Evaluation of Commercial Diagnostic Assays for the Specific Detection of Avian Influenza A (H7N9) Virus RNA Using a Quality-Control Panel and Clinical Specimens in China

A novel avian influenza A H7N9-subtype virus emerged in China in 2013 and threatened global public health. Commercial kits that specifically detect avian influenza A (H7N9) virus RNA are urgently required to prepare for the emergence and potential pandemic of this novel influenza virus. The safety and effectiveness of three commercial molecular diagnostic assays were evaluated using a quality-control panel and clinical specimens collected from over 90 patients with confirmed avian influenza A (H7N9) virus infections. The analytical performance evaluation showed that diverse influenza H7N9 viruses can be detected with high within- and between-lot reproducibility and without cross-reactivity to other influenza viruses (H1N1 pdm09, seasonal H1N1, H3N2, H5N1 and influenza B). The detection limit of all the commercial assays was 2.83 Log₁₀ copies/μl [0.7 Log₁₀TCID₅₀/mL of avian influenza A (H7N9) virus strain A/Zhejiang/DTID-ZJU01/2013], which is comparable to the method recommended by the World Health Organization (WHO). In addition, using a WHO-Chinese National Influenza Center (CNIC) method as a reference for clinical evaluation, positive agreement of more than 98% was determined for all of the commercial kits, while negative agreement of more than 99% was observed. In conclusion, our findings provide comprehensive evidence for the high performance of three commercial diagnostic assays and suggest the application of these assays as rapid and effective diagnostic tools for avian influenza A (H7N9) virus in the routine clinical practice of medical laboratories.     

Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans?

A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.     

Characterization of Influenza A (H7N9) Viruses Isolated from Human Cases Imported into Taiwan

A novel avian influenza A (H7N9) virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave) was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave). The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.     

Establishment of influenza A virus (H6N1) in minor poultry species in southern China

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of those viruses had a common source. Continuing surveillance in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants in 1999; however, the further development of H6N1 viruses has not been investigated since 2001. Here we report influenza virus surveillance data collected in southern China from 2000 to 2005 that show that H6N1 viruses have become established and endemic in minor poultry species and replicate mainly in the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had W312-like hemagglutinin and neuraminidase genes. However, reassortment of internal genes between different subtype virus lineages, including H5N1, H9N2, and other avian viruses, generated multiple novel H6N1 genotypes in different types of poultry. These novel H6N1/N2 viruses are double, triple, or even quadruple reassortants. Reassortment between a W312-like H6N1 virus and an A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest that W312-like viruses may not be the precursors of HK/97 virus but reassortants from an HK/97-like virus and another unidentified H6 subtype virus. These results provide further evidence of the pivotal role of the live poultry market system of southern China in generating increased genetic diversity in influenza viruses in this region.     

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A (H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses (compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.     

Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they 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 reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

Influenza Virus Surveillance in Pakistan during 2008-2011

Background

There is little information about influenza among the Pakistani population. In order to assess the trends of Influenza-like-Illness (ILI) and to monitor the predominant circulating strains of influenza viruses, a country-wide lab-based surveillance system for ILI and Severe Acute Respiratory Illness (SARI) with weekly sampling and reporting was established in 2008. This system was necessary for early detection of emerging novel influenza subtypes and timely response for influenza prevention and control.

Methods

Five sentinel sites at tertiary care hospitals across Pakistan collected epidemiological data and respiratory samples from Influenza-like illness (ILI) and severe acute respiratory illness (SARI) cases from January 2008 to December 2011. Samples were typed and sub-typed by Real-Time RT-PCR assay.

Results

A total of 6258 specimens were analyzed; influenza virus was detected in 1489 (24%) samples, including 1066 (72%) Influenza type A and 423 (28%) influenza type B viruses. Amongst influenza A viruses, 25 (2%) were seasonal A/H1N1, 169 (16%) were A/H3N2 and 872 (82 %) were A(H1N1)pdm09. Influenza B virus circulation was detected throughout the year along with few cases of seasonal A/H1N1 virus during late winter and spring. Influenza A/H3N2 virus circulation was mainly observed during summer months (August-October).

Conclusions

The findings of this study emphasize the need for continuous and comprehensive influenza surveillance. Prospective data from multiple years is needed to predict seasonal trends for vaccine development and to further fortify pandemic preparedness.     

