日本牛鼻炎A型和B型病毒的遗传特征及原代牛呼吸道上皮细胞中病毒RNA扩增的证据

文献信息

中文摘要

背景： 口蹄疫病毒属的牛鼻炎A型病毒（BRAV）和牛鼻炎B型病毒（BRBV）可在牛呼吸道标本中检出。然而，关于日本毒株及基因组规模数据的信息仍然有限。方法：为填补这一空白，我们采用逆转录定量聚合酶链反应对日本牛鼻拭子标本进行BRAV和BRBV筛查，并对阳性标本进行基因组测序。结果：在麻布大学收治的98份牛标本中，以循环阈值（Ct）≤35为界，BRAV和BRBV各检出8份。从4份BRAV阳性标本和1份BRBV阳性标本中获得了近乎完整的基因组序列。系统发育分析显示，4株BRAV毒株属于一个与日本BRAV2毒株H-1不同的BRAV1相关谱系。相比之下，BRBV/25IKH5在BRBV-1组内与LAV/238002聚集在一起。为评估传代后的病毒RNA扩增，将仅BRAV或BRBV阳性且Ct值低的标本接种到Madin-Darby牛肾（MDBK）细胞和原代牛呼吸道上皮细胞上。BRAV/LAV/238037和BRBV/25IKH5的RNA在原代牛呼吸道上皮细胞培养上清中检出，但在MDBK细胞培养上清中未检出，且无明显细胞病变效应。在原代细胞中传代后，接种后72小时，BRAV/LAV/238037的平均Ct值从37.0降至23.9，BRBV/25IKH5的平均Ct值从30.7降至22.7。结论：本研究扩展了牛鼻炎病毒的基因组规模信息，并提供了BRAV和BRBV在原代细胞中传代后RNA扩增的证据。

英文摘要

The aphthoviruses bovine rhinitis A virus (BRAV) and bovine rhinitis B virus (BRBV) can be detected in bovine respiratory specimens. However, information regarding Japanese strains and genome-scale data remains limited. To address this gap, we screened Japanese bovine nasal swab specimens for BRAV and BRBV using reverse transcription-quantitative polymerase chain reaction and subjected positive specimens to genome sequencing. Among 98 specimens from cattle admitted to Azabu University, BRAV and BRBV were detected in 8 each using a cycle threshold (Ct) cutoff ≤35. Near-complete genome sequences were obtained from four BRAV-positive specimens and one BRBV-positive specimen. Phylogenetic analyses revealed that four BRAV strains belonged to a BRAV1-related lineage distinct from the Japanese BRAV2 strain H-1. In contrast, BRBV/25IKH5 clustered with LAV/238002 within the BRBV-1 group. To evaluate viral RNA amplification after passage, specimens with low Ct values that tested positive for BRAV or BRBV alone were inoculated onto Madin-Darby bovine kidney (MDBK) cells and primary bovine respiratory epithelial cells. RNA from BRAV/LAV/238037 and BRBV/25IKH5 was detected in culture supernatants of primary bovine respiratory epithelial cells but not in those of MDBK cells, and there was no apparent cytopathic effect. Following passage in primary cells, mean Ct values decreased from 37.0 to 23.9 for BRAV/LAV/238037 and from 30.7 to 22.7 for BRBV/25IKH5 at 72 hr after inoculation. This study expands genome-scale information on bovine rhinitis viruses and presents evidence of BRAV and BRBV RNA amplification after passage in primary cells.