Zhang Jianqiang, Go Yun Young, Huang Chengjin M, Meade Barry J, Lu Zhengchun, Snijder Eric J, Timoney Peter J, Balasuriya Udeni B R
Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.
Clin Vaccine Immunol. 2012 Aug;19(8):1312-21. doi: 10.1128/CVI.00302-12. Epub 2012 Jun 27.
A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.
开发了马动脉炎病毒（EAV）改良活病毒（MLV）疫苗株的稳定全长cDNA克隆。从该质粒（pEAVrMLV）产生的RNA转录本在转染到哺乳动物细胞后具有感染性，并且所得重组病毒（rMLV）与EAV的亲本MLV疫苗株具有100%的核苷酸同一性。在核衣壳基因中引入了一个沉默核苷酸取代（pEAVrMLVB），通过等位基因鉴别实时逆转录（RT）-PCR检测，使得克隆的疫苗病毒（rMLVB）能够与亲本MLV疫苗以及EAV的其他野毒株和实验室毒株区分开来。体外研究表明，克隆的疫苗病毒rMLVB和亲本MLV疫苗病毒在马内皮细胞中具有相同的生长动力学和蚀斑形态。体内研究证实，克隆的疫苗病毒非常安全，并且在实验免疫的马匹中诱导产生高滴度的抗EAV中和抗体。当用异源EAV KY84株进行攻毒时，rMLVB疫苗病毒在降低病毒血症的程度和持续时间以及病毒排出方面保护了免疫的马匹，但没有抑制马动脉炎（EVA）症状的发展，尽管这些症状的临床严重程度有所降低。疫苗克隆pEAVrMLVB可以进一步操作以提高疫苗效力，以及开发一种用于区分自然感染EAV的动物和接种疫苗动物的标记疫苗。
