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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

PCV2-induced apoptosis.(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, P > 0.05).
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pone.0139457.g007: PCV2-induced apoptosis.(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, P > 0.05).

Mentions: To further explore the possible mechanism by which PCV2 influenced CSFV replication, apoptosis was analyzed by the TUNEL assay and caspase 3/7 enzymatic activities were determined in PK15-CSFV cells with PCV2 infection. In both PK15 and PK15-CSFV cells infected with PCV2, the localization of PCV2-positive cells with TUNEL-positive cells was observed at 72 hpi of PCV2 infection (Fig 7A). The statistical analysis showed that the most predominant cells were TUNEL-PCV2 dual-positive cells in all labeled (TUNEL or immunostained) PK15 and PK15-CSFV cells after infection with PCV2, but the proportions of TUNEL-PCV2 dual-positive PK15 cells and PK15-CSFV cells were not significantly different (Fig 7B, P > 0.05). Subsequently, when cells were inoculated with PCV2 of MOIs, the TUNEL cells increased gradually in a dose-dependent manner (Fig 7C). Correspondingly, caspase 3/7 activities increased significantly in a PCV2 dose-dependent manner in PK15 and PK15-CSFV cells, but there showed no significant difference between PK15 cells and PK15-CSFV cells at the same MOI (Fig 7D, P > 0.05). These results showed that cellular apoptosis was only induced by PCV2 infection in PCV2-CSFV coinfected cells.


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)

PCV2-induced apoptosis.(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, P > 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139457.g007: PCV2-induced apoptosis.(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, P > 0.05).
Mentions: To further explore the possible mechanism by which PCV2 influenced CSFV replication, apoptosis was analyzed by the TUNEL assay and caspase 3/7 enzymatic activities were determined in PK15-CSFV cells with PCV2 infection. In both PK15 and PK15-CSFV cells infected with PCV2, the localization of PCV2-positive cells with TUNEL-positive cells was observed at 72 hpi of PCV2 infection (Fig 7A). The statistical analysis showed that the most predominant cells were TUNEL-PCV2 dual-positive cells in all labeled (TUNEL or immunostained) PK15 and PK15-CSFV cells after infection with PCV2, but the proportions of TUNEL-PCV2 dual-positive PK15 cells and PK15-CSFV cells were not significantly different (Fig 7B, P > 0.05). Subsequently, when cells were inoculated with PCV2 of MOIs, the TUNEL cells increased gradually in a dose-dependent manner (Fig 7C). Correspondingly, caspase 3/7 activities increased significantly in a PCV2 dose-dependent manner in PK15 and PK15-CSFV cells, but there showed no significant difference between PK15 cells and PK15-CSFV cells at the same MOI (Fig 7D, P > 0.05). These results showed that cellular apoptosis was only induced by PCV2 infection in PCV2-CSFV coinfected cells.

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