Influenza in migratory birds and evidence of limited intercontinental virus exchange

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies. Influenza viruses belonging to each of the subtypes H1 through H13 and N1 through N9 were detected in these waterfowl, but H14 and H15 were not found. Viruses of the HP Asian H5N1 subtypes were not detected, and serologic studies in adult mallard ducks provided no evidence of their circulation. The recently described H16 subtype of influenza viruses was detected in American shorebirds and gulls but not in ducks. We also found an unusual cluster of H7N3 influenza viruses in shorebirds and gulls that was able to replicate well in chickens and kill chicken embryos. Genetic analysis of 6,767 avian influenza gene segments and 248 complete avian influenza viruses supported the notion that the exchange of entire influenza viruses between the Eurasian and American clades does not occur frequently. Overall, the available evidence does not support the perpetuation of HP H5N1 influenza in migratory birds and suggests that the introduction of HP Asian H5N1 to the Americas by migratory birds is likely to be a rare event.     

Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses

Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes.     

Characterization of H7 Influenza A Virus in Wild and Domestic Birds in Korea

During surveillance programs in Korea between January 2006 and March 2011, 31 H7 avian influenza viruses were isolated from wild birds and domestic ducks and genetically characterized using large-scale sequence data. All Korean H7 viruses belonged to the Eurasian lineage, which showed substantial genetic diversity, in particular in the wild birds. The Korean H7 viruses from poultry were closely related to those of wild birds. Interestingly, two viruses originating in domestic ducks in our study had the same gene constellations in all segment genes as viruses originating in wild birds. The Korean H7 isolates contained avian-type receptors (Q226 and G228), no NA stalk deletion (positions 69–73), no C-terminal deletion (positions 218–230) in NS1, and no substitutions in PB2-627, PB1-368, and M2-31, compared with H7N9 viruses. In pathogenicity experiments, none of the Korean H7 isolates tested induced clinical signs in domestic ducks or mice. Furthermore, while they replicated poorly, with low titers (10 ^0.7–1.3EID₅₀/50 µl) in domestic ducks, all five viruses replicated well (up to 7–10 dpi, 10 ^0.7–4.3EID₅₀/50 µl) in the lungs of mice, without prior adaptation. Our results suggest that domestic Korean viruses were transferred directly from wild birds through at least two independent introductions. Our data did not indicate that wild birds carried poultry viruses between Korea and China, but rather, that wild-type H7 viruses were introduced several times into different poultry populations in eastern Asia.     

Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.     

Influenza A viruses in American White Pelican (Pelecanus erythrorhynchos)

The role of many wild waterbird species in the ecology and epidemiology of avian influenza viruses (AIV) remains unclear. We report the first isolation of AIV from American White Pelicans (Pelecanus erythrorhynchos; Pelecaniformes) in North America. Two H13N9 AIVs were isolated from hatchling birds in breeding colonies in Minnesota, USA, during 2007 and 2008. Based on molecular sequencing of the hemagglutinin and neuraminidase genes, the 2008 virus was genetically related to AIVs previously isolated from gulls and shorebirds in North America. The 2007 isolate was most related to AIVs from Eurasian gulls and North American ducks, reflecting both global movement of these viruses and reassortment between viruses associated with duck and gull reservoirs.     

Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016–2017 in Northern Japan

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016–2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.

     

Molecular Analysis of H7 Avian Influenza Viruses from Australia and New Zealand: Genetic Diversity and Relationships from 1976 to 2007

Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus isolate (all subtype H7) has enabled comparison of the sequences of each of the genome segments to those of other subtype H7 avian influenza A viruses. The inference of phylogenetic relationships for each segment has been used to develop a model of the natural history of these viruses in Australia. Phylogenetic analysis of the hemagglutinin segment indicates that the Australian H7 isolates form a monophyletic clade. This pattern is consistent with the long-term, independent evolution that is, in this instance, associated with geographic regions. On the basis of the analysis of the other H7 hemagglutinin sequences, three other geographic regions for which similar monophyletic clades have been observed were confirmed. These regions are Eurasia plus Africa, North America, and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3, and H7N7 genomes revealed the same region-based relationships. This pattern of independent evolution of Australian isolates is supported by the results of analysis of each of the six remaining genomic segments. These results, in conjunction with the occurrence of five different combinations of neuraminidase subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest that the maintenance host(s) is nearly exclusively associated with Australia. The single lineage of Australian H7 hemagglutinin sequences, despite the occurrence of multiple neuraminidase types, suggests the existence of a genetic pool from which a variety of reassortants arise rather than the presence of a small number of stable viral clones. This pattern of evolution is likely to occur in each of the regions mentioned above.The emergence of highly pathogenic avian influenza viruses of subtype H5N1 as a potential human pandemic disease threat has focused attention on the roles that wild birds play in the maintenance and distribution of avian influenza viruses (18, 22). Moreover, the H5 and H7 subtypes of avian influenza A virus are major causes of economic loss in poultry production through disease. In Australia, there have been five documented outbreaks of H7 subtype avian influenza A virus disease, with evidence of adaptation to the poultry host being provided by sequence data supporting the presence of high-pathogenicity avian influenza virus (HPAI) isolates in poultry. Waterfowl (Anseriformes order, particularly ducks, geese, and swans) and the waders and gulls (Charadriiformes order, particularly gulls, terns, and waders) have been found to be the major global natural reservoirs of influenza A viruses. Transmission of avian influenza viruses from wild birds to production poultry and geographic spread are dependent upon the migratory behavior of the wild bird reservoir hosts. Members of the Anseriformes and Charadriiformes orders undertake both irregular and regular transcontinental and intercontinental migrations. During these migrations, large numbers of birds congregate at aquatic feeding locations, providing ideal sites for cross-species transmission of avian influenza viruses. A variety of mechanisms have been observed whereby influenza A viruses adapt rapidly. These include genetic shifts facilitated through genome segment reassortment, as well as genetic drift through the insertion, deletion, and substitution of nucleotides. The error-prone RNA replication and a lack of error correction are the causes of drift. In vivo, this results in viral genetic diversity within any viral sample, or a quasispecies, thus providing a pool of closely related variant viruses from enabling events, such as viral adaptation to new hosts (25). Long-term sampling of water birds in North America and Europe has started to elucidate the ecology and biology of the avian influenza A virus types in the natural reservoirs in these regions (8, 18, 22). There is a suggestion that two superfamilies, the Eurasia (which in the context of this paper includes Europe, Asia, and Africa) and the Americas superfamilies, exist; however, the extent of overlap and the rate of transfer of influenza viruses between these two regions are not well-defined. Recent studies suggest that intercontinental virus exchange is slow and limited (17), while a detailed analysis of the differences between H7 hemagglutinin (HA) segments circulating in Europe and China showed that the H7 hemagglutinin segments shared a recent common ancestor and limited sequence divergence on a background of multiple reassortant virus genotypes between 1999 and 2005 (7).Avian influenza A viruses of the Oceania region (Australia, New Zealand, and southwest Pacific) have been far less well studied (3). Australia and New Zealand are at the southern extremity of a number of major bird migration pathways. Waders in the Charadriidae family migrate to south and southeastern Australia and New Zealand from their summer breeding grounds in Arctic regions of Siberia and Alaska, where they freely mix with the same or other species which migrate into the shared breeding grounds of Eurasia and the Americas (30). Pelagic seabirds of the Procellariformes order breed on and around Australian and New Zealand coasts during the southern hemisphere summer and migrate to maritime regions of the northern Pacific associated with Japan, Russia, and Alaska. Some move as far as the west coasts of North and South America (28). Unlike North and South America and Europe, where regular migrations of ducks, geese, swans, etc., are established, the members of the Anatidae family (ducks, etc.) in Australia and New Zealand are mainly endemic residents (30). However, within Australia, ducks undertake long-distance movements in response to water availability. Movements of waterfowl from northern Australia to nearby areas of Southeast Asia are believed to occur but are limited, as suggested by Wallace''s Line (19). Generally, these waterfowl movements have not been well studied (30). The risks to Australian poultry production systems by movement of H5N1 via migratory shorebirds and nomadic wildfowl have been assessed to be low using risk-based analysis techniques (9, 10).Regular and extensive surveillance sampling of migratory birds has been undertaken in North America and northern Europe (17, 18). The findings have shed significant insights into the ecology of the viruses and their hosts (8, 17). In contrast, surveillance sampling of wild birds in Asia and Oceania has been spasmodic and sparse, until the recent emergence of H5N1 highly pathogenic avian influenza virus as a poultry and human disease threat. Spasmodic and small-scale outbreaks of highly pathogenic avian influenza virus have occurred in Australian poultry production flocks located in the southeastern region of the continent. These poultry production areas are concentrated close to large human population centers (26, 33, 34). Each of the Australian outbreaks has been rapidly controlled by slaughter of infected flocks. All have been caused by avian influenza viruses of the H7 subtype, which appear to have entered production poultry from water birds, possibly wild ducks, via contaminated water supplies used on the poultry farms. Disease has occurred on five occasions: 1976 (H7N7), 1985 (H7N7), 1992 (H7N3), 1994 (H7N3), and 1997 (H7N4) (13, 14, 26, 27, 31, 34). National on-farm biosecurity measures have been focused on reducing the likelihood of future outbreaks. The availability of avian influenza virus isolates from poultry and wild birds associated with these outbreaks, along with a small number of subtype H7 avian influenza viruses isolated from wild ducks during recent national surveillance programs in Australia and New Zealand, provided the opportunity to explore the relationships of Australian and New Zealand subtype H7 avian influenza virus isolates with viruses circulating elsewhere in the world.     

