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.     

Studies on antigenic variation in influenza virus. Evidence for multiple antigenic determinants on the hemagglutinin subunits of A-Hong Kong-68 (H3 N2) virus and the A-England-72 strains

Hemagglutinin subunits were isolated from Hong Kong influenza virus (A/Hong Kong/68, H3 N2) and from its antigenic variants, A/England/42/72, and A/Memphis/102/72 which arose as the result of antigenic drift. Immunodiffusion tests showed that the HK/68 subunits possessed at least two different kinds of antigenic determinants while the 1972 variants appeared to possess at least three different determinants. Evidence suggested that the different antigenic determinants were located on the same hemagglutinin subunit and that the virus particles did not possess a mixture of antigenically different subunits.One of the antigenic determinants was common to the hemagglutinin subunits of A/Hong Kong/68, A/England/42/72 and A/Memphis/102/72 viruses. Antibody to this determinant cross-reacted in immunodiffusion, hemagglutination-inhibition, and neutralization tests with all three viruses. Antibodies to another determinant showed no detectable serological cross-reactions between HK/68 and Mem/72 viruses. Thus it appeared that during antigenic drift, Hong Kong influenza virus had undergone an antigenic shift in this “specific” determinant.Antibody to the “specific” determinant on HK/68 hemagglutinin (which did not react at all with Mem/72) did, however, show some cross-reaction with A/England/42/72 hemagglutinin suggesting that this virus was an intermediate in the evolution of the Memphis/72 variant.Despite the occurrence of an antigenic shift in one determinant, peptide maps of the light polypeptides (HA2) from the hemagglutinin subunits of HK/68 and Mem/72 viruses were almost identical and maps of the heavy polypeptides (HA1) showed only a relatively small number of differences.     

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     

Cross-Protection in Mice After Immunization with H2N2, H3N2, and Heq2Neq2 Influenza Virus Strains

Mice were vaccinated with the influenza viruses A/Japan/57 (H2N2), A/Hong Kong/68 (H3N2), and A/Equi/Miami/63 (Heq2Neq2) and the hemagglutinin and neuraminidase recombinants derived from these viruses. After infection with the parent viruses, protection was compared with serological findings. It was found that influenza vaccine protects not only against infection with a strain identical or closely related to the vaccine strain, but against heterologous strains as well. Vaccination with Hong Kong/68 and its neuraminidase recombinant resulted in a heterologous neuraminidase inhibition titer against Japan/57 and in a protection against infection with Japan/57. By contrast, after vaccination with Japan/57 and its neuraminidase recombinant, no relevant heterologous neuraminidase inhibition titer against Hong Kong/68 was observed, whereas a protection against infection with Hong Kong/68 did exist. A cross-protection between Hong Kong/68 and Miami/63, but no relationship in the hemagglutination or neuraminidase inhibition tests, was established in the preinfection sera. A one-way antigenic relationship between these viruses was confirmed by the rise of hemagglutinin or neuraminidase antibodies against Hong Kong/68 in the postinfection sera. No cross-protection or serological relationship existed between Miami/63 and Japan/57. Besides the hemagglutinin and neuraminidase, a third factor, the “mouse-protecting antigen,” was considered to contribute to the protection obtained. According to the protection observed, the mouse-protecting antigen of Hong Kong/68 virus is related to that of Japan/57 as well as Miami/63 virus. The mouse-protecting antigens of both Japan/57 and Miami/63 are related to that of Hong Kong/68.     

Crystallization and peptide maps of neuraminidase “heads” from H2N2 and H3N2 influenza virus strains

Neuraminidase “heads” of two H2N2 influenza virus strains (A/Nederlands/68 and A/Korea/68) and one H3N2 strain (A/Hong Kong/68) were isolated by pronase digestion of recombinant viruses possessing neuraminidase molecules from these strains and H (NWS) hemagglutinin. The neuraminidase heads were purified and crystallized by dialysis against distilled water. Large flat crystals, which differed in appearance between the three strains, were obtained. Tryptic peptide maps of the crystallized neuraminidase heads of the Nederlands/68 and Hong Kong/68 strains were almost identical, suggesting that a virus closely related to the Nederlands/68 (H2N2) strain may have been the source of the neuraminidase for the formation of the H3N2 Hong Kong subtype.     

Properties of Hav6Neq2 and Hswl(H0)Hav2 influenza viruses isolated from waterfowl in southern Turkmenia

Three influenza A virus strains were isolated from shorebirds in October, 1977, in southern Turkmenia, in the vicinities of Tedzhen water reservoir. From a common tern, A/Sterna hirundo/Turkmenia/45/77 strain was isolated with the antigenic formula Hav6Neq2, from a teal and a black-headed gull influenza A/Anas crecca/Turkmenia/4/77 and A/Larus ridibundus/Turkmenia/13/77 strains with previously unknown combination of surface antigens Hswl(H0)Nav2 were recovered. By the molecular weight of the heavy (HA1 59,000 d) and light (HA2 24,000 d) chains of hemagglutinin, the Turkmenian viruses A/Larus ridibundus/Turkmenia/13/77 and A/Anas crecca/Turkmenia/4/77 are similar to each other and to the strains having H0 hemagglutinin: A/PR8/34 (H0N1) and A/Whale/PO/19/76 (H09Nav2). The Turkmenian viruses are characterized by a low content of the light hemagglutinin chain (HA2) which is typical of the viruses with Hsw1 hemagglutinin: A/New Jersey/8/76 (Hsw1N1) and A/SW/Wisk/68 (Hsw1N1).     

