Kaihui Cheng

Domestic cats and dogs are susceptible to H9N2 avian influenza virus

Replication and transmission of avian influenza virus (AIV) in domestic dogs and cats may pose a risk to humans. The susceptibility of cats and dogs to H9N2 influenza virus was evaluated by intranasally or orally inoculating animals with an H9N2 influenza virus. Cats had recoverable virus in respiratory tissues and the olfactory bulb three days post-inoculation and shed H9N2 virus into nasal washes and pharyngeal swabs from day 2 through day 10 post-inoculation. Virus was recovered from respiratory tissues of dogs three days post-inoculation, but was not detected in nasal washes or pharyngeal swabs. While no virus shedding or replication was detected in cats or dogs following consumption of H9N2-infected chicks, one of two cats and one of two dogs seroconverted. Two of three naïve contact cats seroconverted following co-housing with cats that were intranasally inoculated with H9N2 virus, whereas none of the three naïve contact dogs seroconverted. Our results demonstrate that H9N2 AIV can infect domestic cats and dogs via the upper respiratory tract and indicate that cats are more susceptible than dogs to H9N2 AIV. These findings suggest that domestic dogs and cats may serve as host species contributing to the adaptation of H9N2 viruses in mammals.

Molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu, China in 2012.

Between January 2012 and March 2012, the infection rates of porcine epidemic diarrhea virus (PEDV) increased substantially in vaccinated swine herds in many porcine farms in Gansu Province, China. The spike (S) glycoprotein is an important determinant for PEDV biological properties. To determine the distribution profile of PEDV outbreak strains, we sequenced the full-length S gene of five samples from two farms where animals exhibited severe diarrhea and high mortality rates. Five new PEDV variants were identified, and the molecular diversity, phylogenetic relationships, and antigenicity analysis of Gansu field samples with other PEDV reference strains were investigated. A series of insertions, deletions, and mutations in the S gene was found in five PEDV variants compared with classical and vaccine strains. These mutations may provide stronger pathogenicity and antigenicity to the new PEDV variants that influenced the effectiveness of the CV777-based vaccine. Our results suggest that these new PEDV variant strains in Gansu Province might be from South Korean or South China, and the effectiveness of the CV777-based vaccine needs to be evaluated.

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.

Experimental infection of non-human primates with avian influenza virus (H9N2).

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.

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.

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.

Isolation and genetic characterization of H13N8 low pathogenic avian influenza virus from migratory birds in eastern China

Low pathogenic avian influenza virus (LPAIV) is an important zoonotic pathogen. Migratory birds are the natural reservoir for all 16 haemagglutinin (HA) and nine neuraminidase (NA) subtypes of LPAIV. Surveillance of LPAIV in migratory waterfowl and poultry is important for animal and public health. An understanding of the ecology and epidemiology of LPAI viruses in their reservoirs is beneficial for routine surveillance projects. Here, we report the isolation of an H13N8 LPAIV from black-tailed gulls in eastern China. Full genome sequences of this isolate were determined. Genetic analysis of the HA and NA segments of this isolate showed that this H13N8 LPAIV was derived from the Eurasian lineage. Additionally, we speculate that this H13N8 LPAIV was a reassortant between the North American and Eurasian lineages. Interestingly, we identified amino acid motifs responsible for increased virulence or transmission of influenza viruses in mammals. We also found weak but measurable haemagglutination inhibition antibody titers against H13N8 virus in serum samples collected from chickens. These results suggest that continued surveillance for LPAI viruses in migratory birds and poultry is required.

Poultry Infection with Influenza Viruses of Wild Bird Origin, China, 2016

Migratory birds may play a role in transmission of avian influenza virus. We report the infection of black-tailed gulls and chickens in eastern China with avian influenza (H13N2) and (H13N8) viruses. We found that these H13 viruses were transmitted from migratory birds to domestic poultry.