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.     

Protection of chickens against challenge with virulent influenza A viruses of Hav5 subtype conferred by prior infection with influenza A viruses of Hsw1 subtype

Summary Prior infection of six-week-old chickens with influenza A viruses of Hsw 1 haemagglutinin subtype and irrelevant neuraminidase subtypes reduced the deaths and sickness in groups of those birds challenged with A/tern/S. Africa/61 (Hav 5 Nav 2/3) and A/chicken/Scotland/59 (Hav 5 N 1).     

Susceptibility of wild passerines to subtype H5N1 highly pathogenic avian influenza viruses

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have spread throughout many areas of Asia, Europe and Africa, and numerous cases of HPAI outbreaks in domestic and wild birds have been reported. Although recent studies suggest that the dissemination of H5N1 viruses is closely linked to the migration of wild birds, information on the potential for viral infection in species other than poultry and waterfowl is relatively limited. To investigate the susceptibility of terrestrial wild birds to infection with H5N1 HPAI viruses, common reed buntings (Emberiza schoeniclus), pale thrushes (Turdus pallidus) and brown-eared bulbuls (Hypsipetes amaurotis) were infected with A/mountain hawk-eagle/Kumamoto/1/07(H5N1) and A/whooper swan/Aomori/1/08(H5N1). The results showed that common reed buntings and brown-eared bulbuls were severely affected by both virus strains (100% mortality). While pale thrushes did not exhibit any clinical signs, seroconversion was confirmed. In common reed buntings, intraspecies-transmission of A/whooper swan/Aomori/1/08 to contact birds was also confirmed. The findings show that three passerine species; common reed buntings, brown-eared bulbuls and pale thrushes are susceptible to infection by H5N1 HPAI viruses, which emphasizes that continued surveillance of species other than waterfowl is crucial for effective monitoring of H5N1 HPAI virus outbreaks.     

A preliminary panorama of the diversity of N1 subtype influenza viruses

N1 subtype influenza viruses have caused many epidemics and even a few pandemics in humans, pigs and fowls including 1918 human H1N1 pandemic, which killed 20-50 million people and the current avian H5N1 pandemic in the Eastern Hemisphere, which has caused great economic losses and posed a severe threat to human public health. To elucidate the whole diversity of N1 influenza viruses from a dynamic view, 202 neuraminidase (NA) sequences of N1 subtype influenza isolates were selected and analyzed in this study. Our results showed that N1 influenza isolates could be divided into three distinct lineages (Human, Classic Swine and Avian), which largely circulated in the humans, pigs and fowls respectively, though viruses in the Avian lineage could infect mammals and even there was a sublineage in the Avian lineage wholly isolated from pigs. The Avian lineage and the Human lineage, which have existed at least for decades, possibly began divergence around in 1890 through regression analysis. Both of the Human and Avian lineages could be further divided into some sublineages, and the correlation between these lineages (or sublineages) and their isolation places, isolation time, hemagglutinin (HA) subtypes, host species, virulence, or epidemics were discussed. The panorama of the diversity of N1 influenza viruses presented in this study provided a framework for the studies on the evolution and epidemiology of N1 influenza viruses.     

Molecular and pathological characterization of two H5N1 avian influenza viruses isolated from wild ducks

In this study, two H5N1 influenza viruses (HN021 and HN211) were isolated in wild ducks and the characteristics of these viruses were studied systemically. By studying the pathogenesis of both H5N1 isolates, the results showed that HN211 was highly pathogenic in chickens, geese, ducks, and mice, while HN021 was highly pathogenic in chickens and geese but low pathogenic in ducks and mice. Both isolates could replicate in lungs and brains of mice and be transmitted from ducks to ducks. Histopathologic analysis showed that HN211 could cause more severe pathological changes in lungs and brains of infected mice than HN021. Molecular characterization showed that both H5N1 isolates had 20 aa missing in stalk of NA protein and 5 aa missing in NS protein in comparison with most other H5N1 isolates. Phylogenetic analysis indicated that both H5N1 isolates were reassortants from Goose/Guangdong/1/96-like viruses. The results of present study with both H5N1 viruses also suggested that wild ducks may play an important role in maintaining circulation of H5N1 viruses.     

