Flores J, Midthun K, Hoshino Y, Green K, Gorziglia M, Kapikian A Z, Chanock R M
J Virol. 1986 Dec;60(3):972-9. doi: 10.1128/JVI.60.3.972-979.1986.
RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants ("nursery strains") and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity (i.e., each of the four strains tested belonged to a different serotype), the fourth gene appeared to be highly conserved. Similarly, each of the virulent strains tested belonged to a different serotype; nonetheless, there was significant conservation of sequence among the fourth genes of three of these viruses. Significantly, the conserved fourth genes of the nursery strains were distinct from the fourth gene of each of the virulent viruses. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Under these conditions, the fourth genes of the nursery strains were closely related to each other but not to the fourth genes of the virulent viruses. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children.
进行RNA - RNA杂交以评估从患肠胃炎儿童和无症状新生儿中分离出的人类轮状病毒之间的遗传相关性程度。通过体外转录不同测试菌株的病毒核心产生的32P标记单链RNA在两种不同的杂交试验中用作探针：未变性的基因组RNA通过聚丙烯酰胺凝胶电泳分离，原位变性，电泳转移至重氮苄氧基甲基纸（Northern印迹），然后在两种不同的严谨条件下与探针杂交；变性的基因组双链RNA在溶液中与探针杂交，形成的杂交体通过聚丙烯酰胺凝胶电泳鉴定。当在低严谨水平下通过Northern印迹杂交分析时，测试菌株的所有基因都发生了交叉杂交，这为每个相应基因中的一些序列同源性提供了证据。然而，当杂交严谨性提高时，在从无症状新生儿（“托儿所菌株”）中分离出的菌株与从腹泻婴幼儿中分离出的菌株之间检测到基因4序列存在差异。尽管托儿所菌株表现出血清型多样性（即测试的四种菌株中的每一种都属于不同的血清型），但第四个基因似乎高度保守。同样，测试的每种致病菌株都属于不同的血清型；尽管如此，这些病毒中的三种病毒的第四个基因之间存在显著的序列保守性。值得注意的是，托儿所菌株保守的第四个基因与每种致病病毒的第四个基因不同。在溶液中进行RNA - RNA杂交并通过聚丙烯酰胺凝胶电泳分析所得杂交体的实验中，这些结果得到了证实和扩展。在这些条件下，托儿所菌株的第四个基因彼此密切相关，但与致病病毒的第四个基因无关。托儿所菌株的第四个基因与有症状婴幼儿中分离出的菌株的相应基因之间未形成全长杂交体。
