重组犬瘟热病毒rCDV-3的转瓶培养及其在水貂体内的免疫效果评价

Rolling culture of recombinant canine distemper virus rCDV-3 and its immune effects in minks

为克服犬瘟热疫苗生产病毒效价低的瓶颈,本研究在5 L转瓶中培养Vero细胞,接种重组犬瘟热病毒(CDV)(rCDV-3)和野生型CDV(CDV-3),比较二者的病毒滴度。将rCDV-3按10^(6.0)TCID_(50)接种小鼠和按不同剂量(10^(4.0)TCID_(50)~10^(6.0)TCID_(50))接种水貂,同时设立PBS对照组,免疫后观察各组小鼠及水貂的临床症状,并对小鼠称重。测定接种后小鼠(接种后14 d、28 d及56 d)和水貂(接种后7 d、14 d及28 d)血清中和抗体水平。接种后28 d用CDV强毒r HBF-1攻击免疫组和对照组水貂,21 d后采用荧光定量PCR(q PCR)测定攻毒后各组水貂直肠病毒排出量和各脏器病毒载量。结果显示,在5 L转瓶中,rCDV-3和CDV-3最高病毒滴度分别为10^(6.75)TCID_(50)/mL和10^(5.67)TCID_(50)/mL;接种后小鼠和水貂无临床症状,不同剂量感染的小鼠体质量百分比的变化基本一致,各组小鼠和水貂均产生了高水平的中和抗体。水貂攻毒后,免疫组水貂均无临床症状,对照组水貂出现体温升高、排便稀、结膜炎等典型CDV感染的临床症状,并在21 d内全部死亡。qPCR测定水貂直肠排毒量和脏器病毒载量,发现对照组的排毒量显著高于免疫组。对照组水貂心脏、肝脏、直肠和淋巴结的病毒载量均显著高于免疫组,且对照组水貂的心脏和淋巴结病毒载量最高。综上所述,本研究在5 L转瓶培养rCDV-3的增殖能力较强,并可诱导小鼠和水貂产生高水平的保护性体液免疫应答,并能够保护水貂抵抗CDV强毒攻击。表明,rCDV-3扩大培养后滴度高,为犬瘟热疫苗的进一步提质增效奠定基础。

In order to overcome the shortcomings of low titer of the canine distemper vaccine,Vero cells were cultured in 5L bottles rotary flasks,inoculated with recombinant canine distemper virus (rCDV-3) and wild-type CDV-3,and their virus titers were compared The mice and minks were immunized with r CDV-3 at different doses,and a control group was infected with PBS.At 28 days,minks were infected with CDV virulence strain r HBF-1 strain.The antibody levels in each group of mice were detected by neutralization test and virus shedding and viral loading in the organs of minks were measured by q PCR assay.The results showed that the titers of r CDV-3were higher than those of CDV-3,and the highest titers of r CDV-3 or CDV-3 were 10^(6.75)TCID_(50)/m L and 10^(5.67)TCID_(50)/m L,respectively.The immunized mice produced higher levels of antibodies compared to control mice without clinical symptoms.The vaccinated minks had no clinical symptoms and produced higher levels of neutralizing antibodies compared to control group.After the challenge,minks in the immune group showed no clinical symptoms,while minks in the control group showed fever,diarrhea and conjunctivitis,and all died within 21 days.The virus shedding and viral loading in organs of the control minks were higher than immunized group,and the viral loading in the heart and lymph nodes of control minks were the highest.These results indicate that the r CDV-3 cultured in 5L bottles in this study has higher viral titers,which could generate a higher level of humoral immune responses in mice and minks,providing protection against CDV challenge in minks.r CDV-3 has the characteristics of high titer,which lays a foundation for further improving the quality and efficiency of canine distemper virus vaccine.