In this study, the infection with HIV-1, HCV and GBV-C was investigated among 120 IDUs who were recruited in five prefectures of Yunnan province, China. We found the overall GBV-C RNA positive rate (43/120, 35.8%) was agreed with previous reports that GBV-C infection rate among IDUs ranged from 17.9% to 58.0% 
. Among the total recruited subjects, 70 (58.3%) and 103 (85.8%) were found to be anti-HIV and anti-HCV positive, respectively. This accounted for 18.3% of IDUs having the GBV-C/HIV-1/HCV triple infection, 7.5% and 10.0% IDUs having the GBV-C/HIV-1 and GBV-C/HCV dual infection. Significantly, the rate of GBV-C/HIV-1/HCV triple infection was higher (P<0.05) than the rates of GBV-C/HIV-1 and GBV-C/HCV dual infection. Notably, no one was detected to be GBV-C mono-infection. It seems that the transmission of GBV-C though unsafe syringe using is not effective as HCV or HIV-1. In addition, a question is raised – if individuals with HIV-1/HCV dual infection are more susceptible to opportunistic infection such as with GBV-C 
? Yet, an explicit answer to this question requires more analyses. Detection of GBV-C anti-E2 may be only indicative but not conclusive, because it indicates either a past or an active GBV-C infection depending on individuals. In this study, the majority of anti-E2 positive IDUs (29/31) were RNA negative, for whom past GBV-C infection was suggested 
. However, in two IDUs who were clinically confirmed to have AIDS, both GBV-C anti-E2 and GBV-C RNA were positive, for whom active GBV-C infection was approved. Since these two patients were also positive for anti-HIV, other possibilities could exist. A simultaneous detection of the three markers, anti-HIV, GBV-C anti-E2, and GBV-C RNA, may reflect the fact that these individuals have more serious immunity damage; they may have produced higher titer of virus.
For GBV-C genotyping, different methods have been used. These included RT-PCR with specific primers, restriction fragment length polymorphism, and direct DNA sequencing 
. Among these methods, DNA sequencing is the gold standard and the most preferred but requires phylogenetically informative regions to be analyzed. For such a purpose, the highly conserved 5′-UTR or its partial fragment is often concerned 
. Some researchers have argued that phylogenetic analysis of this region may not always supply a solid genotyping result. In contrast, sequencing the complete or partial E2 region can provide stronger information, which is nearly in a complete accordance with sequencing the full-length GBV-C genomes 
. Ultimately, defining a new viral variant or assigning a novel viral group constantly requires the full-length viral genome(s) being characterized. Based on this premise, a stepwise strategy was used in this study to process samples collected from 120 IDUs. Firstly, sequence of GBV-C 5′UTR was screened for all samples and this resulted in 43 samples (35.8%) positive. Of the obtained 43 isolates, 40 (93%) were classified into a new phylogenetic group. Secondly, the complete GBV-C E2 region was amplified in samples positive for GBV-C 5′UTR. This was done to verify the new GBV-C group. From 24 IDUs the E2 region sequences were amplifiable and all were classified into a new GBV-C group we provisionally designated GBV-C genotype 7. Lastly, three full-length GBV-C genomes were characterized; it provides us the ultimate information for defining a new genotype.
Of this study, the GBV-C genotype distribution pattern is vastly different from that previously reported in China where genotype 3 is predominant 
. This pattern looks also different from that observed in Southeast Asia where genotypes 4 and 6 are common 
. Co-analysis of reference 5′UTR sequences showed that GBV-C genotype 7 was also found in Southeast Asia, albeit not common. The basal location of Southeast Asia sequences in phylogenetic tree implies that GBV-C genotype 7 may be indigenous in this region. Recently, an overland drug trafficking route may have exchanged viral strains with those in Southeast Asia. Through Yunnan as a center, this route has been proven to play a critical role in the transmission of HIV-1 and HCV infection from Southeast Asia to the other parts China 
. Mutually, this route may have also spread the infection with GBV-C between Yunnan and its neighboring countries. Up to now, GBV-C genotype 7 has not been found in other provinces marginal to Yunnan, such as Guangxi and Sichuan, where genotype 3 was the only discovered GBV-C strains major due to the lake of recent GBV-C investigation there. However, the spread of this novel GBV-C genotype through modern transmission route could be predicted.
In this study, GBV-C genotype 7 was designated by following the system Mueroff and his colleagues had recommended 
. However, based on the analysis of E2 region sequences and/or full-length genomes, five major GBV-C groups may be proposed. Four groups are represented by genotypes 1, 2, 3 and 5, respectively; while the fifth group is composed of genotypes 4, 6 and the newly designated GBV-C genotype 7. For more than a decade, GBV-C had been well classified into four genotypes: 1–4. This has been established with sequences having origins from exclusive geographic regions: genotype 1 from Africa, genotype 2 from Europe and America, genotype 3 from East Asia, and genotype 4 from Southeast Asia. Recently, genotype 5 has been proposed with sequences from Africa 
. Genotype 6 has been separated from genotype 4 with sequences from Indonesia and Japan 
. Using a similar approach, we also separated an Indonesian isolate, IndHD92, from a previous grouping into genotype 4 as a now grouping into genotype 7 (). Genotypes 4, 6, and 7 can compose a larger genetic group (), which geographically from Southeast Asia or its neighboring region such as Yunnan in the present study. These distribution patterns indicate their common origins. Unlike many other viruses, currently there is a lack of consented and quantified criteria set for GBV-C classification of nucleotide sequences. Even though such a standard is established, it may not always meet the increasing need of classifying new viral variants. For example, new variants are kept being identified for HCV. However, a previously quantified criterion failed to classify them consistently, which had caused a confusion of six or 11 HCV genotypes. For this reason, a consensus paper has been subsequently modified, which now uniformly classifies HCV isolates based on phylogenetic analyses of their genetic sequences, no matter how greater genetic distances may be obtained 
. This wisdom should be also applied for GBV-C classification, we strongly recommend. Following this scheme, all GBV-C isolates would be classified into five major groups to assist for a simpler GBV-C nomenclature. On the other hand, virus genetic variation is a reflection of the continuous mutations accumulated over the past history of viral evolution. Spatial niches with variants of different evolutionary extents do exist. However, due to limited sampling and technical restriction, many of such variants have not been identified. These variants may present continuous genetic variations to fill the gaps between GBV-C genotypes 4, 6, and 7, we strongly believe. As more such variants are characterized, a simpler GBV-C nomenclature may be modified.
A benefit of GBV-C infection on disease progression of AIDS or HIV-1 replication has been documented 
. However, the impact of GBV-C genotypes on the progression of HCV or HIV-1 related diseases remains to be investigated. Although a few reports have described that GBV-C genotype 2 (2a and 2b) and 5 may be in association with a better immunological response among patients co-infected with HIV-1 
, there is a still lack of evidence supporting that GBV-C genetic diversity affects the HIV-1 caused clinical presentation. The identification of GBV-C genotype 7 and its predominance among IDUs enhanced our curiosity: if there is a correlation between GBV-C genotypes and the presence of clinical AIDS markers, which include CD4+
cell count and HIV-1 viral load. In addition, there is a need of investigating the correlation between GBV-C genotypes and HIV-1 subtypes or circulating recombinant forms (CRFs). As cousins of Flaviviridea
, GBV-C and HCV have similar genome organization. A potential association between the two viruses in the context of genetic diversity and evolution also need to be investigated.