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Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages.

Revie D, Alberti MO, Prichard JG, Kelley AS, Salahuddin SZ - Virol. J. (2010)

Bottom Line: Results revealed a number of sequence changes as compared to the serum RNA.The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages.Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, California Lutheran University, Thousand Oaks, California, USA.

ABSTRACT

Background: Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories.

Results: HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3.

Conclusions: Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

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Gel electrophoresis of patient 384 RT-PCR products using various sets of primers. For short time cultures and analyses, we have used cloned cell lines such as P3HR1 and CEM. This was for the purposes of reproducibility and economy. A. Primer sets 9.1a-9.2a (1st PCR) and 37up-318down (2nd PCR). Only 384 serum produced a positive band. CB designates Macrophages purified from cord blood. P3HR1-b and CB-b were supernatants collected at different times than P3HR1 and CB. B. Primer sets 9.1a-9.2a (1st PCR) and 10.1a-10.2 (2nd PCR). Only P3HR1 produced positive bands. C. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 50°C. Only CEM produced positive bands. D. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 55°C. Primary, P3HR1-b, and Cord blood macrophages produced positive bands.
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Figure 2: Gel electrophoresis of patient 384 RT-PCR products using various sets of primers. For short time cultures and analyses, we have used cloned cell lines such as P3HR1 and CEM. This was for the purposes of reproducibility and economy. A. Primer sets 9.1a-9.2a (1st PCR) and 37up-318down (2nd PCR). Only 384 serum produced a positive band. CB designates Macrophages purified from cord blood. P3HR1-b and CB-b were supernatants collected at different times than P3HR1 and CB. B. Primer sets 9.1a-9.2a (1st PCR) and 10.1a-10.2 (2nd PCR). Only P3HR1 produced positive bands. C. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 50°C. Only CEM produced positive bands. D. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 55°C. Primary, P3HR1-b, and Cord blood macrophages produced positive bands.

Mentions: For the purposes of this report, we first made degenerate versions of the primers that were used for HCV-1 [12]. These primers were named 9.1a, 9.2a, and 10.1a (Table 1). Although these sets of primers were suitable for some HCV-3 samples (Figure 2B), we were unable to obtain the appropriate reactivity for the remainder of the samples. We therefore designed another set of primers to work with genotypes 1 and 3 (named 8 up and 347 down for the first PCR; 37 up and 318 down for the second PCR). Unlike our HCV-1 samples, we were unable to find a single set of primers and PCR conditions that always worked with all of the HCV-3 samples. By testing different combinations of primers and temperature conditions, we were able to generate PCR fragments for all of our samples (Figure 2 and Table 2).


Analysis of the 5'UTR of HCV genotype 3 grown in vitro in human B cells, T cells, and macrophages.

Revie D, Alberti MO, Prichard JG, Kelley AS, Salahuddin SZ - Virol. J. (2010)

Gel electrophoresis of patient 384 RT-PCR products using various sets of primers. For short time cultures and analyses, we have used cloned cell lines such as P3HR1 and CEM. This was for the purposes of reproducibility and economy. A. Primer sets 9.1a-9.2a (1st PCR) and 37up-318down (2nd PCR). Only 384 serum produced a positive band. CB designates Macrophages purified from cord blood. P3HR1-b and CB-b were supernatants collected at different times than P3HR1 and CB. B. Primer sets 9.1a-9.2a (1st PCR) and 10.1a-10.2 (2nd PCR). Only P3HR1 produced positive bands. C. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 50°C. Only CEM produced positive bands. D. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 55°C. Primary, P3HR1-b, and Cord blood macrophages produced positive bands.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913957&req=5

Figure 2: Gel electrophoresis of patient 384 RT-PCR products using various sets of primers. For short time cultures and analyses, we have used cloned cell lines such as P3HR1 and CEM. This was for the purposes of reproducibility and economy. A. Primer sets 9.1a-9.2a (1st PCR) and 37up-318down (2nd PCR). Only 384 serum produced a positive band. CB designates Macrophages purified from cord blood. P3HR1-b and CB-b were supernatants collected at different times than P3HR1 and CB. B. Primer sets 9.1a-9.2a (1st PCR) and 10.1a-10.2 (2nd PCR). Only P3HR1 produced positive bands. C. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 50°C. Only CEM produced positive bands. D. Primer sets 8up-347down (1st PCR) and 37up-318down (2nd PCR). First PCR at 55°C. Primary, P3HR1-b, and Cord blood macrophages produced positive bands.
Mentions: For the purposes of this report, we first made degenerate versions of the primers that were used for HCV-1 [12]. These primers were named 9.1a, 9.2a, and 10.1a (Table 1). Although these sets of primers were suitable for some HCV-3 samples (Figure 2B), we were unable to obtain the appropriate reactivity for the remainder of the samples. We therefore designed another set of primers to work with genotypes 1 and 3 (named 8 up and 347 down for the first PCR; 37 up and 318 down for the second PCR). Unlike our HCV-1 samples, we were unable to find a single set of primers and PCR conditions that always worked with all of the HCV-3 samples. By testing different combinations of primers and temperature conditions, we were able to generate PCR fragments for all of our samples (Figure 2 and Table 2).

Bottom Line: Results revealed a number of sequence changes as compared to the serum RNA.The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages.Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, California Lutheran University, Thousand Oaks, California, USA.

ABSTRACT

Background: Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories.

Results: HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3.

Conclusions: Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

Show MeSH
Related in: MedlinePlus