Human Seasonal H3N2 Research Articles

Rapid evolution of the PB1-F2 virulence protein expressed by human seasonal H3N2 influenza viruses reduces inflammatory responses to infection

Influenza A virus (IAV) PB1-F2 protein has been linked to viral virulence. Strains of the H3N2 subtype historically express full-length PB1-F2 proteins but during the 2010–2011 influenza seasons, nearly half of the circulating H3N2 IAVs encoded truncated PB1-F2 protein. Using a panel of reverse engineered H3N2 IAVs differing only in the origin of the PB1 gene segment, we found that only the virus encoding the avian-derived 1968 PB1 gene matching the human pandemic strain enhanced cellular infiltrate into the alveolar spaces of infected mice. We linked this phenomenon to expression of full-length PB1-F2 protein encompassing critical “inflammatory” residues.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis.IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up to the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) that allow the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of proinflammatory cytokines, resulting in viral attenuation in vivo This might represent an adaptation of pH1N1 virus to humans.

Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses.

Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the United States since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U.S. swine population in the mid-1990s, but they are different from both these ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.

Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay.

Background Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube.MethodsClinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay.ResultsThe limitation of detection (LOD) of the multiplex assay was 5.4 × 10−2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10−2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10−2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples.ConclusionsThese findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

The PB2 Subunit of the Influenza A Virus RNA Polymerase Is Imported into the Mitochondrial Matrix.

ABSTRACTThe polymerase basic 2 (PB2) subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localize to the mitochondria. Various roles, including the regulation of apoptosis and innate immune responses to viral infection, have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signaling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we used enhanced ascorbate peroxidase (APEX)-tagged PB2 proteins and electron microscopy to study the localization of PB2 in mitochondria. We found that PB2 is imported into mitochondria, where it localizes to the mitochondrial matrix. We also demonstrated that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we found that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localization of PB2 of human from that of avian influenza A virus strains. We also showed that a virus encoding nonmitochondrial PB2 is attenuated in mouse embryonic fibroblasts (MEFs) compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria.IMPORTANCE The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signaling protein MAVS, implicating PB2 in the regulation of innate immune responses. We found that PB2 is imported into the mitochondrial matrix and showed that amino acid residue 9 is a determinant of mitochondrial import. The presence of asparagine or threonine in over 99% of all human seasonal influenza virus pre-2009 H1N1, H2N2, and H3N2 strains is compatible with mitochondrial import, whereas the presence of an aspartic acid in over 95% of all avian influenza viruses is not, resulting in a clear distinction between human-adapted and avian influenza viruses. These findings provide insights into the interplay between influenza virus and mitochondria and suggest mechanisms by which PB2 could affect pathogenicity.

Structure-Based Tetravalent Zanamivir with Potent Inhibitory Activity against Drug-Resistant Influenza Viruses.

Zanamivir and oseltamivir are principal influenza antiviral drugs that target viral neuraminidase (NA), but resistant viruses containing mutant NAs with diminished drug affinity are increasingly emerging. Using the structural knowledge of both drug-binding sites and their spatial arrangement on the homotetrameric NA, we have developed a tetravalent zanamivir (TZ) molecule that exhibited marked increases in NA binding affinity, inhibition of NA enzyme activity, and in vitro plus in vivo antiviral efficacy over zanamivir. TZ functioned against both human seasonal H3N2 and avian H7N9 viruses, including drug-resistant mutants. Crystal structure of a resistant N9 NA in complex with TZ explained the function, which showed that four zanamivir residues simultaneously bound to all four monomers of NA. The design method of TZ described in this study may be useful to develop drugs or ligands that target proteins with multiple binding sites. The potent anti-influenza activity of TZ makes it attractive for further development.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity.

Novel Reassortant Human-Like H3N2 and H3N1 Influenza A Viruses Detected in Pigs Are Virulent and Antigenically Distinct from Swine Viruses Endemic to the United States.

