The Characterization Of Tembusu Virus And Inhibition Of Tembusu Virus Replication With Recombinant Adenovirus Expressing Shrna In Vitro

Tembusu virus (TMUV), the causative agent of duck hemorrhagic ovaritis (DHO)disease or encephalitis in ducklings, mainly infects20day old aged causes increasingmortality, neurological symptoms, and a severe drop in egg production within1to2weeksafter the ducks were affected. Hyperemia, hemorrhage, degeneration, distortion andlymphocyte infiltration in the ovaries, portal area interstitial inflammation in the livers werethe main pathological changes observed consistently in almost all diseased ducks. Othersymptoms include yolk peritonitis, diarrhea, severe hemorrhages in spleen and pancreas.TMUV which belong to mosquito-borne flavivirus of the Ntaya virus group was knownas disease agents causing severe clinical symptoms in vertebrates and are transmitted bymosquitoes. TMUV share several common aspects like other flaviviruses: common size(40-65nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense,single-stranded RNA of approximately11,000bases), one long open reading frame thatincludes three5’ structural genes and eight3’ non-structural genes, and appearance in theelectron microscope. As the major surface protein of TMUV, the envelope (E) protein isinvolved in many events, such as viral attachment, fusion, penetration, hemagglutination, hostrange and cell tropism. NS5is the largest TMUV protein; NS5is also the most conserved oneacross the genus. Early on a motif of AdoMet-dependent MTases was identified within theN-terminal domain of NS5whereas RdRp motifs were identified in the C-terminal domain ofprotein NS5. In this study, we used different detection methods to determine the TMUV hostrange and the spread of the virus. Moreover, the way of the inhibition of TMUV replicationwere established by recombinant adenovirus expressing shRNA targeting on E and NS5genesof TMUV in HEK293cells.1. Development and application of a semi-nested RT-PCR assay for detection of TMUVAccording to the sequence of NS3gene of flavivirus Bagaza strain published in GenBank,three primers were designed and synthesized. A semi-nested RT-PCR assay for rapiddetection of TMUV was established. A specific277bp fragment was amplified from RNAtemplates of TMUV of Shandong isolates, but no bands were amplified with templatesextracted respectively from duck plagues virus（DPV）, avian influenza virus (AIV) subtype H9, Newcastle disease virus(NDV), infectious bursal disease virus (IBDV)，egg dropsyndrome virus(EDS). Sensitivity of the amplifications by the semi-nested RT-PCR assay was1×105copies/μL and1×102copies/μL, respectively. The sensitivity of the2nd amplificationswas increased by103times and had a high detection rate of clinical samples. These resultssuggested that the semi-nested RT-PCR assay could be used as a method for the diagnosis anddetection of clinical cases, and for molecular epidemiological investigation of TMUV.2. Rapid detection of TMUV by reverse-transcription, Loop-mediated isothermalamplification (RT-LAMP) and the comparison of four nucleic acid detectionmethods of Tembusu virusA sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP)assay was developed for the rapid detection of TMUV infection.The reaction was performedin one step in a single tube at64℃for45min, with SYBR GreenⅠdye added prior toamplification. The detection limit of the RT-LAMP assay was approximately10copies/μl,and no cross reaction with other avian viruses was observed. The assay was evaluated furtherfor the diagnosis of TMUV in field samples and compared with conventional RT-PCR,demonstrating that results of the RT-LAMP assay corresponded to those of conventionalRT-PCR. In conclusion, RT-LAMP with SYBR GreenⅠdye was shown to be a sensitive,simple assay for the rapid diagnosis of TMUV infection in ducks.For the detection of TMUV, we investigated the performances of conventional RT-PCR(C-RT-PCR), semi-nested PCR (SN-RT-PCR), reverse-transcriptase real-time quantitativePCR (Q-RT-PCR), and reverse-transcription loop-mediated isothermal amplification(RT-LAMP) targeting the TMUV-specific NS5gene. To compare the detection sensitivitiesof the four techniques, we used two template systems that used plasmid DNA (plasmiddetection sensitivity), including a partial region of the NS5gene, and genomic RNA (genomicdetection sensitivity) from TMUV-positive cell culture supernatants. The plasmid detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were2×104copies/μl,20copies/μl,2copies/μl, and20copies/μl, respectively. The genomic RNA detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were100pg/tube,100fg/tube,10fg/tube, and100fg/tube, respectively. The TMUV-specific RNA was detectedin cloacal swabs of experimentally infected ducks by these four methods. All control samples (not