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.     

Isolation of a type A influenza virus from an Australian pelagic bird

A type A influenza virus was isolated from a tracheal swab taken from an apparently healthy shearwater bird (mutton bird, Puffinus pacificus chlororhynchus) nesting on Tryon Island off the east coast of Australia. The hemagglutinin subunits of the shearwater virus were of antigenic subtype Hav6, but the neuraminidase subunits were not related antigenically to those of any known virus and represent a new neuraminidase subtype, Nav5. The hemagglutinin and neuraminidase subunits of the shearwater virus were segregated by recombination of the virus with other influenza A viruses. The size and polypeptide composition of these subunits, isolated electrophoretically from the SDS-disrupted recombinant viruses, were similar to those of other influenza viruses.These findings suggest that there may be other avian influenza viruses with novel hemagglutinin or neuraminidase subunits, from which human pandemic strains could arise by genetic recombination.     

Origin and evolutionary pathways of the H1 hemagglutinin gene of avian,swine and human influenza viruses: cocirculation of two distinct lineages of swine virus

Summary The nucleotide sequences of the HA1 domain of the H1 hemagglutinin genes of A/duck/Hong Kong/36/76, A/duck/Hong Kong/196/77, A/sw/North Ireland/38, A/sw/Cambridge/39 and A/Yamagata/120/86 viruses were determined, and their evolutionary relationships were compared with those of previously sequenced hemagglutinin (H1) genes from avian, swine and human influenza viruses. A pairwise comparison of the nucleotide sequences revealed that the genes can be segregated into three groups, the avian, swine and human virus groups. With the exception of two swine strains isolated in the 1930s, a high degree of nucleotide sequence homology exists within the group. Two phylogenetic trees constructed from the substitutions at the synonymous site and the third codon position showed that the H1 hemagglutinin genes can be divided into three host-specific lineages. Examination of 21 hemagglutinin genes from the human and swine viruses revealed that two distinct lineages are present in the swine population. The swine strains, sw/North Ireland/38 and sw/Cambridge/39, are clearly on the human lineage, suggesting that they originate from a human A/WSN/33-like variant. However, the classic swine strain, sw/Iowa/15/30, and the contemporary human viruses are not direct descendants of the 1918 human pandemic strain, but did diverge from a common ancestral virus around 1905. Furthermore, previous to this the above mammalian viruses diverged from the lineage containing the avian viruses at about 1880.     

Host range recombinants of fowl plague (influenza A) virus

Recombinants between the influenza virus strains fowl plague virus (FPV, Hav1N1) and Hong Kong (H3N2) have been isolated which form plaques on MDCK cells but not on chick embryo cells, although they carry the hemagglutinin of FPV. These host range recombinants have been characterized and one of them has been used for a second recombination with virus N (Hav2Neq1) or equi 2 (Heq2Neq2) on chick embryo cells. In this way, recombinants were obtained with a mixed genome which have regained the natural host range of FPV and some pathogenic properties for chicken. The results are discussed as a possible mechanism for a pandemic influenza strain to survive in an animal reservoir by changing its host range by recombination, and to regain the original host range by a second recombination but always keeping the same hemagglutinin.     

Increasing appearance of reassortant influenza B virus in Taiwan from 2002 to 2005

Genetic and antigenic analyses of influenza B virus field strains isolated in Taiwan from 1998 to 2005 were performed. To investigate the molecular evolution of influenza B viruses, sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase genes was performed. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculating with the Yamagata lineage, was identified in Taiwan in March 2001. Concurrently, there was an increasing prevalence of this lineage in many parts of the world, including North America and Europe, during the 2001-2002 season. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain.     

Further studies of the antigenic properties of H3N2 strains of influenza A isolated from swine in South East Asia.

H3N2 strains of influenza A isolated from swine in Hong Kong were compared with human strains of H3N2 influenza A variants in reciprocal HI tests using ferret sera. One isolate from swine was indistinguishable from A/Hong Kong/68, one set of viruses isolated in 1976 and 1977 was most related to A/Hong Kong/68 but was not identical to it, two isolates from 1976 were 'bridging strains' that cross-reacted equally with the contemporary variants A/Victoria/3/75 and A/Texas/1/77, similarly to a small number of recent human isolates, and two isolates from 1977 were similar to A/Victoria/3/75. These general relationships were supported by neuraminidase inhibition tests. The findings confirm and extend previous results indicating that swine may be a reservoir of old and novel variants of influenza A H3N2 strains related to those that infect man.     

Evidence for antigenic variation in influenza A nucleoprotein.