H5N1亚型禽流感病毒基因重排后毒力的变化

[目的]为了探讨高致病性禽流感病毒对水禽致病性差异的分子致病机理.[方法]我们对从野鸭分离到的H5N1亚型禽流感病毒的生物学特性进行鉴定,其中A/mallard/Huadong/Y/2003(Y)是对麻鸭无致病性病毒,而 A/mallard/Huadong/S/2005(S)是对麻鸭高致病性病毒.利用反向遗传技术构建一系列单个和多个基因组合替换基因重排病毒,并验证重排病毒在麻鸭上的致病力.[结果]研究表明,PB2, PB1, PA(3P), HA单基因以及3P基因组合替换的使S病毒对麻鸭的毒力完全致弱,但相应的基因替换后仅使Y病毒对麻鸭的毒力略有上升.两病毒的其它基因对毒力影响较小.[结论]H5N1亚型禽流感病毒对麻鸭的致病力受多基因调控,且这种调控作用在不同病毒骨架上的影响不一致,强毒受影响程度远比弱毒的大.     

The Survey of H5N1 Flu Virus in Wild Birds in 14 Provinces of China from 2004 to 2007

Background

The highly pathogenic H5N1 avian influenza emerged in the year 1996 in Asia, and has spread to Europe and Africa recently. At present, effective monitoring and data analysis of H5N1 are not sufficient in Chinese mainland.

Methodology/Principal Findings

During the period from April of 2004 to August of 2007, we collected 14,472 wild bird samples covering 56 species of 10 orders in 14 provinces of China and monitored the prevalence of flu virus based on RT-PCR specific for H5N1 subtype. The 149 positive samples involved six orders. Anseriformes had the highest prevalence while Passeriformes had the lowest prevalence (2.70% versus 0.36%). Among the 24 positive species, mallard (Anas platyrhynchos) had the highest prevalence (4.37%). A difference of prevalence was found among 14 provinces. Qinghai had a higher prevalence than the other 13 provinces combined (3.88% versus 0.43%). The prevalence in three species in Qinghai province (Pintail (Anas acuta), Mallard (Anas platyrhynchos) and Tufted Duck (Aythya fuligula)) were obviously higher than those in other 13 provinces. The results of sequence analysis indicated that the 17 strains isolated from wild birds were distributed in five clades (2.3.1, 2.2, 2.5, 6, and 7), which suggested that genetic diversity existed among H5N1 viruses isolated from wild birds. The five isolates from Qinghai came from one clade (2.2) and had a short evolutionary distance with the isolates obtained from Qinghai in the year 2005.

Conclusions/Significance

We have measured the prevalence of H5N1 virus in 56 species of wild birds in 14 provinces of China. Continuous monitoring in the field should be carried out to know whether H5N1 virus can be maintained by wild birds.     

