Immunogenicity And Molecular Basis For Attenuation Of BHK-21 Cell-adapted Duck Tembusu Virus

Tembusu virus(TMUV),a member of the the genus flavivirus,is the emerging agent that causes severe egg drop and nervous signs of domestic waterfowls.The infection is endemic in China mainland since it first outbreak in April 2010.The goal of present study was to develop diagnostic methods for seroepidemiological investigation and cell culture-based vaccine candidates for the prevention of the disease in ducks.In the first experiment,a hybriodoma secreting monoclonal antibody(MAb)specific to the TMUV envelope protein E was generated.Based on this MAb,a blocking ELISA was developed for detection of TMUV specific antibodies in multiple poultry species.A total of 2705 serum samples collected from different waterfowl farms during 2009 to 2015 were detected,indicating that TMUV infection occurred sporadically in Northern China.This finding was confirmed by the virus isolation.In the second experiment,TMUV JXSP strain was successfully propagated on BHK-21,inactivated with beta-propiolactone(BPL)and emulsified with mineral oil.The efficacy of different vaccination schedules was assessed in laying ducks and table ducks using virus challenge experiments.Two doses of vaccine provided efficient protection against the virus challenge to avoid the egg production drop in laying ducks.An ELISA demonstrated that 97%(39/40)of ducks seroconverted on day 21 after one dose of the inactivated vaccine and that significant increases in antibody titers against the virus were induced after the second immunization.For table ducks,a single dose of vaccine immunization resulted in a protection index of 87%and significant reduction of viral loads in tissues.Sterilizing immunity can be attained after second immunization.These data provide a scientific basis for the development of an inactivated vaccine for the prevention of duck TMUV infection.To develop a live attenuated vaccine,TMUV JXSP strain was serially passseged on BHK-21 cell culture and the pathogenicity to ducks were assessed by subcutaneous infection with the 150th,280th and 310th generation of the passaged virus separately.Our resuls showed that the virulence of the cell-adapted virus decreased with the passage.The 310th passage of the virus,named as JXSP2-310,was highly attenuated,which was confirmed by infection experiments in both ducklings and laying ducks.Antibody responses of laying ducks were detected 4 days after vaccination with JXSP2-310 via the subcutaneous injection.Ducks were resistant to the virulent virus challenge on day 10 after vaccination,while the unvaccinated ducks exhibited rapid reduction of egg production rate during the experiment period.Genome sequence analysis revealed 23 amino acids mutation occurred in JXSP2-310 in comparison with its parent strain JXSP2-4.To investigate contributions of the genetic mutations to the pathogenicity,the gene encoding the E protein of the virulent parent strain JXSP2-4 was replaced with the corresponding fragment of the attenuated strain JXSP2-310,in which 5 amino acids were found to be mutated.Compared to the wild type strain,the recombinant virus showed attenuation of viulence with significant lower virus loads in tissues of infected ducklings and mice,supporting that the E protein plays a key role in the TMUV pathogenesis.Deep sequence analysis of the the full-length genome of the wild type strain JXSP2-1,BHK-21 cell-adapted virus JXSP2-150,JXSP2-280 and JXSP2-310 revealed the genetic evolution of the virus.It showed that there were 30 nucleotide mutation sites distributed over viral genome of JXSP2-1,indicating it was consist of diverse quasispecies.The nucleotide subsitution variants recovered from cell-adapted virus samples increased with the passage and the quasispecies structure was completely different from the wild type virus.The reslut of diversity indices indicated that quasispecies diversity of attenuated virus was significantly increased in comparison with parental strain.These data demonstrated that gene mutations during the cell culture passage contributed to the virus fitness and impaired its pathogencity in simutaneously.