ANALYSIS OF ALEUTIAN DISEASE VIRUS-INFECTION INVITRO AND INVIVO - DEMONSTRATION OF ALEUTIAN DISEASE VIRUS-DNA IN TISSUES OF INFECTED MINK
ANALYSIS OF ALEUTIAN DISEASE VIRUS-INFECTION INVITRO AND INVIVO - DEMONSTRATION OF ALEUTIAN DISEASE VIRUS-DNA IN TISSUES OF INFECTED MINK
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DOI：
10.1128/jvi.55.3.696-703.1985
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发表时间：
1985-01-01
影响因子：
5.4
通讯作者：
WOLFINBARGER, JB
中科院分区：
文献类型：
--
作者：
BLOOM, ME;RACE, RE;WOLFINBARGER, JB
对阿留申病病毒 (ADV) 感染进行体内和体外分析，以比较细胞培养物和水貂中的病毒复制。最初的实验比较了感染无毒力 ADV-G 毒株或高毒力 Utah I ADV 的克兰德尔猫肾 (CRFK) 细胞的培养物。通过免疫荧光 (IFA) 计算展示 ADV 抗原的细胞百分比来估计 ADV 感染细胞的数量，并确定感染的几个参数。通过斑点印迹 DNA-DNA 杂交估计，受感染的细胞含有大量病毒 DNA（> 105 个基因组/受感染细胞），并且通过 Southern 印迹杂交分析，发现大部分病毒 DNA 为 4.8 千碱基对双链体单体复制形式 (DM DNA)。培养物含有7-67个荧光形成单位(FFU)/感染细胞，并且ADV基因组/FFU比率范围为2×。 103-164次103. IFA 检测到的病毒抗原模式具有特征性的核特征，尽管在相同细胞中经常发现细胞质荧光。因为当比较含有相似数量的感染细胞的培养物时，两种病毒株之间没有发现差异，因此两种病毒在感染细胞培养物中的行为似乎相似。这些数据被用作分析毒力Utah 1 ADV对水貂的感染情况的基础。感染后十天，在脾脏中检测到最高水平的病毒 DNA（373 个基因组/细胞）。肠系膜淋巴结（MLN；750 个基因组/细胞）和肝脏（373 个基因组/细胞）。与感染的 CRFK 细胞形成鲜明对比的是，所有组织中 ADV DNA 的主要种类是单链病毒颗粒 DNA；然而，在 MLN 和脾脏中发现了 4.8 kb 碱基对的 DM DNA。这一观察结果表明，MLN 和脾脏是病毒复制的场所，但肝脏中发现的 DNA 反映了其他地方产生的病毒的隔离。最后一组实验检查了在 Utah I ADV 感染 10 天后取自 9 只水貂的 MLN。所有节点均含有 ADV DNA（46-750 个基因组/细胞），尽管单链病毒粒子 DNA 始终是最丰富的物种，但仍观察到 DM DNA。所有淋巴结均含有对 CRFK 细胞具有感染性的病毒，但当计算基因组/FFU 比率时，来自淋巴结的病毒需要约 .产生 FFU 的基因组比从受感染细胞培养物制备的病毒多 1000 倍。尽管 DM DNA 的存在表明这些 MLN 中发生了病毒复制，但通过对冰冻切片进行的 IFA 无法识别出表现出特征性核 ADV 抗原的细胞。这些实验表明，肠系膜淋巴结是ADV复制的靶器官，但实际复制病毒的细胞数量可能很少。
Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (> 105 genomes/infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). The cultures contained 7-67 fluorescence-forming units (FFU)/infected cell, and the ADV genome/FFU ratio ranged from 2 .times. 103-164 .times. 103. The pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the 2 virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as basis for the analysis of infection of mink by virulent Utah 1 ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes/cell). Mesenteric lymph node (MLN; 750 genomes/cell) and liver (373 genomes/cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8 kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken from 9 mink 10 days after Utah I ADV infection. All of the nodes contained ADV DNA (46-750 genomes/cell), and although single-stranded virion DNA was always the most abundant species, DM DNA was observed. All of the lymph nodes contained virus infectious for CRFK cells, but when the genome/FFU ratio was calculated, virus from the lymph nodes required .apprx. 1000 times more genomes to produce an FFU than did virus prepared from infected cell cultures. Although the presence of DM DNA indicated that virus replication was occurring in these MLN, no cells exhibiting characteristic nuclear ADV antigen could be identified by IFA performed on frozen sections. These experiments suggest that mesenteric lymph node was a target organ for ADV replication, but that the number of cells actually replicating virus was probably small.