Human-like swine H3 influenza A viruses (IAV) were detected by the USDA surveillance system. We characterized two novel swine human-like H3N2 and H3N1 viruses with hemagglutinin (HA) genes similar to those in human seasonal H3 strains and internal genes closely related to those of 2009 H1N1 pandemic viruses. The H3N2 neuraminidase (NA) was of the contemporary human N2 lineage, while the H3N1 NA was of the classical swine N1 lineage. Both viruses were antigenically distant from swine H3 viruses that circulate in the United States and from swine vaccine strains and also showed antigenic drift from human seasonal H3N2 viruses. Their pathogenicity and transmission in pigs were compared to those of a human H3N2 virus with a common HA ancestry. Both swine human-like H3 viruses efficiently infected pigs and were transmitted to indirect contacts, whereas the human H3N2 virus did so much less efficiently. To evaluate the role of genes from the swine isolates in their pathogenesis, reverse genetics-generated reassortants between the swine human-like H3N1 virus and the seasonal human H3N2 virus were tested in pigs. The contribution of the gene segments to virulence was complex, with the swine HA and internal genes showing effects in vivo. The experimental infections indicate that these novel H3 viruses are virulent and can sustain onward transmission in pigs, and the naturally occurring mutations in the HA were associated with antigenic divergence from H3 IAV from humans and swine. Consequently, these viruses could have a significant impact on the swine industry if they were to cause more widespread outbreaks, and the potential risk of these emerging swine IAV to humans should be considered. Pigs are important hosts in the evolution of influenza A viruses (IAV). Human-to-swine transmissions of IAV have resulted in the circulation of reassortant viruses containing human-origin genes in pigs, greatly contributing to the diversity of IAV in swine worldwide. New human-like H3N2 and H3N1 viruses that contain a mix of human and swine gene segments were recently detected by the USDA surveillance system. The human-like viruses efficiently infected pigs and resulted in onward airborne transmission, likely due to the multiple changes identified between human and swine H3 viruses. The human-like swine viruses are distinct from contemporary U.S. H3 swine viruses and from the strains used in swine vaccines, which could have a significant impact on the swine industry due to a lack of population immunity. Additionally, public health experts should consider an appropriate assessment of the risk of these emerging swine H3 viruses for the human population.

Influenza A Viruses of Human Origin in Swine, Brazil.

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil's swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009-2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance.

Pathogenesis and Transmission of Novel Highly Pathogenic Avian Influenza H5N2 and H5N8 Viruses in Ferrets and Mice.

A novel highly pathogenic avian influenza (HPAI) H5N8 virus, first detected in January 2014 in poultry and wild birds in South Korea, has spread throughout Asia and Europe and caused outbreaks in Canada and the United States by the end of the year. The spread of H5N8 and the novel reassortant viruses, H5N2 and H5N1 (H5Nx), in domestic poultry across multiple states in the United States pose a potential public health risk. To evaluate the potential of cross-species infection, we determined the pathogenicity and transmissibility of two Asian-origin H5Nx viruses in mammalian animal models. The newly isolated H5N2 and H5N8 viruses were able to cause severe disease in mice only at high doses. Both viruses replicated efficiently in the upper and lower respiratory tracts of ferrets; however, the clinical symptoms were generally mild, and there was no evidence of systemic dissemination of virus to multiple organs. Moreover, these influenza H5Nx viruses lacked the ability to transmit between ferrets in a direct contact setting. We further assessed viral replication kinetics of the novel H5Nx viruses in a human bronchial epithelium cell line, Calu-3. Both H5Nx viruses replicated to a level comparable to a human seasonal H1N1 virus, but significantly lower than a virulent Asian-lineage H5N1 HPAI virus. Although the recently isolated H5N2 and H5N8 viruses displayed moderate pathogenicity in mammalian models, their ability to rapidly spread among avian species, reassort, and generate novel strains underscores the need for continued risk assessment in mammals. In 2015, highly pathogenic avian influenza (HPAI) H5 viruses have caused outbreaks in domestic poultry in multiple U.S. states. The economic losses incurred with H5N8 and H5N2 subtype virus infection have raised serious concerns for the poultry industry and the general public due to the potential risk of human infection. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals, which is an essential component of pandemic risk assessment. This study demonstrates that the newly isolated H5N2 and H5N8 viruses lacked the ability to transmit between ferrets and exhibited low to moderate virulence in mammals. In human bronchial epithelial (Calu-3) cells, both H5N8 and H5N2 viruses replicated to a level comparable to a human seasonal virus, but significantly lower than a virulent Asian-lineage H5N1 (A/Thailand/16/2004) virus. The results of this study are important for the evaluation of public health risk.

