Zhijun Yu

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.

Fatal H5N6 Avian Influenza Virus Infection in a Domestic Cat and Wild Birds in China

H5N6 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H5N6 virus infection in 2014. Here, we report the first cases of fatal H5N6 avian influenza virus (AIV) infection in a domestic cat and wild birds. These cases followed human H5N6 infections in China and preceded an H5N6 outbreak in chickens. The extensive migration routes of wild birds may contribute to the geographic spread of H5N6 AIVs and pose a risk to humans and susceptible domesticated animals, and the H5N6 AIVs may spread from southern China to northern China by wild birds. Additional surveillance is required to better understand the threat of zoonotic transmission of AIVs.

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.

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.

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.

Identification and Characterization of Porcine Kobuvirus Variant Isolated from Suckling Piglet in Gansu Province, China

Kobuviruses comprise three species, the Aichivirus A, Aichivirus B, and Aichivirus C (porcine kobuvirus). Porcine kobuvirus is endemic to pig farms and is not restricted geographically but, rather, is distributed worldwide. The complete genomic sequences of four porcine kobuvirus strains isolated during a diarrhea outbreak in piglets in the Gansu province of China were determined. Two of these strains exhibited variations relative to the traditional strains. The potential 3C/3D cleavage sites of the variant strains were Q/C, which differed from the Q/S in the traditional porcine kobuvirus genome. A 90-nucleotide deletion in the 2B protein and a single nucleotide insertion in the 3′UTR were found in the variant strains. The VP1 regions of all four porcine kobuviruses in our study were highly variable (81%–86%). Ten common amino acid mutations were found specifically at certain positions within the VP1 region. Significant recombination sites were identified using SimPlot scans of whole genome sequences. Porcine kobuviruses were also detected in pig serum, indicating that the virus can escape the gastrointestinal tract and travel to the circulatory system. These findings suggest that mutations and recombination events may have contributed to the high level of genetic diversity of porcine kobuviruses and serve as a driving force in its evolution.

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.