GB virus C (GBV-C), a human virus of the Flaviviridae family that is structurally and epidemiologically closest to hepatitis C virus (HCV), has been reported to confer beneficial outcomes in HIV-positive patients. However, the prevalence of GBV-C in HIV-positive individuals in Indonesia is unknown. Since GBV-C is more prevalent in anti-HCV positive patients than in anti-HCV negative subjects, transmission of GBV-C and HCV could be by the same method. This study examined the prevalence and molecular characteristics of GBV-C infection in HIV patients in Yogyakarta, Indonesia. The prevalence of GBV-C among HIV patients (n = 125, median age 31 years) based on the 5′UTR region was 111/125 (88.8%), including 39/48 (81.3%) and 72/77 (93.5%) HIV-infected patients with and without HCV infection, respectively. GBV-C isolates were of genotype 2a, 3 and 6 in 58.3%, 12.6% and 28.4% of patients, respectively. Patients with genotype 3 were significantly younger than those with genotypes 2a or 6 (P = 0.001 and P = 0.012, respectively). Genotypes 3 and 6 were significantly associated with injection drug use (P = 0.004 and P = 0.002, respectively) and HCV co-infection (P < 0.001 for both genotypes), indicating a shared transmission route with HCV. In conclusion, the prevalence of GBV-C among HIV-positive patients in Indonesia is high, and three genotypes were detected, namely genotype 2a, 3 and 6.
GB virus C
hepatitis C virus
GB virus C, which was discovered in 1995, is a non-pathogenic human virus belonging to the Flaviviridae family. It is structurally and epidemiologically closest to hepatitis C virus (HCV) [1, 2]. The virus is an enveloped, positive-sense, single-stranded RNA virus (9.4 kb) with lymphotrophic properties [1, 3, 4]. Although some studies have suggested that this virus is associated with non-Hodgkin's lymphoma [5-7], one study did not confirm this association .
Like other lymphotrophic viruses, GBV-C is reportedly transmitted both sexually and parenterally. An earlier report also described vertical transmission from mother to child . Interestingly, that study also showed that GBV-C infection in infants, but not in their mothers, was associated with reduced mother-to-child transmission of HIV .
Because it is transmitted through similar pathways, GBV-C is frequently found in blood-borne infectious diseases, such as HIV, and in HCV-infected subjects . The prevalence of GBV-C infection in injection drug users is reportedly 56% and up to 95% of these subjects had prior infection [11, 12]. Several studies have found that the presence of GBV-C confers beneficial outcomes in patients with HIV and HCV . For example, GBV-C infection is reportedly associated with prolonged survival and a 2.5-fold decrease in mortality in HIV-infected patients and GBV-C clearance is greater in patients with chronic HCV infection [13-15]. GBV-C is also reportedly associated with less severe compensated and decompensated cirrhosis .
So far, only two studies have investigated the prevalence of GBV-C in Indonesian patients with liver diseases, those on maintenance hemodialysis and blood donors [16, 17]. These studies showed that the prevalence of GBV-C RNA was higher in patients on maintenance hemodialysis than in patients with liver diseases [16, 17]. The prevalence of GBV-C in HIV-infected patients in Indonesia and its clinical importance are currently unknown. Therefore, we designed the present study to determine the prevalence of GBV-C in HIV-infected patients and to identify the clinical markers associated with GBV-C infection.
MATERIALS AND METHODS
Sera were collected from patients with HIV visiting Dr Sardjito Hospital, Yogyakarta, Indonesia between April and July 2010 and stored at −30 to −80°C until use. Clinical and other relevant data were collected at the same time. There were 125 patients with an age range of 21–60 years (median, 31 years), including 77 (61.6%) men, 35 (28%) women and 13 (10.4%) transgender persons. Forty-eight patients were anti-HCV antibody-positive, indicating HCV co-infection. Anti-HCV antibodies were detected using a passive serological assay kit (Ortho HCV-Ab PA Test II; Fujirebio, Tokyo, Japan). This study was approved by the Ethics Committees at Kobe University, Japan and at Gadjah Mada University, Indonesia and all subjects gave written informed consent.
