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.
Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane-bound cytosolic enzyme which is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate virus replication. Treatment of Vero cells with SERCA-specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV) replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV and NDV infection induced SERCA expression in Vero cells, which could be blocked by Thapsigargin. Besides inducing enhanced formation of cytoplasmic foci, Thapsigargin was shown to block viral entry into the target cells as well as synthesis of viral proteins. Furthermore, NDV was shown to acquire significant resistance to Thapsigargin upon long-term passage (P) in Vero cells. As compared to the P0 and P70-Control, the fusion (F) protein of P70-Thapsigargin virus exhibited a unique mutation at amino acid residue 104 (E104K), whereas no Thapsigargin-associated mutations were observed in HN gene. To the best of our knowledge, this is the first report describing the virus-supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor.
Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox.
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