一起新生儿病毒性腹泻病原学分析

Etiological analysis of an epidemic of viral diarrhea among newborn infants

目的查明引起新生儿病毒性腹泻疫情的病原并对其进行分子生物学特征分析。方法采用金标法检测轮状病毒抗原,采用乳胶凝集法检测腺病毒;通过聚丙烯酰胺凝胶电泳(PAGE)检测粪便样品中的轮状病毒核酸;利用逆转录-聚合酶链反应(RT-PCR)检测肠道病毒和扩增轮状病毒VP7基因并测序;利用DNAstar和Blast软件对测序结果进行分析。结果11份样品中,金标法检测到轮状病毒阳性样品9份,阳性率为81.82%;腺病毒和肠道病毒检测均为阴性;PAGE法检测到10份轮状病毒阳性样品,电泳带型为4-2-3-2的样品有8份,阳性率为72.73%,另2份阳性样品的电泳条带特殊;8份带型为4-2-3-2的阳性样品均能通过RT-PCR扩增出VP7基因,选择其中3份样品进行测序,测序结果经过比对发现这3个毒株的核苷酸的同源性为99.90%,氨基酸的同源性为100%,与G1型标准株Wa的核苷酸同源性分别为91.30%～91.50%,氨基酸同源性为94.80%;与泰国流行株Thai-2104亲缘关系最近,与中国以前的流行株处于不同的进化分支上。结论引起此次新生儿病毒性腹泻疫情的病原是A组轮状病毒,PAGE带型主要为4-2-3-2,其血清型为G1型。

Objective The study was conducted to identify the etiological agent that induced an epidemic of neonatal viral diarrhea and to analyze its molecular biological characteristics. Methods The rotavirus antigen was detected by dot-immunogold filtration assay, adenovirus by latex agglutination test, rotavirus nucleic acid in stool samples by polyacrylamide gel electrophoresis （PAGE）, and enterovirus by RT-PCR. The VP7 gene of rotavirus was cloned and sequenced by RT- PCR, the results of which were analyzed by DNAstar and Blast. Results Rotavirus was detected in nine out of eleven samples by dot-immunogold filtration assay, the positive rate being 81.82%. Both adenovirus and enterovirus detection were negative. Ten samples were positive for rotavirus detected by PAGE, as eight of them manifested a 4-2-3-2 electrophoresis band, the positive rate being 72.73%. However, the other two positive samples showed distinctively different bands. The VP7 gene could be cloned from the eight samples that had 4-2-3-2 bands by RT-PCR, and three of them were then used for gene sequencing, Sequence analysis revealed that the nucleotide homology of VP7 gene between the three strains was 99.90%, and the amino acid homology was 100%; the VP7 nucleotide homology of them with the serotype G1 standard strain Wa was 91.30%-91.50%, and amino acid homology of VP7 gene 94.80%. Therefore, they had the closest genetic relationship with the Thai-2104 strains, and they were in a different evolutionary branch that separated themselves from the other strains that used to cause epidemics in China. Conclusion It was human rotavirus A that caused the epidemic of neonatal viral diarrhea. The primary banding type of this pathogen, serovar G1, was 4-2-3-2.