Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Adaptive mutations in viral polymerase, which is composed of PB1, PB2, and PA, of avian influenza viruses are major genetic determinants of the host range. In this study, to elucidate the molecular mechanism of mammalian adaptation of avian viral polymerase, we performed cell-based vRNP reconstitution assays and biochemical analyses using purified recombinant viral polymerase complexes. We found that avian viral polymerase from A/duck/Pennsylvania/10,218/84 (DkPen) enhances the viral polymerase activity in mammalian cells by replacing the PA or PB2 gene with that from human influenza virus A/WSN/33 (WSN). Chimeric constructs between DkPen PA and WSN PA showed that the N-terminal endonuclease domain of WSN PA was essential for the mammalian adaptation of DkPen viral polymerase. We also found that the cap-snatching activity of purified DkPen viral polymerase was more than 5 times weaker than that of WSN in vitro in a PB2 Glu627-dependent manner. However, the cap-snatching activity of DkPen viral polymerase was hardly increased by replacing DkPen PA to WSN PA. These results suggest that the activity of viral genome replication may be enhanced in the DkPen reassortant containing WSN PA.     

Targeting of the influenza A virus polymerase PB1-PB2 interface indicates strain-specific assembly differences

Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication. The conserved protein-protein interaction sites have been suggested as potential drug targets. To characterize the PB1-PB2 interface, we fused the PB1-binding domain of PB2 to green fluorescent protein (PB2(1-37)-GFP) and determined its competitive inhibitory effect on the polymerase activity of influenza A virus strains. Coexpression of PB2(1-37)-GFP in a polymerase reconstitution system led to substantial inhibition of the polymerase of A/WSN/33 (H1N1). Surprisingly, polymerases of other strains, including A/SC35M (H7N7), A/Puerto Rico/8/34 (H1N1), A/Hamburg/4/2009 (H1N1), and A/Thailand/1(KAN-1)/2004 (H5N1), showed various degrees of resistance. Individual exchange of polymerase subunits and the nucleoprotein between the sensitive WSN polymerase and the insensitive SC35M polymerase mapped the resistance to both PB1 and PA of SC35M polymerase. While PB2(1-37)-GFP bound equally well to the PB1 subunits of both virus strains, PB1-PA dimers of SC35M polymerase showed impaired binding compared to PB1-PA dimers of WSN polymerase. The use of PA(SC35M/WSN) chimeras revealed that the reduced affinity of the SC35M PB1-PA dimer was mediated by the N-terminal 277 amino acids of PA. Based on these observations, we speculate that the PB1-PA dimer formation of resistant polymerases shields the PB2(1-37) binding site, whereas sensitive polymerases allow this interaction, suggesting different assembly strategies of the trimeric polymerase complex between different influenza A virus strains.     

Mutations in polymerase genes enhanced the virulence of 2009 pandemic H1N1 influenza virus in mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

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.     

PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection

ABSTRACT: BACKGROUND: Although gene exchange is not likely to occur freely, reassortment between the H5N1 highlypathogenic avian influenza virus (HPAIV) and currently circulating human viruses is aserious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported toactivate the influenza replicon activity. METHODS: The replicon activities of PR8 and WSN strains (H1N1) of influenza containing PA fromHPAIV A/Cambodia/P0322095/2005 (H5N1) and the activity of the chimeric RNApolymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (CPA)was then reconstituted and its growth in cells and pathogenicity in mice examined. Theinterferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells werecompared with those of WSN-infected cells. RESULTS: The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and CPAreplicated better than WSN in cells. However, the multi-step growth of C-PA and itspathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, andcaspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cellsbut not in WSN-infected cells. CONCLUSIONS: Apoptosis and interferon were strongly induced early in C-PA infection, which protected theuninfected cells from expansion of viral infection. In this case, these classical host-virusinteractions contributed to the attenuation of this strongly replicating virus.     

Favipiravir-resistant influenza A virus shows potential for transmission

Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.     

Origin of the 1918 Spanish influenza virus: a comparative genomic analysis

To test the avian-origin hypothesis of the 1918 Spanish influenza virus we surveyed influenza sequences from a broad taxonomic distribution and collected 65 full-length genomes representing avian, human and "classic" swine H1N1 lineages in addition to numerous other swine (H1N2, H3N1, and H3N2), human (H2N2, H3N2, and H5N1), and avian (H1N1, H4N6, H5N1, H6N1, H6N6, H6N8, H7N3, H8N4, H9N2, and H13N2) subtypes. Amino acids from all eight segments were concatenated, aligned, and used for phylogenetic analyses. In addition, the genes of the polymerase complex (PB1, PB2, and PA) were analyzed individually. All of our results showed the Brevig-Mission/1918 strain in a position basal to the rest of the clade containing human H1N1s and were consistent with a reassortment hypothesis for the origin of the 1918 virus. Our genome phylogeny further indicates a sister relationship with the "classic" swine H1N1 lineage. The individual PB1, PB2, and PA phylogenies were consistent with reassortment/recombination hypotheses for these genes. These results demonstrate the importance of using a complete-genome approach for addressing the avian-origin hypothesis and predicting the emergence of new pandemic influenza strains.     

Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.     

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.     

The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avian or human influenza virus internal genes. Twenty-four reassortant viruses were isolated and first evaluated for infectivity in avian (primary chick kidney [PCK]) and mammalian (Madin-Darby canine kidney [MDCK]) tissue culture lines. Reassortant viruses with two specific constellations of viral polymerase genes exhibited a significant host range restriction of replication in mammalian (MDCK) tissue culture compared with that in avian (PCK) tissue culture. The viral polymerase genotype PB2-avian (A) virus, PB1-A virus, and PA-human (H) virus was associated with a 900-fold restriction, while the viral polymerase genotype PB2-H, PB1-A, and PA-H was associated with an 80,000-fold restriction of replication in MDCK compared with that in PCK. Fifteen reassortant viruses were subsequently evaluated for their level of replication in the respiratory tract of squirrel monkeys, and two genetic determinants of attenuation were identified. First, reassortant viruses which possessed the avian influenza virus nucleoprotein gene were as restricted in replication as a virus which possessed all six internal genes of the avian influenza A virus parent, indicating that the nucleoprotein gene is the major determinant of attenuation of avian-human A/Pintail/79 reassortant viruses for monkeys. Second, reassortant viruses which possessed the viral polymerase gene constellation of PB2-H, PB1-A, and PA-H, which was associated with the greater degree of host range restriction in vitro, were highly restricted in replication in monkeys. Since the avian-human influenza reassortant viruses which expressed either mode of attenuation in monkeys replicated to high titer in eggs and in PCK tissue culture, their failure to replicate efficiently in the respiratory epithelium of primates must be due to the failure of viral factors to interact with primate host cell factors. The implications of these findings for the development of live-virus vaccines and for the evolution of influenza A viruses in nature are discussed.     

Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA''s reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.     

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.     

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96–99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.     

Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA.

The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera. To obtain these monospecific antisera, portions of cloned cDNAs encoding the individual polymerase proteins (PB1, PB2, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion proteins were injected into rabbits. Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB1 and PB2 are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells. Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus. Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins. In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins. To determine whether a polymerase protein is transported into the nucleus individually, PB1 was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector. PB1 alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus. This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB1 protein and that the PB1 protein may contain an intrinsic signal(s) for nuclear transport.     

Origin and molecular characterization of the human-infecting H6N1 influenza virus in Taiwan

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain’s pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.     

Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.