Antigenic, genetic, and pathogenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from dead whooper swans (Cygnus cygnus) found in northern Japan in 2008

In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.     

Characterization of a highly pathogenic H5N1 avian influenza virus isolated from ducks in Eastern China in 2011

This study describes the characterization of seven H5N1 avian influenza viruses from domestic ducks in Eastern China in 2011. Phylogenetic analysis showed these viruses were closely related to an H5N1 virus circulating in wild birds in Hong Kong. Some characteristics of these viruses were similar to those of an H5N1 strain that circulated in China and Vietnam (2003-2004). The virulence of three isolates was examined in chickens and mice, and they were found to be highly pathogenic in chickens but showed low pathogenicity in mice. These results suggest that continued H5N1 surveillance in poultry should be used as an early warning system for avian influenza outbreaks.     

Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.     

Genetic variety of influenza A virus in the populations of wild birds in the south of Western Siberia

Influenza A virus variants belonging to H3 and H4 subtypes were isolated from wild ducks inhabiting in the south of Western Siberia. Phylogenetic analysis of hemagglutinin (HA) gene of these viruses has revealed that H3 isolates are closely related to those isolated from the bird inhabiting in West Europe (A/Teal/Germany/wv01r/01, A/Duck/Ukraine/1/63) and China (A/Aquatic bird/Hong Kong/399/99); and those isolated from the birds inhabiting in Germany (A/Garganey/Germany/wv157k/01, A/Teal/Germany/wv153k/01). Thus, closely related influenza A virus variants circulate in the populations of the wild birds inhabiting in greatly spaced regions of Eurasia.     

Characterisation of a highly pathogenic H5N1 clade 2.3.2 influenza virus isolated from swans in Shanghai, China

In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007-2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.     

Characterization of the H5N1 influenza virus isolated during an outbreak among wild birds in Russia (Tuva Republic) in 2010

A study of the basic biological properties of H5N1 subtype strain isolated during an outbreak among wild birds in Russia in 2010 is presented. The study was carried out using conventional methods according to the WHO recommendations. H5N1 influenza virus isolated in Siberia belonged to clade 2.3.2 of the hemagglutinin gene, and phylogenetic analysis was performed. The antigenic characteristics and the basic genetic markers of biological properties were studied. It was shown that all strains were highly pathogenic for chickens and white mice. Thus, it was shown that, in Russia, the 2010 H5N1 virus phylogenetically closely related to Asian variants caused epizootic among wild birds. The potential danger of this variant of the virus for humans was confirmed by different methods. We discussed the possibility of formulating the natural focus of H5N1 influenza.     

Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: Role of passerine birds in maintenance or transmission?

In 1985 a fowl plague-like disease occurred in chickens in Lockwood, Victoria, Australia and caused high mortality. An H7N7 influenza virus was isolated from the chickens (A/Chicken/Victoria/1/85); additionally, an antigenically similar virus was isolated from starlings (A/Starling/Victoria/5156/85) and serological evidence of H7N7 virus infection was found in sparrows. Antigenic analysis with monoclonal antibodies to H7, oligonucleotide mapping of total vRNA, and sequence analysis of the HA genes established that the chicken and starling influenza viruses were closely related and probably came from the same source. There was high nucleotide sequence homology (95.3%) between the HA genes of A/Chick/Vic/85 and a fowl plague-like virus isolated from chickens in Victoria 9 years earlier [A/Fowl/Vic/76 (H7N7)]. The sequence homologies indicated that the A/Chick/Vic/85 and A/Fowl/Vic/76 were derived from a common recent ancestor, while another recent H7N7 virus, Seal/Mass/1/80 originated from a different evolutionary lineage. Experimental infection of chickens and starlings with A/Chick/Vic/1/85 (H7N7) was associated with high mortality (100%), transmission to contact birds of the same species, and virus in all organs. In sparrows one-third of the birds died after infection and virus was isolated from most organs; transmission to contact sparrows did not occur. In contrast, the H7N7 virus replicated in ducks and spread to contact ducks but caused no mortality. These studies establish that the host species plays a role in determining the virulence of avian influenza viruses, and provide the first evidence for transmission of virulent influenza viruses between domestic poultry and passerine birds. They support the hypothesis that potentially virulent H7N7 influenza viruses could be maintained in ducks where they cause no apparent disease and may sometimes spread to other wild birds and domestic poultry.     

