Weiyang Sun

Lowly pathogenic avian influenza (H9N2) infection in Plateau pika (Ochotona curzoniae), Qinghai Lake, China

Avian influenza viruses (AIVs) are globally important contagions. Several domestic mammals can be infected with AIVs and may play important roles in the adaptation and transmission of these viruses in mammals, although the roles of wild mammals in the natural ecology of AIVs are not yet clear. Here, we performed a serological survey of apparently healthy Plateau pikas at Qinghai Lake in China to assess the prevalence of exposure to AIVs. Ninety-two of 293 (31%) of wild Plateau pikas possessed serum antibodies against a lowly pathogenic avian influenza (LPAI) H9N2 virus. Experimental inoculation of Plateau pikas with a LPAI H9N2 virus resulted in productive viral replication in respiratory tissues without prior adaptation. Our findings suggest that Plateau pikas represent a natural mammalian host to H9N2 AIVs and may play a role in the ongoing circulation of H9N2 viruses at Qinghai Lake in China. Surveillance for AIV infection in Plateau pika populations and other mammals that have close contact with the Plateau pikas should be considered.

PB2-E627K and PA-T97I substitutions enhance polymerase activity and confer a virulent phenotype to an H6N1 avian influenza virus in mice.

H6N1 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H6N1 virus infection in 2013. Here, we set out to elucidate viral determinants critical to the pathogenesis of this virus using a mouse model. We found that the recombinant H6N1 viruses possessing both the PA-T97I and PB2-E627K substitutions displayed the greatest enhancement of replication in vitro and in vivo. Polymerase complexes possessing either PB2-E627K, PA-T97I, and PB2-E627K/PA-T97I displayed higher virus polymerase activity when compared to the wild-type virus, which may account for the increased replication kinetics and enhanced virulence of variant viruses. Our results demonstrate that PB2-E627K and PA-T97I enhance the ability of H6N1 virus to replicate and cause disease in mammals. Influenza surveillance efforts should include scrutiny of these regions of PB2 and PA because of their impact on the increased virulence of H6N1 AIVs in mice.

Adaptive amino acid substitutions enhance the virulence of a reassortant H7N1 avian influenza virus isolated from wild waterfowl in mice

H7 avian influenza viruses (AIVs) have caused a number of human infections, highlighting the pandemic potential of them. However, the factors that promote their replication in mammals remain poorly understood. Here, we generated mouse-adapted variants of a reassortant H7N1 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the variants were reduced >10,000-fold compared to the parental virus. Adapted variants displayed enhanced replication kinetics in vitro and vivo, and were capable of replicating in multiple organs. Analysis of the variant virus genomes revealed amino acid changes in the PB2 (E627K), HA (H3 numbering; E114K, G205E, and G218E), and NA (S350N) proteins. Notably, some amino acid changes have been identified in natural H7 isolates. Our results implicate a number of amino acid substitutions that collectively enhance the ability of a wild bird-origin H7N1 AIV to replicate and cause severe disease in mice.

Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice

Although H7N7 AIVs primarily circulate in wild waterfowl, documented cases of human infection with H7N7 viruses suggest they may pose a pandemic threat. Here, we generated mouse-adapted variants of a wild waterfowl-origin H7N7 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the adapted variants were reduced >5000-fold compared to the parental virus. Mouse-adapted variants viruses displayed enhanced replication in vitro and in vivo, and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H7N7 AIVs in mice. Genomic analysis of the adapted variant viruses revealed amino acid changes in the PB2 (E627K), PB1 (R118I), PA (L550M), HA (G214R), and NA (S372N) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H7N7 virus to replicate and cause severe disease in mammals.

A PB1 T296R substitution enhance polymerase activity and confer a virulent phenotype to a 2009 pandemic H1N1 influenza virus in mice.

While the 2009 pandemic H1N1 virus has become established in the human population as a seasonal influenza virus, continued adaptation may alter viral virulence. Here, we passaged a 2009 pandemic H1N1 virus (A/Changchun/01/2009) in mice. Serial passage in mice generated viral variants with increased virulence. Adapted variants displayed enhanced replication kinetics in vitro and vivo. Analysis of the variants genomes revealed 6 amino acid changes in the PB1 (T296R), PA (I94V), HA (H3 numbering; N159D, D225G, and R226Q), and NP (D375N). Using reverse genetics, we found that a PB1-T296R substitution found in all adapted viral variants enhanced viral replication kinetics in vitro and vivo, increased viral polymerase activity in human cells, and was sufficient for enhanced virulence of the 2009 pandemic H1N1 virus in mice. Therefore, we defined a novel influenza pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.

