Evaluation of GBV-C / HVG viremia in HIV-infected women / Avaliação da viremia por GBV-C/HGV em mulheres infectadas pelo HIV

The present study aimed at standardizing a real-time quantitative polymerase chain reaction assay to evaluate the presence of GBV-C/HGV RNA. A "TaqMan" assay using primers and probe derived from the 5¢ NCR region was developed and validated. Two hundred and fifty-three plasma samples from HIV-infected women were tested for GBV-C viremia and antibody against the envelope protein 2. GBV-C RNA was detected in 22.5 percent of the patients whereas the antibody was identified in 25.3 percent of the cohort. Detection of viral RNA and of antibodies was mutually exclusive. Viral loads showed a mean of 1,777 arbitrary units / mL, being 1.1 and 13,625 arbitrary units / mL respectively the lowest and highest values measured. We conclude that the real-time quantitative polymerase chain reaction method developed is appropriate for the investigation of GBV-C RNA since it was shown to be highly specific and sensitive, as well as requiring few steps, preventing contamination and providing additional information as to the relative viremia of carriers, a parameter that must be included in studies evaluating the co-factors influencing the clinical outcome of HIV/AIDS.

Este estudo teve como objetivo o desenvolvimento de método de PCR em Tempo Real para a determinação da viremia do vírus GBV-C. Ensaio baseado em primers e sonda "TaqMan" derivados da região 5' não-codificante deste vírus foi padronizado, validado e aplicado em uma série de 253 amostras de plasma de pacientes HIV+. Além do PCR em tempo real, as amostras foram submetidas a um ensaio imunoenzimático anti-E2 e a um nested-PCR. Das 253 amostras testadas, 64 foram positivas para o anticorpo anti-E2 (25,3 por cento), enquanto 57 amostras foram concordantemente RNA positivas pelo nested-PCR e PCR em tempo real (22,5 por cento), perfazendo um índice total de exposição de 48 por cento (25.3 + 22.5). A carga viral teve média de 1.777 UA/mL (13.625 - 1.1UA/mL). Foi obtida metodologia simples, rápida e de boa sensibilidade e especificidade, permitindo a quantificação do RNA do vírus GBV-C com reprodutibilidade. A metodologia permite a análise simultânea de grande número de amostras, sendo apropriada para estudos clínicos. A prevalência de exposição a este agente na população feminina HIV+ estudada é alta, provavelmente decorrente da via sexual comum de transmissão dos agentes.

[Evaluation of touchdown nested PCR to circumvent spurious priming and increase specificity during HIV and GBV-C gene amplification]

Primer-Template hybridization temperature is one of the important parameters in Nested PCR optimization. Unlike instant temperature for sequence amplification in routine PCR process, Touchdown PCR is a modified form of standard PCR that employs a range of annealing temperature. This study intended to develop a Touchdown Nested PCR in order to circumvent spurious priming and enhancing specify during gene amplification. This is an experimental study conducted at Tarbiat Modarres University of Tehran during 2008-2009. Study samples were collected from Digestive Diseases Research Centre at Shariati Hospital and HIV research center-Imam Khomeini Hospital. After extracting the nucleic acid, primer designing for HIV and GBV-C and c-DNA synthesis; Nested PCR was performed on negative and positive samples using standard and touchdown protocols. The intended band was observed in all positive samples. No band was observed in any human and viral negative control samples. After electrophoresis of PCR products, non specific band were seen in HIV and GBV-C samples during standard PCR. Using the touchdown protocol, undesirable bands were omitted or significantly decreased. In the present study, despite the formation of uncalled bands in standard reaction; using the touchdown method led to omission of non-specific bands without any significant effect on the final products. As for its simplicity, cost and time saving, it seems that using this method is a rational and economical way for fast optimization of PCR reactions

Occurrence & nucleotide sequence analysis of hepatitis G virus in patients with acute viral hepatitis & fulminant hepatitis.

BACKGROUND & OBJECTIVE: Association of hepatitis G virus (HGV) with acute viral hepatitis (AVH) and fulminant hepatitis (FH) is not clearly understood.This study was designed to asses the occurrence of HGV infection and its relationship with other hepatotropic viruses in patients with FH and AVH and also to determine the nucleotide sequence of HGV isolates. METHODS: The study included 100 patients of FH and 125 of AVH on the basis of clinical examination, liver function test and serology for hepatitis A, B, C and E virus. HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing for 4 randomly selected samples followed by phylogenetic analysis. RESULTS: Of the 100 patients with FH, 30 were negative for hepatitis viruses A, B, C and E by serology (non A-non E) while 60 were negative in the AVH group. In the non A-non-E hepatitis group, HGV was positive in 16.66 per cent (5/30) cases of FH, 10 per cent (6/60) cases of AVH and 6 per cent (6/100) of healthy controls. The difference in HGV seropositivity between FH and AVH patients was statistically not significant compared to healthy controls, while HBV and HCV infections were significant. The four isolates sequenced seemed to be of same type and close to Chinese strain of HGV (Y13755.1 Y13756.1 Y15407, and U67782) on phylogeny. INTERPRETATION & CONCLUSION: In HGV infection was not found to be clinically significant as well as nonpathogenic in the patients of FH and AVH and appeared to be an innocent bystander in the course of the disease. The four sequenced HGV isolates showed close pairing with Chinese strains.

