Whole Genome Sequencing Of A Torque Teno Virus In Rodent And Establishment Of A Screening Model For Antiviral Drugs Targeting HRSV In Vitro

Objective:In recent years,viral infectious diseases have led an important impact on human health,so diagnosis and treatment of viral infectious diseases have become an important part of clinical treatment.In this study,based on the high-throughput genome sequencing analysis,a local endemic strain of Rodent Torque teno virus?RoTTV?was founded in Rattus norvegicus,Haikou City,Hainan Province.In order to study the potential threat of rodent-derived TTV in Hainan Province,whole-genome sequencing and phylogenetic analysis be performed,and a SYBR Green I based real-time PCR detection assay for RoTTV was established.At the same time for the viral infectious diseases treatment,a cell screening model for anti-Human Respiratory syncytial virus?HRSV?was established,and the screening model was used to screen Natural product library to provide new antiviral drugs.Methods:1.Complete Genome Sequence of a Rodent Torque teno virus:Based on the high-throughput genome sequencing analysis of the RoTTV strain,RoTTV 3-HMU 1was founded.The whole genome sequence was amplified by PCR amplification.The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control.The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.2.Establishment of the inhibition of Human Respiratory syncytial virus A?HRSV-A?by cell in vitro antiviral experiment:To develop a Hep-2 cell-based assay that measures inhibition of HRSV by CCK-8 assay or conventional CPE method.Then the two methods for screening model for antiviral medicine were compared.A more accurate screening model for antiviral drugs targeting HRSV in vitro was selected.The model for antiviral drugs targeting HRSV in vitro was used to screen natural product library and finally the antiviral effect of the drug were determined by measuring the TI of the effective drugs.Results:1.After viral DNA was subjected by high-throughput Sequencing,19,863 TTV-related reads was obtained.The contigs were alligned with the NCBI nucleiotide database and 5 contigs with an N 50 of 361 bp was matched to RoTTV 3-HMU 1,covering the 68%of the whole RoTTV 3-HMU 1 genome.The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced.Phylogenetic analysis indicated that the virus belongs to the RoTTV 3 genotype.To establish the real-time PCR detection method of RoTTV 3,the standard curve generated had a wide dynamic range from 1×10²¹×10⁸copies/mL,with a linear correlation?R²=1.000?.The melting curve analysis using SYBR Green?showed only one specific melting peak and no primer-dimmers represented.The detection limit was 100copies/reaction.2.Positive drugs that have already been published was used to evaluate the difference between CCK-8 method and traditional CPE method in screening antiviral medicines.The results showed that the positive drugs did have antiviral effect which is in consistent with report.However,the CCK-8 method was more sensitive,convenient and objective than traditional CPE method.HRSV drug screening provides an effective platform.Using the established inhibition of Human Respiratory syncytial virus A?HRSV-A?by cell in vitro antiviral experiment,7 effective compounds were screened out from 395compounds in the natural product library,and the best compound was EGCG with the TD₅₀ was 39.93?g/m L,EC₅₀ was 2.039?g/m L and the TI was 19.6,which showed that EGCG could inhibit HRSV-A.There was a significantly difference between the drugs group and the virus group?P<0.05?,indicating that EGCG has an antiviral activity in vitro for HRSV-A.Conclusion:1.The genomic sequence of RoTTV carried by rodent was successfully sequenced,phylogenetic analysis indicated that the virus belongs to the RoTTV 3 genotype.2.The real-time PCR detection method of RoTTV 3 was established.The standard curve generated had a wide dynamic range from 1×10²¹0⁸ copies/mL,with a linear correlation?R²=1.000?.The melting curve analysis using SYBR Green?showed only one specific melting peak and no primer-dimers represanted.The detection limit was 100 copies/reaction.3.CCK-8method and traditional CPE method have the same effect on the antiviral drugs targeting HRSV-A in vitro.4.Exposing Hep-2 cells with different concentration of EGCG could inhibit HRSV-A.