Zhang Z, Guo J, Ni Y, Bazer F W, Giavedoni L, de la Concha-Bermejillo A
Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, USA.
Arch Virol. 2003 Aug;148(8):1485-506. doi: 10.1007/s00705-003-0123-8.
AdUTPase gene ( du) deleted ovine lentivirus (OvLV(Deltadu)) mutant, derived from Visna/maedi virus (VMV) molecular clone KV1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein ( egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV(Deltadu-egfp). OvLV(Deltadu) reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p < 0.05). By contrast, OvLV(Deltadu-egfp) RT activity and virus titer were lower than for KV1772 and OvLV(Deltadu) (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV(Deltadu-egfp) at a low level. OvLV(Deltadu-egfp) retained egfp after 10 passages in cell culture.OvLV(Deltadu-egfp) was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV(Deltadu-egfp)-inoculated lambs, but by contrast to the in vitro experiments OvLV(Deltadu-egfp) lost the insert. Priming with OvLV(Deltadu-egfp) did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV(Deltadu-egfp)-primed lambs. OvLV serum antibody titers increased steadily in OvLV(Deltadu-egfp)-inoculated lambs, but in a lamb from which OvLV(Deltadu-egfp) was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV(Deltadu-egfp) is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV(Deltadu-egfp) as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.
构建了源自维斯纳/梅迪病毒（VMV）分子克隆KV1772的腺嘌呤核苷三磷酸酶基因（du）缺失的绵羊慢病毒（OvLV(Deltadu)）突变体。随后，将优化的绿色荧光蛋白（egfp）编码区的一个拷贝融合到病毒pol开放阅读框（ORF）中缺失的du位点，以产生病毒突变体OvLV(Deltadu-egfp)。与KV1772和OvLV-85/34毒株相比，OvLV(Deltadu)在山羊滑膜（GSM）细胞培养物中的逆转录酶（RT）活性和感染性病毒滴度未受影响（p<0.05）。相比之下，OvLV(Deltadu-egfp)的RT活性和病毒滴度低于KV1772和OvLV(Deltadu)（p<0.05）。OvLV-85/34在绵羊单核细胞衍生巨噬细胞（SMDM）中的RT活性高于KV1772、OvLV(Deltadu)和OvLV(Deltadu-egfp)（p<0.05）。OvLV(Deltadu)和OvLV(Deltadu-egfp)中防止dUTP错误掺入新合成DNA的能力受到破坏（p<0.05）。免疫沉淀表明OvLV(Deltadu-egfp)低水平表达GFP。OvLV(Deltadu-egfp)在细胞培养中传代10次后仍保留egfp。从四只接种OvLV(Deltadu-egfp)的羔羊中的三只的外周血单核（PBMN）细胞中，在GSM细胞中重新分离出OvLV(Deltadu-egfp)，但与体外实验相反，OvLV(Deltadu-egfp)丢失了插入片段。用OvLV(Deltadu-egfp)进行预激发不能预防致病性OvLV的感染，但在假感染的接触对照羔羊中，细胞相关病毒血症高于用OvLV(Deltadu-egfp)预激发的羔羊。在接种OvLV(Deltadu-egfp)的羔羊中，OvLV血清抗体滴度稳步上升，但在未重新分离出OvLV(Deltadu-egfp)的一只羔羊中，仅在用OvLV-85/34攻击后，抗体滴度才超过阴性/阳性临界值。由于OvLV(Deltadu-egfp)在体外致病性减弱、在体内复制并刺激抗体反应，后续实验需要探讨使用OvLV(Deltadu-egfp)作为减毒活病毒疫苗来保护绵羊免受致病性OvLV攻击时引发疾病的可能性。