Detection of GB virus C RNA by reverse transcription polymerase chain reaction and sequencing
Total RNA was extracted from serum using QIAamp Ultrasense Virus or QIAamp Viral Mini kits (Qiagen, Tokyo, Japan). RT-PCR was performed using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and specific primers. Hemi-nested PCR to amplify the 5′UTR region was performed using two oligonucleotide primers, S1 (5′-CAC TGG GTG CAA GCC CCA GAA-3′) and GBVCE1wb2 (5′-CAG GGC GCA ACA GTT TGT GAG-3′) for the first PCR, and another primer, designated 4R (5′-CGG AGC TGG GTG GCC CCA TGC-3′) instead of the E1wb2 primer for the second PCR . Thermocycling conditions involved 40 cycles of denaturation (94°C for 20 s), annealing (55°C for 30 s) and extension (72°C for 45 s), followed by extension at 72°C for 10 min . The primer sets used to amplify the E1 region were the same as described in a previous study . The cycling temperatures used for both the first and second rounds were 1 min at 94°C, 1 min at 45°C and 2 min at 72°C . The PCR products were subjected to 2% agarose (Agarose S; Nippon Gene, Tokyo, Japan) gel electrophoresis and ethidium bromide staining, and then purified by ExoSAP-IT (USB, Cleveland, OH, USA). Direct base sequencing was performed by the dideoxy chain termination method with BigDye version 3.1 (Applied Biosystems, Foster City, CA, USA). Sequencing was performed using an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems).
Phylogenetic analysis of the GBV-C 5′-UTR region was performed using MEGA-4 software  and sequences were aligned using CLUSTALX software . Sequences were also analyzed using the BLAST algorithm (http://www.ncbi.nlm.nih.gov) and BioEdit . Genotyping was performed by phylogenetic analysis based on 5′-UTR sequences that represent separated geographic regions and have been published elsewhere: U44402, U45966, D87255, U36380, U59545, U59529, U59531, U63715, AB013193, AB018650, AB018654, D87712, D87714, AB013189, AB018648, AB018651, AB018656, AB021287, AB026062, AB026065, AF131111, AF131112, AB003292, AB026076, AB026077, HQ331233 and HQ331235. The distances of nucleotide sequences were computed using the Jukes–Cantor method. Neighbor–joining trees were developed using the method proposed by Saitou and Nei . The bootstrap consensus tree was inferred from 1000 replicates.
Data were analyzed by X2 or Fisher's test. Values of P < 0.05 were considered statistically significant. Statistical analyses were performed using SPSS software version 17.0 (SPSS, Chicago, IL, USA).
Prevalence of GB virus C
The GBV-C 5′-UTR sequence was detected in 111 (88.8%) HIV-infected patients, 39/48 (81.3%) of whom were HIV-infected and 72/77 (93.5%) without HCV infection. Clear sequences were obtained in 95 (85.6%) patients.
GB virus C genotype
Alignment of the representative 5′-UTRs of GBV-C showed marked variability and several conserved regions (Fig. 1). Some nucleotide substitutions and deletions differentiated genotype 6 from Indonesia with genotype 6 isolated from Japan (AB003292) and the other genotypes. Based on the GBV-C 5′-UTR sequence alignment, a phylogenetic tree was developed to determine the relationship between the isolated genotypes. In the present study, the GBV-C isolates could be classified into genotypes 2a, 3 and 6 in 56 (58.9%), 12 (12.6%) and 27 (28.4%) patients with HIV, respectively (Fig. 2).
Mean evolutionary distances were determined to calculate the extent of nucleotide variations within and between different GBV-C genotypes (Table 1). This table shows that the mean intra-genotype distances were all shorter than the inter-genotype distances. Therefore, GBV-C could be classified into seven genotypes. The mean distances within subtypes 2a and 2b were shorter than those between these genotypes. Isolates from the USA and Europe (genotypes 2a and 2b) showed less variation (mean intra-subtype distance 0.010 and 0.007, respectively,) than did isolates from Africa and Asia. The mean intra-subtype distances for genotypes 1 and 5, which are widely distributed in Africa, were 0.062 and 0.027, respectively, whereas mean intra-subtype distances for genotypes 3, 4, 6 and 7, which are indigenous to Asia, ranged from 0.019 to 0.032. In terms of nucleotide distances, genotype 6 from Indonesia was closest to genotype 4 (0.078), which is mainly localized to Myanmar and Vietnam , followed by genotype 3 (0.086) from East Asia [25-27] and genotype 7 (0.092) from China .
Amplification of an approximately 280 bp of E1 region was also performed to confirm the previous genotyping by 5′UTR region. Ten samples of genotype 6, ten of genotype 3 and eight of genotype 2a were selected. Of these samples, six (60%), two (20%) and one (12.5%), respectively, were successfully amplified for the E1 region by nested PCR.
