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During the 1988/1989 influenza season, five antigenic reassortant influenza A (H1N2) viruses not previously isolated from man were isolated in Hebei province, People's Republic of China. All isolates contained haemagglutinins (HAs) and neuraminidases (NAs) which were antigenically similar to those of the recent Russian (H1N1) and Hong Kong influenza A (H3N2) viruses, respectively. The results of antigenic and nucleotide sequence analyses revealed that the genes encoding the polymerase, nucleoprotein, NA, matrix and non-structural proteins of the reassortant A/Hebei/24/89 (H1N2) virus were derived from the H3N2 parent virus, whereas its HA gene was from the H1N1 parent virus. The nucleotide sequences of the HA (encoding the HA1 subunit) and NA genes of the reassortant viruses were also determined. Phylogenetic trees constructed from these data by the neighbour-joining method revealed that the HA gene of the reassortant virus was closely related to those of recent human H1N1 viruses, whereas the NA gene was related to a recent human Hong Kong (H3N2) virus lineage.

Nineteen strains of Newcastle disease virus (NDV) isolated from wild ducks in Japan were placed into 4 distinct antigenic groups on the basis of their reactivities to 8 monoclonal antibodies against the HN molecule of NDV in hemagglutination inhibition tests. The NDV strains of duck origin were antigenically distinct from NDV-B1 and NDV-Miyadera originated from chickens, and varied in their virulence to chicken embryos. No apparent correlation was found between the antigenicity of the HN molecule and virulence.

Two H3N2 strains of influenza A virus isolated from pigs in Japan and Thailand were characterized antigenically and genetically. A/swine/Wadayama/5/69 (isolated in Japan in 1969) was antigenically similar to a human strain, A/Aichi/2/68, while A/swine/Bangkok/9/78 (a swine isolate in Thailand) was closely related to A/Bangkok/36/78, a contemporary human isolate. Gel electrophoresis and oligonucleotide mapping of the virus RNA revealed genetic similarity between A/swine/Wadayama/5/69 and A/Aichi/2/68, and between A/swine/Bangkok/9/78 and A/Bangkok/36/78, suggesting the derivation of swine H3N2 strains from contemporary human strains.

Six avian paramyxoviruses isolated from wild and domestic ducks in the United States, Hong Kong and Japan were characterized antigenically and genetically. All viruses examined were shown to be antigenically closely related. Oligonucleotide patterns of duck/Miss/334 and duck/Miss/406 were apparently distinguishable from those of duck/Miss/116 and duck/Miss/320 isolated in the same area of the United States. On the other hand, two viruses isolated from a domestic duck in Hong Kong (duck/Hong Kong/D3/75) and from a domestic duck in Japan (duck/Tokyo/41/78) were genetically very similar to that of duck/Miss/116, suggesting that these three viruses represent a genetically homogeneous group and may be of the same origin.

We performed antigenic analysis of the haemagglutinin and neuraminidase subunits of a recombinant virus (A/swine/Kanagawa/2/78) isolated from a pig in Japan in 1978, using a series of monoclonal antibodies to H1 (Hsw1) haemagglutinin and N2 neuraminidases of H2N2 and H3N2 viruses. Results obtained in haemagglutination inhibition tests with five monoclonal antibodies to the haemagglutinin of A/NJ/8/76 (H1N1) revealed that the haemagglutinin of three H1N1 and the recombinant viruses were indistinguishable from that of A/NJ/8/76. The neuraminidase of A/swine/Kanagawa/2/78 was found to be antigenically similar to A/Kumamoto/22/76 (H3N2, A/Victoria/3/75-like strain). The oligonucleotide maps of the entire RNAs of H1N1, H1N2 and H3N2 viruses showed that A/swine/Kanagawa/2/78 (H1N2) virus was more similar to swine (H1N1) virus than to A/Kumamoto/22/76 (H3N2) virus. Radioactive cDNA was prepared by reverse transcription of the recombinant virus RNA using a dodecadeoxyribonucleotide primer and used in DNA-RNA hybridization experiments. The results obtained in molecular hybridization based on blotting procedures showed that all cDNA segments except gene 6 hybridized efficiently with RNAs of swine (H1N1) influenza virus. The sixth cDNA segment was homologous to the corresponding RNA segment of H3N2 virus. The genetic relatedness of A/swine/Kanagawa/2/78 (H1N2) with either A/swine/Kanagawa/4/78 (H1N1) or A/Kumamoto/22/76 (H3N2) was clearly established by hybridization between the cDNA segment probes and viral RNA. It was concluded that the neuraminidase gene of A/swine/Kanagawa/2/78 (H1N2) was derived from a human H3N2 virus, while the seven other genes were from a swine H1N1 virus.

