Journal: PLOS ONE

Article Title: Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

doi: 10.1371/journal.pone.0289863

Figure Lengend Snippet: Normal PK-15 cells, PK-15 Ifnar1 k/o cells, or PK-15 Stat2 k/o cells treated with various concentrations of pig IFNβ were infected with HIV-1–based reporter virus. Infectivity was calculated as relative light units (RLU) 2 days after infection. Relative infectivity was calculated in comparison that in untreated cells. The results are presented as the mean and standard deviation of quadruplicate measurements from one assay, and they are representative of at least three independent experiments. Differences were examined by one-way ANOVA followed by Tukey’s test. **** p < 0.0001.

Article Snippet: Human kidney-derived Lenti-X 293T cells (TaKaRa, Cat# Z2180N) and porcine kidney-derived PK-15 cells (Japanese Collection of Research Bioresources Cell Bank, Cat# JCRB9040) were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458–16) supplemented with 10% fetal bovine serum (FBS) and 1× penicillin–streptomycin (Pe/St; Nacalai Tesque, Cat# 09367–34).

Techniques: Infection, Virus, Comparison, Standard Deviation

Journal: PLOS ONE

Article Title: Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

doi: 10.1371/journal.pone.0289863

Figure Lengend Snippet: ( a ) Induction of ISG mRNAs in normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells was measured by qRT–PCR 1 day after IFNβ treatment. The results are presented as the mean and standard deviation of sextuplicate measurements from one assay, and they are representative of at least three independent experiments. Differences between 100 ng/mL IFNβ-treated cells and untreated cells were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, ns (not significant). ( b ) The data presented in Fig 2A were used to compare ISG induction among normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells. Multiple comparisons were examined by one-way ANOVA followed by Tukey’s test. **** p < 0.0001. ( c ) Expression of ISG15 protein (15 kDa) in normal PK-15, PK-15 Ifnar1 k/o, and PK-15 Stat2 k/o cells was determined via western blotting, as depicted in the lower panel. The quantity of input protein was visualized using the Total Protein Detection Module, as depicted in the upper panel.

Article Snippet: Human kidney-derived Lenti-X 293T cells (TaKaRa, Cat# Z2180N) and porcine kidney-derived PK-15 cells (Japanese Collection of Research Bioresources Cell Bank, Cat# JCRB9040) were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458–16) supplemented with 10% fetal bovine serum (FBS) and 1× penicillin–streptomycin (Pe/St; Nacalai Tesque, Cat# 09367–34).

Techniques: Quantitative RT-PCR, Standard Deviation, Two Tailed Test, Expressing, Western Blot

Journal: PLOS ONE

Article Title: Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

doi: 10.1371/journal.pone.0289863

Figure Lengend Snippet: Replication of IAV ( a ) and AKAV ( b ) was examined in normal PK-15 cells, PK-15 Ifnar1 k/o cells, and PK-15 Stat2 k/o cells. Cells were pretreated with 0 or 100 ng/mL IFNβ before infection. Viral RNA levels in the culture supernatant were quantified by qRT–PCR 2 days after infection. The relative values were calculated in comparison to that in non-pretreated cells. The results are presented as the mean and standard deviation of sextuplicate measurements from one assay. Differences between 100 ng/mL IFNβ-treated cells and untreated cells were examined by a two-tailed, unpaired Student’s t- test. *** p < 0.001, ** p < 0.05, ns (not significant).

Article Snippet: Human kidney-derived Lenti-X 293T cells (TaKaRa, Cat# Z2180N) and porcine kidney-derived PK-15 cells (Japanese Collection of Research Bioresources Cell Bank, Cat# JCRB9040) were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458–16) supplemented with 10% fetal bovine serum (FBS) and 1× penicillin–streptomycin (Pe/St; Nacalai Tesque, Cat# 09367–34).

Techniques: Infection, Quantitative RT-PCR, Comparison, Standard Deviation, Two Tailed Test

Journal: PLOS ONE

Article Title: Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

doi: 10.1371/journal.pone.0289863

Figure Lengend Snippet: The replication of IAV ( a ) and AKAV ( b ) was examined in normal PK-15, PK-15 Ifnar1 k/o, and PK-15 Stat2 k/o cells. Before infection, the cells were transfected with 0 or 100 ng/mL poly (I:C). Viral RNA levels in the culture supernatant were quantified via qRT–PCR 2 days after viral infection. The relative values were calculated with respect to non-pretreated cells. The results are presented as the mean and standard deviation of eight repeated measurements from one assay. Differences between 100 ng/mL poly (I:C)-treated cells and untreated cells were examined via a two-tailed, unpaired Student’s t- test. **** p < 0.0001, ** p < 0.01, and * p < 0.05, ns (not significant).

Article Snippet: Human kidney-derived Lenti-X 293T cells (TaKaRa, Cat# Z2180N) and porcine kidney-derived PK-15 cells (Japanese Collection of Research Bioresources Cell Bank, Cat# JCRB9040) were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458–16) supplemented with 10% fetal bovine serum (FBS) and 1× penicillin–streptomycin (Pe/St; Nacalai Tesque, Cat# 09367–34).

