Influenza Viruses In Poultry Research Articles

Endemicity of H9N2 and H5N1 avian influenza viruses in poultry in China poses a serious threat to poultry industry and public health

The H9N2 and H5N1 avian influenza viruses (AIVs) have been circulating in poultry in China and become endemic since 1998 and 2004, respectively. Currently, they are prevalent in poultry throughout China. This endemicity makes them actively involved in the emergence of the novel lineages of other subtypes of influenza viruses, such as the well-known viruses of the highly pathogenic avian influenza (HPAI) H5N2 and the 2013 novel H7N7, H7N9 and H10N8 subtypes, thereby threatening both the poultry industry and public health. Here, we will review briefly the prevalence and evolution, pathogenicity, transmission, and disease control of these two subtypes and also discuss the possibility of emergence of potentially virulent and highly transmissible AIVs to humans.

Characterization of clade 2.3.4.4 highly pathogenic H5 avian influenza viruses in ducks and chickens

Worldwide dissemination of reassortant variants of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses has posed a great threat to the poultry industry. Here, we systematically characterized the H5N2, H5N6 and H5N8 influenza viruses in poultry and compared them with those of previous clade 2.3.4 H5N1 virus. All the three H5 subtype reassortants caused systematic infection in ducks, and exhibited efficient direct transmission in ducks. All of them were highly pathogenic in chickens; however, the H5 reassortants have reduced virulence compared to the parental H5N1 virus. Antigenicity analysis revealed that the current vaccines that are widely used in China may fail to confer protection against the H5 reassortants.

Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adoption in mice.

The worldwide circulation of H9N2 avian influenza virus in poultry, the greater than 2.3 % positive rate for anti-H9 antibodies in poultry-exposed workers, and several reports of human infection indicate that H9N2 virus is a potential threat to human health. Here, we found three mutations that conferred high virulence to H9N2 virus in mice after four passages. The PB2-E627K substitution rapidly appeared at the second passage and played a decisive role in virulence. Polymerase complexes possessing PB2-E627K displayed 16.1-fold higher viral polymerase activity when compared to the wild-type virus, which may account for enhanced virulence of this virus. The other two substitutions (HA-N313D and HA-N496S) enhanced binding to both α2,3-linked and α2,6-linked sialic acid receptors; however, the HA-N313D and N496S substitutions alone decreased the virulence of mouse-adapted virus. Furthermore, this mouse-adapted virus was still not transmissible among guinea pigs by direct contact (0/3 pairs). Our findings show that adaption in mice enhanced the viral polymerase activity and receptor-binding ability, which resulted in a virulent phenotype in mice but not a transmissible phenotype in guinea pigs, indicating that host factors play an important role in adaptive evolution of influenza in new hosts.

Novel reassortant highly pathogenic H5N6 avian influenza viruses in poultry in China

We characterized two novel highly pathogenic H5N6 influenza viruses isolated from Chinese poultry in 2013. Genomic analysis showed that both isolates were reassortants, and derived their genes from H5 and H6 subtype viruses found in poultry in China. The virulence of the two isolates was examined in chickens and mice, and both isolates were found to be highly pathogenic in chickens and only moderately virulent for mice. Our results show that continued circulation of these viruses could endanger both avian species and humans.

Emergence and evolution of H10 subtype influenza viruses in poultry in China.

The cases of human infections with H10N8 viruses identified in late 2013 and early 2014 in Jiangxi, China, have raised concerns over the origin, prevalence, and development of these viruses in this region. Our long-term influenza surveillance of poultry and migratory birds in southern China in the past 12 years showed that H10 influenza viruses have been introduced from migratory to domestic ducks over several winter seasons at sentinel duck farms at Poyang Lake, where domestic ducks share their water body with overwintering migratory birds. H10 viruses were never detected in terrestrial poultry in our survey areas until August 2013, when they were identified at live-poultry markets in Jiangxi. Since then, we have isolated 124 H10N8 or H10N6 viruses from chickens at local markets, revealing an ongoing outbreak. Phylogenetic analysis of H10 and related viruses showed that the chicken H10N8 viruses were generated through multiple reassortments between H10 and N8 viruses from domestic ducks and the enzootic chicken H9N2 viruses. These chicken reassortant viruses were highly similar to the human isolate, indicating that market chickens were the source of human infection. Recently, the H10 viruses further reassorted, apparently with H5N6 viruses, and generated an H10N6 variant. The emergence and prevalence of H10 viruses in chickens and the occurrence of human infections provide direct evidence of the threat from the current influenza ecosystem in China. After the outbreak of avian-origin H7N9 influenza viruses in China, fatal human infections with a novel H10N8 virus were reported. Utilizing data from 12 years of influenza surveillance in southern China, we showed that H10 viruses were regularly introduced by migratory ducks to domestic ducks on Poyang Lake, a major aggregative site of migratory birds in Asia. The H10 viruses were maintained and amplified in domestic ducks and then transmitted to chickens and reassorted with enzootic H9N2 viruses, leading to an outbreak and human infections at live-poultry markets. The emergence of the H10N8 virus, following a pathway similar to that of the recent H7N9 virus, highlights the role of domestic ducks and the current influenza ecosystem in China that facilitates influenza viruses moving from their reservoir hosts through the live-poultry system to cause severe consequences for public health.

Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City, China

Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response.

Occurrence and reassortment of avian influenza A (H7N9) viruses derived from coinfected birds in China.

Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.

Surveillance of avian influenza virus in poultry and wild birds in Ethiopian Rift Valley Lakes

Surveillance of avian influenza virus in poultry and wild birds in Ethiopian Rift Valley Lakes

Role of receptor binding specificity in influenza A virus transmission and pathogenesis

The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glycans present on the apical surface of host cells can function as receptors for the influenza virus hemagglutinin (HA) protein. Avian and human influenza viruses typically have a different sialic acid (SA)-binding preference and only few amino acid changes in the HA protein can cause a switch from avian to human receptor specificity. Recent experiments using glycan arrays, virus histochemistry, animal models, and structural analyses of HA have added a wealth of knowledge on receptor binding specificity. Here, we review recent data on the interaction between influenza virus HA and SA receptors of the host, and the impact on virus host range, pathogenesis, and transmission. Remaining challenges and future research priorities are also discussed.

An experimental model to analyse the risk of introduction of a duck-originated H5 low-pathogenic avian influenza virus in poultry through close contact and contaminative transmission

Aquatic wild birds are often carriers of low-pathogenic avian influenza viruses (LPAIVs). If H5 and H7 LPAIVs are transmitted to poultry and have the opportunity to circulate, a highly pathogenic AIV may arise. Contact with aquatic wild birds is one of the most important ways in which these LPAIVs can be introduced into poultry flocks. In this study, the transmissibility of a duck-originated H5 LPAIV between ducks and chickens was analysed in a series of animal experiments, using different transmission routes. Results indicate that the outcome of virus intake by chickens exposed to infectious ducks depends on the way the virus is presented. Faecally contaminated drinking water proved to be the most efficient route by which the virus can be transmitted to chickens. The results from this study also suggest that some duck-originated H5 LPAIVs may be introduced to poultry but do not have the potential to become established in poultry populations.

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

Surveillance for Influenza Viruses in Poultry and Swine, West Africa, 2006–2008

To determine the extent of animal influenza virus circulation in Côte d'Ivoire, Benin, and Togo, we initiated systematic year-round active influenza surveillance in backyard birds (predominantly chickens, guinea fowl, and ducks) and pigs. A total of 26,746 swab specimens were screened by using reverse transcription PCR. Animal influenza prevalence was estimated at 0 (95% CIs for each of the 2 study years 0-0.04% to 0-1.48% [birds] and 0-0.28% to 0-5% [pigs]). In addition, 2,276 serum samples from the same populations were negative for influenza-specific antibodies. These data indicate that the environments and host populations previously identified as harboring high levels of influenza virus in Southeast Asia do not do so in these 3 countries. The combination of climate and animal density factors might be responsible for what appears to be the absence of influenza virus in the backyard sector of the 3 countries.

Avian influenza surveillance reveals presence of low pathogenic avian influenza viruses in poultry during 2009-2011 in the West Bengal State, India

IntroductionMore than 70 outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 have been reported in poultry in the western and north-eastern parts of India. Therefore, in view of the recent HPAI H5N1 outbreaks in poultry, active AI surveillance encompassing wild, resident, migratory birds and poultry was undertaken during 2009–2011 in the State of West Bengal.MethodsA total of 5722 samples were collected from West Bengal; 3522 samples (2906 fecal droppings + 616 other environmental samples) were from migratory birds and 2200 samples [1604 tracheal, cloacal swabs, environmental samples, tissue samples + 596 blood (serum)] were from domestic ducks and poultry. All tracheal, cloacal and environmental samples were processed for virus isolation. Virus isolates were detected using hemagglutination assay and identified using hemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR) assays. Sequencing and phylogenetic analysis of partial region of the hemagglutinin and neuraminidase genes was done. Intravenous pathogenicity index assays were performed in chickens to assess pathogenicity of AI virus isolates. Serum samples were tested for detection of antibodies against AI viruses using HI assay.ResultsA total of 57 AI H9N2, 15 AI H4N6 and 15 Newcastle Disease (NDV) viruses were isolated from chickens, from both backyard and wet poultry markets; AI H4N6 viruses were isolated from backyard chickens and domestic ducks. Characterization of AI H9N2 and H4N6 viruses revealed that they were of low pathogenicity. Domestic ducks were positive for antibodies against H5 and H7 viruses while chickens were positive for presence of antibodies against AI H9N2 and NDV.ConclusionsIn the current scenario of HPAI H5N1 outbreaks in West Bengal, this report shows presence of low pathogenic AI H9N2 and H4N6 viruses in chickens and domestic ducks during the period 2009–2011. This is the first report of isolation of H4N6 from India. Antibodies against AI H5 and H7 in ducks highlight the probable role of domestic ducks in the transmission of AI viruses. Human infections of H9N2 have been reported from China and Hong Kong. This necessitates implementation of prevention and control measures to limit the spread of AI viruses.

