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.

Fig 1. Detection and isolation of PPRV and FMDV from the clinical specimens.

(a) Detection of PPRV and FMDV-specific genomes from the clinical specimens: Virus(es) recovered from the clinical specimen along with a negative control (MEM only) were tested for FMDV and PPRV-specific gene segments by PCR. (b) Virus isolation: Confluent monolayers of cocultured BHK-21/Vero cells were infected with virus recovered from a single specimen (salivary discharge) for 6 hours (h) followed by washing with PBS and addition of fresh media. At 5–7 days post-infection, cells were freeze-thawed and the resulting cell culture supernatant (BP1) was used to reinfect fresh cells. Such blind passages continued until appearance of CPE. (c) Detection of PPRV and FMDV-specific genome in mock-infected and virus-infected cell culture supernatants at BP6.

Fig 2. PPRV purification and characterization.

(a) Plaque assay at lower passage (BP6). (b) Plaque assay at higher passage (BP15). (c) Plaque purification: eleven different plaques were picked and resuspended in 500 μl MEM. (d) Amplification of PPRV (upper panel) and FMDV (lower panel)-specific gene segments from individual plaques by PCR. (e) qRT-PCR for detection of PPRV and FMDV-specific genomes in plaque purified PPRV (purity check).

Fig 3. FMDV purification and characterization.

RNA was extracted from 400 μl of the cell culture supernatant collected from either the mock-infected or BP15 virus-infected cells, followed by transfection in BHK-21 cells. (a) Cytopathic effect in mock-RNA-transfected and virus RNA-transfected BHK-21 cells at 48 hpt. (b) Amplification of FMDV-specific (upper panel) but not PPRV-specific (lower panel) gene segments by PCR in transfected cell culture supernatants. GMEM and virus mixture (BP15) were used respectively as negative and positive controls for RNA isolation and PCR. (c) BHK-21 cells were infected, in triplicates, at MOI of 1 for 1 h followed by washing and addition of fresh GMEM. The infectious progeny virus particles released in the infected cell culture supernatants at indicated time points were quantified by determining TCID₅₀ in BHK-21 cells. (d) Plaque formation by purified FMDV.

Fig 4. Copersistence of FMDV/PPRV in cocultured BHK-21/Vero cells.

Confluent monolayers of cocultured BHK-21/Vero cells or single cell types (Vero or BHK-21 cells) were infected, in triplicates (12 well cell culture plate), with 100 μl of the cell culture supernatant (BP15) for 2 h followed by washing with PBS and addition of fresh media. Virus release in the infected cell culture supernatant (cocultured BHK/Vero cells) at indicated time points was quantified by determining TCID₅₀ (a). Infected cell culture supernatants from Vero (b) and BHK-21 (c) cells at indicated time points were tested for PPRV and FMDV-specific genomes by qRT-PCR. The viral RNA levels, expressed as threshold cycle (CT) values, were analyzed to determine relative fold change in RNA copy number over 2 hpi. Error bars indicate SD. Statistical analysis was conducted with Student’s t test (*** = P<0.001).

Fig 5. PPRV interferes FMDV replication.

(a) FMDV plaque assay in BHK21 cells (b) Confluent monolayers of BHK-21 cells were infected, in triplicates, with PPRV at MOI of 1. At 12 hpi, the cells were superinfected with FMDV at MOI of 1 for 1 h followed by washing with PBS and addition of fresh media. The infectious progeny virus particles released in the supernatant at indicated time points were quantified by determining TCID₅₀ in BHK-21 cells. (b) BHK-21 cells were transfected, in triplicates, with PPRV, NDV, RV or mock (cellular) RNA. At 6 hpt, cells were washed with PBS and infected with FMDV at MOI of 1 followed by washing with PBS and addition of fresh media. Infectious progeny virus particles released in the infected cell culture supernatants were quantified by determining TCID₅₀. Results shown are the averages from at least three independent experiments. Error bars indicate SD. Statistical analysis was conducted with Student’s t test (*** = P<0.001, * = P<0.05).

Fig 6. FMDV interferes with PPRV replication.

Confluent monolayers of Vero cells were infected, in triplicates, with a single virus (PPRV or FMDV) or coinfected with PPRV/FMDV, each at MOI of 1 for 2 h followed by washing with PBS and addition of fresh media. Virus released in the supernatants at indicated time points were quantified by (a) determination of TCID₅₀. (b) Relative fold change in RNA copy number (over 2 hpi).

Fig 7. Fate of long-term in vitro viral coinfections.

(a) Vero cells were infected with indicated combinations of the viruses at MOI of 1. Infected cell culture supernatant was harvested at 7–8 days post-infection or earlier if >50% CPE was observed and 200 μl of it was used for next passage. Five such sequential passages were made. The infected cell culture supernatant from 5^th passage was subjected for amplification of respective viral genome by PCR. (b) Growth curve of PPRV. Confluent monolayers of Vero cells were infected, in triplicates, with PPRV at MOI of 1 for 2 h followed by washing with PBS and addition of fresh media. The infectious progeny virus particles released in the supernatant at indicated time points were quantified by determination of TCID₅₀ in Vero cells. (c) Growth curve of NDV. Confluent monolayers of Vero cells were infected, in triplicates, with NDV for 1 h at MOI of 1 followed by washing with PBS and addition of fresh media. The infectious progeny virus particles released in the supernatant at indicated time points were quantified by determining TCID₅₀ in Vero cells. (d) Growth curve of BPXV. Confluent monolayers of Vero cells were infected, in triplicates, with BPXV at MOI of 1 for 1 h followed by washing with PBS and addition of fresh media. The infectious progeny virus particles released in the supernatant at indicated time points were quantified by determining TCID₅₀ in Vero cells.

Fig 8. Extinction of standard FMDV genome at higher passage.

(a) Detection of FMDV genome in mock-infected, BP15-infected, BP15.ST (anti-PPRV serum treated BP15)-infected or BP15.ST.P3 (Sequential 3 time passage of BP15.ST)-infected cell culture supernatant. (b) Detection of FMDV genome in mock-infected, BP15-infected and BP19-infected cell culture supernatant. (c) Detection of FMDV genome in mock-infected, purified FMDV(P0)-infected and six time passaged FMDV (P6)-infected cell culture supernatant.