一株变异型猪流行性腹泻病毒的分离鉴定及其灭活疫苗的免疫效力试验

doi:10.16431/j.cnki.1671-7236.2020.11.028

摘要/Abstract

摘要： 本研究旨在分离猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）变异株，通过悬浮培养工艺制备成高效价的PEDV灭活疫苗。2017年从中国多个规模化猪场采集腹泻病死仔猪的小肠及其内容物200份，通过RT-PCR方法进行PEDV检测并测序，筛选一株PEDV变异株，将其在2 L反应器里悬浮培养的Vero细胞上进行病毒分离与传代培养，收获的病毒液鉴定后测定TCID₅₀，经甲醛灭活后加入氢氧化铝胶佐剂配制成PEDV灭活疫苗，对其物理性状、稳定性、黏度、无菌等进行检验，检验合格后免疫妊娠母猪及所产仔猪，对其安全性和免疫效力进行研究。结果显示，200份病料中有86份为PEDV阳性，将筛选的PEDV变异株病料在Vero细胞上传至第5代时出现细胞病变，传至第10代收获病毒液，经鉴定后确定为PEDV变异毒株，并命名为PEDV-GF10株。收获的病毒液浓缩后测得病毒滴度可达1×10^8.0 TCID₅₀/mL。疫苗检验合格后在母猪产前40和25 d时试验组后海穴肌内注射4 mL疫苗，空白组不免疫，结果显示试验组与空白组母猪的生产情况无明显差异，所产3日龄仔猪分别免疫不同剂量后体温无显著差异，表明该疫苗对母猪和仔猪均安全性良好。随机挑选试验组与空白组母猪所产3日龄仔猪各20头，分别口服4 mL PEDV-F10病毒培养物，空白组母猪所产仔猪在攻毒24 h后PEDV发病率为100%，抗体均为阴性；试验组母猪所产仔猪只有10%出现了轻微的腹泻症状，仔猪获得了高达90%保护率，且仔猪被动免疫后抗体能持续至35 d以上。以上结果表明，PEDV-GF10变异株通过悬浮细胞培养后病毒滴度显著提高，研制的PEDV-GF10株灭活疫苗安全有效，能够对中国的PEDV变异株达到有效防控，为国内PED防控提供了理论依据。

关键词: 猪流行性腹泻; 病毒分离; 悬浮培养; 灭活疫苗

Abstract: This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.

Key words: porcine epidemic diarrhea; virus isolation; suspension culture; inactivated vaccine

中图分类号:

S828

常小云, 赵润泽, 同珂, 陈少贤, 刘国平. 一株变异型猪流行性腹泻病毒的分离鉴定及其灭活疫苗的免疫效力试验[J]. 中国畜牧兽医, 2020, 47(11): 3651-3658.

CHANG Xianyun, ZHAO Runze, TONG Ke, CHEN Shaoxian, LIU Guoping. Isolation and Identification of a Variant Porcine Epidemic Diarrhea Virus and the Immune Efficacy of Its Inactivated Vaccine[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3651-3658.

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