight influenza virus A strains were isolated from 269 cloacal swabs taken from wild ducks (Anas platyrhynchos), shot during their autumn migrations in the years 1978-1981. One strain was identified as subtype A-H3N8N6 (Hav7Neq2Nav1), the remaining seven as subtype A-H4N6 (Hav4Nav1).     

Intestinal influenza: replication and characterization of influenza viruses in ducks.

Influenza A viruses isolated from the cloaca of naturally infected feral ducks replicate in the lungs and in the cells lining the intestinal tract of feral and domestic ducks. Despite the low pH of the gizzard, the duck influenza viruses reach the intestines via the digestive tract and are found in high concentration in the feces. The viruses retain infectivity in fecal material for at least 30 days at 4° and for 7 days at 20°. The morphology of one strain of intestinal duck influenza virus (Hav7 Neg2) that had never been passed in embryonated eggs and was isolated from the feces was roughly spherical and fairly uniform in size and shape. However, another strain of duck influenza virus studied (Hav3 Nav6) was predominantly filamentous, suggesting that the morphology of influenza viruses in their natural hosts varies from strain to strain. After passage in the chick embryo each strain retained the morphological characteristics found in the feces. In contrast to duck influenza viruses, representative human influenza viruses of the HON1, H3N2, and Hswl Nl subtypes replicate only in the upper respiratory tract of ducks. The duck influenza viruses are more stable to low pH than human strains and retain infectivity for over 30 days in nonchlorinated river water at 0° and for 4 days at 22°. The susceptibility of ducks to infection with human and avian strains of influenza virus and the possibility of transmission to animal species through the water supply suggests that ducks may be important in the ecology of influenza viruses. The possibility of “intestinal influenza” virus vaccines is considered.