Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Next generation sequencing (NGS) analysis of the recovered JFH1-based 4d Core-NS5A (C5A) virus. The 4d(C5A)-3m recombinant virus, which contains JFH1 NS5B and UTRs, recovered from the 2nd passage (P2), was subjected to NGS analysis (full coding sequence is split into three equal parts in the figure). The SNP frequencies (%) for mutations that developed ≥20% are shown with corresponding HCV proteins (Core: 1–191; E1: 192–383; E2: 384–745; p7: 746–808; NS2: 809–1025; NS3: 1026–1656; NS4A: 1657–1710; NS4B: 1711–1971; NS5A: 1972–2415; NS5B: 2416–3006). The green boxes indicate mutations that were originally introduced in the 4d(C5A)-3m recombinant. P2D8, day 8 in the 2nd passage. aa, amino acid. The region of JFH1-NS5B is indicated with blue bar.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Next-Generation Sequencing, Recombinant, Sequencing

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Efficacy of PIs and NS5A inhibitors against HCV genotype 4a (ED43) full-length and 4d(C5A) culture viruses. Huh7.5 cells were seeded on 96-well plates overnight, then infected with the indicated viruses for 24 h. The cells were subsequently treated with specific inhibitors for an additional 48 h before analysis. Values are means of triplicates ± SEM. The ED43cc 4a virus was included for comparison. Mean EC 50 (nM) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Infection

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Differences in amino acids (aa) of NS5A domain I (aa 1-200) of analyzed 4d patient sequences and culture infectious DH13 clones compared with other HCV genotype 4 sequences.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Clone Assay

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 and 4d DH13 Core-NS5A (C5A) recombinant viruses to human monoclonal antibodies, AR3A and AR5A. ( A ): Infectivity titers in supernatants for selected time points of indicated viruses were determined after transfection of Huh7.5 cells (see legend of A). # These data were also shown in A. ( B ): The SNP frequencies (%) were shown for 4d(C5A)-12m virus. The virus recovered from the 2nd passage was NGS analyzed. Only mutations that developed in ≥20% of the virus population are shown. The green boxes indicate the originally introduced mutations in 4d(C5A)-12m recombinant. The region of JFH1-NS5B is indicated with the blue bar. ( C ): The indicated viruses were subjected to a 5-fold dilution series of either AR3A (top) or AR5A (bottom) from 50 µg/mL to 0.000128 µg/mL. The virus–antibody mixes and virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained, and the number of FFUs per well were counted. Values are means of quadruplicates ±SEM normalized to the values of 8 replicates of virus only. The data were analyzed by using four-parameter curve fitting to obtain sigmoidal dose–response curves. Mean EC 50 (μg/mL) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Transfection

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 (ED43/JFH1) and 4d DH13 Core-NS5A (C5A) JFH1-based recombinant viruses to plasma obtained from HCV infected patients. The indicated viruses were subjected to a single 1/50 (vol/vol) dilution ( A – C ) or a 5-fold dilution series from 1/50 to 1/19,531,250 (vol/vol) of the indicated patient plasma ( D , E ). The virus–plasma mixes and the virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained and the number of FFUs per well were counted. Values are means of quadruplicates ± SEM normalized to the values of 6 replicates of virus only for single-dose neutralization or 8 replicates of virus only for dilution series neutralization. The data of dilution series neutralization were analyzed using four-parameter curve fitting to obtain a sigmoidal dose–response curve. AA, pt3, and pt9: plasma collected from patients infected with HCV genotype 4a. pt2, pt6: plasma collected from patients infected with HCV genotype 4d. Mean EC 50 (vol/vol) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Neutralization

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 and 4d Core-NS5A (C5A) JFH1-based recombinant viruses to plasma taken at different time points from a patient with chronic HCV genotype 4d infection. The indicated viruses were subjected to 5-fold dilution series from 1/50 to 1/19,531,250 (vol/vol) of plasma collected from the HCV genotype 4d infected patient (initial infection with DH13; reinfected) at the indicated sampling dates. The virus–antibody mixes and virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained and the number of FFUs per well were counted. Values are means of quadruplicates ±SEM normalized to the values of 8 replicates of virus only. The data were analyzed using four-parameter curve fitting to obtain sigmoidal dose–response curves. Mean EC 50 (vol/vol) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Sampling

