Renshaw R W, Soiné C, Weinkle T, O'Connell P H, Ohashi K, Watson S, Lucio B, Harrington S, Schat K A
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
J Virol. 1996 Dec;70(12):8872-8. doi: 10.1128/JVI.70.12.8872-8878.1996.
Chicken infectious anemia virus (CIAV) is a unique infectious agent with an amino acid composition that has been found to be remarkably conserved even in isolates from different parts of the world. We have characterized field isolates of CIAV which vary significantly in terms of their abilities to replicate in culture, demonstrating a biological difference between isolates. Two sublines of MDCC-MSB1 cells that differ in their abilities to support CIAV were identified. In the MSB1(S) subline the CIA-1 isolate of CIAV was found to be less cytopathogenic than the prototype Cux-1(C) isolate; the MSB1(L) subline, which supports Cux-1(C) replication, was found to be nonpermissive for CIA-1. Alignments of the VP1 sequences of previously examined isolates with those of the field isolates CIA-1 and L-028 and the culture-adapted ConnB isolate revealed a previously unreported hypervariable region spanning amino acid positions 139 to 151. Chimeras of Cux-1(C) and CIA-1 were constructed to examine the potential for this region to affect cytopathogenicity. Transfer of a 316-bp region of Cux-1(C) open reading frame 1 into CIA-1 produced a virus with a cytopathogenic profile typical of Cux-1(C), indicating that one or both of the amino acid differences at positions 139 and 144 affect the rate of replication or the spread of infection. Transfection experiments with additional chimeras indicated that the inability of CIA-1 to replicate in MSB1(L) cells is mediated by a larger region of the genome which contains the hypervariable region in addition to upstream amino acid differences. Analysis of chimeras excluding the entire region of open reading frame 1 suggested the presence of a secondary mediator in the progression of infection in culture that was localized to a region containing a single nucleotide difference which results in amino acid differences in both VP2 (V-153) and the nuclear localization signal of VP3 (C-118). Immunofluorescence assays indicated an increased cytoplasmic distribution of VP3 and a general lack of VP3-associated apoptotic bodies in infections of CIA-1 and chimeras containing V-153 or C-118, as opposed to a primarily nuclear distribution and association with well-formed apoptotic bodies in Cux-1(C)-infected cells.
鸡传染性贫血病毒（CIAV）是一种独特的感染因子，其氨基酸组成即便在来自世界不同地区的分离株中也被发现具有显著的保守性。我们已对CIAV的野外分离株进行了特性分析，这些分离株在培养物中的复制能力差异显著，表明不同分离株之间存在生物学差异。我们鉴定出了两个在支持CIAV能力上存在差异的MDCC-MSB1细胞亚系。在MSB1(S)亚系中，发现CIAV的CIA-1分离株比原型Cux-1(C)分离株的细胞致病性更低；支持Cux-1(C)复制的MSB1(L)亚系被发现对CIA-1不敏感。将先前检测的分离株的VP1序列与野外分离株CIA-1和L-028以及适应培养的ConnB分离株的VP1序列进行比对，发现了一个先前未报道的高变区，该区域跨越氨基酸位置139至151。构建了Cux-1(C)和CIA-1的嵌合体，以研究该区域影响细胞致病性的可能性。将Cux-1(C)开放阅读框1的一个316 bp区域转移到CIA-1中，产生了一种具有典型Cux-1(C)细胞致病特征的病毒，这表明139位和144位的一个或两个氨基酸差异会影响复制速率或感染传播。用其他嵌合体进行的转染实验表明，CIA-1在MSB1(L)细胞中无法复制是由基因组中一个更大的区域介导的，该区域除了上游氨基酸差异外还包含高变区。对排除开放阅读框1整个区域的嵌合体进行分析表明，在培养物感染过程中存在一个次要介导因子，其定位于一个包含单个核苷酸差异的区域，该差异导致VP2（V-153）和VP3的核定位信号（C-118）中都出现氨基酸差异。免疫荧光分析表明，在CIA-1以及含有V-153或C-118的嵌合体感染中，VP3的细胞质分布增加，且普遍缺乏与VP3相关的凋亡小体，相反，在Cux-1(C)感染的细胞中，VP3主要分布在细胞核中，并与形成良好的凋亡小体相关。
