Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses

The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian-human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs.     

NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.     

Reassortment between seasonal H1N1 and pandemic (H1N1) 2009 influenza viruses is restricted by limited compatibility among polymerase subunits

Reassortment is important for influenza virus evolution and the generation of novel viruses with pandemic potential; however, the factors influencing reassortment are still poorly understood. Here, using reverse genetics and a replicon assay, we demonstrated that a mixed polymerase complex containing a pandemic (H1N1) 2009 influenza virus PB2 on a seasonal H1N1 virus background has reduced polymerase activity, leading to impaired virus viability. Adaptation of viruses containing the mixed polymerase complex resulted in compensatory mutations in PB1. Taken together, our results identify the cooperation between PB2 and PB1 as an important restricting factor for reassortment of influenza viruses.     

Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers

The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.     

Compatibility among polymerase subunit proteins is a restricting factor in reassortment between equine H7N7 and human H3N2 influenza viruses

Reassortment is an important driving force for influenza virus evolution, and a better understanding of the factors that affect this process could improve our ability to respond to future influenza pandemics and epidemics. To identify factors that restrict the generation of reassortant viruses, we cotransfected human embryonic kidney cells with plasmids for the synthesis of viral RNAs of both A/equine/Prague/1/56 (Prague; H7N7) and A/Yokohama/2017/03 (Yokohama; H3N2) viruses together with the supporting protein expression plasmids. Of the possible 256 genotypes, we identified 29 genotypes in 120 randomly plaque-picked reassortants examined. Analyses of these reassortants suggested that the formation of functional ribonucleoprotein (RNP) complexes was a restricting factor, a finding that correlated with the activities of RNP complexes composed of different combinations of the proteins from the two viruses, as measured in a minigenome assay. For at least one nonfunctional RNP complex (i.e., Prague PB2, Prague PB1, Yokohama PA, and Prague NP), the lack of activity was due to the inability of the three polymerase subunit proteins to form a heterotrimer. Adaptation of viruses possessing a gene encoding a chimera of the PA proteins of the two viruses and the remaining genes from Prague virus resulted in compensatory mutations in the PB2 and/or PA protein. These results indicate substantial incompatibility among the gene products of the two test viruses, a critical role for the RNP complex in the generation of reassortant viruses, and a functional interaction of PB2 and PA.     

Identification of reassortant pandemic H1N1 influenza virus in Korean pigs

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.     

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.     

The RNA Polymerase PB2 Subunit of Influenza A/HongKong/156/1997 (H5N1) Restrict the Replication of Reassortant Ribonucleoprotein Complexes

Background

Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood.

Methodology/Principal Findings

Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated.

Conclusions/Significance

Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes.     

Ubiquitous reassortments in influenza A viruses

The influenza A virus is a negative-stranded RNA virus composed of eight segmented RNA molecules, including polymerases (PB2, PB1, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (MP), and nonstructure gene (NS). The influenza A viruses are notorious for rapid mutations, frequent reassortments, and possible recombinations. Among these evolutionary events, reassortments refer to exchanges of discrete RNA segments between co-infected influenza viruses, and they have facilitated the generation of pandemic and epidemic strains. Thus, identification of reassortments will be critical for pandemic and epidemic prevention and control. This paper presents a reassortment identification method based on distance measurement using complete composition vector (CCV) and segment clustering using a minimum spanning tree (MST) algorithm. By applying this method, we identified 34 potential reassortment clusters among 2,641 PB2 segments of influenza A viruses. Among the 83 serotypes tested, at least 56 (67.46%) exchanged their fragments with another serotype of influenza A viruses. These identified reassortments involve 1,957 H2N1 and 1,968 H3N2 influenza pandemic strains as well as H5N1 avian influenza virus isolates, which have generated the potential for a future pandemic threat. More frequent reassortments were found to occur in wild birds, especially migratory birds. This MST clustering program is written in Java and will be available upon request.     

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing

Background

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance.

Methodology/Principal Findings

A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses.

Conclusions/Significance

In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.     

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.     

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.     

PB2 residue 158 is a pathogenic determinant of pandemic H1N1 and H5 influenza a viruses in mice

Influenza A viruses are human and animal pathogens that cause morbidity and mortality, which range from mild to severe. The 2009 H1N1 pandemic was caused by the emergence of a reassortant H1N1 subtype (H1N1pdm) influenza A virus containing gene segments that originally circulated in human, avian, and swine virus reservoirs. The molecular determinants of replication and pathogenesis of H1N1pdm viruses in humans and other mammals are poorly understood. Therefore, we set out to elucidate viral determinants critical to the pathogenesis of this novel reassortant using a mouse model. We found that a glutamate-to-glycine substitution at residue 158 of the PB2 gene (PB2-E158G) increased the morbidity and mortality of the parental H1N1pdm virus. Results from mini-genome replication assays in human cells and virus titration in mouse tissues demonstrated that PB2-E158G is a pathogenic determinant, because it significantly increases viral replication rates. The virus load in PB2-E158G-infected mouse lungs was 1,300-fold higher than that of the wild-type virus. Our data also show that PB2-E158G had a much stronger influence on the RNA replication and pathogenesis of H1N1pdm viruses than PB2-E627K, which is a known pathogenic determinant. Remarkably, PB2-E158G substitutions also altered the pathotypes of two avian H5 viruses in mice, indicating that this residue impacts genetically divergent influenza A viruses and suggesting that this region of PB2 could be a new antiviral target. Collectively, the data presented in this study demonstrate that PB2-E158G is a novel pathogenic determinant of influenza A viruses in the mouse model. We speculate that PB2-E158G may be important in the adaptation of avian PB2 genes to other mammals, and BLAST sequence analysis identified a naturally occurring human H1N1pdm isolate that has this substitution. Therefore, future surveillance efforts should include scrutiny of this region of PB2 because of its potential impact on pathogenesis.     

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.     

Reassortment events among swine influenza A viruses in China: implications for the origin of the 2009 influenza pandemic

That pigs may play a pivotal role in the emergence of pandemic influenza was indicated by the recent H1N1/2009 human pandemic, likely caused by a reassortant between viruses of the American triple-reassortant (TR) and Eurasian avian-like (EA) swine influenza lineages. As China has the largest human and pig populations in the world and is the only place where both TR and EA viruses have been reported to cocirculate, it is potentially the source of the H1N1/2009 pandemic virus. To examine this, the genome sequences of 405 swine influenza viruses from China were analyzed. Thirty-six TR and EA reassortant viruses were identified before and after the occurrence of the pandemic. Several of these TR-EA reassortant viruses had genotypes with most segments having the same lineage origin as the segments of the H1N1/2009 pandemic virus. However, these viruses were generated from independent reassortment events throughout our survey period and were not associated with the current pandemic. One TR-EA reassortant, which is least similar to the pandemic virus, has persisted since 2007, while all the other variants appear to be transient. Despite frequent reassortment events between TR and EA lineage viruses in China, evidence for the genesis of the 2009 pandemic virus in pigs in this region is still absent.     

Molecular basis of efficient replication and pathogenicity of H9N2 avian influenza viruses in mice

H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.     

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.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.