Prevalence and subtypes of Influenza A Viruses in Wild Waterfowl in Norway 2006-2007

The prevalence of influenza A virus infection, and the distribution of different subtypes of the virus, were studied in 1529 ducks and 1213 gulls shot during ordinary hunting from August to December in two consecutive years, 2006 and 2007, in Norway. The study was based on molecular screening of cloacal and tracheal swabs, using a pan-influenza A RT-PCR. Samples found to be positive for influenza A virus were screened for the H5 subtype, using a H5 specific RT-PCR, and, if negative, further subtyped by a RT-PCR for the 3'-part of the hemagglutinin (HA) gene, encompassing almost the entire HA2, and the full-length of the neuraminidase (NA) gene, followed by sequencing and characterization. The highest prevalence (12.8%) of infection was found in dabbling ducks (Eurasian Wigeon, Common Teal and Mallard). Diving ducks (Common Goldeneye, Common Merganser, Red-breasted Merganser, Common Scoter, Common Eider and Tufted Duck) showed a lower prevalence (4.1%). In gulls (Common Gull, Herring Gull, Black-headed Gull, Lesser Black-headed Gull, Great Black-backed Gull and Kittiwake) the prevalence of influenza A virus was 6.1%. The infection prevalence peaked during October for ducks, and October/November for gulls. From the 16 hemagglutinin subtypes known to infect wild birds, 13 were detected in this study. Low pathogenic H5 was found in 17 dabbling ducks and one gull.     

H7亚型禽流感病毒与禽类和人类的关系

2013年在中国首次发生了H7N9亚型流感病毒感染人事件,已经证实H7N9型禽流感是一种新型禽流感,是全球首次发现感染人类的新亚型流感病毒,以往这种病毒只在野生鸟类存在和传播。H7N9型禽流感病毒属于H7亚型中的一种,全球感染人的H7亚型病毒主要分为两大支系,即北美支系和欧亚支系,感染人的流感亚型也主要集中在H7N7,H7N3,H7N2等亚型上。为了清晰的了解H7亚型病毒的来龙去脉,本文重点讨论了A亚型流感病毒的宿主分布、H7亚型病毒感染禽类和人类的历史、H7亚型病毒的生物学特性以及未来研究展望。     

Identification of a new genotype of Torque Teno Mini virus

Background

Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens.

Findings

Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only.

Conclusion

We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.     

Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014–2015

A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015. Based on the genetic and phylogenetic analyses, the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA, a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin. The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain (A/Alberta/01/2014). Further analysis showed that similar viruses have been circulating in wild birds in China, Russia, Dubai (Western Asia), Bulgaria and Romania (Europe), as well as domestic poultry in some regions of Africa. The affected areas include the Central Asian, East Asian-Australasian, West Asian-East African, and Black Sea/Mediterranean flyways. These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds, thus posing a serious threat to wild birds and potentially humans.

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Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.     

A Live Attenuated H7N7 Candidate Vaccine Virus Induces Neutralizing Antibody That Confers Protection from Challenge in Mice,Ferrets, and Monkeys