inoculated) were negative by the four methods. The sensitivities of RT-PCR,SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs samples taken fromducks within2weeks of severe egg-drop were38/69(55.1%),52/69(75.4%),57/69(82.6%),and55/69(79.7%), respectively. All evaluated assays were100%specific for TMUV. Inconclusion, given its combined sensitivity, specificity, and speed, RT-LAMP is the preferredassay for the detection of TMUV.3. Characterization of TMUV isolated from different hostFor the characterization of TMUV isolated from different host, the sample from68housesparrows,35pools of mosquitoes,73laying hens,132egg-laying ducks, and112ducklingswere collected at different sites in Shandong province in2010-2012. The samples were firsttested by RT-PCR. Then the positive tissues were used for TMUV isolation and identification.The detection rates of NS3-based RT-PCR were66%(45/68),60%(21/35),33%(24/73),62%(82/132), and69%(77/112), respectively. Finally, a TMUV isolate were obtained fromevery kind of samples. BLAST results showed that the NS5sequences of five isolatesdetermined in this study had nucleotide homology above99.5%. The TMUV-SDHS hadnucleotide homology above99%with YY5and BYD strains, and ranging from87.0%to91%with the published abroad TMUV sequences in GenBank. The TMUV-SDHS isolates werehighly pathogenic to50-week-old healthy Cherry Valley Pekin ducks, and the pathologicchanges were similar to the clinical cases. The results showed that the TMUV infection wasvery common in Shandong province; the TMUV infection not only in egg-laying ducks and ducklings, but also in laying hens and house sparrows without pathologic changes; housesparrows and mosquitoes carrying the TMUV may play an important role in transmitting thevirus among birds; the infections in house sparrows is important for overwintering of TMUV.4. Analysis of the complete genome of TMUVIn this study, we obtained a complete genome sequence of TMUV using RT-PCR andRACE techniques. TMUV genome is single-stranded RNA,10,990nucleotides in length andcontaining a single open reading frame (3410amino acids) encoding11viral proteins with5’and3’nontranslated regions (NTRs) of142and618nt, respectively. We characterized theopen reading frame (ORF) with respect to gene sizes, cleavage sites, potential glycosylationsites. The different genomic regions of virus were also compared with those of five other flaviviruses including Japanese encephalitis virus, West Nile virus, dengue-2virus, yellowfever virus, Tick-borne encephalitis virus and Bagaza virus. TMUV demonstrated the highestsimilarity to Bagaza virus. The result of entire ORF scanning shows TMUV was close toBagaza viruses in genetic relatedness. The nucleotide sequences of conserved sequences (CS)of TMUV are most identical to the corresponding consensus CSs. Thus, including therepeated CSs (RCSs), the CS organization (in5’-3’ direction) in the3’-UTR isRCS3-CS3-RCS2-CS2-CS1for TMUV. These data demonstrate that TMUV is a unique virusamong the mosquito-borne flaviviruses and also provide a useful reference for a criticallyimportant study to determine why TMUV is a serious pathogen for ducks.5. Adenovirus-mediated RNA interference against TMUV replication in vitroRNA interference (RNAi) is the process of mRNA degradation that is induced bydouble-stranded RNA in a sequence-specific manner, which is called post transcripitionalgene silence (PTGS). RNAi has revolutionary approaches in the study of gene function andbecome a powerful new tool to identify the function of gene. Importantly, it is great hopefulto be a new therapy strategy for virus diseases.Firstly, the results of fluorescence microscopic observation, flow cytometry revealed,westernblot, and virus infection revealed that all the shRNA vector could inhibit E or NS5fusion protiein in293T cells in the screening test of shRNA. The inhibition effect was highestin E1(75.8%) and NS52(68.7%). But in inhibition of the virus replication, the NS52washighest in all six shRNA vector(86%).Secondly, we constructed the recombinant adenovirus vector with shRNA-NS52usingAdMax system. The expressing kit shRNA-NS52transferred from pSilencer to pDC312forthe construction of shuttle vector. There combinant shuttle plasmid, adenovirus genomicplasmid pBHGloxΔE1were transfected into HEK293cells to construct the recombinantadenovirus pAd-NS52. The recombinant adenovirus vectors of pAd-NS52and pAd-CMVwas successfully constructed with titer of2.1×109PFU/ml.Finally, we infected HEK293cells with recombinant adenovirus pAd-NS52. After24h,the cells were challenged with TMUV. The TMUV RNA was detected by Real-time RT-PCRat48h post-infection of TMUV. The result showed that pAd-NS52could inhibit thereplication of TMUV and caused a significant reduction in TMUV viral RNA production (88%). The antiviral effect was highest at500MOI dose.