Limits...
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

Infectivity of PCV2 and CSFV in different porcine cell lines.3D4/31, ST and PK15 cell lines were inoculated with PCV2 at MOI = 1, 4, 7 and 10 or with CSFV at MOI = 0.03 and 0.1, respectively. After culture for 72 h, cells were fixed and stained for IFA, and then four fields were randomly chosen to count the percentage of the positive cells. Meanwhile, the cells were freeze-thawed to obtain virus stocks for determining virus titers. The percentage of PCV2-postive cells (A), PCV2 titer (B), percentage of CSFV-positive cells (C) and CSFV titer (D) were determined in 3D4/31, ST and PK15 cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4592061&req=5

pone.0139457.g001: Infectivity of PCV2 and CSFV in different porcine cell lines.3D4/31, ST and PK15 cell lines were inoculated with PCV2 at MOI = 1, 4, 7 and 10 or with CSFV at MOI = 0.03 and 0.1, respectively. After culture for 72 h, cells were fixed and stained for IFA, and then four fields were randomly chosen to count the percentage of the positive cells. Meanwhile, the cells were freeze-thawed to obtain virus stocks for determining virus titers. The percentage of PCV2-postive cells (A), PCV2 titer (B), percentage of CSFV-positive cells (C) and CSFV titer (D) were determined in 3D4/31, ST and PK15 cells.

Mentions: In order to select a cell line sensitive to infection with PCV2 and CSFV, three widely used porcine cell lines PK15, ST and 3D4/31 were inoculated with PCV2 or CSFV at different MOIs. After the cells were infected with PCV2 at the MOIs, average percentages of PCV2-positive cells were counted by selecting four random fields of view of the tested cells, and progeny virus titers were determined in PK15 cells. The results indicated that all three porcine cell lines could be infected with PCV2 (Fig 1A and 1B). However, the susceptibility of different cell lines to infection varied. The PK15 cell line had the highest virus-positive cell percentages and progeny virus titers in stocks, compared with those of the ST and 3D4/31 cell lines. Moreover, the percentage of PCV2-positive cells and titer of progeny virus increased with the increase of MOI in different cell lines. These results showed that the PK15 cell line was the most permissive for PCV2 and could be infected in a PCV2 dose-dependent manner.


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)

Infectivity of PCV2 and CSFV in different porcine cell lines.3D4/31, ST and PK15 cell lines were inoculated with PCV2 at MOI = 1, 4, 7 and 10 or with CSFV at MOI = 0.03 and 0.1, respectively. After culture for 72 h, cells were fixed and stained for IFA, and then four fields were randomly chosen to count the percentage of the positive cells. Meanwhile, the cells were freeze-thawed to obtain virus stocks for determining virus titers. The percentage of PCV2-postive cells (A), PCV2 titer (B), percentage of CSFV-positive cells (C) and CSFV titer (D) were determined in 3D4/31, ST and PK15 cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139457.g001: Infectivity of PCV2 and CSFV in different porcine cell lines.3D4/31, ST and PK15 cell lines were inoculated with PCV2 at MOI = 1, 4, 7 and 10 or with CSFV at MOI = 0.03 and 0.1, respectively. After culture for 72 h, cells were fixed and stained for IFA, and then four fields were randomly chosen to count the percentage of the positive cells. Meanwhile, the cells were freeze-thawed to obtain virus stocks for determining virus titers. The percentage of PCV2-postive cells (A), PCV2 titer (B), percentage of CSFV-positive cells (C) and CSFV titer (D) were determined in 3D4/31, ST and PK15 cells.
Mentions: In order to select a cell line sensitive to infection with PCV2 and CSFV, three widely used porcine cell lines PK15, ST and 3D4/31 were inoculated with PCV2 or CSFV at different MOIs. After the cells were infected with PCV2 at the MOIs, average percentages of PCV2-positive cells were counted by selecting four random fields of view of the tested cells, and progeny virus titers were determined in PK15 cells. The results indicated that all three porcine cell lines could be infected with PCV2 (Fig 1A and 1B). However, the susceptibility of different cell lines to infection varied. The PK15 cell line had the highest virus-positive cell percentages and progeny virus titers in stocks, compared with those of the ST and 3D4/31 cell lines. Moreover, the percentage of PCV2-positive cells and titer of progeny virus increased with the increase of MOI in different cell lines. These results showed that the PK15 cell line was the most permissive for PCV2 and could be infected in a PCV2 dose-dependent manner.

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