Molecular Epidemic Analysis Of Duck Tembusu Virus In China

Duck tembusu virus (DTMUV) is a novel infectious agent that caused severe egg drop in duck and huge economic loss in duck-breeding industry. To illustrate the molecular characteristics and genome profiles of DTMV, and help for the prediction and controlment of the disease, we carried out the molecular epidemic analysis of duck Tembusu virus in China.We collected187and252clinical samples suspected of containing DTMUV from nine provinces in china during2010and2011, respectively. By using reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for DTMUV, forty-two out of187samples in2010and thirty-seven out of252samples in2011were test to be positive for DTMUV. The positive rate of the samples decreased from22.5%in2010to14.7%in2011, and most of positive samples were collected from Sheldrake duck which was66.7%in2010and40.5%in2011, respectively. Except from the breeding duck and egg-laying duck, the positive samples also came from meat duck, meat goose and egg-laying chicken.A fragment of NS5gene of DTMUV was amplified for molecular variance analysis of DTMUV. NS5gene fragments of nine strains obtained in this study were alignment with those that retrieved from GenBank database. Results showed that NS5genes’ diversity of the different virulent strains that isolatd from different time、region and host had no correlation and the sequences obtained in this study shared a high identity of more than98%with those of the contemporaneous isolates, and shared a similarity with mosquito-vector Tembusu viruses deposit in GenBank between87.6%-92.0%. Phylogenetic analysis based on NS5gene illustrated that all of the contemporaneous DTMUV isolated from poultry were gathered into the same clade, while the Tembusu virus Sitiawan strain isolated from chicken in2000in Malaysia is closed with Mosquito-vector Tembusu viruses.To obtain the profile of the whole genome sequence of DTMUV,9sets of primers were designed for determination of the whole genome sequence of FJMH220strain. The5’ end and3’end sequence of viral genome were acquired by using rapid amplification of cDNA ends (RACE) methods. Results showed that the genome structure of FJMH220strain was the same as that of Tembusu virus which has been deposited in GenBank. The genome of FJMH220strain is10,990nucleotides (nt) in length and encode a putative ployprotein of3,426amino acids. The length of the5’and3’noncoding region (NCR) of the genome is94and618nt, respectively. Identity analysis showed that FJMH220strain shared a highest similarity with the DTMUV isolated at the same periods in other places; the polyprotein shared more than98.4%nucleotides identity and more than99.2%amino acid identity with these isolates, and shared72.1%nucleotides identity and81.7%amino acid identity with Bagaza virus which belongs to the same Ntaya virus group. Phylogenetic analysis showed FJMH220strain cluster into Ntaya virus antigen group and formed an subcluster group with other contemporaneous DTMUV isolates.The present results indicated that DTMUV is still prevalent in poultry in china although the positive rate is higher in2010than in2011, and the virus host range continues to expand, but NS5genes’ diversity of the different virulent strains that isolatd from different time、region and host had no correlation. The genome sequence of DTMUV isolate determined in this study share a highest identification with other strains isolated at the same time in genome composition and sequence. Intensive surveillance and genetic analysis of DTMUV will contribute to predication of emergence of novel viruses with potential threat, selection of DTMUV vaccine strains and developing pandemic vaccines and have great theoretical significance to the disease prediction and control.