Yoshizawa Nobuki, Saigo Tomotaka, Suzuki Tohru
Ehime Prefectural Livestock Disease Diagnostic Center, 743-1 Tanokubo, Toon, Ehime 791-0212, Japan.
Fukushima Prefectural Chuo Livestock Hygiene Service Center, 114-12 Arayashiki, Gampoji, Tamagawa, Ishikawa, Fukushima 963-6311, Japan.
J Virol Methods. 2025 Sep;337:115188. doi: 10.1016/j.jviromet.2025.115188. Epub 2025 May 20.
Outbreaks of diarrhea and respiratory symptoms caused by bovine coronavirus (BCoV) have been reported worldwide, leading to significant economic losses. It is important to genetically analyze the spike (S) gene of this virus as it is closely linked to its antigenicity and virulence, in order to quickly understand the molecular characteristics of circulating BCoVs. A previous study showed that BCoVs can be easily genotyped using reverse transcription (RT)-polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis using AvaII and EcoO65I enzymes targeted on the polymorphic region in the S1 subunit of the S gene. However, we found that a total number of 19 BCoV strains, including three BCoV strains isolated in this study and 16 Japanese BCoV strains available in GenBank, could not be genotyped by the original RT-PCR/RFLP analysis in silico. This study aimed to modify the original RT-PCR/RFLP assay by introducing additional restriction enzymes (AvaII, EcoO65I, AfaI, and Bsp1286I) to more accurately genotype recent Japanese BCoV strains without sequencing. As a result, the newly designed RT-PCR/RFLP analysis we established led to 98.3 % (291 of 296 BCoV strains used in this study) highly improved the accuracy of genotyping of BCoV. Therefore, this modified RT-PCR/RFLP method is useful for rapidly monitoring BCoVs, as it is a simple, easy, and inexpensive analysis that does not require sequencing.
牛冠状病毒（BCoV）引起的腹泻和呼吸道症状疫情已在全球范围内报道，导致重大经济损失。对该病毒的刺突（S）基因进行基因分析很重要，因为它与其抗原性和毒力密切相关，以便快速了解流行的BCoV的分子特征。先前的一项研究表明，使用针对S基因S1亚基多态性区域的AvaII和EcoO65I酶，通过逆转录（RT）-聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）分析可以轻松对BCoV进行基因分型。然而，我们发现包括本研究中分离的3株BCoV菌株和GenBank中可用的16株日本BCoV菌株在内的总共19株BCoV菌株，无法通过原始的RT-PCR/RFLP分析在计算机上进行基因分型。本研究旨在通过引入额外的限制性酶（AvaII、EcoO65I、AfaI和Bsp1286I）来修改原始的RT-PCR/RFLP检测方法，以便在不进行测序的情况下更准确地对近期日本BCoV菌株进行基因分型。结果，我们建立的新设计的RT-PCR/RFLP分析使BCoV基因分型的准确性提高了98.3%（本研究中使用的296株BCoV菌株中的291株）。因此，这种改良的RT-PCR/RFLP方法对于快速监测BCoV很有用，因为它是一种简单、易行且廉价的分析方法，不需要测序。
