Characterization of an avian influenza virus of subtype H4N6 isolated from ducks in the northern China

In 2010, an H4N6 avian influenza virus (AIV) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China. This virus was named A/duck/Shandong/1/2010 (H4N6) (DK/SD/1/2010 hereafter). The gene sequence of the virus was determined, and genetic and evolutionary analyses were conducted by combining related sequences in GenBank. Results indicated that seven genes of DK/SD/1/2010 (PB2, PB1, PA, HA, NP, M, and NS) originate from the Eurasian lineage. Another gene, the NA gene, originated from both the Eurasian and the North American lineages. The amino acid sequence near the cleavage site of DK/SD/1/2010 HA (PKKASR↓GLF) corresponded to the characteristics of AIV of low pathogenicity. Animal inoculation tests showed that the virus cannot replicate in chickens and mice. Therefore, DK/SD/1/2010 may be a recombinant virus formed by influenza virus genes from different sources through complicated restructuring and evolution in ducks that is avirulent to chickens and mice.     

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.     

Genetic analysis of H3 subtype influenza viruses isolated from domestic ducks in northern China during 2004–2005

The broad distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a global threat to both human and veterinary health. In this present study, six H3N8 influenza viruses isolated from domestic ducks during 2004–2005 in northern China were genetically and phylogenetically characterized. Sequence analysis showed that HA, NA, and M genes of all the six H3N8 isolates had a close relationship with those of Equine/Jilin/1/89 (H3N8) virus, which once caused outbreak in equine populations in northern China. The PB2 and PA genes of the viruses possessed the highest similarities with highly pathogenic avian H5N1 influenza viruses currently circulating in this region. These findings emphasize the importance of avian influenza virus surveillance in this region for understanding the genesis and emergency of novel reassortants with pandemic potential.     

Characterization of a new avian-like influenza A virus from horses in China.

In March 1989 a severe outbreak of respiratory disease occurred in horses in the Jilin and Heilongjiang provinces of Northeast China that caused up to 20% mortality in some herds. An influenza virus of the H3N8 subtype was isolated from the infected animals and was antigenically and molecularly distinguishable from the equine 2 (H3N8) viruses currently circulating in the world. The reference strain A/Equine/Jilin/1/89 (H3N8) was most closely related to avian H3N8 influenza viruses. Sequence comparisons of the entire hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and NS genes along with partial sequences of the three polymerase (PB1, PB2, PA) genes suggest that six of the eight gene segments (PA, HA, NP, NA, M, NS) are closely related to avian influenza viruses. Since direct sequence analysis can only provide a crude measure of relationship, phylogenetic analysis was done on the sequence information. Phylogenetic analyses of the entire HA, NP, M, and NS genes and of partial sequences of PB1, PB2, and PA indicated that these genes are of recent avian origin. The NP gene segment is closely related to the gene segment found in the newly described H14 subtype isolated from ducks in the USSR. The A/Equine/Jilin/1/89 (H3N8) influenza virus failed to replicate in ducks, but did replicate and cause disease in mice on initial inoculation and on subsequent passaging caused 100% mortality. In ferrets, the virus caused severe influenza symptoms. A second outbreak of influenza in horses in Northeast China occurred in April 1990 in the Heilongjiang province with 48% morbidity and no mortality. The viruses isolated from this outbreak were antigenically indistinguishable from those in the 1989 outbreak and it is probable that the reduced mortality was due to the immune status of of the horses in the region. No influenza was detected in horses in Northern China in the spring, summer, or fall of 1991 and no influenza has been detected in horses in adjacent areas. Our analysis suggests that this new equine influenza virus in horses in Northeast China is the latest influenza virus in mammals to emerge from the avian gene pool in nature and that it may have spread to horses without reassortment. The appearance of this new equine virus in China emphasizes the potential for whole avian influenza viruses to successfully enter mammalian hosts and serves as a model and a warning for the appearance of new pandemic influenza viruses in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012

To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade 2.3.2.1 and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.     