The hemagglutinin-neuraminidase (HN) subunits of NDV and NDV-like isolates were analyzed antigenically by monoclonal antibodies to the HN of Miyadera and Taka viruses. In immuno-double-diffusion (IDD) tests, all NDVs examined gave clear lines of precipitation with some of the potent monoclonal antibodies, but it was difficult to determine with certainty the immunological properties of HN subunits due to a rare disagreement with the results obtained in other immunological tests. Monoclonal antibodies used in the tests were found to show different immunological reactivities with the viruses. Monoclonal antibodies belonging to the 1st group (1/29) inhibited the hemagglutinating (HA) activity of all strains but not the neuraminidase (NA) activity. The second monoclonal antibody (5/205) inhibited both the HA and NA activities of the restrictive NDV strains, indicating antigenic changes in HN molecules. However, the inhibitory activity of this monoclone to neuraminidase appeared to be greatly diminished when neuraminyl lactose was used as substrate. Although the 3rd type of monoclonal antibody (5/220) showed HI activity against several strains, this antibody did not inhibit NA activity of any viruses. The remaining monoclone to the HN of Taka virus inhibited the HA activity of all reference strains of NDV and many NDV-like isolates but did not affect NA activity. Two inhibitory activities of four monoclonal antibodies against different viruses, HI and hemolysis-inhibition, were not always consistent with inhibition of virus growth. HI and NI tests with the above four monoclonal antibodies showed that the strains tested fell into five antigenic groups according to their reaction patterns with mouse hybridoma antibodies.

Virus isolation and serological studies on swine sera collected during 1973 to 1978 showed that H1N1 (Hsw1N1) influenza viruses first appeared in the swine population of Japan about May 1977. With the exception of one strain, both haemagglutinin and neuraminidase subunits of all the H1N1 viruses isolated from swine in Japan and from pigs imported from North America were antigenically indistinguishable from those of A/NJ/8/76 virus, suggesting the introduction of swine influenza virus into Japan with imported pigs from North America as breeding stock. Antigenic analysis of a recombinant virus by neuraminidase-inhibition tests with specific antisera to the isolated neuraminidases of A/Victoria/3/75 and A/Aichi/2/68 revealed that the neuraminidase antigen of the recombinant virus, A/swine/Kanagawa/2/78 (H1N2), was closely related to those of A/Tokyo/6/73 (H3N2) and A/Kumamoto/22/76 (H3N2) viruses.

The objective of this international collaborative study was to compare recent swine isolates of influenza viruses and determine whether significant antigenic differences among isolates from different areas of the world could be detected. H1N1 viruses isolated from pigs, birds and humans in 12 different countries were compared in haemagglutination-inhibition assays with post-infection ferret sera and monoclonal antibodies to H1N1 strains. Using A/NJ/8/76 as the reference strain, we found that recent swine isolates from Hong Kong, Italy, Japan, and the USA possess a haemagglutinin virtually indistinguishable from that of viruses typically associated with pigs, i.e., A/NJ/8/76. In contrast, recent swine isolates from several European countries (Belgium, Denmark, France, Federal Republic of Germany, and Spain) were distinguishable from A/NJ/8/76, as demonstrated by tests in the various laboratories. These studies suggest that the H1N1 viruses in pigs are antigenically heterogeneous and that the circulation of particular variants is associated with the geographical location of the animals. These results raise the question of whether these viruses originated from the same source, i.e., pigs, and have undergone antigenic drift or, alternatively, were introduced from other hosts, such as birds.

Specific antisera for the isolated HN proteins of eight reference strains of avian paramyxoviruses could be prepared in guinea pigs by intraperitoneal injection of guinea pig red blood cells (GRBC) coated with purified HN proteins. In the hemagglutination inhibition (HI) tests, all reference strains reacted strongly with each homologous antiserum to the isolated HN showing that a low level of cross-reactivity among the reference strains was greatly diminished by using specific antisera. Immuno-double-diffusion (IDD) tests showed that all antisera except those to turkey/Wisconsin/68 and duck/Hong Kong/D3/75 gave single well-defined lines only with the homologous viruses. The remaining two antisera developed a single definite precipitin line together with weak lines with homologous virus. Two isolates in Japan were clearly identified in HI and IDD tests with specific antisera to the HN subunits of the reference strains suggesting that the antisera were useful for identification of avian paramyxovirus isolates. Two isolates in Japan, H-70 from a munia-bird and Y-7 from a duck were found to have HN proteins related closely to those of finch/N. Ireland/Bangor/73 and duck/Hong Kong/199/77, respectively.