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of highly pathogenic (HP) A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs). Intranasal (i.n.) administration of a single dose of the H7N7 NL/03 ca vaccine virus fully protected mice from lethal challenge with homologous and heterologous H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03 ca vaccine induced neutralizing antibodies in serum and provided complete protection from pulmonary replication of homologous and heterologous wild-type H7 challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine elicited an antibody response in one of three AGMs that was completely protected from pulmonary replication of the homologous wild-type H7 challenge virus. The contribution of CD8⁺ and/or CD4⁺ T cells to the vaccine-induced protection of mice was evaluated by T-cell depletion; T lymphocytes were not essential for the vaccine-induced protection from lethal challenge with H7 wt viruses. Additionally, passively transferred neutralizing antibody induced by the H7N7 NL/03 ca virus protected mice from lethality following challenge with H7 wt viruses. The safety, immunogenicity, and efficacy of the H7N7 NL/03 ca vaccine virus in mice, ferrets, and AGMs support the evaluation of this vaccine virus in phase I clinical trials.Highly pathogenic avian influenza (HPAI) is a disease of poultry that is caused by H5 or H7 avian influenza viruses and is associated with up to 100% mortality (2). Influenza A H7 subtype viruses from both Eurasian and North American lineages have resulted in more than 100 cases of human infection since 2002 in the Netherlands, Italy, Canada, the United Kingdom, and the United States. These cases include outbreaks of HPAI H7N7 virus in the Netherlands in 2003 that resulted in more than 80 cases of human infection and one fatality; HPAI H7N3 virus in British Columbia, Canada, in 2004 that resulted in two cases of conjunctivitis; a cluster of human infections of low-pathogenicity avian influenza (LPAI) H7N2 virus in the United Kingdom in 2007 that resulted in several cases of influenza-like illness and conjunctivitis; and a single case of respiratory infection in New York in 2003 (3-6, 17, 27).Due to an unprecedented geographic spread of H5 subtype viruses since 2003 and the continued occurrence of sporadic cases of H5N1 infections in humans, much emphasis has been placed on the pandemic threat posed by H5 subtype viruses. However, H7 subtype viruses also have significant pandemic potential. Humans are immunologically naïve to the H7 avian influenza viruses (16), and LPAI H7 subtype viruses circulating in domestic poultry and wild birds in Eurasia and North America have the potential to evolve and acquire an HP phenotype either by accumulating mutations or by recombination at the hemagglutinin (HA) cleavage site resulting in a highly cleavable HA that is a virulence motif in poultry (30, 33, 34). Recent work also suggests that contemporary North American lineage H7 subtype viruses, isolated in 2002 to 2003, are partially adapted to recognize α2-6-linked sialic acids, which are the receptors preferred by human influenza viruses and are preferentially found in the human upper respiratory tract (7). Moreover, coinfection and genetic reassortment of RNA genomes between H7 avian influenza viruses and human influenza viruses, including the seasonal H1N1 and H3N2 and pandemic H1N1 viruses, could result in the generation of reassortant viruses with the capacity to efficiently transmit among people and result in a pandemic. Domesticated birds may serve as important intermediate hosts for the transmission of wild-bird influenza viruses to humans, as may pigs, as evidenced by human infections with swine-origin 2009 pandemic H1N1 influenza virus throughout the world.Vaccination is the most effective method for the prevention of influenza. However, technical limitations result in delays in the rapid generation and availability of a strain-specific vaccine against an emerging pandemic virus. The emergence of antigenically distinct virus clades poses a substantial challenge for the design of vaccines against H5N1 viruses because of the possible need for clade-specific vaccines (1). Similar challenges are present for the generation of H7 subtype vaccine candidates, because antigenically distinct H7 subtype viruses, including North American lineage H7N2 and H7N3 and Eurasian lineage H7N7 and H7N3 viruses, have caused human disease. The successful control of H7 influenza virus in poultry has been achieved by stamping out and by vaccination of poultry (9). Vaccines for human use against both lineages of H7 influenza virus are under development, and candidate vaccines have been evaluated in preclinical and clinical studies (14, 23, 29, 42).We have previously analyzed the antigenic relatedness among H7 viruses from Eurasian and North American lineages using postinfection mouse and ferret sera (22). Among 10 H7 viruses tested, A/Netherlands/219/03 (H7N7) virus induced the most broadly cross-neutralizing antibodies (Abs) (22). Based on the phylogenetic relationships and its ability to induce broadly cross-neutralizing antibodies in mice and ferrets, we selected the A/Netherlands/219/03 (NL/03) (H7N7) virus from the Eurasian lineage for vaccine development. We used reverse genetics to generate a live attenuated cold-adapted (ca) H7N7 candidate vaccine virus bearing a modified HA, a wild-type (wt) neuraminidase (NA) gene from the NL/03 wt virus, and the six internal protein gene segments from the cold-adapted (ca) influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (AA ca) (H2N2). The immunogenicity and protective efficacy against challenge with HP and LP H7 viruses from the Eurasian and North American lineages of the reassortant H7N7 NL03/AA ca vaccine virus were evaluated in mice, ferrets, and African green monkeys (AGMs).     

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.     

