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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

Replication of PCV2 and CSFV in PK15-CSFV cells.PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID50 in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID50 of PCV2. (B) Genomic copies of PCV2. (C) TCID50 of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; ns, P > 0.05; *P < 0.05; **P < 0.01).
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pone.0139457.g006: Replication of PCV2 and CSFV in PK15-CSFV cells.PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID50 in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID50 of PCV2. (B) Genomic copies of PCV2. (C) TCID50 of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; ns, P > 0.05; *P < 0.05; **P < 0.01).

Mentions: Thus far, we had demonstrated that PCV2 could infect PK15-CSFV cells at the same efficient infection rate as in PK15 cells. To investigate if the PCV2 progeny replicated in PK15-CSFV was mature and infectious and to determine the ability of cells to harbor CSFV, PCV2 and CSFV titers were determined by measuring TCID50 and viral genomic copies in PK15-CSFV cells. As shown in Fig 6A and 6B, infectious PCV2 was efficiently produced in both PK15 and PK15-CSFV cells. No difference (P > 0.05) in titers of the PCV2 progeny was observed between PK15 and PK15-CSFV cells inoculated with PCV2 at the same MOI, indicating that PCV2 could replicate well in PK15 cells irrespective of the presence or absence of replicating CSFV. Therefore, we concluded that no significant exclusion of PCV2 occurred in its superinfection with CSFV.


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)

Replication of PCV2 and CSFV in PK15-CSFV cells.PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID50 in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID50 of PCV2. (B) Genomic copies of PCV2. (C) TCID50 of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; 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.g006: Replication of PCV2 and CSFV in PK15-CSFV cells.PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID50 in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID50 of PCV2. (B) Genomic copies of PCV2. (C) TCID50 of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; ns, P > 0.05; *P < 0.05; **P < 0.01).
Mentions: Thus far, we had demonstrated that PCV2 could infect PK15-CSFV cells at the same efficient infection rate as in PK15 cells. To investigate if the PCV2 progeny replicated in PK15-CSFV was mature and infectious and to determine the ability of cells to harbor CSFV, PCV2 and CSFV titers were determined by measuring TCID50 and viral genomic copies in PK15-CSFV cells. As shown in Fig 6A and 6B, infectious PCV2 was efficiently produced in both PK15 and PK15-CSFV cells. No difference (P > 0.05) in titers of the PCV2 progeny was observed between PK15 and PK15-CSFV cells inoculated with PCV2 at the same MOI, indicating that PCV2 could replicate well in PK15 cells irrespective of the presence or absence of replicating CSFV. Therefore, we concluded that no significant exclusion of PCV2 occurred in its superinfection with CSFV.

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