Short Communication: Evaluation of GB Virus C/Hepatitis G Viral Load Among HIV Type 1-Coinfected Patients in São Paulo,Brazil

Introduction

A lthough first considered a hepatotropic virus, unlike hepatitis C virus (HCV), GB virus C/hepatitis G virus (GBV-C/HGV) infection does not show any hepatotoxic effect. ^1,2 Infection by this virus occurs worldwide, ³ primarily in people who have frequent exposure to blood products, such as intravenous drug users, polytransfused individuals, and individuals on hemodialysis. ⁴ By sharing the same routes of transmission, GBV-C/HVG infection is also very common among HIV-infected patients. ⁵

Recent studies have shown that coinfection of HIV-positive individuals with GBV-C/HGV is associated with a slower progression to AIDS, including higher CD4⁺ T lymphocyte counts, reduced HIV viral load, and increased survival after the development of AIDS, compared with patients who are infected only by HIV. ^{6 –8} However, other studies failed to demonstrate any beneficial effect in the course of HIV infection in coinfected patients. ^9,10 Some studies showed that GBV-C/HGV viral replication occurs not in hepatocytes but in lymphocytes; perhaps this fact could explain why the virus does not cause liver disease and, in some ways, may interfere with the viral replication of HIV. ^5,11 In vitro studies suggest that GBV-C/HGV E2 protein interacts with CD4⁺ T cells, increasing the secretion of RANTES and internalizing the coreceptor CCR5 surface, leading to a block for the entry of HIV into cells. ¹² In this study we developed a TaqMan system for quantification of GBV-C/HGV plasma viral load to attempt to correlate these data with the virological and immunological status in coinfected GBV-C/HIV and monoinfected HIV individuals.

Material and Methods

One hundred and two whole blood samples of HIV-1-positive individuals from the Immunodeficiency Control Center of the Federal University of São Paulo (CCDI/UNIFESP) were collected from April 2006 to April 2007. Informed written consent was obtained from all the patients and the study was approved by the Ethics Committees and the Institutional Review Board of the Federal University of São Paulo [#1296/05].

For sample analysis we performed quantification of CD4⁺ and CD8⁺ T cells (flow cytometry, FACSCalibur-BD, Franklin Lakes, NJ), HIV viral load (COBAS AMPLICOR HIV-1 MONITOR, Roche (Roche Products Chemicals and Pharmaceuticals SA.), and anti-E2 research (ELISA–Roche). For GBV-C/HGV load plasma, primers were designed based on the alignment of 119 sequences of the 5' UTR region and 7 sequences of the GBV-C/HGV genome obtained from GenBank (Sequencher, version 4.5, Gene Codes Corporation).

The sequences of primers were analyzed in BLAST (www.ncbi.nlm.nih.gov/BLAST/). Sequences obtained were forward: 5' CGG CCA AAA GGT GGT GGA 3' (−450 to −432); reverse: 5' AAG CTT ACC CTA CAC TAG TGG 3' (−384 to −363); and probe: 5'-FAM-ATG CCA GGG CGT GGT TGG AAA GTA TC-TAMRA-3' (−426 to −400). To construct the curve for absolute quantification of Real-Time PCR, a fragment of the 5' UTR region of GBV-C/HGV was cloned into TOPO TA, Cloning Version Q (Invitrogen, CA, USA) and purified by the Plasmid Kit QIAfilter Midi Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

The plasmid was linearized by a single cut restriction enzyme and was performed in an in vitro transcription reaction (T7 Transcription DuraScribe, Epicentre Biotechnologies). Serial dilution (1:10) was done by creating a standard curve with nine orders of magnitude (from 10¹⁰ to 10² copies/ml). RNA from clinical samples was extracted using the QIAamp Viral RNA kit (Qiagen, Valencia, Spain) as instructed by the manufacturer. The GBVC/HGV load plasma was performed using the SuperScript III Platinum One-Step qRT-PCR System (Invitrogen, CA, USA), according to the manufacturer's instructions.

Reactions were performed in iCycler (Bio-Rad Laboratories, Hercules, CA) under the following conditions: 95°C for 3 min followed by 45 cycles of 94°C for 20 s and 60°C for 40 s. Patients were divided into four groups: Group 1: HIV patients and HIV/HCV (n=51); Group 2: HIV patients, HIV/HCV, and anti-E2 positive (n=29); Group 3: HIV patients, HIV/HCV, and GBV-C PCR positive (n=20); and Group 4: HIV patients, HIV/HCV PCR positive, and GBV-C anti-E2 positive (n=2). To calculate the prediction of GBV-C/HGV viral load and linearization of qPCR we used the Excel program. For statistical analysis we used ANOVA, Pearson correlation tests, and Spearman (the Statistical Package for the Social Sciences—SPSS).

