A novel genotype of GB virus C: its identification and predominance among injecting drug users in Yunnan, China

Abstract

GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5'UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5'UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a "non-pathogenic virus".

Figure 1. Map of the Yunnan Province in the People's Republic of China.

Gray areas mark the five prefectures the 120 IDUs were recruited. GBV-C positive rates were shown in percentages.

Figure 2. Phylogenetic tree based on 5′UTR sequences of 378 nt in length.

The sequences corresponding to nucleotide positions -210 to 148 in the genome of prototype GBV-C isolate U36380. The known six GBV-C genotypes are indicated with number 1–6. Reference GBV-C sequences is indicated with isolate names followed by their Genbank accession numbers. The neighbor-joining tree was constructed using the Jukes-Cantor distances. Bootstrap values are shown in bold.

Figure 3. Phylogenetic tree reconstructed with GBV-C E2 region sequences.

These sequences were each 1004 bp in length, corresponding to nt 610–1613 in the U36380 genome. Otherwise all the labels are as the same as shown in figure 2.

Figure 4. Rooted phylogenetic tree based on the full-length GBV-C genome sequences of 48 isolates.

The isolate GBV-C tro (Genbank accession no. AF070476) was used as an outgroup. The symbol//indicates a trimmed branch. Putative recombinant isolates are boxed with dashed lines. Otherwise all the labels are as the same as shown in figure 2.

Figure 5. Phylogenetic tree based on partial 5′UTR region sequences.

These sequences were each 187 nt in length, corresponding to nucleotides -398∼-202 in the genome of GBV-C isolate U36380. The tree was constructed using the Minimum Evolution method. Except for G7, all the isolates were adopted from the previous report by Muerhoff et al. [2006].

Figure 6. Analysis on amino acid sequences of E2 region.

The entropy score (a mathematical measure of variability) at amino acid positions 75-348 within E2 region, analyzed with 158 GBV-C isolates. The gray bar indicates the amino acid positions starting from 75 and ending at 348. Within the gray bar a blue area indicates the three putative GBV-C E2 peptides inhibited HIV infection corresponding to aa 133–156, 289–306 and 325–342. Five red and 18 blue dots linking under the gray bar represent the 23 positions having the entropy score ≥0.4 (regarded as high variability) hat is measure by the scale on the left. A curve under the gray bar plots the entropy score against each amino acid position that is numbered by the ruler at the bottom. The blue area of the gray bar is further expanded; it shows an alignment of the consensus amino acid sequences from seven GBV-C genotypes spanning the blue area. In the alignment, the reported HIV-1 entry blocking peptide domain of GBV-C E2 region are cited as the consensus sequence. Symbol * indicates the site with variation lower than 10%, which considered as conservation site. For comparison, 21 reference strains of genotype 1, 64 strains of genotype 2, 26 strains of genotype 3, 2 strains of genotype 4, 18 strains of genotype 5, 2 strains of genotype 6 and 25 strains of our discoved genotype 7 were apopted. The pink little tri-angle, diamond and disk represent the mutation in this site account for 10%-20%, 20-50% and more than 50% of total cited strains in this genotype, respectively.