Isolation,Identification,Epidemiological Survey In Sichuan And Chongqing And In Vitro Growth Characteristic Of Goose Pegivirus

The pathogens of the sick geese from Sichuan province and Chongqing municipality were dectected in this research.Bacterial and common viral infections(goose parvovirus(GPV),goose circovirus(Go CV),goose astrovirus(Go Ast V),goose coronavirus(GCo V),goose paramyxovirus(GPMV),avian influenza virus(AIV))were excluded by bacterial culture and polymerase chain reaction(PCR)detection.To identify the possible pathogen causing the disease,material isolated from intestine and liver homogenate of dead geese was used to infect nine-day-old goose embryos through allantoic cavity injection.The putative pathogen was passaged for 10 times after which viral DNA and RNA were extracted from allantoic fluid and analysed using de novo sequencing by BGI(Shenzhen,China).Then a new RNA virus was found.Using NCBI Genbank to compare the virus sequence and genome structure and analyze the phylogeny,the virus was classified as a new species within the genus Pegivirus of the family Flaviviridae,and named goose pegivirus(GPgV).Epidemiological survey of GPgV in Sichuan and Chongqing:Based on the conserved NS3 gene of GPgV,the quantitative real-time reverse transcription PCR(q RT-PCR)and nested RT-PCR assays were established,which were applied to quantitative detection of viral genome and infection rate of clinical samples,respectively.According to the epidemiological survey of gosling viral diseases in some goose farms in Sichuan and Chongqing in2018?2019,goslings infected with GPgV,GPV,Go CV and Go Ast V.The total positive detection rate of viruses reached 85.7%.The positive detection rate of GPgV was the highest(44.9%),showing a high degree of mixed infection(36.8%),mainly mixed with GPV and Go CV.Artificial infection experiment of GPgV:24 three-day-old goslings were intravenously injected with the allantoic fluid from GPgV-infected goose embryos and fed in a waterfowl isolator for 30 days.The weight of goslings was measured every three days.GPgV infection was found to significantly retard weight gain in the goslings at 19,22,25 days post-infection(dpi).Over the course of the study,two infected goslings died,one at 8 dpi and another at18 dpi.The q RT-PCR detection showed that the goslingswhich died at 8 dpi was GPgV negative.When goslings were sampled at 30 dpi,6 out of 22 infected goslings showed moderate enlargement of the spleen,and 9 out of 22 infected goslings showed a hyperemic and moderately swollen thymus.The q RT-PCR detection showed that GPgV was widely distributed in thymus,spleen,liver,pancreas,duodenum,rectum,and bursa of Fabricius.The highest viral genome copy numbers were found in the immune tissues,such as the spleen and thymus.Immunohistochemical(IHC)tests were performed and mouse anti-GPgV E2monoclonal antibody was used in the experiment.And positive signals were detected equably in the spleen and thymus.Proliferation of GPgV in vitro:GPgV was serially passaged 10 times in nine-day-old goose embryos.The mortality varied from 0 to 66.7%and the death of infected embryos mainly occurred at 59?174 h post inoculation(hpi).The proliferation of GPgV in the 11^th,12^th,13^th and 14^th passages increased,with the average genome copy numbers in the allantoic fluid ranged from 2.53×10⁶ to1.13×10¹⁰ copies/ml.In the 14^th passage,the average viral load slightly declined at 24?120 hpi,and gradually increased with incubation time at 120?192hpi.Goose embryo fibroblasts(GEFs)were infected with the 12^th passage GPgV goose allantoic fluid and passaged 8 times.GPgV caused no cytotoxicity on GEFs,and no cytopathic effect(CPE)was observed.GPgV genome copies gradually increased with passages,with the copy numbers ranged from 2.02×10⁷ to 3.78×10⁸ copies/ml.The indirect immunofluorescence assay(IFA)detection with mouse anti-GPgV E2 monoclonal antibody confirmed the proliferation of GPgV in GEFs.The growth curve of GPgV on GEFs showed that in the infected cells,the viral genome copy number gradually increased with incubation time and reached a plateau at 72?168 hpi.In the cell culture supernatant,no increase in virus genome copy number could be detected up to 48 hpi.From this point onward,the viral genome copy number increased rapidly and reached a plateau at 120?168 hpi.Meanwhile,GPgV could not proliferate in nine-day-old duck embryos,duck embryo fibroblasts(DEFs),hamster kidney cells(BHK-21),bat lung cells(Tb1Lu),isolated goose peripheral blood lymphocytes(GPBLs)and spleen lymphocytes(GSLs).In summary,the first non-mammalian pegivirus was isolated and identified in this research.The virus was named goose pegivirus to reflect its host origin and is potentially a causative agent,or one of causative agents,of gosling immune injury in Sichuan province and the Chongqing municipality of China.The prevalence of GPgV and other major viruses in goslings in Sichuan and Chongqing in 2018?2019 was investigated.The clinicopathological changes and viral tissue distribution of GPgV in artificially infected goslings were clarified,as well as the viral growth characteristics in vitro,which laid the foundation for further research on the pathogenic mechanism of GPgV.