Antigenic variability of avian influenza virus A/H13, isolated in the USSR

The antigenic structure of influenza H13 viruses isolated from wild birds in the USSR in 1976-1985 was studied. Antiserum against the reference A/gull/Maryland/704/77 (H13N6) strain was used to demonstrate the antigenic variations among the viruses. The homology of nucleotide sequences in the region 99-215 for the two A/H13N6 strains, A/gull/Maryland/704/77 and A/great black-headed gull/Astrakhan/227/84, were 75% and 86%, respectively. The 9-base segment deletion in A/grey black-headed gull/Astrakhan/277/84 was observed. Comparison of the predicted amino acid sequences of the strains' hemagglutinin in the appropriate region (amino acids 2-40) revealed 5 replacements (86% homology). Two replacements of arginine by lysine and asparagine by serine in positions 15 and 16, respectively, are the most significant. The latter replacement is accompanied by a change in the glycosylation site and might alter its three-dimensional structure. Further studies of the isolate genome are under way.     

Character of apathogenic influenza a viruses found in Moscow, Russia

An influenza virus A/gull/Moscow/3100/2006 (H6N2) was isolated from gull’ faeces within the precincts of Moscow in autumn 2006. Nucleotide sequence of complete genome (GenBank, EU152234-EU152241) and genotype (K, G, D, 6B, F, 2D, F, 1E) for this virus were determined. Phylogenetic analysis suggests that the H6N2 virus derived by numerous reassortment between viruses, which have been circulating among different birds in Europe since 1999 and in South-East Asia (NA gene) for last years. Some of these viruses probably were introduced by migratory birds from South-East Asia earlier. The strain A/gull/Moscow/3100/2006 is nonpathogenic for chicken embryos and mice and induces specific antibody production in mice. Similar to all avian influenza viruses A/gull/Moscow/3100/2006 binds to Neu5Acα2–3Gal receptors but reveals higher affinity for fucosylated sialosugars (SLex) in contrast to the duck viruses, as was shown in receptor specificity assay and clarified due to modeling the accommodation of SLex into receptor binding site of duck and gull influenza virus hemagglutinin.     

不同亚型禽流感病毒红细胞凝集特性分析

目的:了解不同亚型禽流感病毒与不同动物来源红细胞的凝集特性,为流感环境样本监测工作选择更适宜的检测用红细胞。方法:选取2009-2016年我国禽流感环境监测中分离到的不同亚型的禽流感毒株,采用红细胞凝集试验,选用5种动物红细胞(鸡、火鸡、豚鼠、马和绵羊)进行检测;应用流式细胞仪检测不同动物红细胞表面唾液酸受体的表达及类...     

Avian influenza virus isolates from wild birds replicate and cause disease in a mouse model of infection

The direct transmission of highly pathogenic avian influenza (HPAI) viruses to humans in Eurasia and subsequent disease has sparked research efforts leading to better understanding of HPAI virus transmission and pathogenicity in mammals. There has been minimal focus on examining the capacity of circulating low pathogenic wild bird avian influenza viruses to infect mammals. We have utilized a mouse model for influenza virus infection to examine 28 North American wild bird avian influenza virus isolates that include the hemagglutinin subtypes H2, H3, H4, H6, H7, and H11. We demonstrate that many wild bird avian influenza viruses of several different hemagglutinin types replicate in this mouse model without adaptation and induce histopathologic lesions similar to other influenza virus infections but cause minimal morbidity. These findings demonstrate the potential of wild avian influenza viruses to directly infect mice without prior adaptation and support their potential role in emergence of pandemic influenza.     

H13 influenza viruses in wild birds have undergone genetic and antigenic diversification in nature

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.     

Reassortment of American and Eurasian genes in an influenza A virus isolated from a great black-backed gull (Larus marinus), a species demonstrated to move between these regions

The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.     

Genetically destined potentials for N-linked glycosylation of influenza virus hemagglutinin

The addition of oligosaccharide side chains to influenza virus hemagglutinin (HA) is believed to facilitate viral escape from immune pressure in the human population. To determine the implicit potentials for acquisition of N-linked glycosylation, we analyzed the genetic background of 16 subtypes of avian influenza virus, some of which may be potential pandemic viruses in the future. We found a significant difference among HA subtypes in their genomic sequences to produce N-glycosylation sites. Notably, recently circulating avian influenza viruses of the H5 and H9 subtypes may have rather greater capacities to undergo mutations associated with glycosylation of HA than past pandemic viruses. We hypothesize that influenza viruses maintained in natural reservoirs could have different potentials for sustained circulation, depending on their HA subtypes, if introduced into the human population.     

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.     