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Next generation sequencing (NGS) analysis of the recovered JFH1-based 4d Core-NS5A (C5A) virus. The 4d(C5A)-3m recombinant virus, which contains JFH1 NS5B and UTRs, recovered from the 2nd passage (P2), was subjected to NGS analysis (full coding sequence is split into three equal parts in the figure). The SNP frequencies (%) for mutations that developed ≥20% are shown with corresponding HCV proteins (Core: 1–191; E1: 192–383; E2: 384–745; p7: 746–808; NS2: 809–1025; NS3: 1026–1656; NS4A: 1657–1710; NS4B: 1711–1971; NS5A: 1972–2415; NS5B: 2416–3006). The green boxes indicate mutations that were originally introduced in the 4d(C5A)-3m recombinant. P2D8, day 8 in the 2nd passage. aa, amino acid. The region of JFH1-NS5B is indicated with blue bar.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Next-Generation Sequencing, Recombinant, Sequencing

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Efficacy of PIs and NS5A inhibitors against HCV genotype 4a (ED43) full-length and 4d(C5A) culture viruses. Huh7.5 cells were seeded on 96-well plates overnight, then infected with the indicated viruses for 24 h. The cells were subsequently treated with specific inhibitors for an additional 48 h before analysis. Values are means of triplicates ± SEM. The ED43cc 4a virus was included for comparison. Mean EC 50 (nM) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Infection

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Differences in amino acids (aa) of NS5A domain I (aa 1-200) of analyzed 4d patient sequences and culture infectious DH13 clones compared with other HCV genotype 4 sequences.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Clone Assay

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 and 4d DH13 Core-NS5A (C5A) recombinant viruses to human monoclonal antibodies, AR3A and AR5A. ( A ): Infectivity titers in supernatants for selected time points of indicated viruses were determined after transfection of Huh7.5 cells (see legend of A). # These data were also shown in A. ( B ): The SNP frequencies (%) were shown for 4d(C5A)-12m virus. The virus recovered from the 2nd passage was NGS analyzed. Only mutations that developed in ≥20% of the virus population are shown. The green boxes indicate the originally introduced mutations in 4d(C5A)-12m recombinant. The region of JFH1-NS5B is indicated with the blue bar. ( C ): The indicated viruses were subjected to a 5-fold dilution series of either AR3A (top) or AR5A (bottom) from 50 µg/mL to 0.000128 µg/mL. The virus–antibody mixes and virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained, and the number of FFUs per well were counted. Values are means of quadruplicates ±SEM normalized to the values of 8 replicates of virus only. The data were analyzed by using four-parameter curve fitting to obtain sigmoidal dose–response curves. Mean EC 50 (μg/mL) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Transfection

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 (ED43/JFH1) and 4d DH13 Core-NS5A (C5A) JFH1-based recombinant viruses to plasma obtained from HCV infected patients. The indicated viruses were subjected to a single 1/50 (vol/vol) dilution ( A – C ) or a 5-fold dilution series from 1/50 to 1/19,531,250 (vol/vol) of the indicated patient plasma ( D , E ). The virus–plasma mixes and the virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained and the number of FFUs per well were counted. Values are means of quadruplicates ± SEM normalized to the values of 6 replicates of virus only for single-dose neutralization or 8 replicates of virus only for dilution series neutralization. The data of dilution series neutralization were analyzed using four-parameter curve fitting to obtain a sigmoidal dose–response curve. AA, pt3, and pt9: plasma collected from patients infected with HCV genotype 4a. pt2, pt6: plasma collected from patients infected with HCV genotype 4d. Mean EC 50 (vol/vol) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Neutralization

Journal: Viruses

Article Title: Novel HCV Genotype 4d Infectious Systems and Assessment of Direct-Acting Antivirals and Antibody Neutralization

doi: 10.3390/v14112527

Figure Lengend Snippet: Sensitivity of HCV genotype 4a ED43 Core-NS2 and 4d Core-NS5A (C5A) JFH1-based recombinant viruses to plasma taken at different time points from a patient with chronic HCV genotype 4d infection. The indicated viruses were subjected to 5-fold dilution series from 1/50 to 1/19,531,250 (vol/vol) of plasma collected from the HCV genotype 4d infected patient (initial infection with DH13; reinfected) at the indicated sampling dates. The virus–antibody mixes and virus only were added to Huh7.5 cells for 4 h prior to washing and addition of fresh medium. At 48 h post-infection, cells were immunostained and the number of FFUs per well were counted. Values are means of quadruplicates ±SEM normalized to the values of 8 replicates of virus only. The data were analyzed using four-parameter curve fitting to obtain sigmoidal dose–response curves. Mean EC 50 (vol/vol) values with 95% confidence intervals (95% CI) are shown.

Article Snippet: To analyze viral viability of recombinants, in vitro RNA transcripts were transfected into Huh7.5 cells using lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), and the presence of HCV antigen positive cells were determined by immunostaining with anti-HCV NS5A antibody [ ] and anti-HCV core antibody (Enzo life sciences, Farmingdale, NY, USA).

Techniques: Recombinant, Infection, Sampling