Nucleoprotein (NP) antigens isolated from sodium sarcosyl detergent-disrupted influenza A virus particles by cellulose acetate electrophoresis were used to prepare specific immune sera. Antigenic analysis of the nucleoproteins by immuno-double-diffusion and antibody absorption tests revealed antigenic differences in the nucleoprotein antigens of human A/PR8/34 (HON1) virus and viruses of the Hong Kong (H3N2) subtype. The nucleoprotein antigens of avian A/duck/Ukraine/1/63 (Hav7Neg2) and A/swine/Iowa/15/30 (Hsw1Nl) viruses resembled more closely the NP of A/PR8/34 virus than the NP of human H3N2 strains. Antigenic analysis of recombinant strains prepared from A/PR8/34 and H3N2 parent viruses indicated that the parental origin of the NP antigens could be clearly identified. Identification of the nucleoproteins of parental and recombinant influenza A viruses by migration rate analysis of NP polypeptides and RNA genes by electrophoresis on polyacrylamide gels gave results which correlated with the antigenic characterization of their NP antigens. The findings suggested that antigenic “drift” occurs in the nucleoproteins of human influenza A viruses.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

Isolation of influenza A viruses with a new antigenic formula from wild birds

Three hemagglutinating agents were isolated from mixed pools of the viscera from black-headed gulls (Larus ridibundus) and robin (Erithacus rubecula) collected in the Byelorussian SSA and the Kaliningrad region of the RSFSR. Typing of the viruses by double immunodiffusion technique revealed antigenic relationships of the viruses with swine hemagglutinin (Hsw1) and human hemagglutinin H0. One of the strains had neuraminidase N2 the other two Nav2. An analysis of the polypeptide composition of the virus showed the molecular weights of the heavy (HA1) and light (HA2) hemagglutinin chains to be similar in both strains (about 50,000 and 25,000 daltons, respectively). The strains had a low content of the light hemagglutinin chain (HA2) which is typical for viruses having Hsw1 hemagglutinin.     

Polygenic virulence factors involved in pathogenesis of 1997 Hong Kong H5N1 influenza viruses in mice

Virulence factors of influenza A (H5N1) viruses collected in 1997 from mammalian hosts were examined using a BALB/c mouse model. Fifteen amino acid (aa) residues in four influenza virus genes which correlated with high- and low-pathogenic phenotypes in mice were identified by analyzing sequence alignments. In addition to these specific residues, the effects of aa residue 627 of the PB2 gene, and the hemagglutinin (HA) and neuraminidase (NA) genes were also investigated using a reverse genetics system established with representative viruses of low (A/Hong Kong/486/97) and high (A/Hong Kong/483/97) pathogenicity for mice. None of 15 aa residues alone had any effect on virulence. The HA and NA genes had a synergistic effect on virulence and the absence of a glycosylation site at aa154 in the HA gene also increased virulence of virus. Multiple genes are involved in virulence of Hong Kong H5N1 influenza A viruses for mice with the presence of lysine at aa627 in the PB2 gene exhibiting a significantly larger effect than the HA and NA genes.     

Characterization of DNA complementary to nucleotide sequences adjacent to poly(A) at the 3'-terminus of the avian sarcoma virus genome.

The temperature-sensitive influenza virus A/Hong Kong/68 (H3N2) ts-1[E] has been used as a prototype live attenuated influenza virus vaccine. Using recently developed techniques to map the genome of influenza viruses and to “genotype” influenza virus recombinants, the temperature-sensitive lesions in the virus were identified. These defects, responsible for the attenuation of the virus, are located in the genes for the P3 protein and the nucleoprotein and are associated with virus-specific RNA synthesis. Hong Kong/68 (H3N2) ts-1[E] virus can also serve as a donor of “attenuation characteristics” for the selection of recombinant strains which have different surface antigens and may be used as vaccine strains in the future. The temperature-sensitive mutations of Hong Kong/68 (H3N2) ts-1[E] virus were previously transferred to recombinant viruses carrying the HO hemagglutinin. The RNAs of 8 of these temperature-sensitive recombinants were analyzed. One of these viruses, R1, classified in group 1 of the Hong Kong mutant virus set was found to possess a ts defect only in the P3 protein. R8, a member of group 2 of the Hong Kong mutant virus set had a ts mutation in the nucleoprotein.     

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.     

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans.     

Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.

Analysis of the sequences of all eight RNA segments of the influenza A/G oose/Guangdong/1/96 (H5N1) virus, isolated from a sick goose during an outbreak in Guangdong province, China, in 1996, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997. However, the remaining genes showed greater similarity to other avian influenza viruses. Notably, the neuraminidase gene did no have the 19-amino-acid deletion in the stalk region seen in the H5N1 Hong Kong viruses and the NS gene belonged to allele B, while that of the H5N1 Hong Kong viruses belonged to allele A. These data suggest that the H5N1 viruses isolated from the Hong Kong outbreaks derived their HA genes from a virus similar to the A/Goose/Guangdong/1/96 virus or shared a progenitor with this goose pathogen.