Multiple amino acid substitutions involved in the adaptation of H6N1 avian influenza virus in mice.

H6N1 avian influenza viruses (AIVs) are one of the most abundantly detected avian influenza virus subtype, and a human H6N1 infection case has been reported in 2013. H6N1 AIVs may pose a potential human risk, however, the factors that promote the replication of H6N1 viruses in mammals remain poorly understood. Here, we generated mouse-adapted variants of a H6N1 virus (A/Mallard/SanJiang/275/2007) to identify adaptive changes that confer enhanced virulence to H6N1 viruses in mammals. After eight sequential passages in mice, the mouse lethal doses (MLD50) of the variants were reduced >1000-fold compared to the parental virus. We found that the variants displayed the greatest enhancement of replication in vitro and in vivo, and also were capable of replicating in the brains of infected mice. These observations suggest that enhanced growth characteristics and modified cell tropism may contribute to increased virulence of H6N1 AIVs in mice. Sequencing of the variants revealed amino acid changes in the PB2 (E627K), PA (T97I), and HA (N394T) proteins. Our results suggest that these mutations involved in the enhancement of the ability of H6N1 virus to efficient replicate and cause severe disease in mammals.

Adaptive amino acid substitutions enhance the virulence of a novel human H7N9 influenza virus in mice

To identify molecular features that confer enhanced H7N9 virulence in mammals, we independently generated three mouse-adapted variants of A/Shanghai/2/2013 (H7N9) by serial passage in mice. The mouse lethal doses (MLD50) of the mouse-adapted variants were reduced >1000-100000-fold when compared to the parental virus. Adapted variants displayed enhanced replication kinetics in vivo, and were capable of replicating in multiple organs. Analysis of adapted viral genomes revealed a total of 14 amino acid changes among the three variant viruses in the PA (T97I, K328R, P332T, and Q556R), HA (H3 numbering; A107T, R220I, L226Q, and R354K), NP (A284T and M352I), NA (M26I, N142S, and G389D), and M1 (M128R) proteins. Notably, many of these adaptive amino acid changes have been identified in naturally occurring H7 isolates. Our results identify amino acid substitutions that collectively enhance the ability of a human H7N9 virus to replicate and cause severe disease in mice.

The innate immunity of guinea pigs against highly pathogenic avian influenza virus infection

H5N1 avian influenza viruses are a major pandemic concern. In contrast to the highly virulent phenotype of H5N1 in humans and many animal models, guinea pigs do not typically display signs of severe disease in response to H5N1 virus infection. Here, proteomic and transcriptional profiling were applied to identify host factors that account for the observed attenuation of A/Tiger/Harbin/01/2002 (H5N1) virulence in guinea pigs. RIG-I and numerous interferon stimulated genes were among host proteins with altered expression in guinea pig lungs during H5N1 infection. Overexpression of RIG-I or the RIG-I adaptor protein MAVS in guinea pig cell lines inhibited H5N1 replication. Endogenous GBP-1 expression was required for RIG-I mediated inhibition of viral replication upstream of the activity of MAVS. Furthermore, we show that guinea pig complement is involved in viral clearance, the regulation of inflammation, and cellular apoptosis during influenza virus infection of guinea pigs. This work uncovers features of the guinea pig innate immune response to influenza that may render guinea pigs resistant to highly pathogenic influenza viruses.

Multiple adaptive amino acid substitutions increase the virulence of a wild waterfowl-origin reassortant H5N8 avian influenza virus in mice

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD50) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo, and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice.

Autophagy is highly targeted among host comparative proteomes during infection with different virulent RABV strains

Rabies virus (RABV) is a neurotropic virus that causes serious disease in humans and animals worldwide. It has been reported that different RABV strains can result in divergent prognoses in animal model. To identify host factors that affect different infection processes, a kinetic analysis of host proteome alterations in mouse brains infected with different virulent RABV strains was performed using isobaric tags for a relative and absolute quantification (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach, and this analysis identified 147 differentially expressed proteins (DEPs) between the pathogenic challenge virus standard (CVS)-11 strain and the attenuated SRV9 strain. Bioinformatics analyses of these DEPs revealed that autophagy and several pathways associated with autophagy, such as mammalian target of rapamycin (mTOR) signaling, p70S6K signaling, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress and superoxide radical degradation, were dysregulated. Validation of the proteomic data showed that attenuated SRV9 induced more autophagosome accumulation than CVS-11 in an in vitro model. Our findings provide new insights into the pathogenesis of RABV and encourage further studies on this topic.