Results

At first we developed a TaqMan to perform the load plasma GBVC/HGV. The limit of detection of the assay was 100 copies/ml. The dynamic range was reached between 10⁹ and 10² copies of GBV-C/ml (Pearson's coefficient, r ² =0.99). The reaction efficiency was 90.9%, the slope was −3.56, and the intercept was 45 (Fig. 1).

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FIG. 1.

GB virus C (GBV-C) RT qPCR standard curve showing linearity over a range of eight orders of magnitude. The least-squares regression was calculated from plots of measured CT (y axis) versus input plasmid DNA over a range of 10² to 10⁹/ml (x axis). The correlation coefficient was 0.99 and the slope of the line was –3.5.

The reproducibility of the test was assessed by intraassay performed in quadruplicate. The coefficients of variation were 2.27% and 0.7% for the quantification of 10⁹ and 10², respectively. Of the 102 patients who participated in this study, 57.8% were male and the mean age was 42±9 years. All of them were infected by HIV-1, 41.1% had coinfection with HCV, and 85.3% of patients were on HAART. In relation to viral load, 67.6% showed undetectable HIV load, and of the subjects with detectable viral load, the average was 4.49 log₁₀ (2.79 log₁₀ to 5.84 log₁₀ copies/ml).

All of the patients were evaluated for the presence and quantification of RNA and the presence of GBV-C/HGV anti-E2. Of the 102 patients, 21.5% showed GBVC/HGV plasma load by TaqMan qPCR, with an average of 3.62±0.65 log₁₀ copies/ml (range 2.54 to 5 log₁₀ copies/ml). In relation to anti-E2, 26.4% were positive, indicating past infection by GBV-C/HGV. Only two patients had both markers. Although we found a lower median viral load in HIV patients coinfected by GBV-C/HGV compared to the other groups, there was no significance when applied to statistical tests (p≤0.46). Also there was no statistical significance when means of CD4⁺ count (p≤0.29), CD8⁺ T cells (p≤0.64), and age (p≤0.33) were compared between the groups (Table 1).

There was no correlation between HIV viral load and GBV-C/HGV viral load (Spearman correlation coefficient=−0.237) in the group of coinfected patients and also there was no correlation between GBV-C/HGV viral load and CD4⁺ count (Pearson's coefficient r ² =0.243). There was no correlation between GBV-C/HGV viral load count and CD8⁺ T cells when the Pearson's coefficient was −0.007.

Discussion

Because GBV-C/HGV shares the same transmission routes as HIV, GBV-C/HGV infection is very common among HIV-infected patients. Some studies have shown that GBV-C/HGV infection in these patients could be associated with decreased mortality and improved clinical outcomes compared to HIV-monoinfected patients. ⁴ It is known that there could be many other factors associated with survival such as virological, immunological, and host genetic that contribute to the course of HIV infection.

The development of molecular techniques for the detection and quantification of GBV-C/HGV can improve our understanding of this interaction. Assessment of viral loads of both agents involved in a coinfection provides useful benchmarks for establishing interactions among the various factors involved in this complex relationship. The reproducibility of RT qPCR evaluated by intraassay using the coefficient of variation (CV) showed values of 2.27% and 0.7% for 10⁹ and 10² dilutions.

Castelain and colleagues obtained a CV of 1.8% and Sauleda and colleagues a value of 1.25% for the higher dilutions. ^13,14 Thus, we demonstrate that our RT qPCR for GBV-C has high reproducibility. There was no statistical significance when comparing the groups with the presence or absence of markers of GBV-C/HGV infection in relation to HIV viral load, lymphocyte count, and CD4⁺ and CD8⁺ T cells.

This can be explained by the number of patients included in the study. Nevertheless, we found that the group presenting GBV-C/HGV RNA showed a discrete number of patients with undetectable HIV load and lower median viral load than the other groups. Considering that there was no difference in the percentage of patients under HAART in both groups, these data may indicate some impact of GBV-C/HGV infection in HIV replication, as suggested by most authors. ^{5 –8}

Our study suggests the possibility that there may be an impact on HIV viral load in patients coinfected with GBV-C/HGV. However, more studies are needed to clarify the molecular and cellular mechanisms that may be involved in this viral interaction, previously reported in other studies, hopefully to contribute to the development of new targets for drugs against HIV.