利用Real-time PCR对外周血白细胞中EIAV前病毒及血浆中病毒RNA含量的测定

Quantitative determination of viral RNA in plasma and proviral DNA in PBMC by real-time PCR following inoculation of horses with different EIAVs

利用Real_timePCR和Real_timeRT_PCR方法对马传染性贫血病(EIA)驴白细胞弱毒疫苗(DLA_EIAV)、DLA_EIAV感染性分子克隆衍生毒(vOK8226)、强弱毒嵌合病毒(vOKVltr)以及EIA强毒接种马后不同时期外周血白细胞中前病毒含量及血浆中病毒含量进行了监测,结果发现直接攻击强毒的2匹马及1匹接种vOK8226后再用强毒攻击的马血浆中病毒含量快速升高,伴随着明显的临床反应,最后均以死亡结束。其它免疫接种马在攻击强毒后均获得保护,没有发病,马血浆中有低水平病毒存在,攻毒后3个月血浆中检测不到病毒,说明感染马体内病毒的大量增殖与疾病的进程有着直接的关系。而外周血白细胞中前病毒的含量在各试验马各时期均能检测到,说明EIAV以前病毒的形式潜伏在感染马体内,疫苗的免疫只能控制发病,而不能清除感染的病毒。

The provirus load in PBMC and virus load in plasma of the experiment horses were detected by Real_time PCR or Real_time RT_PCR at different time following inoculation with equine infectious anemia virus(EIAV) donkey leucocyte attenuated vaccine (DLA_EIAV), a virus derived from infectious molecular clone of DLA_EIAV(vOK8226),a chimeric virus based on vOK8226 with substitution of LTR from virulent EIAV(vOKVltr),and a virulent EIAV.It was found that the virus load in plasma increased quickly in two horses inoculated with virulent EIAV and one horse inoculated with vOK8226 and challenged with virulent virus later,the three horses developed typical EIA symptom and died of high fever.Other four horses inoculated with DLA_EIAV,vOK8226 or vOKVltr were challenged with the virulent EIAV, they were all protected clinically,very low virus loads were detected in plasma, and three months after challenge the virus loads decreased to undetectable level.This indicated that the large duplication of the virus in blood is directly related with the progress of the disease. Provirus DNA in PBMC was detected in all experiment horses in all period of infection,indicating that EIAV present in infected horses as provirus, the immunization can protect animal from clinical disease, but can not eliminate the virus.