Novel Avian Influenza A (H7N9) Virus Induces Impaired Interferon Responses in Human Dendritic Cells

In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced “cytokine storm” seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

Background

Avian influenza A (H7N9) virus has emerged recently and continues to cause severe disease with a high mortality rate in humans prompting the development of candidate vaccine viruses. Live attenuated influenza vaccines (LAIV) are 6:2 reassortant viruses containing the HA and NA gene segments from wild type influenza viruses to induce protective immune responses and the six internal genes from Master Donor Viruses (MDV) to provide temperature sensitive, cold-adapted and attenuated phenotypes.

Methodology/Principal Findings

LAIV candidate A/Anhui/1/2013(H7N9)-CDC-LV7A (abbreviated as CDC-LV7A), based on the Russian MDV, A/Leningrad/134/17/57 (H2N2), was generated by classical reassortment in eggs and retained MDV temperature-sensitive and cold-adapted phenotypes. CDC-LV7A had two amino acid substitutions N123D and N149D (H7 numbering) in HA and one substitution T10I in NA. To evaluate the role of these mutations on the replication capacity of the reassortants in eggs, the recombinant viruses A(H7N9)RG-LV1 and A(H7N9)RG-LV2 were generated by reverse genetics. These changes did not alter virus antigenicity as ferret antiserum to CDC-LV7A vaccine candidate inhibited hemagglutination by homologous A(H7N9) virus efficiently. Safety studies in ferrets confirmed that CDC-LV7A was attenuated compared to wild-type A/Anhui/1/2013. In addition, the genetic stability of this vaccine candidate was examined in eggs and ferrets by monitoring sequence changes acquired during virus replication in the two host models. No changes in the viral genome were detected after five passages in eggs. However, after ten passages additional mutations were detected in the HA gene. The vaccine candidate was shown to be stable in the ferret model; post-vaccination sequence data analysis showed no changes in viruses collected in nasal washes present at day 5 or day 7.

Conclusions/Significance

Our data indicate that the A/Anhui/1/2013(H7N9)-CDC-LV7A reassortant virus is a safe and genetically stable candidate vaccine virus that is now available for distribution by WHO to vaccine manufacturers.     

A Cross-Sectional Study of Avian Influenza in One District of Guangzhou, 2013

Since Feb, 2013, more than 100 human beings had been infected with novel H7N9 avian influenza virus. As of May 2013, several H7N9 viruses had been found in retail live bird markets (LBMs) in Guangdong province of southern China where several human cases were confirmed later. However, the real avian influenza virus infection status especially H7N9 in Guangzhou remains unclear. Therefore, a cross-sectional study of avian influenza in commercial poultry farms, the wholesale LBM and retail LBMs in one district of Guangzhou was conducted from October to November, 2013. A total of 1505 cloacal and environmental samples from 52 commercial poultry farms, 1 wholesale LBM and 18 retail LBMs were collected and detected using real-time RT-PCR for type A, H7, H7N9 and H9 subtype avian influenza virus, respectively. Of all the flocks randomly sampled, 6 farms, 12 vendors of the wholesale LBM and 18 retail LBMs were type A avian influenza virus positive with 0, 3 and 11 positive for H9, respectively. The pooled prevalence and individual prevalence of type A avian influenza virus were 33.9% and 7.9% which for H9 subtype was 7.6% and 1.6%, respectively. None was H7 and H7N9 subtype virus positive. Different prevalence and prevalence ratio were found in different poultry species with partridges having the highest prevalence for both type A and H9 subtype avian influenza virus. Our results suggest that LBM may have a higher risk for sustaining and transmission of avian influenza virus than commercial poultry farms. The present study also indicates that different species may play different roles in the evolution and transmission of avian influenza virus. Therefore, risk-based surveillance and management measures should be conducted in future in this area.     

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.     

Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses

Background

Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches.

Results

The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference.

