Detection Of The Novel Human Pegivirus Type 2 In China And Preliminary Exploration Of Its Infectious Clone

BackgroundHuman Pegivirus type 2(HPgV-2)was first reported in the United Kingdom and the United States in 2015.Like HPgV(GBV-C),evolutionary analysis revealed that HPgV-2 belongs to the pegivirus genus of the Flaviviridae.At present,mostly of studies related to HPgV-2 were observational to describe the population distribution variation of HPgV-2.It was found that HPgV-2 mainly transmitted through blood,especially intravenous drug injection.The virus mainly infects HCV positive individuals,and the RNA positive rate was between 0.29%and 2.4%.There were rarely reports about the detection of HPgV-2 RNA in healthy blood donors,HBV infected individuals and HIV-1 infected individuals.At present,there is no report reveal the cytotropism and pathogenicity of HPV-2.During recent 20 years,Pegivirus from animals have been successively identified.Like other species of Pegivirus,HPgV-2 has nit been isolated and cultured in vitro,which directly obstructs the ananlysis of infection mechanism and biological characteristics.Research purpose and significanceThis study was aimed to:(1)Establish serological and nucleic acid detection methods of HPgV-2 virus,and explore the epdemiologycal characteristics of HPgV-2 infection.(2)Amplify the whole genom of HPgV-2 and analyze the gene characteristics and molecular evolution of HPgV-2.(3)Explore the potential pathogenicity and phagocytosis of HPgV-2.(4)Try to establish the culture system of HPgV-2 in vitro and further study the biological characteristics and the relationship with HCV or HIV-1.Research contents and methods1.Population survey(1)Objective to establish serological and nucleic acid detection methods of HPV-2 and invesgate the distribution characteristics of HPV-2 in different populations.(2)The nearly full-length genome of HPV-2 was amplified,and the sequence variation and molecular evolution of HPV-2 were analyzed.2.Pathogenicity and phagocytosis research of HPgV-2.(1)Comparing the serological indexes of liver function in patients with positive and negative HPgV-2 to receal the impact of hepatitis C patients with HPgV-2 infection on liver injury.(2)The infection status of HPV-2 and HCV/HIV-1 co-infection was observed.The relationship between HPgV-2 and liver injury was studied by comparing the serum indexes of liver function and liver pathological changes before and after HCV clearance.(3)Liver tissues and PBMCs were obtained during the follow-up period and detected by immunohistochemistry and in situ hybridization to confirm the phagocytosis of HPgV-2.3.In vitro culture system(1)The whole genome sequences of the vector and HPgV-2 were analyzed to determine the single enzyme restriction sites.The sequence of the monoclonal sites was synthesized and the vector containing the monoclonal sites were constructed.(2)The whole genome sequence of HPgV-2 was amplified by PCR,and the amplified productions were constructed into the vector by restriction enzyme linked method.(3)The whole genome RNA of HPgV-2 was obtained by linearization and in vitro transcription.(4)HPgV-2 RNA was transfected into cells by liposome transfection method.HPgV-2 nucleic acid and antigen were tested to identify whether the virus was successfully rescued in vitro.Results1.Infection characteristics and genom variation of HPgV-2(1)Construction of HPgV-2 antigen and nucleic acid detection methodIn this study,three polypeptides(P4,P9,P16)of HPgV-2 genome were synthesized.An enzyme linked immunosorbent assay was established to detect anti-HPgV-2 antibody in serum.Nest-PCR for HPgV-2 5 'UTR and NS3 regions and q-PCR were designed according to the reported genome sequence.(2)Analysis of gene variation of HPgV-2We designed RT-PCR method to amplify the complete coding region and most of the non-coding regions of HPgV-2.We found that the variation of HPgV-2 strains was very small(93.6-97.8%).At the same time,we found the evolutionary relationship of HPgV-2 virus is closest to that of Pegivirus from bats and rodants.(3)Analysis of infection characteristics of HPgV-2A total of 8198 cross-sectional serum samples were collected.The results showed that HPV-2 was associated with HCV and HIV-1 infection.The serological positive rate of HPV-2 in HCV population(1.23%)was 8.23 times higher than that in healthy blood donors.In HCV/HIV-1 co-infected population,the HPgV-2 serological positive rate(8.91%)was 59.66 times higher than that of healthy blood donors and 7.25 times higher than that of HCV population;The HPgV-2 RNA positive rate(3.47%)was 12.08 times higher than that of HCV(0.29%).2.Infection status,pathogenicity and phagocytosis of HPgV-2 infection in human(1)HPgV-2 causes persistent and resolved infectionIn this study,240 cases from Nanfang Hospital and 512 cases from Guangzhou Eighth People's hospital were collected,and the HPgV-2 RNA positive patients were observed longitudinally.We observed persistent and stable HPgV-2 infection in patients for more than 5 years.At the same time,we found that DAAs could inhibite HCV but not HPgV-2.HPgV-2 remained stable in patients during the follow-up period of half a year after HCV clearance.(2)Influence of HPgV-2 infection on liver injury(liver index,pathological)We used case-control study to compare the liver indexes of healthy blood donors with positive and negative HPgV-2 infection.We found that co-infection of HPgV-2 and HCV did not increase ALT level,although there was slightly decrease,there was no statistical difference(P=0.766).There was no significant difference in ALT level between HPgV-2 RNA positive and negative blood donors(P=0.298),Positive and negative HPgV-2 antibody detection and antibody level(OD?1.0 or OD<1.0)had no effect on ALT level(P=0.623).At the same time,through the comparison of patients with HPgV-2/HCV co-infection before and after receiving DAAs treatment,we found that when the HCV was cleared,there was no obvious aggravation of liver indexes and pathological characteristics caused by HPgV-2.(3)Tissue tropism of HPgV-2The positive and negative RNA of HPgV-2 and HCV in liver tissue and PBMCs were detected by RNA in situ hybridization and PCR amplification.The results showed that HPgV-2 did not infect the liver cells,but infected the peripheral blood lymphocytes.The positive and negative RNA positive cells were 8%-10%and 5%-6%,respectively.At the same time,we also detected HCV positive RNA(6%-11%)and negative RNA(4%-6%)in PBMCs.We further sorted CD4+,CD 8+T lymphocytes and B lymphocytes of PBMCs by flow cytometry,enriched and amplified positive and negative RNA of HPgV-2 and HCV,and found that HPgV-2 and HCV mainly infected B lymphocyte subsets.3.Construction of HPgV-2 infectious cloneWe amplified the whole genome of HPgV-2 in segments and linked it into the skeleton vector one by one through a single restriction site.The preliminary results of cell transfection showed that the full-length clone constructed by us could save HPgV-2,but could not produce sustained and effective infection.Conclusions1.There is HPgV-2 infection in Chinese population and the gene sequences of HPgV-2 are highly conserved.HPgV-2 infection is associated with HCV,and HIV-1 infection can increase HPgV-2 infection rate in HCV population.2.HPgV-2 can form stable and resolved infection status;DAAs drugs may can't inhibit the replication of HPgV-2;HPgV-2 can form a persistent infection state in patients independently.3.HPgV-2 does not infect liver cells and cause obvious liver damage;HPgV-2 is a lymph phagocytic virus,and mainly infects B lymphocytes.4.The full-length clone of HPgV-2 genome was preliminarily constructed,which can rescue some infectious virus particles in vitro.