Establishment Of The Reverse Genetics System Of Tembusu Virus MM1775 Strain And Biological Characterization Of The Rescued Virus

The newly emerged duck Tembusu viral disease caused by duck Tembusu virus(DTMUV) had resulted in a huge economic loss to the Chinese duck industry since 2010. The infectious disease was characterized by severe egg production decline of laying ducks, with other symptoms such as high fever, loss of appetite, dyskinesia and even death. Tembusu virus MM1775 strain, the original isolation, was isolated in mosquitoes in Malaysia.Several Tembusu virus strains were isolated in the following decades, but there were few animals infected by Tembusu viruses.Biological characteristics of early mosquito-origined Tembusu viruses, such as replication, pathogenicity and transmissibility in different animals remain unknown. The knowledge on the differences in the biological characteristics between early TMUV strain and DTMUV will help us understand pathogenic mechanism of TMUV.In this study, the complete genome sequences of TMUV MM1775 strain were obtained to compare the differences in the genome sequences between TMUV MM1775 strain and DTMUV FX2010 strain. 5’ and 3’ terminal sequences of MM1775 were obtained by using 5’ and 3’ RACE techniques. Ten segments of RT-PCR products, overlapping at the 5’ and the 3’ ends, were purified for sequencing, and then assembled to gain the whole genome sequences of MM1775. Compared with FX2010, there were 5, 4, 16, 21, 16, 4, 8, 4, 5, or 18 amino acid residue changes in C, pr M, E, NS1, NS2 A, NS2 B, NS3, NS4 A, NS4 B or NS5 protein, respectively. The different pathogenicity of the two virus strains might be determined by these changes. Three segment of c DNA, covering the complete genome of MM1775, were inserted into three plasmids respectively by cloning and sub-cloning methods. The full-length c DNA, with T7 promoter sequence added at 5’ends, obtained by fusion-PCR using the above three recombinant plasmids as templates was transcribed in vitro to produce the virus RNA. The virus RNA was transfected into DF-1 cells to rescue virus. Cytopathic effect(CPE) was found at 48 hours post-transfection. When 90% transfected-cells showed CPEs, the supernatant was collected to infect fresh DF-1 cells. Specific green fluorescence could be detected in the newly infected cells by indirect immunofluorescence(IFA) and specific RT-PCR products could be obtained from the supernatants at 72 hours post-infection, suggesting the success of TMUV MM1775 rescue. Sequence analysis showed the rescued virus have no unexpected nucleotide changes compared with parental virus.In this study, we compared the differences in the replication and pathogenicity between TMUV MM1775 rescued strain(R-MM1775) and the newly emerged DTMUV FX2010 strain in SPF embryos, mice and ducks. R-MM1775 strain and FX2010 strain showed similar pathogenicity for the 9-day-old SPF embryos. The dead embryos showed severely swollen, congested and hemorrhaged. Mice inoculated i.n. with 105.0TCID50 of R-MM1775 showed severe weight loss at 6-10 days post-inoculation(dpi) and died at 10-12 dpi. High titers of virus could be detected in lungs and brains of mice at 4 dpi. However, mice inoculated i.n. with 105.0TCID50 of FX2010 strain showed no apparent weight decline and survived during experiments and vruses could be detected in the lungs of mice but no viruses were detected in the brains of mice at 4 dpi. For the ducks inoculated i.m. with 103.5TCID50 of R-MM1775 strain, high titers of virus were detected in the spleens, and low titers of virus were detected in livers and ovaries, and no virus was detected in other tissues at 4 dpi. However, the ducks inoculated i.m. with 103.5TCID50 of FX2010 strain showed systemic infection and high titers virus could be detected in most tissues of ducks at 4 dpi. For the ducks inoculated i.n. with 103.5TCID50 of DTMUV, virus could be detected in 2/3 MM1775 inoculated-ducks, while high titers of virus could be detected in most tissues of FX2010 inoculated-ducks. The R-MM1775 strain could not transmit from inoculated ducks to contact ducks while FX2010 strain could transmit from inoculated ducks to contact ducks. These results indicated that early isolation of Tembusu virus has limited transmissibility among ducks compared with DTMUV FX2010 strain.In conclusion, a stable infectious c DNA of TMUV MM1775 strain was constructed to facilitate further studies of TMUV pathogenesis. Moreover, there were significant differences in the pathogenicity between mosquito-origined Tembusu virus MM1775 rescued strain and duck-origined Tembusu virus FX2010 strain for the mice and ducks.