Study On The Protective Effect Of The Duck Tembusu Virus Inactivated Vaccine On The Epidemic Srains

Objective:Duck Tembusu virus disease is a highly contagious disease caused by Tembusu virus,which is characterized by a sharp decline in duck egg production.The disease has caused great economic losses to domestic duck breeding industry,and has been popular in China for more than ten years.At present,vaccines have been listed for the prevention of duck Tembusu virus disease in China,but on the other hand,cases of immunization failure in duck farmers have occurred occasionally in recent two years.Over the years,researchers have continuously isolated duck Tembusu virus epidemic strains from diseased ducks around the country.It is an urgent problem to analyze the variation of the epidemic strains to clarify whether the existing vaccine has a protective effect on the epidemic strains.In this study,two batches of inactivated duck Tembusu virus vaccines were prepared using EB66 cells cultured by serum-free suspension technology,and the protective efficacy of the two batches of vaccines was evaluated using the epidemic strains isolated in 2020.Methods:The infected ducks were inoculated with 6-day-old SPF chicken embryos,and the dead embryos were collected.The supernatant was homogenized and inoculated with C6/36 cells to harvest the virus.The virus was identified as duck Tembusu virus by hemagglutination test and RT-PCR;Genome sequencing and analysis of the identified virus;The vaccine strain of duck Tembusu virus（HB strain）preserved by Baoding Rip Biotechnology Co.,Ltd.was inoculated into EB66 cells,and the virus was harvested by suspension culture.After inactivation,purification and concentration,two batches of inactivated vaccines were prepared;After immunizing 42-year-old Cherry Valley ducks with two batches of vaccines,the challenge protection experiment was carried out with the newly isolated JA-2020 strain,and the efficacy of the two batches of vaccines was preliminarily evaluated through observation of clinical symptoms,determination of serum antibody,blood virus isolation and virus PCR detection.Results:A strain of duck Tembusu virus named JA-2020 was successfully isolated.The viral hemagglutination value was 1:64,and the ELD₅₀was 10^5.9/0.1 m L;The whole genome sequencing and E gene phylogenetic tree analysis showed that this strain had the closest genetic relationship with Tembusu virus isolate GX2012,with 97.87%homology,and the farthest genetic relationship with Tembusu virus isolate GX2015,with 96.11%homology;The amino acid sequences of E gene varied from 409,564,660,686 and 774.Two batches of EB66 cell source（HB strain）inactivated vaccines（numbered as ZS202001 and ZS202005）were prepared by using the virus cultured by cell suspension culture technology.The hemagglutination values were 1:128 and 1:256,the formaldehyde contents were 0.05%and 0.06%,and the viscosity was 52.72 cP and 47.83cP,respectively.The inactivated tests were qualified;There was no adverse reaction after the two batches of vaccines were inoculated with 42-day-old Cherry Valley ducks,and the two batches of vaccines could significantly increase the serum antibody level;Cherry Valley ducks were challenged with chest intramuscular injection 21 days after the enhanced immunization.There were no obvious clinical symptoms in the immunized ducks,and no virus was isolated from the serum collected 2 days after the challenge.In the control group,Cherry Valley ducks showed clinical symptoms such as decreased food intake,increased eye secretion,and positive-isolated virus and virus PCR detection.Conclusion:A strain of duck Tembusu virus was isolated in this experiment,and the sequence of this strain was different from that of the existing vaccine strain by whole genome sequencing analysis.Two batches of EB66 cell-derived inactivated vaccine were prepared to meet the requirements with high safety;The prepared inactivated vaccine had good protective effect against duck Tembusu virus epidemic strain.