Evidence of persistence and multiple genetic modifications of H7N7 low-pathogenic avian influenza virus in wild mallards in Poland provided by phylogenetic studies

Genetic characterization of the whole genome of four avian influenza H7N7 viruses isolated in three successive winter seasons (2007 to 2009) from wild mallards in three cities in Poland was performed. All of the tested strains were of low pathogenicity and no molecular marker associated with an increased adaptation to poultry, mammals or resistance to antiviral drugs was found. The major outcome of the phylogenetic studies was that the isolate A/mallard/Poland/446/09 (detected in December 2009) shared a recent common ancestor with A/mallard/Poland/41/09 (isolated in February 2009) in relation to HA and PB1 genes, with A/mallard/Poland/16/09 (found in January 2009) regarding NA and NS genes, and with A/mallard/Poland/01/08 (recovered in December 2007) as regards the NS gene. Interestingly, A/mallard/Poland/16/09 and A/mallard/Poland/446/09 were isolated at the same sampling site almost exactly 1 year apart, which points to resident population of mallards (and other resident waterfowl) as responsible for the perpetuation of avian influenza virus (AIV) in the given area between successive winters. On the other hand, the ornithological data discussed in detail in the paper strongly suggest that the virus transmission between close sites but located in different urban areas is most probably achieved by migratory birds, a fact additionally supported by a close relatedness between different gene segments of Polish H7N7 and AIV detected in wild birds in Europe. A high heterogeneity of the gene pool found in the study is indicative of frequent reassortment events. Additionally, two H7N7 isolates were shown to possess selected genes closely related to AIV detected in domestic poultry in Italy and the Czech Republic. The present study corroborates the importance of active surveillance in wild birds as a valuable tool for early warning of avian influenza in poultry.     

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.     

Amino acid substitutions in low pathogenic avian influenza virus strains isolated from wild birds in Korea

Wild birds are natural hosts and reservoirs for influenza A viruses. However, many species, such as many waterfowl, are asymptomatic when infected and so facilitate the generation of viral genetic diversity. Mutations of key genes affect the replicability, pathogenicity, transmissibility, and antiviral resistance of influenza A viruses. In this study, we isolated avian influenza (AI) viruses from wild bird fecal samples and analyzed changes in amino acids over time and geographic region to monitor the biological change of the AI virus. Between 2014 and 2016, we collected 38,921 fresh fecal samples from major wild bird habitats located throughout Korea and isolated 123 AI viruses. We subsequently selected 22 amino acid sites to analyze for changes. These sites included ten sites associated with replication, ten sites associated with pathogenicity, three sites associated with transmission, and seven sites associated with antiviral resistance. We found substitution rates of 71.7% at the C38Y amino acid site within the polymerase basic protein 1 (PB1) gene, 66.7% at the D222G site within the hemagglutinin (HA) 1 gene, and 75.6% at the A184 site within the nucleoprotein (NP) gene. Alterations of the PB1, HA1, and NP genes are closely associated with increased pathogenicity in chickens and mammals. The remaining sites of interest exhibited few modifications. In this study, we confirmed that AI viruses circulating among wild birds in Korea consistently exhibit modifications at amino acid sites linked with replication and pathogenicity.     

Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia

A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.     

Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy

Since the "bird flu" incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002-2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived "in toto" from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.     

Epidemiology of influenza in Lower Saxony during the period 1968–1978 with particular emphasis on subtypes A(H3N2) and A(H1N1) in winter 1977–78

The influenza surveillance in Lower Saxony is based mainly on laboratory investigations, and especially those which involve the isolation of influenza viruses throughout the year. These investigations have provided information on the circulation of influenza viruses and of the antigenic drift during the Hong Kong period which resulted in many different variants. Since the advent of the Hong Kong virus subtype, a high excess mortality was observed only in the winter of 1969-70. In most of the other years there was a low excess mortality which coincided with the circulation of the influenza A viruses in the population. During the last winter 1978, influenza A viruses were not isolated before February. In February and March, the two distinct subtypes H3N2 and H1N1 circulated simultaneously. The influenza A(H1N1) virus attacked only children and young adults whereas the patients infected with influenza A(H3N2) virus ranged over all age groups.     

