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.     

Characterization of a novel highly pathogenic H5N2 avian influenza virus isolated from a duck in eastern China

During surveillance for avian influenza viruses (AIVs) in live-poultry markets (LPMs) in eastern China in 2013, one H5N2 AIV was isolated from a duck. Phylogenetic analysis showed that the hemagglutinin of this strain belongs to clade 2.3.4 and received its genes from H5, H3 and H6 AIVs of poultry in China. The virulence of this strain was examined in chickens and mice, and it was found to be highly pathogenic in chickens but demonstrated moderate pathogenicity in mice. These results suggest that active surveillance of AIVs in LPMs should be used in an early warning system for avian influenza outbreaks.     

Novel reassortant H10N7 avian influenza viruses isolated from chickens in Eastern China

BackgroundSince 2004, the H10N7 subtype avian influenza virus (AIV) has caused sporadic human infections with variable clinical symptoms world-wide. However, there is limited information pertaining to the molecular characteristics of H10N7 AIVs in China.ObjectiveTo more fully characterize the genetic relationships between three novel H10N7 strains isolated from chickens in Eastern China and the strains isolated from birds throughout Asia, and to determine the pathogenicity of the H10N7 isolates in vivo.Study designAll eight gene segments from the Chinese H10N7 strains were sequenced and compared with AIV strains available in GenBank. The virulence of the three isolates was determined in chickens and mice.ResultsThree H10N7 subtype avian influenza viruses were isolated from chickens in live poultry markets in Eastern China in 2014: (1) A/chicken/Zhejiang/2C66/2014(H10N7) (ZJ-2C66), (2) A/chicken/Zhejiang/2CP2/2014(H10N7) (ZJ-2CP2), and (3) A/chicken/Zhejiang/2CP8/2014(H10N7) (ZJ-2CP8). Phylogenetic analysis indicated that the viruses contained genetic material from H10, H2, H7, and H3 AIV strains that were circulating at the same time. The reassortant H10N7 viruses were found to be minimally pathogenic in chickens and moderately pathogenic in mice. The viruses were able to replicate in mice without prior adaptation.ConclusionThese results suggest that H10N7 surveillance in poultry should be used as an early warning system for avian influenza outbreaks. The novel strains identified here may post a threat to human health in the future if they continue to circulate.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia

The reported transmission of avian H9N2 influenza viruses to humans and the isolation of these viruses from Hong Kong poultry markets lend urgency to studies of their ecology and pathogenicity. We found that H9N2 viruses from North America differ from those of Asia. The North American viruses, which infect primarily domestic turkeys, replicated poorly in inoculated chickens. Phylogenetic analysis of the hemagglutinin and nucleoprotein genes indicated that the Asian H9N2 influenza viruses could be divided into three sublineages. Initial biological characterization of at least one virus from each lineage was done in animals. Early isolates of one lineage (A/Chicken/Beijing/1/94, H9N2) caused as high as 80% mortality rates in inoculated chickens, whereas all other strains were nonpathogenic. Sequence analysis showed that some isolates, including the pathogenic isolate, had one additional basic amino acid (A-R/K-S-S-R-) at the hemagglutinin cleavage site. Later isolates of the same lineage (A/Chicken/Hong Kong/G9/97, H9N2) that contains the PB1 and PB2 genes similar to Hong Kong/97 H5N1 viruses replicated in chickens, ducks, mice, and pigs but were pathogenic only in mice. A/Quail/Hong Kong/G1/97 (H9N2), from a second lineage that possesses the replicative complex similar to Hong Kong/97 H5N1 virus, replicated in chickens and ducks without producing disease signs, was pathogenic in mice, and spread to the brain without adaptation. Examples of the third Asian H9N2 sublineage (A/Chicken/Korea/323/96, Duck/Hong Kong/Y439/97) replicated in chickens, ducks, and mice without producing disease signs. The available evidence supports the notion of differences in pathogenicity of H9N2 viruses in the different lineages and suggests that viruses possessing genome segments similar to 1997 H5N1-like viruses are potentially pathogenic in mammals.     

Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

H5 and H7 avian influenza viruses can become highly pathogenic in chickens after interspecies transmission. These viruses have transmitted directly to humans from birds in Eurasia and Africa (H5N1), the Netherlands (H7N7), and Canada (H7N3). Here we report antigenic, sequence, and phylogenetic analyses of H7N3 viruses isolated from chickens in Pakistan from 1995 to 2002. We compared the pathogenic and zoonotic potential of the Pakistani viruses in avian and mammalian hosts. In chickens, all of the isolates showed high pathogenicity with poor transmissibility to contact birds. Viral shedding from the trachea and cloaca was equivalent, but cloacal shedding occurred longer; dissemination of virus into the tissues was widespread. In contrast, the viruses replicated poorly in 6-week-old mallard ducks. In mammalian hosts, of the two Pakistani H7N3/02 viruses that caused weight loss, one also caused 40% mortality in mice without prior adaptation, and preliminary experiments in ferrets showed significant virus multiplication in the lungs, intestine, and conjunctiva. We conclude that the H7N3/02 isolates from Pakistan show limited antigenic drift and have evolved slowly during their 8-year circulation in chickens; however, these viruses have the potential to infect mammals.     

A comparison of the pathogenicity of avian and swine H5N1 influenza viruses in Indonesia

Highly pathogenic avian influenza H5N1 viruses are circulating in many countries. We recently discovered that these viruses have been transmitted to pigs on multiple occasions in Indonesia. To investigate whether avian H5N1 influenza viruses adapted to mammals through their introduction into pigs, we examined the growth of avian and swine isolates in cell culture and compared their pathogenicity in mice. We found that swine isolates were less virulent to mice than avian isolates, suggesting that the viruses became attenuated during their replication in pigs. Continuous surveillance of H5N1 viruses among pigs is clearly warranted.     

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.     

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.     

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.     

Zoonotic potential of highly pathogenic avian H7N3 influenza viruses from Pakistan

H5 and H7 avian influenza viruses can become highly pathogenic in chickens after interspecies transmission. These viruses have transmitted directly to humans from birds in Eurasia and Africa (H5N1), the Netherlands (H7N7), and Canada (H7N3). Here we report antigenic, sequence, and phylogenetic analyses of H7N3 viruses isolated from chickens in Pakistan from 1995 to 2002. We compared the pathogenic and zoonotic potential of the Pakistani viruses in avian and mammalian hosts. In chickens, all of the isolates showed high pathogenicity with poor transmissibility to contact birds. Viral shedding from the trachea and cloaca was equivalent, but cloacal shedding occurred longer; dissemination of virus into the tissues was widespread. In contrast, the viruses replicated poorly in 6-week-old mallard ducks. In mammalian hosts, of the two Pakistani H7N3/02 viruses that caused weight loss, one also caused 40% mortality in mice without prior adaptation, and preliminary experiments in ferrets showed significant virus multiplication in the lungs, intestine, and conjunctiva. We conclude that the H7N3/02 isolates from Pakistan show limited antigenic drift and have evolved slowly during their 8-year circulation in chickens; however, these viruses have the potential to infect mammals.     

Reassortment and modification of hemagglutinin cleavage motif of avian/WSN influenza viruses generated by reverse genetics that correlate with attenuation

Avian influenza associated with H9N2 and H5N1 subtypes of avian influenza viruses (AIVs) has raised great concerns in China. To study this problem, reverse genetics has been employed. Three reassortants, rgH9N2, rgH5N1 and rgH5N2, were prepared and compared. Their hemagglutinin (HA) and neuraminidase (NA) genes originated from Chinese AIV isolates of H9N2 or H5N1 subtype, while the rest of their genes were derived from A/WSN/33(H1N1) virus (WSN). In the H5 HA reassortants, the multibasic cleavage site was converted to a monobasic one. The results demonstrated that the reassortants did not produce CPE on MDCK cells in the absence of trypsin, showed egg-adaptation phenotype and stability of HA and NA during consecutive egg passages, and were not lethal to chickens and mice. However, the rgH5N1 reassortant exhibited a residual virulence in terms of lethality to chick embryos and pathogenesis in chickens. It can be concluded that (i) the genetic modification of H5 HA attenuated the H5 reassortants, (ii) the presence of internal WSN proteins contributed to the attenuated properties of the reassortants independently on H5 HA, and (iii) also the overall genome composition contributed to virulence differences. This report provides further contribution of reverse genetics to the knowledge of virulence of influenza viruses.     

