| Tembusu virus(TMUV) is a contagious pathogen of waterfowls including ducks and geese, with symptoms of high fever, loss of appetite and fall in egg production. It was originally isolated in eastern of China in 2010. Since then, the disease was reported in all major duck breeding areas of China. The main pathological changes observed in the affected ducks are ovarian hyperemia, hemorrhage, degeneration, macrophage and lymphocyte infiltration, distortion and hyperplasia. Morbidity was usually high(up to 90%), and mortality varied from 5% to 10%. To date, duck TMUV disease caused an enormous loss on many egg-laying and breeder duck farms in China.TMUV virion was 50 nm in diameter, coated with the capsule membrane and a 10,990 nucleotides single-stranded positive-sense genomic RNA inside. The translation product of viral genome is a polyprotein, containing 3 structural proteins( capsid, pr M, envelope) and 7 nonstructural proteins(NS1, NS2 A, NS2 B, NS3,NS4 A, NS4 B, NS5), with 94 nt in 5’ UTR and 618 nt in 3’ UTR in both terminus of the ORF sequence. Previous study showed that the untranslated regions of flaviviruses play important role in viral replication, RNA translation and virulence. In our study, two waterfowl-origin TMUV strains were isolated and identified. The full-length of genomic of both isolates were sequenced and analyzed. An infectious clone of TMUV ZC strain was established to research on the structural function of genome, function of viral proteins and the mechanism of molecular pathogenesis. Deletion of conserved sequences and point mutations of the second terminal nucleotide of 3’ UTR using the ZC infectious clone and fusion PCR technology to research on the biological function of these elements in 3’ untranslational regions.(1) Establishment of TMUV Taqman-based real-time FQ-RT-PCRAccording to the sequence of NS1 gene of TMUV isolates published in Genbank, a pair of primers and a taqman probe inside were designed and synthesized. A Taqman-based real-time fluorescence quantitative RT-PCR rapid detection of TMUV was established. The results showed that the standard curve of the assay was: y=-2.9227x+42.083 and R2=0.9972. There were no amplified products with templates extracted respectively from duck plagues virus(DPV), avian influenza virus(AIV) subtype H9, Newcastle disease virus(NDV), duck hepatitis virus(DHAV), duck reovirus(DRV) and duck circovirus(DuCV) which could affect ducks. Sensitivity of the amplifications by the taqman Q-RT-PCR was 29 copies/μL. Viral genome in air or swabs colud be well detected by the assay. The disadvantage is the need of Real-Time PCR system.(2) Isolation, identification and whole genomic sequence analysis of two TMUV isolatesTwo TMUV strains were isolated from a breeding ducks farm in Zoucheng county and a geese farm in Linqu county which were suspected infected with TMUV. According to the full-length genome sequences of TMUV isolates in Genbank database, six pairs of primer were designed to overlap the TMUV genome cDNA sequence, and the 5’ UTR sequence was amplified by 5’ race PCR. 9-day-old duck embryos were inoculated with the two TMUV isolates and the ELD50 of those were 10-4.2/mL(LQ) and 10-3.6/mL(ZC), respectively. 5-day-old ducklings showed neurological symptoms and mortality post infected with two strains and monitored for seven days. The homologous analysis of full length genome sequences between the two isolates and other TMUV isolates showed the homologous between SX1 strain and the two ioslates were nearest and the rate were 99.1% and 99%, respectively. The amino acid homology phylogenetic analysis of E and NS1 proteins amone these TMUV ioslates were conserved and no significant differences were observed. The furthest homology with most TMUV isolates were FJMH220, AH2011 GX2013 G strains which were isolated from muscovy in south of China. It indicated that further study should be focused to examine the relationship between TMUV islolates and the hosts.(3) Establishment of TMUV ZC strain reverse genetics systemAccording the the full-length genomic sequence of ZC sstrain, genome sequences of TMUV isolates published in Genbank database and the restriction sites analysis of vector and TMUV genome cDNA sequences, seven pairs of primers were designed to amplify the full length TMUV genome cDNA sequence. The multiple cloning sites of low copy plasmid pWSK29 were replaced by a new sequence which contained the right MCS used for digestion and ligation and were named pWSK29 C. Seven amplified fragments were cloned into pWSK29 C, with T7 promotor, enhanced element TAA and Apa I restriction site added in 5’ UTR, and T7 terminator sequence with Avr II upstream and Not I downstream in 3’ UTR. The 10495 nt G mutation was used as a molecular marker and the recombinant plasmid were named pWSK29C-DTMUV. The vector was linearized by Not I and then transcripted to infectious RNA by T7 RNA polymerase in vitro. RNA was purified and transfected into BHK21 cells. Cells were cultured 72 h and thawed three times, then centrifuged. The supernatant was harvested. The results showed that TMUV ZC strain infectious clone was correctly constructed. The rescued virus could reacted with TMUV Mab 12B1 and the green fluorescence was visible. The 10495 nt was G in the rescued virus. Eld50 of the rescued virus was 10-3.8/mL. Ducklings observed the same symptoms infected with rescued virus and parent virus. The one-step growth curves of two strains and ducklings survival curves were similar.(4) Preliminary Study of 3 ’untranslated region functionsTo construct the infectious clones of CS sequences deletion and second terminal nucletide mutation in 3’ UTR of ZC strain plasmid using pWSK29C-DTMUV skeleton, the recombinant strains were rescued and identified. Fusion PCR was used for fragment mutation. All operations were the same as the original rescued strain. The results showed that all of the virus strain were successfully rescued except the CS1 sequences delta strain. It indicated that mutation C to G of the second nucletide in 3’ terminal did Not block the proliferation of TMUV, and C to A, T or delta could seriously block the TMUV genome replication. No significant differences were observed between Delta of RCS2 and RCS3 strains and parent strain and CS2 and CS3 delta could block the proliferation of TMUV. Further study should focused to the pathgen of the mutation strains. |
PDF Full Text Request