Identification of conserved nucleotide sequences within the GB virus C 5′-untranslated region: Design of PCR primers for detection of viral RNA

doi:10.1016/0166-0934(96)02088-5

Journal of Virological Methods

Volume 62, Issue 1, October 1996, Pages 55-62

https://doi.org/10.1016/0166-0934(96)02088-5 Get rights and content

Abstract

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5′-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

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Cited by (54)

GB virus C
2003, Perspectives in Medical Virology
GBV-C is a flavivirus-like enveloped RNA virus measuring 50-100 nm in diameter and bands in sucrose at a peak density of 1.08-1.13 g/cm3. The isolated agent was not a single virus, but two related viruses, termed GB virus A (GBV-A) and GB virus B (GBV-B). Further, molecular studies designed to determine the prevalence of these viruses in the human population resulted in the isolation of a third related virus, GB virus C (GBV-C). This virus was later described as hepatitis G virus. It possesses long, non-translated sequences both upstream and downstream, the ORF, 533 and 312 nucleotides, respectively. Determination of the genome-length sequences of the GB viruses revealed that each genetically related to one another and to hepatitis C virus (HCV). Currently, GBV-A and GBV-B have only been detected in non-human primates, while GBV-C has only been detected naturally in humans. GBV-C encodes at least two distinct envelope proteins (E1 and E2) and evidence suggests further post-translational cleavage of E2. Both proteins contain sugar moieties that substantially increase their molecular mass and a number of cysteine residues present suggests that they possess extensive tertiary structure.
Genetic diversity of GBV-C/HGV strains among HIV infected-IVDU and blood donors from Buenos Aires, Argentina
1999, Virus Research
GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5′ UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5′ UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.
The entire nucleotide sequence of a TT virus isolate from the United States (TUS01): Comparison with reported isolates and phylogenetic analysis
1999, Virology
A nonenveloped and single-stranded DNA virus designated TT virus (TTV) has been reported from Japan in association with hepatitis of unknown etiology. Very recently, the prototype TTV isolate (TA278) of genotype 1 is proven to have a circular genome with 3852 nucleotides. A TTV isolate (TUS01) was recovered from a blood donor in the United States, and its entire circular nucleotide sequence of 3818 nucleotides was determined. It possessed two open reading frames coding for 761 and 156 amino acids, respectively. TUS01 shared 60.5% of the nucleotide sequence with the TA278 isolate from Japan that was longer by 35 nt. The sequence of the noncoding region of 1203 nt was conserved with a similarity of 83.4%. Sequence preservation was much lower for the two open reading frames; nucleotide and amino acid sequences were 54.8 and 37.0% similar, respectively, for one and 55.5 and 38.8% similar for the other. By comparison of a partial sequence of 222 nucleotides among 239 TTV isolates available from various countries, at least 11 genotypes with sequence divergence of >30% were recognized. TUS01 was deduced to be of genotype 11, which has not been reported before. Conserved sequences in the noncoding region could be used as primers for sensitively detecting TTV DNA by polymerase chain reaction. Divergent sequences in coding regions would be useful as primers for distinguishing various TTV genotypes.
Prevalence of GBV-C/hepatitis G virus viraemia among blood donors, health care personnel, chronic non-B non-C hepatitis, chronic hepatitis C and hemodialysis patients in Egypt
1999, Journal of Virological Methods
A new RNA virus, designated GBV-C/hepatitis G virus (HGV) has been identified recently. To evaluate the prevalence of GBV-C/HGV infection among Egyptians, five groups were enrolled in this study: group I, healthy blood donors (82); group II, health care personnel (30); group III, chronic non-B non-C hepatitis patients (63); group IV, chronic hepatitis C patients (100); group V, renal dialysis patients (79). GBV-C/HGV-RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from 5′-non coding region of GBV-C/HGV. GBV-C/HGV-RNA was detected in 57 of 354 tested sera with an overall prevalence of 16.1%. Meanwhile, isolated GBV-C/HGV infection was detected in 16/57 (28.1%), GBV-C/HGV coinfection with hepatitis C virus (HCV) in 37/57 (64.9%) and with hepatitis B virus (HBV) in 4/57 (7.6%) of cases. The highest prevalence was encountered among dialysis patients reaching 30% followed by chronic hepatitis C (14%), blood donors (12.2%), chronic non-B non-C hepatitis (11.1%), whereas the lowest