Selection of H5N1 Influenza Virus PB2 during Replication in Humans

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.Given the continued circulation of highly pathogenic H5N1 avian influenza viruses and their sporadic transmission to humans, the threat of a pandemic persists. However, for H5N1 influenza viruses to be efficiently transmitted among humans, amino acid substitutions in the avian viral proteins may be necessary.Two positions in the PB2 protein affect the growth of influenza viruses in mammalian cells (3, 11, 18): the amino acid at position 627 (PB2-627), which in most human influenza viruses is lysine (PB2-627Lys) and most avian viruses is glutamic acid (PB2-627Glu), and the amino acid at position 701. PB2-627Lys is associated with the efficient replication (16) and high virulence (5) of H5N1 viruses in mice. Moreover, an H7N7 avian virus isolated from a fatal human case of pneumonia possessed PB2-627Lys, whereas isolates from a nonfatal human case of conjunctivitis and from chickens during the same outbreak possessed PB2-627Glu (2).The amino acid at position 701 in PB2 is important for the high pathogenicity of H5N1 viruses in mice (11). Most avian influenza viruses possess aspartic acid at this position (PB2-701Asp); however, A/duck/Guangxi/35/2001 (H5N1), which is highly virulent in mice (11), possesses asparagine at this position (PB2-701Asn). PB2-701Asn is also found in equine (4) and swine (15) viruses, as well as some H5N1 human isolates (7, 9). Thus, both amino acids appear to be markers for the adaptation of H5N1 viruses in humans (1, 3, 17).Massin et al. (13) reported that the amino acid at PB2-627 affects viral RNA replication in cultured cells at low temperatures. Recently, we demonstrated that viruses, including those of the H5N1 subtype, with PB2-627Lys (human type) grow better at low temperatures in cultured cells than those with PB2-627Glu (avian type) (6). This association between the PB2 amino acid and temperature-dependent growth correlates with the body temperatures of hosts; the human upper respiratory tract is at a lower temperature (around 33°C) than the lower respiratory tract (around 37°C) and the avian intestine, where avian influenza viruses usually replicate (around 41°C). The ability to replicate at low temperatures may be crucial for viral spread among humans via sneezing and coughing by being able to grow in the upper respiratory organs. Therefore, the Glu-to-Lys mutation in PB2-627 is an important step for H5N1 viruses to develop pandemic potential.However, there is no direct evidence that the substitutions of PB2-627Glu with PB2-627Lys and PB2-701Asp with PB2-701Asn occur during the replication of H5N1 avian influenza viruses in human respiratory organs. Therefore, here, we directly analyzed the nucleotide sequences of viral genes from several original specimens collected from patients infected with H5N1 viruses.     

The role of wild birds in the spread of influenza viruses

Eggs deposited by different migrating wild bird species in pond farm areas in Hungary were examined for yolk antibodies to different variants of human A/H3N2 influenza virus. Antibodies to Victoria/75 and Texas/77 occurred in 17.9 and 32.0% of gull eggs, and 5.6 and 16.4% of common tern eggs, respectively, while antibodies to A/H1N1/77 occurred in roughly similar proportions (10.2 and 13.4%) in the eggs of both species. Infection of the gull and tern populations of given areas by human and avian influenza A viruses differed greatly in two consecutive hatching periods. While in 1978 7.6 and 1.1% of the gull and tern eggs, respectively, contained antibodies to the avian subtype Havl, no such antibodies were found in 1977. Subtype A/H3N2/Texas/77 virus was isolated from adult gulls and 1-3 weeks old gull chicks, and subtype H1N1 virus from mallard ducks. Three months before the onset of the Texas/77 epidemic, 95% of SPF chickens, and 71-81% of chickens hatched 3 months after termination of the A/H1N1/77 epidemic, had had HI, VN and SRH antibodies to the Texas/77 strain and A/H1N1/77 strains, respectively.     

A 27-Amino-Acid Deletion in the Neuraminidase Stalk Supports Replication of an Avian H2N2 Influenza A Virus in the Respiratory Tract of Chickens