Antigenic structure of hemagglutinins of influenza H1N1 (Hsw1N1) virus isolated from humans and ducks

The method of specific adsorption followed by the use of antisera in HI test and competitive enzyme immunoassay was used to study the antigenic composition of hemagglutinins (HA) Hsw1 in influenza viruses isolated in 1982 from humans in Bulgaria and in 1976 in Canada from ducks as well as their antigenic relationships with HA of Hsw1 variant isolated from swine and man. Hemagglutinins of Hsw1 strains isolated from man in Bulgaria and Alma-Ata were found to be similar to HA of A/New Jersey/8/76 virus in two determinants and with hemagglutinin of the classic virus of swine in three determinants. The HA of A/duck/Alberta/35/76 virus was similar in three determinants to HA of A/New Jersey/8/76 virus and in two determinants with other Hsw1 variants. The similarities and differences in antigenic determinants of HA in Hsw1 viruses isolated from man and animals attest to their common origin and different modes of variability.     

Comparative characteristics of hemagglutinins of influenza A viruses with antigenic structure Hsw1N1 isolated from man and animals

Competitive radioimmunoassay was used to study the antigenic composition of hemagglutinin of Hsw1N1 viruses isolated from man in comparison with hemagglutinin Hsw1 of influenza virus of swine and ducks. The data of oligonucleotide analysis of the 4th RNA segment coding for hemagglutinin in these viruses are presented. It has been shown that in Alma-Ata, 1984-1985, influenza viruses Hsw1N1 were isolated with the antigenic structure of hemagglutinin and with the hemagglutinin gene identical with those of the classical influenza virus of swine A/Swine/Iowa/15/30 but differing from virus A/New Jersey/8/76.     

Radioimmunological study of the hemagglutinin of influenza viruses subtype H1N1

Radioimmunoassay were carried out with influenza A/Cambridge/46, A/FM/1/47, A/England/1/51, A/Denwer/1/57, and A/USSR/90/79 of the H1N1 subtype to determine the antigenic determinants of influenza A/USSR/90/77 virus hemagglutinin. The test system included 125I-labeled hemagglutinin of A/USSR/90/77 virus and homologous antiserum to this virus. The most marked antigenic similarity was found between A/USSR/90/77 and A/USSR/90/79 viruses. The A/Cambridge/46 and A/Denwer/1/57 viruses showed clear-cut differences in the antigenic determinants from the A/USSR/90/77 virus.     

Antiviral etiotropic chemicals: efficacy against influenza A viruses A subtype H5N1

The paper analyzes data of an experimental study of the efficacy of antiviral agents (amantadine, remantadine, ozeltamivir, zanamivir, arbidol, ribavirin) in the cultured cells and on a model of murine influenza pneumonia against influenza A viruses subtype H5N1. It also gives data on their use in the treatment of human beings during avian influenza outbreak. The mechanism of action of the agents, pharmacokinetics, adverse reactions, and their potential resistance are considered.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

Antigenic specificity of neuraminidase and structure of neuraminidase gene of influenza A(H1N1) serotype Hsw1N1 viruses isolated from man and animals

The enzyme immunoassay and neuraminidase activity inhibition test using polyclonal and monospecific antineuraminidase sera were employed to establish the similarities and differences in the antigenic structure of neuraminidase of influenza A viruses (H1N1), serovariant Hsw1N1, isolated from man in Alma-Ata (USSR), 1984, New Jersey (USA), 1976, and Pazardjik (BPR), 1982, as well as from swine and birds. Oligonucleotide mapping revealed significant structural differences in the genes coding for neuraminidase of Hsw1N1 viruses. The experimental results indicate a high degree of the enzyme variability in this group of viruses.     

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.     