Genetic characterization of H5N1 influenza viruses isolated from chickens in Indonesia in 2010

Since 2003, highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry in Indonesia every year, producing the highest number of human victims worldwide. However, little is known about the H5N1 influenza viruses that have been circulating there in recent years. We therefore conducted surveillance studies and isolated eight H5N1 viruses from chickens. Phylogenic analysis of their hemagglutinin and neuraminidase genes revealed that all eight viruses belonged to clade 2.1.3. However, on the basis of nucleotide differences, these viruses could be divided into two groups. Other viruses genetically closely related to these two groups of viruses were all Indonesian isolates, suggesting that these new isolates have been evolving within Indonesia. Among these viruses, two distinct viruses circulated in the Kalimantan islands during the same season in 2010. Our data reveal the continued evolution of H5N1 viruses in Indonesia.     

Highly pathogenic avian influenza viruses with low virulence for chickens in in vivo tests.

Four avian influenza viruses have been recognized that have genetic coding for highly pathogenic avian influenza viruses, but do not show virulence for chickens. The two different mechanisms that prevent this potential being expressed have been determined for A/chicken/Pennsylvania/1/83 (H5N2) and A/goose/Guandong/2/96 (H5N1), but neither of these applies to A/turkey/England/87-92BFC/91 (H5N1) or A/chicken/Texas/298313/04 (H5N2).     

Characterization of An Avian Influenza Virus of Subtype H7N2 Isolated from Chickens in Northern China

An H7N2 avian influenza virus was isolated from chickens during routine surveillance in northern China in 2002. To understand the origin of this virus, we completely sequenced its genome. The PB1, PA, HA, and M genes of this virus were highly homologous with those of the wild bird virus A/Africa starling/Eng-Q/983/79 (H7N1). The NP and NS genes were closely related to those of two other wild bird viruses isolated 30 years ago. The closest relatives of the PB2 and NA genes of the virus were those of the A/swine/Germany/2/81 (H1NI) and A/Leningrad/134/57 (H2N2), respectively. Animal inoculation tests showed that the virus cannot replicate efficiently in chickens. However, after intranasal inoculation, the virus induced 20% weight loss and replicated well in the lungs of mice. The virus was also recovered from the hearts and brains of the mice. These results suggest that the influenza virus isolated in chickens in northern China in 2002 originated in wild birds and may pose a threat for both avian species and mammalian hosts.     

Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice

A/duck/Guangxi/53/2002 (DKGX/53) and A/duck/Fujian/01/2002 (DKFJ/01) are H5N1 avian influenza viruses that are lethal in chickens. In mice, however, DKFJ/01 is highly pathogenic, whereas DKGX/53 displays low pathogenicity. In this study, we used reverse genetics to demonstrate that two amino acid residues at positions 30 and 215 of the M1 protein of these two viruses are important determinants for pathogenicity in mice. We thus firstly prove the M1 protein contributes to the virulence of H5N1 viruses in mice, and the amino acid residues shown to attenuate the virulence could be targeted in influenza virus candidates for live vaccine development.     

Distribution of avian influenza H5N1 viral RNA in tissues of AI-vaccinated and unvaccinated contact chickens after experimental infection

Avian influenza due to highly pathogenic avian influenza (HPAIV) H5N1 virus is not a food-borne illness but a serious panzootic disease with the potential to be pandemic. In this study, broiler chickens were vaccinated with commercial H5N1 or H5N2 inactivated vaccines prior to being challenged with an HPAIV H5N1 (clade 2.2.1 classic) virus. Challenged and non-challenged vaccinated chickens were kept together, and unvaccinated chickens served as contact groups. Post-challenge samples from skin and edible internal organs were collected from dead and sacrificed (after a 14-day observation period) birds and tested using qRT-PCR for virus detection and quantification. H5N1 vaccine protected chickens against morbidity, mortality and transmission. Virus RNA was not detected in the meat or edible organs of chickens vaccinated with H5N1 vaccine. Conversely, H5N2 vaccine did not confer clinical protection, and a significant virus load was detected in the meat and internal organs. Phylogenetic analysis showed that the H5N1 virus vaccine and challenge virus strains are closely related. The results of the present study strongly suggest a need for proper selection of vaccines and their routine evaluation against newly emergent field viruses. These actions will help to reduce human exposure to HPAIV H5N1 virus from both infected live birds and slaughtered poultry. In addition, rigorous preventive measures should be put in place in order to minimize the public-health risks of avian influenza at the human-animal interface.     

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

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