The events and mechanisms that lead to interspecies transmission of, and host adaptation to, influenza A virus are unknown; however, both surface and internal proteins have been implicated. Our previous report highlighted the role that Japanese quail play as an intermediate host, expanding the host range of a mallard H2N2 virus, A/mallard/Potsdam/178-4/83 (H2N2), through viral adaptation. This quail-adapted virus supported transmission in quail and increased its host range to replicate and be transmitted efficiently in chickens. Here we report that of the six amino acid changes in the quail-adapted virus, a single change in the hemagglutinin (HA) was crucial for transmission in quail, while the changes in the polymerase genes favored replication at lower temperatures than those for the wild-type mallard virus. Reverse genetic analysis indicated that all adaptive mutations were necessary for transmission in chickens, further implicating quail in extending this virus to terrestrial poultry. Adaptation of the quail-adapted virus in chickens resulted in the alteration of viral tropism from intestinal shedding to shedding and transmission via the respiratory tract. Sequence analysis indicated that this chicken-adapted virus maintained all quail-adaptive mutations, as well as an additional change in the HA and, most notably, a 27-amino-acid deletion in the stalk region of neuraminidase (NA), a genotypic marker of influenza virus adaptation to chickens. This stalk deletion was shown to be responsible for the change in virus tropism from the intestine to the respiratory tract.Of the 16 known hemagglutinin (HA) subtypes, only 3 (H1, H2, and H3) have established stable lineages in humans. The H2N2 virus caused a pandemic in 1957 and circulated in the human population until reassortment of the H2N2 virus with an avian H3 virus resulted in the H3N2 pandemic of 1968 (36). Since then, H2N2 viruses have been absent from the human population; however, the H2 subtype has been repeatedly isolated in wild-bird surveillance, and its HA has been found to be antigenically similar to the H2 pandemic virus HA (23, 25, 36). An H2 influenza virus containing human-like receptor specificity was recently isolated as an H2N3 avian-swine reassortant. This virus caused disease and was transmitted in swine and ferrets (24), indicating that this subtype continues to circulate and mutate and can cross the species barrier to mammals. The repeat introduction of a novel H1N1 pandemic this past year (12, 37) highlights the need to understand the mechanisms of introduction, adaptation, and transmission of avian H2N2 influenza viruses in terrestrial birds and potentially mammalian species.Our previous study built on reports that Japanese quail (Coturnix coturnix) play an important role as an intermediate host in the adaptation of avian influenza viruses to land-based birds (38). Japanese quail are typically more susceptible to aquatic influenza viruses than other terrestrial poultry. These viruses establish infection in the respiratory tract, and shedding occurs via aerosol (2, 19, 26, 34, 38, 43). Quail have been implicated in the transmission of avian influenza viruses, such as H5N1 and H9N2 viruses, which have crossed the species barrier to infect humans (9, 14, 15, 22, 28). The susceptibility of quail to multiple subtypes and their role in interspecies transmission led to their removal from live-bird markets in Hong Kong in 2000; however, they continue to be an integral part of live-bird markets throughout the world. Their role as potential intermediate hosts requires further study to identify important molecular markers in the adaptation via quail of avian viruses to other terrestrial poultry, and possibly to humans.The molecular determinants of the host range and pathogenesis of influenza A viruses have been linked to multiple regions of the 11 genes, most notably those encoding the viral surface glycoproteins (HA and neuraminidase [NA]) and the polymerase proteins (PB2, PB1, PA, and NP). However, a comprehensive map of the various determinants remains incomplete, and the molecular mechanisms involved are unclear. In our previous report, we demonstrated that through the use of quail as an intermediate host, a mallard H2N2 influenza virus, A/mallard/Potsdam/178-4/83 (mall/178), which in its wild-type (wt) form was unable to be transmitted in quail or to establish an efficient infection in chickens, was able, in its adapted form (qa-mall/178), not only to be transmitted to sentinel quail but also to replicate to efficient levels in the chicken intestinal tract and to be transmitted to sentinel cagemates via the fecal-oral route. This adaptation was the result of six serial passages of lung homogenates in quail that led to six amino acid changes in four genes (38). Here we present data confirming the role that Japanese quail play in the transmission of this mall/178 H2N2 virus in land-based birds. Reverse genetics studies confirmed that the amino acid changes produced during the adaptation in quail were necessary for the infection of chickens with this virus and for its transmission in chickens. Further adaptation of the qa-mall/178 H2N2 virus in chickens, aimed at establishing replication in the respiratory tract, resulted in a deletion in the stalk region of the NA, which supported replication in the chicken trachea and lung. This 27-amino-acid deletion in the stalk region of the N2 NA is characteristic of the adaptation of aquatic influenza viruses to domestic poultry, particularly chickens (3, 5, 29). Our work indicates that through the use of quail as an intermediate host, this mallard H2N2 virus is able to further adapt within an additional terrestrial poultry species, potentially improving its chances of expanding its host range further.