Impacts Of The Poly(A) Length On Viral Replication Of DHAV-1

Duck viral hepatitis(DVH)type A,a highly contagious and lethal infectious disease which was caused by duck hepatitis A virus(DHAV),can rapidly spreads infection in young ducklings aged 1 to 3 weeks.The dead ducklings exhibited obvious opisthotonus and liver damage that characterized with enlargement and hemorrhage.DVH is distributed worldwide,and is also considered as the major disease in duck industry.Even though people start to pay attention to DVH in recent years and the complete genome of DHAV was known to public,researchers still focused on study on etiology and detection methods,and the cognition on viral replication and infection mechanism is still limited in the histology and serological levels of last century.Many studies have shown that the viral replication and infectivity efficiency has intimate relationship with 5? untranslate region(5'UTR)and 3' untranslate region(3'UTR).The poly(A)tail also play important role in determine viral replication and infectivity efficiency.Yet up to now,there was no report about the relativity between poly(A)tail and viral replication and infectivity efficiency of DHAV-1.We have constructed a infectious clone of DHAV-1 before and the rescued virus derived from it has similar replication and infectivity efficiency compared to parental strain.But the in vitro transcription has raised experimental complexity and cost.In this study,we constructed a DNA-Launched infectious clone by adding two ribozyme sequences with self-cleaving activity on both terminals of viral genome based on the foundation of RNA-Launched infectious clone.The rescued virus could be harvested by transfected directly with recombinant plasmid.We also constructed a series of DNA-Launched infectious clones with various length of poly(A)tail by designing primers to reveal the relationship between the length of poly(A)tail and viral replication and infectivity of DHAV-1.1.Construction of DNA-Launched infectious clone of DHAV-1 strain LY0801Four pairs of specific primers were designed to amplify four fragments covering the entire viral genome,which was then assembled into vector based on multiple clone site region of DHAV-1 and vector.The hammerhead ribozyme sequence and hepatitis delta virus ribozyme were added on both terminals of viral genome.The base T at 3042 position was mutated to C to create a new BamHI restriction enzyme site,which was then treated as a genetic marker to distinguish parental and rescued virus.The recombinant plasmid of DNA-Launched infectious of DHAV-1 was then confirmed by sequencing and no mutation of nucleotide and amino acid was found exclude the base C at 3042 position.The recombinant plasmid was then quantitated and transfected directly into BHK-21 cells,the positive and negative control was also conducted.At 60 hpt,cell culture was collected and treated with three freeze-thaw cycles to propagate blank BHK-21 cells.After three times passages,the IFA/RT-PCR assays were conducted to confirm that the rescued virus was harvested by transfected directly into BHK-21 cells.2.Bioactivity analysis of the rescued virusThe cell culture was collected and treated with three freeze-thaw cycles to propagate blank BHK-21 cells at 60 hpt.After three times passages,the rescued virus was used to propagate blank BHK-21 cells in six-plate well.Cells and supernatant was collected separately every 12 h at 24 hpt and the parental virus and PBS was conducted as positive and negative control.The quantitative real time fluorescence PCR was conducted to detect viral copies number at each detection point.The growth characteristics of cells and supernatant indicated that rescued and parental virus shared similar replication and virulence efficiency.In order to measure virulence of rescued virus to young duckling,60 young ducklings were divided into three groups: parental group,rescued group and negative control group and then inoculated intramuscularly with 0.25 ml of 104 TCID50 of rDHAV-1,104 TCID50 of pDHAV-1 of strain LY0801 and aseptic PBS(pH7.2),respectively.Survival curve indicated that rescued virus and parental virus shared similar virulence to young duckling.The dead ducklings showed obvious clinical symptoms of duck viral hepatitis.In order to confirm that the rescued virus has similar tissue tropism,100 healthy ducklings were divided into two groups and then inoculated intramuscularly with 0.25 ml of 104 TCID50 of rDHAV-1,104 TCID50 of pDHAV-1 of strain LY0801,respectively.The heart,liver,kidney,spleen,thymus and Bursa of bursa were collected after 1h,6h,12 h,18h,24 h,48h and 72 h,separately.The viral RNA was extracted and then used for qRT-PCR to detect viral copies number in each tissue.The detected results showed that rescued virus has similar tissue tropism with parental virus.3.The length of poly(A)tail has impact on viral replication and infectivity efficiencyIn this study,we constructed a series of DNA-Launched infectious clones with various length of poly(A)tail consisted of 0/5/10/15/20/25/30/40 adenine(s)by designing specific primers.After transfected the series of DNA-Launched infectious clones with various length of poly(A)tail into BHK-21 cells,supernatant and cells were collected separately and then used for RNA extraction.During the RNA extraction assay,the DNasI was used to digest the residual recombinant plasmid DNA.The DHAV-1 genomic RNA was quantified by a Taqman real-time RT-PCR.The viral RNA copies in BHK-21 cells category peaked at 24 hpt and began to decrease in the next 24 hours.At 48 hpt to 60 hpt,DHAV-1 genomic RNA start to increase.Unlike the growth characteristic in cells,the genomic RNA copies of the rescued viruses reached one peak of replication in the supernatants at 48 hpt and start to decrease since then.The viral copies number in cells represent the replication efficiency of rescued virus,and the growth curve of rescued virus derived from series of DNA-Launched infectious clones with various length of poly(A)tail indicated that the rescued virus with a poly(A)tail consisted 25 adenines has highest replication efficiency.Since there was no DHAV-1 genomic RNA in supernatant at first,therefore the viral copies number in supernatant represents the infectivity efficiency and the experimental data showed that the rescued virus with a poly(A)tail consisted 20 adenines has highest infectivity efficiency.In addition,the growth curve of cell culture has similar growth characteristic with cell category indicated us that the viral copies number in cells category were way much higher than that in supernatant.4.Growth characteristic of rescued virus derived from poly(A)0 groupAfter we transfected a series of DNA-Launched infectious clones into BHK-21 cells,we found that the viral copies number in poly(A)0 group start to increase at 48 hpt.In order to identify whether the DHAV-1 could replicate in BHK-21 cells without poly(A)tail,we transfected the poly(A)0 group DNA-Launched infectious clones into BHK-21 cells.The cell culture was collected and treated with three freeze-thaw cycles and used to infect blank BHK-21 cells.After three times passages,the RT-PCR,IFA assay was conducted and the positive results indicated us that the rescued virus was harvested in poly(A)0 group.The success of rescued virus derived from poly(A)0 group DNA-Launched infectious clone showed clear point that the poly(A)tail was not absolutely required for viral replication of DHAV-1.In order to measure the growth and infectivity characteristic of rescued DHAV-1 without poly(A)tail,the transfected product was serially passaged in duck embryo and BHK-21 cells for 20 generations.The 3'-Full RACE Core Set with PrimeScript? RTase was used to identify the accurate number of adenines of the poly(A)tail in each generation.Cell culture and alantoic fluid were used for RNA extraction which was then used for viral copies number detection by the Taqman real-time RT-PCR.The experimental data showed that the genomic RNA of DHAV-1 could give rise to infectious particle and could replicate in duck embryos and BHK-21 cells,yet had a lower replication and infectivity efficiency compared to parental group.After serially passaged in duck embryos and BHK-21 cells,the tailless DHAV-1 could replicate in BHK-21 cells and duck embryo with a restored poly(A)tail of 12 residues since passage 4.This experimental data showed that the poly(A)tail was not absolutely required for viral replication and infectivity of DHAV-1,and the tailless genomic DHAV-1 could give rise to infectious viral particle with a restored poly(A)tail consisted 12 adenines.