Techniques: Infection, Transfection, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: PLOS ONE

Article Title: Generation of porcine PK-15 cells lacking the Ifnar1 or Stat2 gene to optimize the efficiency of viral isolation

doi: 10.1371/journal.pone.0289863

Figure Lengend Snippet: ( a ) PK-15 Ifnar1 k/o cells were infected with retroviral vectors expressing IFNAR1 (WT), IFNAR1 (W70C), or ZsGreen protein. After selection with G418, Ifnar1 expression was measured by qRT–PCR 1 day after infection. The results are presented as the mean and standard deviation of octuplicate measurements from one assay, and they are representative of at least three independent experiments. Multiple comparisons were examined by one-way ANOVA followed by Tukey’s test. **** p < 0.0001. ( b ) PK-15 Ifnar1 k/o and PK-15 Stat2 k/o cells infected with retroviral vectors expressing IFNAR1 (WT), IFNAR1 (W70C), or ZsGreen protein were treated with 0 or 100 ng/mL IFNβ for 24 h. Cells were superinfected with AKAV. Viral RNA levels in the culture supernatant were quantified by qRT–PCR 2 days after infection. The relative values were calculated in comparison to that in non-pretreated cells. The results are presented as the mean and standard deviation of septuplicate measurements from one assay, and they are representative of at least three independent experiments. Differences between 0 and 100 ng/mL IFNβ-treated cells were examined by a two-tailed, unpaired Student’s t- test. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns (not significant). ( c ) Induction of ISG mRNAs in PK-15 Ifnar1 k/o cells infected with retroviral vectors expressing IFNAR1 (WT), IFNAR1 (W70C), or ZsGreen protein was measured by qRT–PCR 1 day after IFNβ treatment. The results are presented as the mean and standard deviation of sextuplicate measurements from one assay, and they are representative of at least three independent experiments. Multiple comparisons were examined by one-way ANOVA followed by Tukey’s test. *** p < 0.001, ** p < 0.01, ns (not significant).

Article Snippet: Human kidney-derived Lenti-X 293T cells (TaKaRa, Cat# Z2180N) and porcine kidney-derived PK-15 cells (Japanese Collection of Research Bioresources Cell Bank, Cat# JCRB9040) were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Kyoto, Japan, Cat# 08458–16) supplemented with 10% fetal bovine serum (FBS) and 1× penicillin–streptomycin (Pe/St; Nacalai Tesque, Cat# 09367–34).

Techniques: Infection, Expressing, Selection, Quantitative RT-PCR, Standard Deviation, Comparison, Two Tailed Test

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Respiratory sign scores of pigs dually inoculated with Mycoplasma hyopneumoniae /PCV2a ( ▲ ), M. hyopneumoniae /PCV2b ( ▲ ), and M. hyopneumoniae /PCV2d ( ▲ ), and pigs singularly inoculated with PCV2a ( ● ), PCV2b ( ● ), and PCV2d ( ● ), and negative control pigs ( ● ). Variation is expressed as the standard deviation. Different superscripts (a, b and c) indicate significant ( p < 0.05) difference among seven groups.

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Negative Control, Standard Deviation

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Body weight and average daily weight gain (ADWG) data (mean ± standard deviation) from pigs dually infected with Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2), and pigs singularly infected with PCV2 at 28 days of age (−14 days post-inoculation, dpi) and 63 days of age (21 dpi).

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Standard Deviation, Infection, Negative Control

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Porcine circovirus type 2 (PCV2)-specific ELISA antibody levels in serum of pigs dually inoculated with Mycoplasma hyopneumoniae /PCV2a ( ▲ ), M. hyopneumoniae /PCV2b ( ▲ ), and M. hyopneumoniae /PCV2d ( ▲ ), and pigs singularly inoculated with PCV2a ( ● ), PCV2b ( ● ), and PCV2d ( ● ), and negative control pigs ( ● ). Variation is expressed as the standard deviation. Serum samples are considered positive for PCV2 antibodies if the optical density (OD) is greater than 0.3 (red dotted line) Different superscripts (a and b) indicate significant ( p < 0.05) difference among 7 groups.

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Mean values of the genomic copy number of porcine circovirus type 2 (PCV2) DNA in serum of pigs dually inoculated with Mycoplasma hyopneumoniae /PCV2a ( ▲ ), M. hyopneumoniae /PCV2b ( ▲ ), and M. hyopneumoniae /PCV2d ( ▲ ), and pigs singularly inoculated with PCV2a ( ● ), PCV2b ( ● ), and PCV2d ( ● ), and negative control pigs ( ● ). Variation is expressed as the standard deviation. Different superscripts (a, b, and c) indicate significant ( p < 0.05) difference among seven groups.

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Negative Control, Standard Deviation

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Mean values of the genomic copy number of Mycoplasma hyopneumoniae (Mhp) in larynx of pigs dually inoculated with M . hyopneumoniae /PCV2a ( ▲ ), M. hyopneumoniae /PCV2b ( ▲ ), and M. hyopneumoniae /PCV2d ( ▲ ), and pigs singularly inoculated with PCV2a ( ● ), PCV2b ( ● ), and PCV2d ( ● ), and negative control pigs ( ● ). Variation is expressed as the standard deviation. Different superscripts (a and b) indicate significant ( p < 0.05) difference among 7 groups.

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Negative Control, Standard Deviation

Journal: Pathogens

Article Title: A Comparison of Pathogenicity and Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with Mycoplasma hyopneumoniae and PCV2

doi: 10.3390/pathogens10080979

Figure Lengend Snippet: Pathology data (mean ± standard deviation) from pigs dually inoculated with Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2), and pigs singularly inoculated with PCV2 at 21 days post-inoculation.

Article Snippet: At 0 dpi (42 days of age), pigs in the three M. hyopneumoniae /PCV2 groups were each inoculated intranasally with either 3 mL of PCV2a (SNUVR100032 strain, 5th passage in PCV-free PK15 cell lines, PCV-free PK cell line was kindly provided by WOOGENE B&G Ltd., Seoul, Korea), PCV2b (SNUVR202155, 5th passage in PCV-free PK15 cell lines), or PCV2d (SNUVR202003, 5th passage in PCV-free PK15 cell lines) inoculum containing 1.2 × 10 5 50% tissue culture infective dose (TCID 50 /mL), based on their groups.

Techniques: Standard Deviation, Negative Control