Yuping Hua

Molecular characterization and phylogenetic analysis of the reticuloendotheliosis virus isolated from wild birds in Northeast China.

To analyze the status of reticuloendotheliosis (RE) infection of wild birds in China, 585 samples from wild birds collected in Liaoning, Jilin and Heilongjiang provinces China were investigated and analyzed. The sampled birds represent 3 orders and more than 40 species. Virus isolation and PCR amplification showed that some of the wild birds were infected with REV, and 10 REV strains were isolated. The gp90 gene from each of the 10 REV strains was amplified, cloned, and sequenced. Sequence analysis indicated that the gp90 genes of the 10 REV strains isolated in this study were more similar at the nucleotide level with the northeast Chinese strains HLJR0901 and HLJR0801 and some REV strains found in the US and Taiwan than with the early Chinese REV isolate HA9901. Furthermore, phylogenetic analysis indicated that the gp90 genes of the 10 REV strains were more similar to the REV subtype III-representing strain (CSV) than to strains 170A (subtype I) or SNV (subtype II). This is the first study to investigate the status of wild birds infected with REV. The results of this paper will not only provide necessary information for further understanding the evolution of REV, but they also identify the potential role of wild birds in REV transmission and furthers our understanding of the ecology of REV in wild bird species.

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.

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.

First isolation of reticuloendotheliosis virus from mallards in China

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission.

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.

Pathogenesis and Phylogenetic Analyses of Two Avian Influenza H7N1 Viruses Isolated from Wild Birds

The emergence of human infections with a novel H7N9 influenza strain has raised global concerns about a potential human pandemic. To further understand the character of other influenza viruses of the H7 subtype, we selected two H7N1 avian influenza viruses (AIVs) isolated from wild birds during routine surveillance in China: A/Baers Pochard/Hunan/414/2010 (BP/HuN/414/10) (H7N1) and A/Common Pochard/Xianghai/420/2010 (CP/XH/420/10) (H7N1). To better understand the molecular characteristics of these two isolated H7N1 viruses, we sequenced and phylogenetically analyzed their entire genomes. The results showed that the two H7N1 strains belonged to a Eurasian branch, originating from a common ancestor. Phylogenetic analysis of their hemagglutinin (HA) genes showed that BP/HuN/414/10 and CP/XH/420/10 have a more distant genetic relationship with A/Shanghai/13/2013 (H7N9), with similarities of 91.6 and 91.4%, respectively. To assess the replication and pathogenicity of these viruses in different hosts, they were inoculated in chickens, ducks and mice. Although, both CP/XH/420/10 and BP/HuN/414/10 can infect chickens, ducks and mice, they exhibited different replication capacities in these animals. The results of this study demonstrated that two low pathogenic avian influenza (LPAI) H7N1 viruses of the Eurasian branch could infect mammals and may even have the potential to infect humans. Therefore, it is important to monitor H7 viruses in both domestic and wild birds.

Complete Nucleotide Sequence of Avian Paramyxovirus Type 6 Strain JL Isolated from Mallard Ducks in China

ABSTRACT A new strain of avian paramyxovirus type 6 (APMV-6), JL, has been isolated from mallard ducks in China, and its complete genome has been sequenced and analyzed. This work is the first announced complete genome sequence of APMV-6 from mallards.

Amino Acid Substitutions Associated with Avian H5N6 Influenza A Virus Adaptation to Mice

At least 15 cases of human beings infected with H5N6 have been reported since 2014, of which at least nine were fatal. The highly pathogenic avian H5N6 influenza virus may pose a serious threat to both public health and the poultry industry. However, the molecular features promoting the adaptation of avian H5N6 influenza viruses to mammalian hosts is not well understood. Here, we sequentially passaged an avian H5N6 influenza A virus (A/Northern Shoveler/Ningxia/488-53/2015) 10 times in mice to identify the adaptive amino acid substitutions that confer enhanced virulence to H5N6 in mammals. The 1st and 10th passages of the mouse-adapted H5N6 viruses were named P1 and P10, respectively. P1 and P10 displayed higher pathogenicity in mice than their parent strain. P10 showed significantly higher replication capability in vivo and could be detected in the brains of mice, whereas P1 displayed higher replication efficiency in their lungs but was not detectable in the brain. Similar to its parent strain, P10 remained no transmissible between guinea pigs. Using genome sequencing and alignment, multiple amino acid substitutions, including PB2 E627K, PB2 T23I, PA T97I, and HA R239H, were found in the adaptation of H5N6 to mice. In summary, we identified amino acid changes that are associated with H5N6 adaptation to mice.

Construction and characterization of a recombinant reticuloendotheliosis virus expressing enhanced green fluorescent protein

Reticuloendotheliosis virus (REV) causes an immunosuppressive and oncogenic disease in chickens and other birds. In this study, based on an infectious clone of REV, named HLJR0901, a recombinant virus containing the enhanced green fluorescence protein (EGFP) gene was constructed by inserting the EGFP expression cassette downstream of the 3′ terminus of the viral env gene. An EGFP-tagged REV that stably expresses EGFP was rescued. This visible recombinant REV could contribute to the further understanding of the molecular mechanism involved in the replication and pathogenicity of REV.