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.

Virological and Immunological Outcomes of Coinfections

“…However, in such instances, at least one of the viruses can be purified by plaque assay (14). Moreover, we have witnessed conditions where despite formation of CPE (in mixed culture), none of the coinfecting viruses formed plaques (14), though subsequent higher passage of the mixed culture allowed plaque formation by one of the viruses (14).…”

“…If the culture conditions are more permissible for the adventitious virus, it is likely that it will exclude the targeted agent on high passage, thereby resulting in failure of the targeted isolation of a known virus. Even under conditions where both the coinfecting viruses are able to persist until the appearance of CPE, it is not mandatory that both of them will participate in the formation of CPE (14). However, in such instances, at least one of the viruses can be purified by plaque assay (14).…”

“…Even under conditions where both the coinfecting viruses are able to persist until the appearance of CPE, it is not mandatory that both of them will participate in the formation of CPE (14). However, in such instances, at least one of the viruses can be purified by plaque assay (14). Moreover, we have witnessed conditions where despite formation of CPE (in mixed culture), none of the coinfecting viruses formed plaques (14), though subsequent higher passage of the mixed culture allowed plaque formation by one of the viruses (14).…”

“…Depending on the nature of coinfecting viruses, strategies for virus purification from mixed culture vary (Table 1) and may include (i) elimination of the enveloped viruses by treatment with the organic solvents (25), (ii) hemagglutination to separate a hemagglutinating virus, (iii) endpoint dilution assay to purify multiple agents, (iv) antibody (Ab) neutralization to eliminate other confecting viruses (26), (v) acid/alkali treatment if one of the viruses is more susceptible to extreme pH, (vi) plaque assay to purify single or multiple viruses, and (vii) transfection of the viral RNA mixture into target cells, which allows amplification (production) of only positive-sense RNA viruses, thereby eliminating negative-sense RNA viruses from the mixed culture (14).…”

“…When individual cells are coinfected, one virus usually influences replication of the other, a phenomenon termed viral interference. The result can be clearance (exclusion) of one virus but persistence of the other (14). Viral interference may be mediated by factors such as interferons (IFNs), defective interfering (DI) particles, production of trans-acting proteases, cellular factors, and nonspecific double-stranded RNA (dsRNA) (1).…”