Lower seroreactivity to European than to North American H3N2 swine influenza viruses in humans, Luxembourg, 2010

Seroreactivity to H3N2 swine influenza viruses (SIVs)was evaluated in serum samples collected from 843 people aged 0 to 100 years in 2010 in Luxembourg.Sera were analysed by haemagglutination inhibition(HI) and virus neutralisation (VN) assays targeting a European H3N2 SIV, a North American H3N2 variant of swine origin (H3N2v) and human seasonal H3N2 viruses isolated in 1975, 1995 and 2005. HI antibodies(titre ≥ 10) against European H3N2 SIV were almost exclusively detected in those born before 1990, of whom 70% were seropositive. HI antibodies against H3N2v were predominantly found in those born before 2000, with 86% seropositive. Titres against the North American H3N2v were higher than against the European H3N2 SIV. VN patterns were similar, but with higher rates and titres. We also demonstrated lower seroreactivity to European H3N2 SIV than to North American H3N2v virus. Finally, we found a strong correlation between HI titres against the European H3N2SIV and H3N2v and their respective human ancestors,A/Victoria/3/75 and A/Nanchang/933/95. This finding and the minimal contacts between humans and pigs in Luxembourg suggest that anti-SIV antibodies inhuman serum samples reflect serological cross-reactivity with historical human H3N2 viruses. Our findings help assess the pandemic risk of H3N2 SIV.

Serological survey of canine H3N2, pandemic H1N1/09, and human seasonal H3N2 influenza viruses in cats in northern China, 2010-2014.

BackgroundThe close contact between cats and humans poses a threat to public health because of the potential zoonotic transmission of influenza viruses to humans. Therefore, we examined the seroprevalence of pandemic H1N1/09, canine H3N2, and human H3N2 viruses in pet cats in northern China from 2010 to 2014.FindingOf 1794 serum samples, the seropositivity rates for H1N1/09, canine H3N2, and human H3N2 were 5.7%, 0.7%, and 0.4%, respectively. The seropositivity rate for H1N1/09 in cats was highest in 2010 (8.3%), and then declined continuously thereafter. Cats older than 10 years were most commonly seropositive for the H1N1/09 virus.ConclusionsOur findings emphasize the need for continuous surveillance of influenza viruses in cats in China.

Computational study of interdependence between hemagglutinin and neuraminidase of pandemic 2009 H1N1.

Influenza type A viruses are classified into subtypes based on their two surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA protein facilitates the viral binding and entering a host cell and the NA protein helps the release of viral progeny from the infected cell. The complementary roles of HA and NA entail their collaboration, which has important implications for viral replication and fitness. The HA protein from early strains of pandemic 2009 H1N1 of swine origin preferentially binds to human type receptors with a weak binding to avian type receptors. This virus caused several human deaths in December 2013 in Texas, USA, which motivated us to investigate the changes of genetic features that might contribute to the surged virulence of the virus. Our time series analysis on the strains of this virus collected from 2009 to 2013 implied that the HA binding preference of this virus in USA, Europe, and Asia has been the characteristic of swine H1N1 virus since 2009. However, its characteristic of seasonal human H1N1 and its binding avidity for avian type receptors both were on steady rise and had a clear increase in 2013 with American strains having the sharpest surge. The first change could enhance the viral transmission and replication in humans and the second could increase its ability to cause infection deep in lungs, which might account for the recent human deaths in Texas. In light of HA and NA coadaptation and evolutionary interactions, we also explored the NA activity of this virus to reveal the functional balance between HA and NA during the course of virus evolution. Finally we identified amino acid substitutions in HA and NA of the virus that were critical for the observed evolution.

Mammalian adaptive mutations of the PA protein of highly pathogenic avian H5N1 influenza virus.

Highly pathogenic H5N1 influenza A viruses continue to circulate among avian species and cause sporadic cases of human infection. Therefore, the threat of a pandemic persists. However, the human cases of H5N1 infection have been limited mainly to individuals in close contact with infected poultry. These findings suggest that the H5N1 viruses need to acquire adaptive mutations to gain a replicative advantage in mammalian cells to break through the species barrier. Many amino acid mutations of the polymerase complex have been reported to enhance H5N1 virus growth in mammalian cells; however, the mechanism for H5N1 virus of adaptation to humans remains unclear. Here, we propose that the PA of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010 [36285]) increased the ability of an avian H5N1 virus (A/chicken/Vietnam/TY31/2005 [Ck/TY31]) to grow in human lung epithelial A549 cells. The five PA amino acid substitutions V44I, V127A, C241Y, A343T, and I573V, which are rare in H5N1 viruses from human and avian sources, enhanced the growth capability of this virus in A549 cells. Moreover, these mutations increased the pathogenicity of the virus in mice, suggesting that they contribute to adaptation to mammalian hosts. Intriguingly, PA-241Y, which 36285 encodes, is conserved in more than 90% of human seasonal H1N1 viruses, suggesting that PA-241Y contributes to virus adaptation to human lung cells and mammalian hosts. Many amino acid substitutions in highly pathogenic H5N1 avian influenza viruses have been shown to contribute to adaptation to mammalian hosts. However, no naturally isolated H5N1 virus has caused extensive human-to-human transmission, suggesting that additional, as-yet unidentified amino acid mutations are needed for adaptation to humans. Here, we report that five amino acid substitutions in PA (V44I, V127A, C241Y, A343T, and I573V) contribute to the replicative efficiency of H5N1 viruses in human lung cells and to high virulence in mice. These results are helpful for assessing the pandemic risk of isolates and further our understanding of the mechanism of H5N1 virus adaptation to mammalian hosts.

Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice.

In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity. Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.

Identification of PB2 mutations responsible for the efficient replication of H5N1 influenza viruses in human lung epithelial cells.

Highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry worldwide, resulting in sporadic infections in humans with approximately 60% mortality. However, efficient transmission of H5N1 viruses among humans has yet to occur, suggesting that further adaptation of H5N1 viruses to humans is required for their efficient transmission among humans. The viral determinants for efficient replication in humans are currently poorly understood. Here, we report that the polymerase PB2 protein of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, virus 36285) increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, virus Wb/AH82) in human lung epithelial A549 cells (however, the reassortant virus did not replicate more efficiently than human 36285 virus). Furthermore, we demonstrate that the amino acid residues at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible for its efficient replication in A549 cells. PB2 residues 249G and 339M, which are found in the human H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly, PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data are of value to H5N1 virus surveillance. Highly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the species barrier between avian species and humans. When H5N1 viruses replicate in human respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans through continuous selective pressure. Several amino acid mutations have been shown to be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic risk of circulating avian viruses and for further functional analysis of PB2.

Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: Modeling studies of antibody and receptor binding

The envelope protein hemagglutinin (HA) of influenza viruses is primarily associated with host antibody and receptor interactions. The HA protein is known to maintain a functional balance with neuraminidase (NA), the other major envelope protein. Prior to 2007–2008, human seasonal H1N1 viruses possessing the NA H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. Subsequently, secondary mutations that compensate for the deleterious effect of the NA H274Y mutation have been identified. The molecular mechanism of how the defect could be counteracted by these secondary mutations is not fully understood. We studied here the effect of three such mutations (T86K, K144E and R192K) in the HA protein, which are located at either the HA receptor binding site or in the H1N1 antigenic sites. Molecular docking and dynamics studies showed that, of the three mutations, the R192K mutation could have mediated neutralizing antibody escape and decreased receptor binding affinity, either or both of which may have contributed to increased viral fitness. The study suggests the molecular basis of enhanced viral fitness induced by secondary mutations in the evolution of oseltamivir-resistant influenza strains.

Canine susceptibility to human influenza viruses (A/pdm 09H1N1, A/H3N2 and B).

We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naïve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.

Serological report of pandemic and seasonal human influenza virus infection in dogs in southern China.

From January to July 2012, we looked for evidence of subclinical A (H1N1) pdm09 and seasonal human influenza viruses infections in healthy dogs in China. Sera from a total of 1920 dogs were collected from Guangdong, Guangxi, Fujian and Jiangxi provinces. We also examined archived sera from 66 dogs and cats that were collected during 2008 from these provinces. Using hemagglutination inhibition (HI) and microneutralization (MN) assays, we found that only the dogs sampled in 2012 had elevated antibodies (≥ 1:32) against A(H1N1)pdm09 virus and seasonal human influenza viruses: Of the 1920 dog sera, 20.5 % (n = 393) had elevated antibodies against influenza A(H1N1) pdm09 by the HI assay, 1.1 % (n = 22), and 4.7 % (n = 91) of the 1920 dogs sera had elevated antibodies against human seasonal H1N1 influenza virus and human seasonal H3N2 influenza virus by the HI assay. Compared with dogs that were raised on farms, dogs that were raised as pets were more likely to have elevated antibodies against A(H1N1)pdm09 and seasonal human influenza viruses. Seropositivity was highest among pet dogs, which likely had more diverse and frequent exposures to humans than farm dogs. These findings will help us better understand which influenza A viruses are present in dogs and will contribute to the prevention and control of influenza A virus. Moreover, further in-depth study is necessary for us to understand what roles dogs play in the ecology of influenza A.

Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus

To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.