Research On The Molecular Biological Characteristics And Gene Function Of Avian Gyrovirus 2

Avian gyrovirus 2(AGV2)was the second member of the viral genus Gyrovirus to be reported in 2011,and was reported in China in 2015 for the first time.Not only in poultry,had PCR detection identified the DNA of AGV2/HGyV in the blood of healthy humans,transplant patients,and HIV-positive patients.Moreover,we know little about this new found virus.So AGV2 poses a significant potential threat to humans and poultry due to its global dissemination and infectiousness.Our research group performed an epidemiologic investigation of AGV2 covering most parts of China from April 2015 to April 2016.Briefly,448 poultry samples(282 in 2015,and 166 in 2016)were collected from 15 provinces.Of the analysed samples,55(12.28%)(45 in 2015;10 in 2016)were positive for AGV2 and were distributed in 11 provinces.Only 10(18.19%)samples showed a single infection;the remaining(81.82%)were mixed infections.Then analysing the temporal and spatial distribution of AGV2,and we found that 85.45%positive cases were from north China,and more samples were positive in autumn than other season.In addition,we detected the situation of AGV2 genome mixed in commercially available poultry vaccines of north China.We found that 23(27.71%)different vaccines produced by 11 different manufactures were AGV2-positive,and 78.26%(18/23)positive results were related to NDV and IBV.Partial nucleotide sequence analysis showed different viral strains were mixed in these vaccines.The investigation result challenged the safety of available vaccines and raise threaten of AGV2 infection.We used three overlapping polymerase chain reactions(PCRs)to map the whole genome of AGV2.We then modelled the evolutionary history of these novel sequence data in the context of related sequences from GenBank.We analysed the viral protein characteristics of the different phylogenetic groups and explored differences in evolutionary trends between Chinese strains and strains from other countries.We obtained 17 avian-sourced AGV2 whole genomes from different regions of China from 2015 to 2016.Phylogenetic analyses of these Chinese AGV2 sequences and related sequences produced four distinct groups(A-D)with significant bootstrap values.We also built phylogenies using predicted viral protein sequences.We found a potential hypervariable region in VP1 at sites 288-314,and we identified the amino acid changes responsible for the distinct VP2 and VP3 groups.Three new motifs in the AGV2 5'-UTR direct repeat(DR)region were discovered and grouped.Based on the conserved sequence,we established a novel real-time PCR method exhibited a high specificity for AGV2.The detection limit of the new method was as low as 100 viral DNA copies,which is 10 times more sensitive than conventional PCR.Coefficients of variation(CVs)of both inter-assay and intra-assay reproducibility tests were below 1%.Using this new method,15/82(17.24%)chicken-derived samples and 8/29(27.58%)human-derived samples were determined to be AGV2-positive,which represents significant detection rates higher than those of conventional PCR(12.20%and 18.29%,respectively;P<0.01).The distribution and viral loads of AGV2 in clinical chickens were assessed using the real-time PCR-based method.AGV2 was found to be widely distributed among different tissues and organs,with particularly high viral loads in the lungs and blood cells.We isolated the virus via infection with 1-day-old SPF(Specific Pathogen Free)chickens for the first report worldwide,and we also obtained the photos of AGV2 particle via electron microscope.And then,we preliminary explored the pathogenicity of AGV2.Coinfection of AGV2 isolation HLJ1508 and avirulent CAV can cause the mortality of 56.67%,with a serious hemorrhagic-aplastic anemia syndrome.The coinfection caused the significant hepatosplenomegaly and thymic atrophy;obvious hemorrhage in glandular stomach and pale aplastic bone marrow.In addition,we proved the anemia and immunosuppression via the result of blood routine test.The research on the gene function of AGV2 included 5'-UTR and the viral proteins.Firstly,we studied the function of AGV2 5'-UTR.We found the promoter sequence and proved the function of the initiation transcription via a series of dual-fluorescence reporter system,furthermore,we proved the function of DR regions as an enhancer to improve the ability of transcription.More interesting,we observed that different DR combinations had different ability of transcription,which may contributed the pathogenicity of difference AGV2 isolations.Secondly,we investigated the function of AGV2 VP2 and VP3 in cancer cells.We observed that these two viral proteins had the similar subcellular localization,nucleus,in Hela cells.We detected that these two proteins both can cause over 50%of Hela cells to apoptosis by flow cytometry,but they may have different pathways to induce apoptosis based on the different Cytochrome-C quantity released by mitochondria.Interestingly,these two proteins both can cause DNA damage which characterized by the up-regulated of the expression of ?H2AX,and the up-regulated expression of Chk2 suggested these two viral proteins may be involved the ATM-Chk2 pathway.The results obtained in these researches established the preliminary understanding of the molecular biological characteristics and pathogenicity of AGV2,and provided many materials for further unveiling the biology of AGV2 and genus Gyrovirus.