prevalence rate of 6.6% was detected among health care personnel. Nucleotide sequence analysis in three Egyptians confirmed that these PCR products were derived from GBV-C/HGV genome and all isolates classified into US/European type (type 2) of GBV-C/HGV genotypes. The risk factors of all cases were non-transfusion parenteral exposure, e.g. reusing syringes, dental treatment, surgery, invasive medical maneuvers, with an exception of renal dialysis patients who have had repeated blood transfusion. It is concluded that there is a relatively high prevalence of GBV-C/HGV-RNA among different Egyptian groups compared to international figures. The main risk factors were direct percutaneous exposure rather than blood transfusion. The Egyptian GBV-C/HGV isolates are very similar to the American isolate PNF 2161.
Intraspousal transmission of GB virus C/hepatitis G virus in an hepatitis C virus hyperendemic area in Japan
1999, American Journal of Gastroenterology
OBJECTIVE: An immunoassay for antibodies against an hepatitis G virus (HGV) protein (anti-E2) was recently developed that might serve as a useful marker for diagnosing recovery from HGV infection.
METHODS: We investigated the intraspousal transmission of GB virus C/hepatitis G virus (GBV-C/HGV) using both reverse transcription hemipolymerase chain reaction (RT-hemi-PCR for the 5′ untranslated region) and a recently developed anti-E2.
RESULTS: Thirty-two GBV-C/HGV-infected index subjects were selected from an hepatitis C virus hyperendemic area in Japan. Of the 32 subjects, seven (6.4%) were GBV-C/HGV RNA-positive, 24 (21.8%) were anti-E2-positive, and one (0.9%) was both GBV-C/HGV RNA- and anti-E2-positive. Among the 32 spouses of these subjects, GBV-C/HGV RNA, anti-E2, and both GBV-C/HGV RNA and anti-E2 positivity were detected in 0, 6, (18.8%), and one (3.1%) spouses, respectively (the total prevalence of GBV-C/HGV was 7 spouses [21.9%]). Thus, the intraspousal transmission of GBV-C/HGV was undeniable in these seven couples. The respective positive rates of 175 sex- and age-matched controls were 7 (4.0%), 26 (14.9%), and 0 (the total prevalence of GBV-C/HGV was 34 [19.4%]). No significant difference in positive rates was observed between the subjects/spouses and the controls. Five spouses among the seven couples who were positive for any of GBV-C/HGV markers had parenteral risk factors such as blood transfusion, acupuncture, and major surgery.
CONCLUSION: Based on these observations, we cannot draw a definitive conclusion that intraspousal transmission of GBV-C/HGV had occurred among these seven couples.
Hepatitis G virus infection in Chinese patients with chronic hepatitis B, C or non-A-E: Clinical and molecular aspects
1999, Hepatology Research
In the present study we investigated the epidemiology, clinical significance and molecular characteristics of hepatitis G virus (HGV) infection in Chinese patients with chronic hepatitis B, C or non-A-E. Based on the detection of HGV RNA by reverse transcription polymerase chain reaction, 17 of 70 patients (24%) with chronic post-transfusion hepatitis C were coinfected with HGV. In contrast, none of 21 patients with non-A-E hepatitis had detectable HGV RNA. Among nine patients with severe chronic hepatitis caused by HBV/HCV coinfection, two were HGV RNA positive without affecting the outcome. Sustained biochemical response to interferon-alpha was similar in patients with HCV infection only (22/51, 43%) and in patients with HCV/HGV coinfection (7/17, 41%). Among ten Chinese HGV isolates, the nucleotide sequence homology in the 5′-UTR was 91–100% as compared to 84–95% between the Chinese HGV isolates and those from other countries. No core gene was found in the Chinese HGV isolates examined. In conclusion, about 20% of Chinese patients with chronic hepatitis C are coinfected with HGV. Coinfection did not affect the natural course of chronic hepatitis C or the response to antiviral therapy. Sequence analyses revealed a high degree of homology among Chinese HGV isolates. HGV infection does not appear to play a major clinical role in Chinese patients with chronic hepatitis B, C or non-A-E.

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Research paperIdentification of conserved nucleotide sequences within the GB virus C 5′-untranslated region: Design of PCR primers for detection of viral RNA

Abstract

J. Virol. Methods

Lancet

Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay

Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma

J. Clin. Microbiol.

Cloning and genomic organization of a new virus associated with human hepatitis

J. Med. Virol.

Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent

Science

Research paper
Identification of conserved nucleotide sequences within the GB virus C 5′-untranslated region: Design of PCR primers for detection of viral RNA