Genetic characterization of an H5N1 avian influenza virus with neurovirulence in ducks

In this report, an H5N1 avian influenza virus, A/duck/Hubei/hangmei01/2006, which could lead to acute disease including neurovirulence and mortality in ducks, was isolated in brains of domestic ducks in spring of 2006. Molecular characterization of the genes revealed that this virus harbored the common characteristics of a highly pathogenic avian influenza (HPAI). Phylogenetic analyses demonstrated that this virus was a member of the Fujian-like virus sublineage. All eight genes except NA and PB2 had the closest genetic relatives to the human influenza virus A/China/GD01/2006. It might indicate that the virus A/duck/Hubei/hangmei01/2006 originated from southern China, resulting from the wild bird migration or poultry transportation, and indicate that more surveillance upon evolution and transmission of influenza viruses in ducks was urgent. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of avian influenza viruses isolated from domestic ducks in Vietnam in 2009 and 2010

In the surveillance of avian influenza in Vietnam, 26 H9N2, 1 H3N2, 1 H3N8, 7 H4N6, 3 H11N3, and 1 H11N9 viruses were isolated from tracheal and cloacal swab samples of 300 domestic ducks in April 2009, and 1 H9N6 virus from 300 bird samples in March 2010. Out of the 27 H9 virus isolates, the hemagglutinins of 18 strains were genetically classified as belonging to the sublineage G1, and the other nine belonged to the Korean sublineage. Phylogenetic analysis revealed that one of the 27 H9 viruses was a reassortant in which the PB2 gene belonged to the Korean sublineage and the other seven genes belonged to the G1 sublineage. Three representative H9N2 viruses were intranasally inoculated into ducks, chickens, pigs, and mice. On the basis of experimental infection studies, it was found that each of the three viruses readily infected pigs and replicated in their upper respiratory tracts, and they infected chickens with slight replication. Viruses were recovered from the lungs of mice inoculated with two of the three isolates. The present results reveal that H9 avian influenza viruses are prevailing and genetic reassortment occurs among domestic ducks in Vietnam. It is recommended that careful surveillance of swine influenza with H9 viruses should be performed to prepare for pandemic influenza.     

Genetic evolution of swine influenza A (H3N2) viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.     

Genetic Conservation of Hemagglutinin Gene of H9 Influenza Virus in Chicken Population in Mainland China

The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995–2002 were genetically analyzed. All the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage (except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.     

Isolation and characterization of an H9N2 influenza virus isolated in Argentina

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) - herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.     

Isolation of a genotypically unique H5N1 influenza virus from duck meat imported into Japan from China

An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed >97% nucleotide identity with their counterparts from recent H5N1 viruses, but that the remaining two genes (PB2 and NP) were derived from other unknown viruses. This duck meat isolate was highly pathogenic to chickens upon intravenous or intranasal inoculation, replicated well in the lungs of mice and spread to the brain, but was not as pathogenic in mice as H5N1 human isolates (with a dose lethal to 50% of mice (MLD50)=5x10(6) 50% egg infectious doses [EID50]). However, viruses isolated from the brain of mice previously infected with the virus were substantially more pathogenic (MLD50=approximately 10(2) EID50) and possessed some amino acid substitutions relative to the original virus. These results show that poultry products contaminated with influenza viruses of high pathogenic potential to mammals are a threat to public health even in countries where the virus is not enzootic and represent a possible source of influenza outbreaks in poultry.     

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 H5N1 influenza A viruses isolated from domestic green-winged teal

Two avian influenza virus strains, A/domestic green-winged teal/Hunan/67/2005 (H5N1) (D-GWT/67) and A/domestic green-winged teal/Hunan/79/2005 (H5N1) (D-GWT/79), were isolated from healthy domestic green-winged teals (Anas crecca) in Hunan Province, South China. Genomic analysis showed that both isolates were reassortants. The hemagglutinin (HA) genes of the two isolates were closely related to that of an H5N1 strain isolated from tree sparrow (A/tree sparrow/Henan/1/04). The neuraminidase (NA) genes and the internal protein genes of both isolates were closely related to those from A/chicken/Shantou/4231/2003-like (H5N1) viruses, with exception of the matrix (M) gene of D-GWT/79, which was closely related to that of the H7N3 strain A/mallard/Netherlands/12/2000 isolated from wild mallard duck. The virulence of the two isolates was examined in chickens, ducks, and mice. Both strains were found to be highly pathogenic in chickens and ducks, but showed low pathogenicity in mice. These findings contribute to the realization that domestic green-winged teals carrying the H5N1 virus may play an important role in transmitting the virus among birds.     

