Kumar Naveen, Barua Sanjay, Riyesh Thachamvally, Chaubey Kundan K, Rawat Krishan Dutt, Khandelwal Nitin, Mishra Anil K, Sharma Nitika, Chandel Surender S, Sharma Shalini, Singh Manoj K, Sharma Dinesh K, Singh Shoor V, Tripathi Bhupendra N
Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
PLoS One. 2016 May 26;11(5):e0156110. doi: 10.1371/journal.pone.0156110. eCollection 2016.
Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV) and foot-and-mouth disease virus (FMDV) mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE) of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP). PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture) transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV) and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus) in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of viruses, but could also help in establishing better guidelines for trading animals that could transmit further infections and epidemics in disease free nations.
从混合感染中成功纯化多种病毒仍然是一项挑战。在本研究中，我们调查了山羊中的小反刍兽疫病毒（PPRV）和口蹄疫病毒（FMDV）混合感染情况。在第6次盲传（BP）时，在共培养的Vero/BHK-21细胞中观察到了病毒的细胞病变效应（CPE），而不是在单一细胞类型中。通过空斑试验可从病毒混合物中纯化出PPRV，但无法纯化出FMDV。在BHK-21细胞中进行病毒RNA（混合物）转染产生了FMDV病毒粒子，但未产生PPRV病毒粒子，这是我们首次成功采用的一种从病毒混合物中消除负链RNA病毒的策略。在传代水平BP8、BP15和BP19时，分别观察到了FMDV的不同表型，如复制能力正常但不产生细胞病变、产生细胞病变但空斑形成有缺陷以及产生细胞病变但空斑形成和标准FMDV基因组均有缺陷，因此在细胞培养系统中使病毒分离变得复杂。未发现混合感染在PPRV和FMDV中诱导任何显著的抗原性和基因多样性。此外，我们首次证明了PPRV和FMDV之间的病毒干扰。预先转染PPRV RNA，但不转染新城疫病毒（NDV）和轮状病毒RNA，导致BHK-21细胞中FMDV的复制减少，这表明PPRV RNA诱导的干扰是特异性针对FMDV的。在Vero细胞中长期共感染一些急性致病病毒（PPRV、FMDV、NDV和水牛痘病毒的所有可能组合）时，在大多数情况下，其中一种共感染病毒在第5代传代时被排除，这表明长期共感染可能会改变病毒的持续性。据我们所知，这是首次记录的关于FMDV和PPRV自然混合感染的证据。该研究不仅为分离和纯化两种在流行病学和经济上重要的病毒组提供了简单可靠的方法，还有助于为在无病国家交易可能传播进一步感染和疫情的动物制定更好的指导方针。