Conclusion

The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

     

Continuing evolution of H9N2 influenza viruses in Southeastern China

H9N2 influenza viruses are panzootic in domestic poultry in Eurasia and since 1999 have caused transient infections in humans and pigs. To investigate the zoonotic potential of H9N2 viruses, we studied the evolution of the viruses in live-poultry markets in Hong Kong in 2003. H9N2 was the most prevalent influenza virus subtype in the live-poultry markets between 2001 and 2003. Antigenic and phylogenetic analysis of hemagglutinin (HA) showed that all of the 19 isolates found except one belonged to the lineage represented by A/Duck/Hong Kong/Y280/97 (H9N2). The exception was A/Guinea fowl/NT184/03 (H9N2), whose HA is most closely related to that of the human isolate A/Guangzhou/333/99 (H9N2), a virus belonging to the A/Chicken/Beijing/1/94-like (H9N2) lineage. At least six different genotypes were recognized. The majority of the viruses had nonstructural (and HA) genes derived from the A/Duck/Hong Kong/Y280/97-like virus lineage but had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses isolated in 2001. Viruses of all six genotypes of H9N2 found were able to replicate in chickens and mice without adaptation. The infected chickens showed no signs of disease, but representatives of two viral genotypes were lethal to mice. Three genotypes of virus replicated in the respiratory tracts of swine, which shed virus for at least 5 days. These results show an increasing genetic and biologic diversity of H9N2 viruses in Hong Kong and support their potential role as pandemic influenza agents.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA₁ allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.     

Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.

An avian influenza A virus, A/Mallard/NY/6750/78(H2N2), was restricted in in replication in the respiratory tract of squirrel monkeys. Avian-human influenza A reassortant viruses possessing the six RNA segments coding for nonsurface proteins (i.e., internal genes) of this avian virus were as restricted in replication in squirrel monkeys as their avian influenza parent. These findings indicated that restriction of replication of the avian influenza virus is a function of one or more of its internal genes. For an investigation of which of the avian influenza genes was responsible for restricted replication in the respiratory tract of primates, reassortant viruses were produced that contained human influenza virus surface antigens from the A/Udorn/72(H3N2) virus and one or more of the internal genes derived from the avian influenza virus parent. Avian-human reassortant influenza A viruses containing only the nucleoprotein or matrix protein RNA segment from the avian influenza virus parent were as restricted in their growth as an avian-human influenza reassortant virus containing each of the six avian influenza internal genes. In addition, an avian-human influenza reassortant virus possessing only the avian RNA 1 and nonstructural genes (which by themselves do not specify restricted replication) manifested a significant reduction of virus replication in squirrel monkey tracheas. Thus, the avian nucleoprotein and matrix genes appear to play a major role in the host range restriction exhibited by the A/Mallard/78 virus and its reassortants, but the combination of RNA 1 and nonstructural genes also contributes to restriction of replication.     

Phylogeography of Avian influenza A H9N2 in China

Background

During the past two decades, avian influenza A H9N2 viruses have spread geographically and ecologically in China. Other than its current role in causing outbreaks in poultry and sporadic human infections by direct transmission, H9N2 virus could also serve as an progenitor for novel human avian influenza viruses including H5N1, H7N9 and H10N8. Hence, H9N2 virus is becoming a notable threat to public health. However, despite multiple lineages and genotypes that were detected by previous studies, the migration dynamics of the H9N2 virus in China is unclear. Increasing such knowledge would help us better prevent and control H9N2 as well as other future potentially threatening viruses from spreading across China. The objectives of this study were to determine the source, migration patterns, and the demography history of avian influenza A H9N2 virus that circulated in China.

Results

Using Bayesian phylogeography framework, we showed that the H9N2 virus in mainland China may have originated from the Hong Kong Special Administrative Region (SAR). Southern China, most likely the Guangdong province acts as the primary epicentre for multiple H9N2 strains spreading across the whole country, and eastern China, most likely the Jiangsu province, acts as an important secondary source to seed outbreaks. Our demography inference suggests that during the long-term migration process, H9N2 evolved into multiple diverse lineages and then experienced a selective sweep, which reduced its genetic diversity. Importantly, such a selective sweep may pose a greater threat to public health because novel strains confer higher fitness advantages than strains being replaced and could generate new viruses through reassortment.

Conclusion

Our analyses indicate that migratory birds, poultry trade and transportation have all contributed to the spreading of the H9N2 virus in China. The ongoing migration and evolution of H9N2, which poses a constant threat to the human population, highlights the need for a more comprehensive surveillance of wild birds and for the enhancement of biosafety for China’s poultry industry.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1110) contains supplementary material, which is available to authorized users.