Low pathogenic H7 subtype avian influenza viruses isolated from domestic ducks in South Korea and the close association with isolates of wild birds

We characterized low pathogenic avian influenza (LPAI) viruses of the H7 subtype that were isolated from domestic ducks and wild birds in South Korea from 2008 to 2011. A total of 20 H7 viruses were collected from live-bird markets (LBMs), duck farms and wild-bird habitats using avian influenza (AI) surveillance and epidemiological approaches. A phylogenetic analysis of the H7 viruses that were isolated from domestic ducks and wild birds demonstrated that they were separated into 12 genotypes (A-D and Wb-1-8, respectively), indicating genetic diversity. These H7 viruses were related to the recently isolated Eurasian LPAI H7 viruses and various influenza viruses that are circulating in Asia, including southern China and South Korea. The same genotype was not found between domestic poultry and wild-bird isolates; however, most of the H7 viruses in poultry (genotypes B and C) were closely related to the H7 virus isolated from a wild bird (genotype Wb-3). Animal-challenge studies revealed that certain H7 AI viruses replicated well only in chickens or ducks depending on the genotype, indicating that the pathogenicity of H7 viruses has the potential to be altered due to multiple reassortments, and these viruses can potentially expand their host range. Our results are evidence of abundant and frequent reassortment between H7 viruses in poultry and wild birds and emphasize the continuing need to monitor the evolutionary genetics of the influenza virus in poultry and wild birds.

Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus.

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.

Neuraminidase Inhibitor Sensitivity and Receptor-Binding Specificity of Cambodian Clade 1 Highly Pathogenic H5N1 Influenza Virus

The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

New Evidence Suggests Southern China as a Common Source of Multiple Clusters of Highly Pathogenic H5N1 Avian Influenza Virus

Highly pathogenic H5N1 avian influenza is considered an avian disease, although there is some evidence of limited human-to-human transmission of the virus. A global effort is underway to control or eradicate the highly pathogenic H5N1 avian influenza virus in poultry and prevent human exposure, both of which may also reduce the risk of pandemic emergence. Hemagglutinin gene sequences from 215 human H5N1 influenza viruses were used to trace the source and dispersal pattern of human H5N1 influenza viruses on a global scale. A mutation network and phylogenetic analyses of the hemagglutinin gene show that human H5N1 influenza viruses can be clearly divided among 4 clusters across geographic space. On the basis of analysis of the N-glycosylation sites at positions 100 and 170 in the hemagglutinin protein, human H5N1 influenza viruses were also divided into 3 types. When we combined these analyses with geographic information system data analyses, we found that Southern China is often a common source of multiple clusters of H5N1 influenza viruses and that each cluster has different dispersal patterns and individual evolutionary features. In summary, the genetic evidence presented here provides clear evidence for multiple clusters of human H5N1 influenza viruses that initially originated in Southern China.

Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007-2009

To investigate the prevalence and evolution of the H5 subtype highly pathogenic avian influenza (HPAI) viruses circulating in poultry in China during 2007-2009, five molecular epidemiological surveys were carried out. A total of 21, 591 swab samples were collected, and from them 55 H5 HPAI viruses were isolated. None of the 55 viruses carried any known mutations, which can render the virus binding to human SAa2,6Gal receptors. The surveys indicated that live-bird markets, backyard flocks and slaughtering sites were at greater risk of being infected with the viruses during winter, and Clades 2.3.2, 2.3.4 and 7 of the viruses co-circulated in poultry in China during 2007-2009. Viruses within Clades 2.3.2 and 7 have become genetically distinguishable from the viruses isolated before 2007 and antigenically distinguishable from the vaccine strains used in China. Viruses within Clade 2.3.2 have been circulating widely in China and caused a new wave of cross-continental spreading from Asia to Europe.

Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry.

In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.

OP3-2 Spread and evolution of avian influenza virus in poultry and wild-birds in Africa

OP3-2 Spread and evolution of avian influenza virus in poultry and wild-birds in Africa