title: Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion.
abstract: 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.
authors.affiliation: 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.
authors.forename: Naveen
authors.initials: N
authors.lastname: Kumar
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Barua
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Thachamvally
authors.initials: T
authors.lastname: Riyesh
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Kundan K
authors.initials: KK
authors.lastname: Chaubey
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Krishan Dutt
authors.initials: KD
authors.lastname: Rawat
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Nitin
authors.initials: N
authors.lastname: Khandelwal
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Mishra
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Nitika
authors.initials: N
authors.lastname: Sharma
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Surender S
authors.initials: SS
authors.lastname: Chandel
authors.affiliation: Department of Veterinary Physiology and Biochemistry, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India.
authors.forename: Shalini
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Singh
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Dinesh K
authors.initials: DK
authors.lastname: Sharma
authors.affiliation: Division of Animal Health, ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India.
authors.forename: Shoor V
authors.initials: SV
authors.lastname: Singh
authors.affiliation: National Centre for Veterinary Type Culture Collections, ICAR-National Research Centre on Equines, Hisar, Haryana, India.
authors.forename: Bhupendra N
authors.initials: BN
authors.lastname: Tripathi
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D005536:Foot-and-Mouth Disease / Q000453:epidemiology* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015511:Goat Diseases / Q000453:epidemiology* / Q000821:virology; D006041:Goats / Q000235:genetics / Q000821:virology*; D029021:Peste-des-Petits-Ruminants / Q000453:epidemiology* / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics
chemical_list: D012367:RNA, Viral