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In Vitro Coinfection and Replication of Classical Swine Fever Virus and Porcine Circovirus Type 2 in PK15 Cells.

Zhou N, Xing G, Zhou J, Jin Y, Liang C, Gu J, Hu B, Liao M, Wang Q, Zhou J - PLoS ONE (2015)

Bottom Line: However, CSFV reproduction decreased in a PCV2 dose-dependent manner.In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells.Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China.

ABSTRACT
Increasing clinical lines of evidence have shown the coinfection/superinfection of porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV). Here, we investigated whether PCV2 and CSFV could infect the same cell productively by constructing an in vitro coinfection model. Our results indicated that PCV2-free PK15 cells but not ST cells were more sensitive to PCV2, and the PK15 cell line could stably harbor replicating CSFV (PK15-CSFV cells) with a high infection rate. Confocal and super-resolution microscopic analysis showed that PCV2 and CSFV colocalized in the same PK15-CSFV cell, and the CSFV E2 protein translocated from the cytoplasm to the nucleus in PK15-CSFV cells infected with PCV2. Moreover, PCV2-CSFV dual-positive cells increased gradually in PK15-CSFV cells in a PCV2 dose-dependent manner. In PK15-CSFV cells, PCV2 replicated well, and the production of PCV2 progeny was not influenced by CSFV infection. However, CSFV reproduction decreased in a PCV2 dose-dependent manner. In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells. Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells. Taken together, our results demonstrate for the first time the coinfection/superinfection of PCV2 and CSFV within the same cell, providing an in vitro model to facilitate further investigation of the underlying mechanism of CSFV and PCV2 coinfection.

No MeSH data available.


Related in: MedlinePlus

Biological characteristics of PK15-CSFV cells.PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, P > 0.05; *P < 0.05; **P < 0.01).
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pone.0139457.g003: Biological characteristics of PK15-CSFV cells.PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, P > 0.05; *P < 0.05; **P < 0.01).

Mentions: Based on the results above, PK15 and ST cells were selected to establish cell lines harboring replicating CSFV. Cell monolayers at 80–90% confluency were inoculated with the CSFV HCLV strain at the MOI of 0.03 and subcultured at 48 hpi. CSFV-inoculated PK15 cells were designated PK15-CSFV and recorded as F0, while the first set of subcultured cells was labeled as F1, and so forth. PK15-CSFV cells were selected randomly after subculture for 72 h to detect CSFV-positive cells and virus titers. Flow cytometric analysis (Fig 2A) indicated that the percentages of CSFV infected cells gradually increased by serial passaging. As shown in Fig 2B, the indexes of CSFV infection increased gradually at the initial passages (F0 –F7). However, percentages of positive cells, genomic copies and TCID50 of CSFV in PK15-CSFV cells from the 8th passage were maintained at levels of more than 90%, 5 × 105 copies/μl and 10−3.6/0.1 ml, respectively. Virus growth curves showed that CSFV titers increased gradually with the increase in culture time, reached a peak at 48 h (Fig 3A) and remained at a relatively stable level. Thus, the results indicated that CSFV maintained a high infection rate since the 8th passage in PK15-CSFV cells. Similar results were revealed in the ST cell line harboring replicating CSFV (ST-CSFV cells), which reached a nearly 90% positive rate and maintained a stable infection level (106.26 copies/μl and of 104.1 TCID50/0.1 ml) as early as the 2nd passage (data not shown).


In Vitro Coinfection and Replication of Classical Swine Fever Virus and Porcine Circovirus Type 2 in PK15 Cells.

Zhou N, Xing G, Zhou J, Jin Y, Liang C, Gu J, Hu B, Liao M, Wang Q, Zhou J - PLoS ONE (2015)

Biological characteristics of PK15-CSFV cells.PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, P > 0.05; *P < 0.05; **P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592061&req=5

pone.0139457.g003: Biological characteristics of PK15-CSFV cells.PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, P > 0.05; *P < 0.05; **P < 0.01).
Mentions: Based on the results above, PK15 and ST cells were selected to establish cell lines harboring replicating CSFV. Cell monolayers at 80–90% confluency were inoculated with the CSFV HCLV strain at the MOI of 0.03 and subcultured at 48 hpi. CSFV-inoculated PK15 cells were designated PK15-CSFV and recorded as F0, while the first set of subcultured cells was labeled as F1, and so forth. PK15-CSFV cells were selected randomly after subculture for 72 h to detect CSFV-positive cells and virus titers. Flow cytometric analysis (Fig 2A) indicated that the percentages of CSFV infected cells gradually increased by serial passaging. As shown in Fig 2B, the indexes of CSFV infection increased gradually at the initial passages (F0 –F7). However, percentages of positive cells, genomic copies and TCID50 of CSFV in PK15-CSFV cells from the 8th passage were maintained at levels of more than 90%, 5 × 105 copies/μl and 10−3.6/0.1 ml, respectively. Virus growth curves showed that CSFV titers increased gradually with the increase in culture time, reached a peak at 48 h (Fig 3A) and remained at a relatively stable level. Thus, the results indicated that CSFV maintained a high infection rate since the 8th passage in PK15-CSFV cells. Similar results were revealed in the ST cell line harboring replicating CSFV (ST-CSFV cells), which reached a nearly 90% positive rate and maintained a stable infection level (106.26 copies/μl and of 104.1 TCID50/0.1 ml) as early as the 2nd passage (data not shown).

Bottom Line: However, CSFV reproduction decreased in a PCV2 dose-dependent manner.In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells.Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, PR China.

ABSTRACT
Increasing clinical lines of evidence have shown the coinfection/superinfection of porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV). Here, we investigated whether PCV2 and CSFV could infect the same cell productively by constructing an in vitro coinfection model. Our results indicated that PCV2-free PK15 cells but not ST cells were more sensitive to PCV2, and the PK15 cell line could stably harbor replicating CSFV (PK15-CSFV cells) with a high infection rate. Confocal and super-resolution microscopic analysis showed that PCV2 and CSFV colocalized in the same PK15-CSFV cell, and the CSFV E2 protein translocated from the cytoplasm to the nucleus in PK15-CSFV cells infected with PCV2. Moreover, PCV2-CSFV dual-positive cells increased gradually in PK15-CSFV cells in a PCV2 dose-dependent manner. In PK15-CSFV cells, PCV2 replicated well, and the production of PCV2 progeny was not influenced by CSFV infection. However, CSFV reproduction decreased in a PCV2 dose-dependent manner. In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells. Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells. Taken together, our results demonstrate for the first time the coinfection/superinfection of PCV2 and CSFV within the same cell, providing an in vitro model to facilitate further investigation of the underlying mechanism of CSFV and PCV2 coinfection.

No MeSH data available.


Related in: MedlinePlus