Study On Biological Characteristics, Genome Analysis,Diagnosis Methods And Infectious Clone For Newly Emerged Duck Tembusu Virus

In2010, a duck tembusu virus outbreak occurred in China. Duck tembusu virus rapidly spread in the entire duck-breeding area, resulting in a great loss in the duck industry. This virus is one of the major causes of diseases among ducks in China. This study focused on four aspects:biological characteristics of duck tembusu virus, genome sequencing and evolution analysis, establishment of diagnosis methods based on molecular biology and construction of an infectious clone.1. Virus isolation, identification, and characteristic analysisMore than10strains of duck tembusu virus were isolated from infected ducks in China. Physical and chemical characteristics indicated that this virus fails to cause red blood cell agglutination in chickens, ducks, geese, and pigeons. Furthermore, this virus is sensitive to chloroform, sodium deoxycholate, acid and heat. MgCl2does not provide protection. We observed this virus under an electron microscope and found that it is round or oval in shape with a size of50nm. This virus could be isolated from a chick or duck embryo that was sacrificed for approximately72h to120h, at which edema occurred, resulting in bleeding and necrosis of the embryonic liver. This virus could grow well in DEF, Vero, DF-1, BHK-21, and other cell lines with evident lesions. Different virus strains exhibited various modes of adaptations to CEF. For instance, some viruses could adapt to CEF after these strains were subjected to blind passage of three generations. Other strains could not adapt to CEF although these strains were subjected to blind passage of six generations. Animal regression test result showed that the typical symptoms and lesions of this disease could be manifested in different locations, particularly in the brain, subcutaneous tissue, eyes and nasal organs. However, the incubation period of the virus differed in a specific location. For instance, the virus exhibited the shortest incubation period in the brain; by contrast, this virus displayed the longest incubation period in the eyes as well as in the nasal organ and oral cavities.2. Genome sequencing and evolution analysisWe obtained the whole genome sequence of duck tembusu virus BZ-10from a random primer. Based on the genome sequence of BZ-10, we completed the genome sequences of five other duck tembusu virus strains. Analyzed these sequences, we found that the genomes of the six strains are all10,990nt in length, with an open reading frame (ORF) of10,278nt in length encoding a polyprotein3,426amino acid in length. The5’untranslated region (UTR) was94nt in length and the3’UTR was618nt in length. The polyprotein contained10proteins. Five strains had17glycosylation sites except WFZ-12strain, which loses a glycosylation site at the154th amino acid of E protein.Duck tembusu virus was also subjected to genetic evolution analysis. The results revealed that this virus exhibited a close genetic relation to tembumu virus, which was isolated from Malaysian Culex and Sitiawan virus from a diseased chick. The homologies are respectively88.7%and87.3%.The E protein space structure of duck tembusu virus was subjected to simulation analysis. The results showed the E protein contained three functional areas and two glycosylation sites, which are located at the103th and154th amino acid. However, the154th amino acid of WFZ-12strain, which is found in Tembusu virus and Bagaza virus, failed to undergo glycosylation. This result indicates that some mechanisms of infection of duck tembusu virus may change as the disease progressed.Phylogenetic analysis showed that duck tembusu virus could be divided into gene Ⅰ and gene Ⅱ according to their E gene. GeneⅡcould be further divided into two subtypes, Ⅱ.a and Ⅱ.b. All virus strains in were isolated in2010, which possibly hinted the virus had a little mutation in the past two years. However, the serum neutralization test of BZ-10, GX-11, and FX-12, respectively belonging to genes type Ⅰ, Ⅱa, and Ⅱb, showed that the neutralization index was>0.9. This proved that three viruses belonged to the same serum type.3. Establishment of diagnosis methods To detect the pathogen, we performed RT-PCR and fluorescent quantitative PCR (FQ-PCR). Clinical application showed that these two methods are rapid, accurate, highly sensitive, and specific. The limits of identifying the virus by RT-PCR and FQ-PCR are respectively1.0TCIDso/O.1ml and0.1TCID50/0.1ml. FQ-PCR is more sensitive than RT-PCR.In the antibody test, high flux indirect ELISA detection method based on prokaryotic-expression E protein was established successfully. The threshold quantity of this method was0.336, and the corresponding rate was95.0%contrasting serum neutralization test.4. Construction of infectious cloneFive fidelity DNA fragments covering the full genome of DTMUV BZ-10strain were amplified by RT-PCR, and inserted into pBR322vector in order, resulting in the full-length cDNA clone. The sequence homology of cDNA and parental virus strain was99.9%. The non-coding areas of the5’end and the3’end were completely the same. The arrangement of the amino acids in polyprotein was also consistent.The in vitro-transcribed RNA from full-length cDNA was transfected into DF-1cells and the rescued virus was identified using RT-PCR, indirect immunofluorescence assay and restriction enzyme site identification after five generations. The results showed that infectious virus named BZ10-R was rescued successfully. The ELD50of BZ10-R in the allantoic fluid was1046/0.2mL and the TCID50in DF-1was1053/0.2mL. These values were slightly different from those of the parental strain.Because pBR322vector we selected has a limit capacity for exogenous gene, the cDNA clone that we obtained was not stable, thereby causing experimental challenges in subsequent studies.