鸭坦布苏病毒鸡胚弱化毒株的选育

Selection of a Live Chicken Embryo Attenuated Duck Tembusu Virus Vaccine

【目的】将鸭坦布苏病毒BZ_2010株在SPF鸡胚上进行连续传代致弱;旨在选育安全性高、免疫原性好的活疫苗候选毒株。【方法】以SPF鸡胚为增殖宿主,将BZ_2010株连续传代,直至第120代;以1日龄雏鸭和30周龄的产蛋种鸭为试验对象,对第120代次毒株(命名为VC2)的安全性进行评价;以1d雏鸭为试验对象,对VC2株的返强情况进行评价;以18周龄的种鸭为试验对象,对VC2株免疫后的中和抗体进行监测;以25周龄的种鸭为试验对象,对VC2株免疫后的保护效果进行评价;利用RT-PCR方法分别扩增BZ_2010株和VC2株的E基因和N54A基因,进行测序分析。【结果】传代病毒对鸡胚的平均死亡时间逐渐缩短,而病毒毒价有逐)渐提高的趋势,ELD_(50)由第20代的10^(-5.3)/0.1 mL提高到第120代的10^(-5.8)/0.1mL,而且第80代之前提高较快,后期基本稳定。将VC2株通过颈部皮下接种1d雏鸭和肌肉接种30周龄产蛋种鸭,接种后试验鸭无异常临床表现,肝脏也无明显病理变化,这说明VC2株具有良好的安全性。将VC2在1d雏鸭进行连续5次传代,未发现试验鸭有任何异常症状,将第5代组织悬液接种1d雏鸭,采集肝脏进行病理切片观察,发现无明显病理变化,这说明VC2株具有良好的稳定性。基因测序分析结果显示,VC2株E蛋白的第86、157、189、301和312位氨基酸发生改变,而NS4A蛋白只有一个氨基酸发生突变,即第54位氨基酸由F变为L。VC2免疫种鸭后抗体水平上升很快,第4周即可达到高峰,并且维持较长时间;VC2免疫后于第2周和第50周利用强毒株进行攻毒试验,结果VC2免疫组在攻毒后未出现异常症状,粪便正常,产蛋率保持正常,这说明VC2株免疫可对强毒株的攻击产生完全保护。【结论】通过鸡胚连续传代成功获得了一株安全性高、免疫原性好的鸭坦布苏病毒鸡胚弱化毒株。VC2免疫种鸭后抗体水平上升很快,可维持较长时间。攻毒试验结果表明,VC2株免疫可对强毒株的攻击产生完全保护。

【Objective】 Tembusu virus BZ-2010 strain was continuously passaged in specific-pathogen-free embryonic （SPF） eggs in order to select a live attenuated vaccine candidate of good safety and immunogenicity properties. 【Method】 Tembusu virus BZ-2010 strain was cultured for 120 passages in SPF eggs. The safety of the 120th passage viral strain was evaluated with 1-day-old SPF ducklings and 30-week-old egg-laying ducks. The property of virulent return of VC2 viral strain was evaluated with 1-day-old SPF ducklings. The neutralizing antibodies were detected after the 18-week-old breeding ducks were immunized with VC2 strain. The protective effects were evaluated after the 25-week-old breeding ducks were immunized with VC2 strain. E gene and NS4A gene of BZ_2010 and VC2 strains were amplified by RT-PCR and sequenced. 【Result】 The average death time of SPF eggs was shortened by passage virus and viral titer was increased with the escalation of passage times in SPF chicken embryonic eggs. ELD50 of the 20th virus was 10^-5.3/0.1mL and ELD50 of the 120th virus was 10^-5.8/0.1mL.The viral titer reached the plateau at passage 80 and remained unchanged further passages. The experimental ducks showed no clinical symptoms after 1-day-old ducklings and 30-week-old breeding ducks were immunized with VC2 strain by subcutaneous injection and by intramuscular injection, respectively. The results showed that VC2 strain had a good safety. No symptoms appeared in 1-day-old ducklings in which VC2 strain were cultured for 5 passages. 1-day-old ducklings were infected with the 5th tissue suspension and no symptoms were observed in liver pathological section by microscope. The results showed that VC2 strain had a good stability. Sequence analysis revealed that the E protein of Tembusu VC2 evolved amino acid changes in positions 86, 157, 189, 301, and 302, respectively. The NS4A protein of Tembusu VC2 only had one amino acid change in position 54 in that phenylalanine was replaced by Leucine. The level of antibodies rose very quickly, reached the plateau at the 4th week and remained a long time. Ducks were challenged by TMUV virulent strain at 2 and 50 weeks after immunization with VC2 strain in the experimental group. There was no symptom, normal stool, and regular egg production in the vaccinated group after challenge of virulent strain. The results showed that VC2 strain could provide complete protection for the challenge of TMUV virulent strain.【Conclusions】 An attenuated strain of TMUV with good immunogenicity and high safety was acquired through serial passages of SPF chicken embryos. The level of antibodies rose very quickly and remained a long time after immunization of the VC2 attenuated strain. The toxicity attack experiments showed that VC2 could provide complete protection for the challenge of TMUV virulent strain.