Genetic characterization of low pathogenic H5N1 and co-circulating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008

As part of a long-term wild bird monitoring programme, five different low pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards (subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating (same location, same time period) viruses were selected for full genome sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus (A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later in December 2008. All genes of these co-circulating viruses shared common ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses. The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A double infection with two different PA segments from H5N1 and from H5N3 could be observed for the H1N1 sample. The observed gene constellations resulted from multiple reassortment events between viruses circulating in wild birds in Eurasia. Several internal gene segments from these 2008 viruses and the N3 sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the growing sequence evidence of the dynamic nature of the avian influenza natural reservoir in Eurasia, and underline the importance of monitoring AIV in wild birds. Genetic information of potential hazard to commercial poultry continues to circulate in this reservoir, including H5 and H7 subtype viruses and genes related to previous AIV outbreaks.     

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.     

Characterization of recent H5 subtype avian influenza viruses from US poultry.

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.     

Avian influenza viruses in Korean live poultry markets and their pathogenic potential

We surveyed live-poultry markets in Korea in 2003 and isolated 9 H9N2, 6 H3N2, and 1 H6N1 influenza viruses. Antigenic and phylogenetic analyses showed that all 9 H9N2 isolates were of A/Chicken/Korea/25232-96006/96-like lineage (which caused disease in chickens in Korea in 1996) but were different from H9N2 viruses of southeastern China. They had at least 4 genotypes and replicated in chickens but not in mice. The H3N2 and H6N1 viruses were new to Korea and were probably reassortants of avian influenza viruses from southeastern China and recent Korean H9N2 viruses. All 8 segments of the H3N2 viruses formed a single phylogenetic cluster with 99.1 to 100% homology. The H3N2 viruses replicated in chickens and mice without preadaptation, but the H6N1 virus did not. Our results show an increasingly diverse pool of avian influenza viruses in Korea that are potential pandemic influenza agents.     

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.     

Genome-sequenee Analysis of the Pathogenic H5N1 Avian Influenza A Virus Isolated in China in 2004

Analysis of the sequences of the genome of the avian influenza A/chicken/Hubei/327/2004 (H5N1) virus, isolated from a poultry farm during the outbreak of avian influenza (AI) in Hubei Province, central China, in the spring of 2004, revealed that the hemagglutinin (HA) gene of the virus was genetically similar to those of the H5 highly pathogenic avian influenza virus (HPAI). Notably, the neuraminidase gene of the virus had a 20-amino acid deletion in the stalk region and a 5-amino acid deletion in the NS gene which belonged to allele B. Furthermore, the internal genes (PB2, PA, NP, M2) of the A/chicken/Hubei/327/2004 virus with the particular amino acid residues were more closely related to H5N1 viruses of 2000–2003 isolated in Hong Kong and the AIV of Thailand and Vietnam in 2004, but less likely to evolve from the viruses of Hong Kong 1997. Finally, our results demonstrated that the influenza A/chicken/Hubei/327/2004 (H5N1) virus was similar to those of the AI viruses isolated from Hong Kong (2000–2003), Vietnam, and Thailand rather than the viruses from the 1997 lineage of Hong Kong and with closest genetic relatives to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus. These data suggest that the influenza A/chicken/Hubei/327/2004 (H5N1) virus which circulated in central China derived its internal gene from a virus similar to the influenza A/Chicken/Hong Kong/61.9/02 (H5N1) virus.