The generated phylogenetic tree (Fig. 3) exposed the presence of genotypes 2a, 3 and 6. The genotype 6 demarcated in the samples of the present study were in the same group as the IndHD50 isolate from Indonesia. This IndHD50 isolate has been previously defined as genotype 6 by the 5′UTR region [16, 29]. When carefully assessed, genotypes 6 from Indonesian isolates differed from Japanese isolates (AB003292) by this E1 region, even though they were more similar by the 5′UTR region. Phylogenetic analysis by E1 region was not able to clearly distinguish genotypes 2a and 2b in the present study.
Comparison of clinical and other relevant characteristics between patients with GBV-C genotype 2a and patients with GBV-C genotypes 3 or 6
As shown in Table 2, patients with genotype 3 were significantly younger (P = 0.001) than patients with genotype 2a, but not patients with genotype 6. Significantly more patients with genotype 3 than genotype 6 were < 30 years old (P = 0.012; data not shown). Injection drug use and anti-HCV antibody-positive status were significantly associated with genotypes 3 (P = 0.004) and 6 (P = 0.002), respectively. Sexual contact risk factors were more common in patients with genotype 2a than in patients with genotype 6 (P = 0.002). Excluding subjects who were both injection drug users and had sexual contact risk factors, sexual contact risk factors were also significantly associated with genotype 2a rather than genotype 3 (P = 0.022) (data not shown). Higher education and ALT ≥ 40 IU/L were associated only with genotype 6 (P = 0.010 and P = 0.021, respectively). Apart from age, there were no significant differences between genotypes 3 and 6.
|n||Genotype 2a||Genotype 3||Genotype 6||P-valuec|
|56 (%)||12 (%)||27 (%)||2a vs. 3||2a vs. 6|
|Age, years||>30||34 (61)||1 (8)||15 (54)||0.001||0.587|
|Sex||Male||25 (45)||8 (67)||18 (64)||0.166||0.071|
|Educationa||Higher||32 (57)||10 (83)||23 (85)||0.112||0.010|
|IDU||Yes||12 (21)||8 (67)||15 (56)||0.004||0.002|
|Sexual contact risk factors||Yes||47 (84)||7 (58)||14 (52)||0.108||0.002|
|HAARTb||Yes||44 (80)||10 (83)||24 (89)||1.000||0.365|
|HAART durationb||>1 year||20 (45)||6 (60)||16 (67)||0.494||0.113|
|HBsAg||Positive||3 (5)||0 (0)||3 (11)||1.000||0.390|
|Anti-HCV||Positive||5 (9)||8 (67)||16 (59)||<0.001||<0.001|
|CD4 countb, cells/µL||<200||29 (52)||7 (58)||14 (54)||0.771||0.932|
|ALT, IU/L||≥ 40||8 (14)||4 (33)||10 (37)||0.203||0.021|
|APRIb||>0.5||9 (16)||1 (9)||9 (36)||1.000||0.057|
There are two reported studies on GBV-C in various patient subgroups in two cities in Indonesia, Yogyakarta  and Surabaya . The prevalence of GBV-C RNA differed between these two cities in patients with chronic hepatitis (8% vs. 5.7%), liver cirrhosis (5% vs. 11.5%), hepatocellular carcinoma (3% vs. 7%) and patients on maintenance hemodialysis (55% vs.29%) [16, 17]. In Surabaya, the prevalence of GBV-C was 17.8% and 2.7% in blood donors with and without anti-HCV antibodies, respectively (P < 0.001) . It can be inferred from these studies that the prevalence of GBV-C is higher in patients on maintenance hemodialysis than in patients with liver diseases. The present study is the first study in Indonesia to characterize GBV-C among HIV patients. Surprisingly, the majority of HIV-positive patients (88.8%) were positive for GBV-C. This high prevalence, which is similar to that in patients on maintenance hemodialysis, could be attributable to sharing injection devices, a common mode of GBV-C transmission according to previous studies [28, 30, 31].