New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks

During the past years increasing incidences of influenza A zoonosis have made it of uppermost importance to possess methods for rapid and precise identification and characterisation of influenza A viruses. We present here a convenient one-step RT-PCR method that will amplify full-length haemagglutinin (HA) and neuraminidase (NA) directly from clinical samples and from all known subtypes of influenza A. We applied the method on samples collected in September 2003 from a Danish flock of mallards with general health problems and by this a previously undescribed influenza A subtype combination, H5N7, was identified. The HA gene showed great sequence similarity to the highly pathogenic avian influenza A virus (HPAIV) A/Chicken/Italy/312/97 (H5N2); however, the cleavage site sequence between HA1 and HA2 had a motif typical for low pathogenic avian influenza viruses (LPAIV). The full-length NA sequence was most closely related to the HPAIV A/Chicken/Netherlands/01/03 (H7N7) that infected chickens and humans in the Netherlands in 2003. Ten persons with direct or indirect contact with the Danish mallard ducks showed signs of influenza-like illness 2-3 days following the killing of the ducks, but no evidence of influence infections was detected. To our knowledge this is the first report of an H5N7 influenza A virus.     

Genetic characterization of influenza A virus subtype H12N1 isolated from a watercock and lesser whistling ducks in Thailand

Monitoring of influenza A virus (IAV) was conducted in wild bird species in central Thailand. Four IAV subtype H12N1 strains were isolated from a watercock (order Gruiformes, family Rallidae) (n = 1) and lesser whistling ducks (order Anseriformes, family Anatidae) (n = 3). All H12N1 viruses were characterized by whole-genome sequencing. Phylogenetic analysis of all eight genes of the Thai H12N1 viruses indicated that they are most closely related to the Eurasian strains. Analysis of the HA gene revealed the strains to be of low pathogenicity. This study is the first to report the circulation of IAV subtype H12N1 in Thailand and to describe the genetic characteristics of H12N1 in Eurasia. Moreover, the genetic information obtained on H12N1 has contributed a new Eurasian strain of H12N1 to the GenBank database.     

General antigenic determinants in the hemagglutinin composition of influenza viruses H0, H1 and Hsw1

The antigenic properties of influenza type A viruses with hemagglutinin subtypes H0, H1, and Hsw1 were studied by hemagglutination-inhibition test, indirect solid-phase radioimmunoassay and biological neutralization test using highly potent hyperimmune rabbit sera. The results obtained indicate the presence of common antigenic determinants in hemagglutinins of H0, H1 and Hsw1 viruses.     

Emergence of novel reassortant H3N2 influenza viruses among ducks in China

During 2006-2009 influenza virus surveillance, three H3N2 viruses were isolated from ducks in Central China. Sequence and phylogenetic analyses revealed that most segments of these three isolates had high identity with H3N2 swine isolates in South China. However, for M, the three viruses, along with H1N1 swine isolates of North America, formed a cluster; for PB2, two of these isolates fell into the cluster of the H5N1 duck isolates, indicating a reassortment among H3N2, H1N1 swine viruses and H5N1 avian virus. The emergence of H3N2 virus with incorporation of an H5N1 virus gene raises new concerns about the generation of novel viruses that could affect humans.     

Differential host gene responses in mice infected with two highly pathogenic avian influenza viruses of subtype H5N1 isolated from wild birds in Thailand

In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice.     

Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks

The Asian highly pathogenic avian influenza (HPAI) H5N1 viruses have changed from producing no disease or mild respiratory infections in ducks to some strains causing systemic disease and death. Differences in pathogenicity between four of these viruses as well as the effect of host age on the outcome of infection were studied in ducks. Three of the viruses were highly lethal in 2-week-old ducks and induced severe neurological dysfunction. Neurological signs were also observed in 5-week-old ducks inoculated with one of these viruses; however mortality was low. The fourth virus studied did not induce neurological signs in 2-week-old ducks, but did produce moderate mortality. This virus caused no clinical signs or death in 5-week-old ducks. All viruses studied were isolated from oropharyngeal and cloacal swabs, and also from brain, heart, lung and muscle tissues, demonstrating systemic infection. All viruses evaluated transmitted efficiently to contact ducks. Phylogenetic analysis of the viruses studied and other Asian H5N1 HPAI viruses with diverse pathogenicity in ducks, showed changes in several genes, but none clearly associated with pathogenicity. In conclusion, the pathogenicity of circulating H5N1 HPAI viruses in ducks varies depending on the virus strain and the age of the duck and correlates with the level of viral replication in tissues. High titers of virus in organs, high viral shedding, and variable mortality enable ducks to circulate H5N1 HPAI viruses.