Limits...
Viral Load Analysis of Hepatitis C Virus in Huh7.5 Cell Culture System.

Teimourpour R, Meshkat Z, Gholoubi A, Nomani H, Rostami S - Jundishapur J Microbiol (2015)

Bottom Line: Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA.The amount of viral load, which was measured using real-time PCR was 17592 IU/mL.This method could be used in future studies.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran.

ABSTRACT

Background: Previous studies using cell culture systems for the replication of hepatitis C virus have opened new research dimensions, and paved the ways for further and detailed studies of the virus in vitro.

Objectives: The purpose of the present study was to cultivate hepatitis C virus in a cell culture system and evaluate viral amplification.

Materials and methods: In order to propagate hepatitis C virus, cloned whole genome of virus, JFH-1, was used. JFH-1 cDNA was introduced into strain JM109 of Escherichia coli and plasmid, containing the viral genome was purified from transformed bacteria. After XbaI digestion, RNA synthesis was induced using T7 RNA polymerase enzyme. Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA. Finally, Huh-7.5 cell line was infected with replicated virus and viral load was determined using real-time PCR (Polymerase Chain Reaction).

Results: The amount of viral load, which was measured using real-time PCR was 17592 IU/mL.

Conclusions: In the present study, using cell culture, a high titer (in acceptable range) of infectious hepatitis C virus was produced. This method could be used in future studies.

No MeSH data available.


Related in: MedlinePlus

Standard Curve Used to Measure the Concentration of HCV RNA in Unknown SamplesFive standards from 2.5 × 103 to 2.5 × 107 IU/mL were used. Type (column 3): standards and sample arrangements; Ct (column 4): Ct value of standards and sample; Given Conc (IU/mL) (column 5): quantification standard concentrations; Calc Conc (IU/mL) (column 6): concentrations of the standards and sample that was measured by Rotor Gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4537521&req=5

fig19360: Standard Curve Used to Measure the Concentration of HCV RNA in Unknown SamplesFive standards from 2.5 × 103 to 2.5 × 107 IU/mL were used. Type (column 3): standards and sample arrangements; Ct (column 4): Ct value of standards and sample; Given Conc (IU/mL) (column 5): quantification standard concentrations; Calc Conc (IU/mL) (column 6): concentrations of the standards and sample that was measured by Rotor Gene.

Mentions: For viral load analysis, viral RNA was purified form collected supernatant of re-infected Huh-7.5 cells and was measured by real-time PCR. In the final sample, a viral load of 1.76 × 104 IU/mL (r2 = 0.997, Median 1.6 × 104 IU/mL) was detected (Figure 1) compared to the standard concentrations. Sample CT was in conformity with standard 2. This indicates the high accuracy of the test.


Viral Load Analysis of Hepatitis C Virus in Huh7.5 Cell Culture System.

Teimourpour R, Meshkat Z, Gholoubi A, Nomani H, Rostami S - Jundishapur J Microbiol (2015)

Standard Curve Used to Measure the Concentration of HCV RNA in Unknown SamplesFive standards from 2.5 × 103 to 2.5 × 107 IU/mL were used. Type (column 3): standards and sample arrangements; Ct (column 4): Ct value of standards and sample; Given Conc (IU/mL) (column 5): quantification standard concentrations; Calc Conc (IU/mL) (column 6): concentrations of the standards and sample that was measured by Rotor Gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4537521&req=5

fig19360: Standard Curve Used to Measure the Concentration of HCV RNA in Unknown SamplesFive standards from 2.5 × 103 to 2.5 × 107 IU/mL were used. Type (column 3): standards and sample arrangements; Ct (column 4): Ct value of standards and sample; Given Conc (IU/mL) (column 5): quantification standard concentrations; Calc Conc (IU/mL) (column 6): concentrations of the standards and sample that was measured by Rotor Gene.
Mentions: For viral load analysis, viral RNA was purified form collected supernatant of re-infected Huh-7.5 cells and was measured by real-time PCR. In the final sample, a viral load of 1.76 × 104 IU/mL (r2 = 0.997, Median 1.6 × 104 IU/mL) was detected (Figure 1) compared to the standard concentrations. Sample CT was in conformity with standard 2. This indicates the high accuracy of the test.

Bottom Line: Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA.The amount of viral load, which was measured using real-time PCR was 17592 IU/mL.This method could be used in future studies.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran.

ABSTRACT

Background: Previous studies using cell culture systems for the replication of hepatitis C virus have opened new research dimensions, and paved the ways for further and detailed studies of the virus in vitro.

Objectives: The purpose of the present study was to cultivate hepatitis C virus in a cell culture system and evaluate viral amplification.

Materials and methods: In order to propagate hepatitis C virus, cloned whole genome of virus, JFH-1, was used. JFH-1 cDNA was introduced into strain JM109 of Escherichia coli and plasmid, containing the viral genome was purified from transformed bacteria. After XbaI digestion, RNA synthesis was induced using T7 RNA polymerase enzyme. Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA. Finally, Huh-7.5 cell line was infected with replicated virus and viral load was determined using real-time PCR (Polymerase Chain Reaction).

Results: The amount of viral load, which was measured using real-time PCR was 17592 IU/mL.

Conclusions: In the present study, using cell culture, a high titer (in acceptable range) of infectious hepatitis C virus was produced. This method could be used in future studies.

No MeSH data available.


Related in: MedlinePlus