HEV, TTV and GBV-C/HGV markers in patients with acute viral hepatitis

The aim of the present study was to evaluate the prevalence of HEV, TTV and GBV-C/GBV-C/HGV in patients with acute viral hepatitis A, B and non-A-C. We evaluated sera of 94 patients from a sentinel program who had acute hepatitis A (N = 40), B (N = 42) and non-A-C (N = 12); 71 blood donors served as controls. IgM and anti-HEV IgG antibodies were detected by enzyme immunoassay using commercial kits. TTV and GBV-C/HGV were detected by nested PCR; genotyping was done by sequencing and phylogenetic analysis. Anti-HEV IgG was present in 38, 10 and 17 percent of patients with hepatitis A, B and non-A-C. Four patients with hepatitis A and 1 with non-A-C hepatitis also had anti-HEV IgM detected in serum. TTV was detected in 21 percent of patients with acute hepatitis and in 31 percent of donors. GBV-C/HGV was detected in 9 percent of patients with hepatitis, and in 10 percent of donors. We found TTV isolates of genotypes 1, 2, 3, and 4 and GBV-C/HGV isolates of genotypes 1 and 2. Mean aminotransferase levels were lower in patients who were TTV or GBV-C/HGV positive. In conclusion, the detection of anti-HEV IgM in some acute hepatitis A cases suggests co-infection with HEV and hepatitis E could be the etiology of a few cases of sporadic non-A-C hepatitis in Salvador, Brazil. TTV genotype 1, 2, 3 and 4 isolates and GBV-C/HGV genotype 1 and 2 strains are frequent in the studied population. TTV and GBV-C/HGV infection does not appear to have a role in the etiology of acute hepatitis.

High prevalence of GB Virus C/Hepatitis G Virus RNA among Brazilian blood donors

The aim of this study was to investigate the presence of the Hepatitis G Virus on a population of blood donors from São Paulo, Brazil and to evaluate its association to sociodemographic variables. Two RT-PCR systems targeting the putative 5'NCR and NS3 regions were employed and the former has shown a higher sensitivity. The observed prevalence of HGV-RNA on 545 blood donors was 9.7 percent (CI 95 percent 7.4;12.5). Statistical analysis depicted an association with race/ethnicity, black and mulatto donors being more frequently infected; and also with years of education, less educated donors presenting higher prevalences. No association was observed with other sociodemographic parameters as age, gender, place of birth and of residence. DNA sequencing of nine randomly chosen isolates demonstrated the presence of genotypes 1, 2 and 3 among our population but clustering of these Brazilian isolates was not detected upon phylogenetic analysis

The prevalence of viral hepatitis among the Hmong people of northern Thailand.

Sera from 269 Hmong people (102 males and 167 females, with mean age 35.4 years, range 16-63 years) were examined in order to determine the seroprevalence of hepatitis virus infection. The seroprevalence rates for HAV (hepatitis A virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus) and TTV (TT virus) infection were 87.8% (n=140), 76.0% (n=150), 2.0% (n=150), 0.7% (n=150), 6.5% (n=139), 5.3% (n=94) and 25.6% (n=121) respectively. The rate for carriers of HBV (HBsAg) was 13.8% (20.5% in males and 9.6% females) with a peak prevalence in the 21-40 year age group. A high rate of HAV seropositivity was found among the younger subjects. The rate of HEV seroprevalence was low. The prevalence of TTV-DNA was high with no difference between the sexes. HGV-RNA prevalence was low and seen primarily in males. This study indicates that the Hmong people are endemically infected with HAV and HBV infection and should be considered for targeted vaccination. The role of TTV and HGV in producing illness and hepatic disease has yet to be determined in this population.

Prevalence and genotypes of GB Virus C/Hepatitis G virus among blood donors in Central Brazil

A survey was conducted in a blood donor population of Central Brazil aiming to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection and also to analyze the virus genotypes distribution. A total of 241 voluntary blood donors were interviewed at the State Blood Bank in Goiânia, State of Goiás, Brazil. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Seventeen samples were GBV-C/HGV RNA-positive, resulting in a prevalence of 7.1 percent (95 percent CI: 4.2-11.1). A significant trend of GBV-C/HGV RNA positivity in relation to age was observed, with the highest prevalence in donors between 29-39 years old. Ten infected individuals were characterized by reporting parenteral (30 percent), sexual (18 percent), both (6 percent) and intrafamiliar (6 percent) transmission. However, 7 (40 percent) GBV-C/HGV RNA-positive donors did not mention any potential transmission route. RFLP analysis revealed the presence of genotypes 1 and 2 of GBV-C/HGV; more precisely, 10 (58.9 percent) samples were found belonging to the 2b subtype, 4 (23.5 percent) to the 2a subtype, and 3 (17.6 percent) to genotype 1. The present data indicate an intermediate endemicity of GBV-C/HGV infection among this blood donor population, and a predominant circulation of genotype 2 (subtype 2b) in Central Brazil

Hepatitis G virus (HGV): current perspectives.

Five viruses are usually associated with hepatitis in humans: A-E. In addition to these viruses as aetiological agents of hepatitis, there remain a number of patients with hepatitis in whom no virus could be identified. It was therefore postulated that there may be other agents which may be causing hepatitis. Recently, two viruses have been associated with hepatitis: hepatitis G virus (HGV), and transfusion transmissible virus (TTV). Hepatitis G virus (HGV) is a single stranded RNA virus which represents a newly discovered virus belonging to the flavivirus family. HGV is distinct from hepatitis C virus (HCV) and the newly discovered GBV-A and GBV-B agents, while GBV-C represents an isolate of HGV. The structure of the HGV genome resembles that of HCV. HGV replicates in peripheral blood cells, while replication in liver cells has not been observed till date. Diagnosis of HGV infection is mainly by use of polymerase chain reaction (PCR), as serological techniques are still being developed. Epidemiological data indicate that the virus is prevalent throughout the world, including India and is transmitted via blood/blood products, sexually and vertically from infected mothers to children. The relationship between infection with the virus and presence of liver pathology is controversial and has not been proven beyond doubt, as majority of patients with HGV have no detectable evidence of disease.