The choice of primers for gene amplification also influences the detected prevalence of GBV-C. Earlier studies showed that primers for the 5′-UTR region are more sensitive than other widely used primers, which amplify the E2, non-structural 3 and NS5A regions [32, 33]. However, another study investigating GBV-C status in relation to the clinical outcome of HIV-positive patients suggested that at least two different sets of primers that amplify two different regions should be used . Therefore, the prior study having defined GBV-C positivity based on detection of the 5′-UTR and E2 regions could have resulted in a marked difference in HIV viral load between patients identified as GBV-C-positive and -negative . Although the specificity of E2 primers is high, their sensitivity is low; the specificity of primers for the conserved region and length of the sequence to be amplified determines their sensitivity . Previous studies have determined the prevalence of GBV-C by amplifying the NS3 region of the GBV-C genome [16, 17]. In the current study, because the primers are specific for the conserved region, they may have been more sensitive at detecting GBV-C RNA in the wider population, including in HIV-positive patients [18, 19]. The sense primer (S1) and the antisense primer (E1wb2) were designed after aligning the GBV-A and GBV-C genomes because it was thought that targeting the most conserved sequence in these two dissimilar viruses would yield primers targeting a highly conserved region . The other antisense primer (4R) was taken from the absolutely conserved region of the 42 sequences. These factors might help to explain the high prevalence of GBV-C RNA detected in the present study. Furthermore, the 5′-UTR region might discriminate GBV-C into several clusters better than the other regions, supporting its use in genotyping and phylogenetic analysis [16, 29].
The geographical diversity of GBV-C is related to human evolution, which began in ancient Africa and then disseminated to create other populations in Asia and America [34, 35]. A constant geographical cluster with multiple genotypes has been demonstrated; seven genotypes have been identified in different regions. Genotype 1 is the dominant genotype in Africa, genotype 2 is dominant in the USA and Europe, and genotype 3 was first reported in East Asia [25-27]. Studies have since localized genotype 4 to Myanmar and Vietnam , genotype 5 to South Africa , genotype 6 to Indonesia [16, 29] and genotype 7 to Yunnan, China . Genotypes 2, 3, 4 and 6 have been identified in patients in Surabaya, Indonesia . The present study also detected genotypes 2a, 3 and 6 among patients with HIV in Yogyakarta, Indonesia. Genotype 6 is closest to genotypes 4, 3 and 7, which are the dominant genotypes in Asia. However, an earlier study suggested that genotypes 4, 6 and 7 should be reclassified as a single genotype as more GBV-C variants are described .
It seems that genotyping by E1 region supports the data of three genotypes, genotype 2a, 3, and 6, distributed in Indonesian HIV patients in the present study (Fig. 3). However, the genotypes are less reliably distinguished by the E1 region than the 5′UTR region, as documented in an earlier study . It is possible that obtaining a longer segment of the 5′UTR region or the complete genome would achieve more precise results .
In the current study, patients with genotype 3 were significantly younger than those with genotypes 2a and 6 (P = 0.001 and P = 0.012, respectively; data not shown). Patients with genotype 6 had a significantly higher level of education (P = 0.010) and a higher proportion had ALT levels ≥40 IU/L (P = 0.021) than did patients with genotype 2a. Both genotype 3 and 6 were correlated with injection drug use and anti-HCV antibody positive status. However, genotype 2a was more strongly associated with sexual contact risk factors than were genotypes 3 and 6, suggesting that this genotype is transmitted sexually. Studies in Yunnan, a province in southwest China in which blood-borne infections are prevalent because of the high rate of drug trafficking, revealed the presence of several unique HCV genotypes [37, 38]. Genotype 7, a novel GBV-C genotype that was studied in this province and is frequent in injection drug users, is closer to the other GBV-C isolates circulating in Southeast Asia, particularly genotypes 4 and 6 . Additionally, as shown in Table 1, the sequences characteristics of genotype 3 are closer to genotype 6 than to genotype 2a.
In conclusion, the prevalence of GBV-C among HIV-positive patients in Indonesia is high and three genotypes were detected, namely genotypes 2a, 3 and 6. Injection drug use and anti-HCV antibody positive status are associated with transmission of genotypes 3 and 6, but not genotype 2a. Genotypes 3 and 6 are also associated with HCV co-infection, suggesting that these genotypes are transmitted via the same mode as HCV. However, no difference in the CD4-positive cell count between genotypes was found (Table 2). However, this was a cross-sectional study; more rigorous studies are needed to explore these findings because co-infection with GBV-C may have some advantages in subjects with HIV infection.
The authors thank Totok Utoro, MD, PhD, Bambang Sigit Riyanto, MD, Abu Tholib, MD, PhD and Ahmad Hamim Sadewa, MD, PhD for help in sample collection and their support and encouragement. We are also grateful to Ikuo Shoji, MD, PhD for priceless advice concerning preparation of the manuscript. This study was supported by a Grant-in-Aid from the Japanese Initiative for Global Research Network on Infectious Diseases (J-GRID), supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
The authors have no conflicts of interest to declare.