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.     

Transmission and reassortment of avian influenza viruses at the Asian-North American interface

Twenty avian influenza viruses were isolated from seven wild migratory bird species sampled at St. Lawrence Island, Alaska. We tested predictions based on previous phylogenetic analyses of avian influenza viruses that support spatially dependent trans-hemispheric gene flow and frequent interspecies transmission at a location situated at the Asian-North American interface. Through the application of phylogenetic and genotypic approaches, our data support functional dilution by distance of trans-hemispheric reassortants and interspecific virus transmission. Our study confirms infection of divergent avian taxa with nearly identical avian influenza strains in the wild. Findings also suggest that H16N3 viruses may contain gene segments with unique phylogenetic positions and that further investigation of how host specificity may impact transmission of H13 and H16 viruses is warranted.     

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000–2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005–2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.     

Emerging influenza.

In 1918 the Spanish influenza pandemic, caused by an avian H1N1 virus, resulted in over 50 million deaths worldwide. Several outbreaks of H7 influenza A viruses have resulted in human cases, including one fatal case. Since 1997, the outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype have affected a wide variety of mammals in addition to poultry and wild birds. Here, we give an overview of the current knowledge of the determinants of pathogenicity of these three subtypes of avian influenza A virus in mammals. Common mechanisms for acquisition of virulence and replication of these avian influenza viruses in mammals are becoming apparent. Therefore, monitoring these and additional genetic changes upon zoonotic infections is important. Identification of genetic changes responsible for transmission between mammals will be an important task for the near future.     

An avian influenza A(H11N1) virus from a wild aquatic bird revealing a unique Eurasian-American genetic reassortment

Influenza surveillance in different wild bird populations is critical for understanding the persistence, transmission and evolution of these viruses. Avian influenza (AI) surveillance was undertaken in wild migratory and resident birds during the period 2007–2008, in view of the outbreaks of highly pathogenic AI (HPAI) H5N1 in poultry in India since 2006. In this study, we present the whole genome sequence data along with the genetic and virological characterization of an Influenza A(H11N1) virus isolated from wild aquatic bird for the first time from India. The virus was low pathogenicity and phylogenetic analysis revealed that it was distinct from reported H11N1 viruses. The hemagglutinin (HA) gene showed maximum similarity with A/semipalmatedsandpiper/Delaware/2109/2000 (H11N6) and A/shorebird/Delaware/236/2003(H11N9) while the neuraminidase (NA) gene showed maximum similarity with A/duck/Mongolia/540/2001(H1N1). The virus thus possessed an HA gene of the American lineage. The NA and other six genes were of the Eurasian lineage and showed closer relatedness to non-H11 viruses. Such a genetic reassortment is unique and interesting, though the pathways leading to its emergence and its future persistence in the avian reservoir is yet to be fully established.     

Experimental infection of dogs with H6N1 avian influenza A virus

H6N1 avian influenza A viruses, which have spread across North America, Europe and Asia, have been shown to be infectious not only for birds but also for mammals. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. Replication of H6N1 avian influenza A viruses in dogs may facilitate their adaptation in humans because dogs are often in close contact with humans. However, the susceptibility of dogs to these viruses is unknown. To address this question, we infected beagles intranasally (i.n.) with an H6N1 avian influenza A virus that was isolated from a mallard. Inoculation of this virus into beagles resulted in the virus being detectable in the lung and seroconversion with no clinical signs except for a fever at 1 day post-inoculation (dpi). In addition, the virus was transiently shed from the nose and in the feces of the infected beagles. Our results suggest that dogs can be subclinically infected with H6N1 avian influenza A viruses, which, like H7N9, have low pathogenicity in birds and may serve as an intermediate host to transfer this virus to humans. Certain actions may be taken to prevent the potential transmission of these viruses, including the development of H6N1 avian influenza vaccines for prevention.