Human influenza a viral genes responsible for the restriction of its replication in duck intestine

Although influenza A viruses are occasionally transmitted from one animal species to another, their host range tends to be restricted. Currently circulating human influenza A viruses are thought to have originated from avian viruses, yet none of these strains replicate in duck intestine, a major site of avian virus replication. Although the hemagglutinin (HA) and neuraminidase (NA) genes are known to restrict human virus replication in ducks, the contribution of the other viral genes remains unknown. To determine the genetic basis for host range restriction of the replication of human influenza A virus in duck intestine, we first established a reverse genetics system for generating A/Memphis/8/88 (H3N2) (Mem/88) and A/mallard/New York/6750/78 (H2N2) (Mal/NY) viruses from cloned cDNAs. Using this system, we then attempted to generate reassortant viruses with various combinations of candidate genes. We were able to generate single-gene reassortants, which possessed PB2, NP, M, or NS from Mem/88, with the remainder from Mal/NY. Despite unsuccessful production of other single-gene reassortants from Mem/88, we did generate reassortant viruses comprised of both the HA and the NA, all three polymerase genes (PB2, PB1, and PA), or all polymerase genes and the NP gene from Mem/88, with the rest derived from Mal/NY. Among these reassortants, only those possessing the M or NS gene from Mem/88 and the remainder from Mal/NY replicated in duck intestine. These results indicate incompatibility between the genes of avian and human influenza A viruses and indicate that all genes other than the M and NS restrict replication of human influenza A virus in duck intestine.     

The interrelations of the human and avian influenza viruses A(H2) determined by the mathematical processing of data on the antigenic structure of their hemagglutinin]

Mathematical methods were used to analyse the data on the antigenic specificity of H2 subtype hemagglutinin of human and avian influenza A viruses. This approach allowed the evaluation of possible evolutional relationships in this little-studied group of viruses. Influenza A (H2) viruses isolated from birds in the USA were found to represent a sufficiently isolated group, whereas European avian strains (A/duck/Germany/1215/73, A/pintail duck/Primor'e/695/76, A/duck/Marseilles/46/76) were close to "human" viruses. The A/Leningrad/1468/65, A/laughing gull/New Jersey/75/85, and A/pintail duck/Alberta/2728/77 strains represent marked antigenic variants apparently rather far gone as a result of hemagglutinin drift.     

Attachment of infectious influenza A viruses of various subtypes to live mammalian and avian cells as measured by flow cytometry

At present there is much interest in the cell tropism and host range of influenza viruses, especially those of the H5N1 subtype. We wished to develop a method that would enable investigation of attachment of infectious virus through the interaction of the hemagglutinin molecule and live mammalian and avian cells and the subsequent infection of these cells. To this end, influenza viruses of various HA subtypes were constructed that either carry the green fluorescent protein (GFP) instead of the neuraminidase protein, or that express GFP in the cytoplasm of infected cells. The HA genes were derived from influenza viruses A/PR/8/34 (H1N1), A/Netherlands/178/95 (H3N2) and A/Vietnam/1194/04 (H5N1). Using these pairs of viruses, attachment and post-attachment events in the virus replication cycle can be distinguished. In general, the expression of NeuAc(alpha2-3)Gal or NeuAc(alpha2-6)Gal receptors on the cells tested corresponded with the attachment of the viruses that were studied with respect to predicted receptor specificity. Virus attachment was not always predictive for efficient infection of the cells.     

Detection of antibodies using recombinant strains of influenza viruses in the sera of various species of birds circulating in the territories of the Ukrainian and Azerbaijan SSRs

Examinations of blood sera from different species of birds trapped in the Ukrainian and Azerbaijan SSRs using diagnostic preparations from the influenza A/sea gull/Maryland/704/77 virus strain and a recombinant R117 derived from it revealed the presence of antibodies to hemagglutinin H13. The diagnostic preparation produced from the recombinant strain was found to be more active in the detection of antibodies in avian sera.     

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.     

Persistence of Q strain of H2N2 influenza virus in avian species: Antigenic,biological and genetic analysis of avian and human H2N2 viruses

Summary The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5^–/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific -inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42° C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42° C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957.With 2 Figures     

Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method

This study presents an interconnected approach for circumventing two inherent limitations associated with studies defining the natural history of influenza A viruses in wild birds. The first limiting factor is the ability to maintain a cold chain from specimen collection to the laboratory when study sites are in more remote locations. The second limiting factor is the ability to identify all influenza A virus HA subtypes present in an original sample. We report a novel method for molecular subtyping of avian influenza A virus hemagglutinin genes using degenerate primers designed to amplify all known hemagglutinin subtypes. It was shown previously that templates larger than 200 bp were not consistently amplifiable from ethanol-fixed cloacal swabs. For this study, new primer sets were designed within these constraints. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating hemagglutinin subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different hemagglutinin subtypes. This study further demonstrates the complex ecobiology of avian influenza A viruses in wild birds.     

动物源性和人源性甲型流感病毒(H1N1)血凝素(HA)的特征分析

目的 研究动物源性和人源性甲型H1N1流感病毒(influenza A virus)血凝素(hemagglutinin,HA)的特征,以探讨动物源性和人源性甲型H1N1流感病毒血凝素之间的关系.方法 从美国生物信息中心(NCBI)下载禽(鸟)源、猪源、人源的甲型H1N1流感病毒血凝素氨基酸序列,使用Clustal W2.0生物学软件比较上述血凝素氨基酸序列,并建立甲型H1N1流感病毒血凝素氨基酸序列的进化树.结果 2009年分离的人源性甲型流感病毒血凝素氨基酸序列同源性非常高,达到了99%～100%,而2009年分离的人源性甲型流感病毒血凝素氨基酸序列和禽(鸟)源,猪源的甲型流感病毒血凝素氨基酸序列之间的同源性非常低,只有77%～90%(只有猪源ABW36355和2009年分离的人源甲型流感病毒血凝素氨基酸序列同源性为90%,余同源性为77%～83%);蛋白生物进化树表明禽源(鸟)、猪源、人源的甲型流感病毒血凝素氨基酸序列明显分为3个大的分支.2009年分离的人源性甲型流感病毒血凝素氨基酸序列(ADA71154除外)与疫情前分离得到的人源的血凝素氨基酸序列同源性很低(79%～80%),并且进化树分为3个分支.结论 2009年流行的甲型H1N1流感病毒是一种新的流感病毒,病毒的血凝素氨基酸序列之间的同源性非常高,而和猪、禽(鸟)源的甲型H1N1流感病毒的血凝素氨基酸序列之间的同源性非常低,从这一层面上来讲,目前流行的甲型流感病毒的血凝素的基因并不是猪源和禽源,和疫情前人源的血凝素比对的结果也表明,2009年流行的甲型H1N1流感病毒也并不是直接源于2009年以前的人源流感H1N1病毒,而应该是另有来源.     

Evolutionary pathways of the PA genes of influenza A viruses

Nucleotide sequences of the PA genes of influenza A viruses, isolated from a variety of host species, were analyzed to determine the evolutionary pathways of these genes and the host specificity of the genes. Results of maximum parsimony analysis of the nucleotide sequences indicate at least five lineages for the PA genes. Those from human strains represent a single lineage, whereas the avian genes appear to have evolved as two lineages--one comprising genes from many kinds of birds (e.g., chickens, turkeys, shorebirds, and ducks) and the other comprising only genes from gulls. H3N2 swine influenza virus PA genes are closely related to the currently circulating duck virus PA gene. By contrast, the H1N1 swine and equine virus PA genes appear to have evolved along independent lineages. Comparison of predicted amino acid sequences disclosed 10 amino acid substitutions in the PA proteins of all avian and H3N2 swine viruses that distinguished them from human viruses. The H1N1 swine viruses seem to be chimeras between human and avian viruses and they contain 8 amino acids not shared by other viruses. The equine viruses also appear to show their own amino acid substitutions. These findings indicate that the PA genes of influenza A viruses have evolved in different pathways defined by apparently unique amino acid substitutions and host specificities. They also indicate that influenza A viruses have been transmitted from avian to mammalian species.     

Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus

Two influenza A viruses whose hemagglutinin (HA) did not react with any of the reference antisera for the 13 recognized HA subtypes were isolated from mallard ducks in the USSR. Antigenic analysis by hemagglutination inhibition and double immunodiffusion tests showed that the HAs of these viruses are similar to each other but distinct from the HAs of other influenza A viruses. Nucleotide sequence analysis showed that these HA genes differ from each other by only 21 nucleotides. However, they differ from all other HA subtypes at the amino acid level by at least 31% in HAI. Thus, we propose that the HAs of these viruses [A/Mallard/Gurjev/263/82 (H14N5) and A/Mallard/Gurjev/244/82 (H14N6) belong to a previously unrecognized subtype, and are designated H14. Unlike any other HAs of influenza viruses, the H14 HAs contained lysine at the cleavage site between HA1 and HA2 instead of arginine. Experimental infection of domestic poultry and ferrets with A/Mallard/Gurjev/263/82 (H14N5) showed that the virus is avirulent for these animals. Based on comparative sequence analysis of different HA genes, it is suggested that differences of 30% or more at the amino acid level in HA1 constitute separate subtypes. Phylogenetic analysis of representatives of each HA subtype showed that H14 is one of the most recently diverged lineages while H8 and H12 branched off early during the evolution of the HA subtypes. These latter two subtypes (H8 and H12) have been isolated very infrequently in recent years, suggesting that these old subtypes may be disappearing from the influenza reservoirs in nature.     

Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes

Summary Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.     

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.