Hain length of 20 million states under HKY+G4. In each case, MCMC chains were run for sufficient time to achieve convergence. Uncertainty in the data was described by 95 high-probability density (HPD) intervals. Convergence of trees was checked using Tracer v1.5 (available at: http://beast.bio.ed.ac.uk/Tracer). The inferred trees were visualized using FigTree ver. 1.3.1 (available at: http://tree. bio.ed.ac.uk/software/figtree/). We utilized the Bayesian skyline plot (BSP) as a coalescent prior to inferring the population dynamics of GBV-C within the HIV infected individual. We randomly selected 10 HIV infected patients representing different geographic region of Hubei province and performed the Bayesian coalescent analysis on each set of sequences representing each patient and evaluated the BSP patterns. The estimated population size reflects the effective population size of GBV-C in each patient. Therefore, the unit of BSP should be the viral effective population size through time. To determine the putative role of MedChemExpress CTX-0294885 positive selection (v.1) in the GBV-C viral diversity within each patient, we performed sitespecific positive selection analysis using Fixed- Effect Likelihood (FEL) via the Datamonkey web server [46]. Site with Pvalue,0.05 were considered to be under positive selection. The ML approach implemented in CODEML of PAML package version 3.15[47] was also used to detect the sites under positive selection in each patient. The codon-based substitution models (M7, M8) implemented in the CODEML allows the dN/dS to vary among sites. The likelihood ratio test (LRT) was used to compare M7 model that assume no positive selection (dN/dS,1)Table 2. Detection of recombination in complete E2 sequences by six different methods.Recombination Event Number 1 2 3aBreakpoint Positionsa 636-32 1106-493 662 – 1106 536 -Recombinant Sequence(s) ZX_M_15_014 ZX_M_15_020 JL_M_29_42 JL_M_29_RDP 6.33E-19 8.61E-10 4.95E-12 NSGENECONV 3.60E-13 1.CX-4945 18E-09 1.44E-08 7.35E-Maxchi 1.04E-13 1.96E-13 1.70E-09 8.92E-Chimaera 1.37E-13 3.98E-08 1.28E-09 5.79E-SiSscan 4.09E-17 6.61E-13 2.57E-11 1.14E-3Seq 6.53E-23 1.80E-05 3.38E-22 NSBreakpoint Positions Relative to U36380. NS: Not significant at p = 0.0005. doi:10.1371/journal.pone.0048417.tIntra-Host Dynamics of GBV-C in HIV PatientsFigure 2. Phylogenetic tree inferred from the complete E2 sequence data showing GBV-C variants in each HIV-infected subjects formed a unique cluster and emerged as a unique lineage with strong statistical support. Sequences representing each genotype were used as references for genotype identification. Sequences with GenBank accession numbers were the reference sequences. Isolates shaded in grey colors were the recombinant sequences (Table 2). Patients YXX_M_11 and JL_M_29 together formed a unique cluster. All the variants of JL_M_29 clustered together and appeared to emerge from a single GBV-C variant of YXX_M_11. GBV-C in patients QC_M_5, XA_M_20, and JZ_M_26 appearedIntra-Host Dynamics of GBV-C in HIV Patientsto be monophyletic and therefore shared the common ancestor. Bootstrap support 70 were shown at the base of the node. Each patient was coded with geographic region, sex, and a unique patient number. doi:10.1371/journal.pone.0048417.gwith the M8 model that assume positive selection (dN/dS.1). Sites with Bayes Empirical Bayes (BEB) posterior probabilities .95 were considered to be under positive selection.population 1662274 within JL_M_29 was emerged from a founding population (Fig. 2; Tab.Hain length of 20 million states under HKY+G4. In each case, MCMC chains were run for sufficient time to achieve convergence. Uncertainty in the data was described by 95 high-probability density (HPD) intervals. Convergence of trees was checked using Tracer v1.5 (available at: http://beast.bio.ed.ac.uk/Tracer). The inferred trees were visualized using FigTree ver. 1.3.1 (available at: http://tree. bio.ed.ac.uk/software/figtree/). We utilized the Bayesian skyline plot (BSP) as a coalescent prior to inferring the population dynamics of GBV-C within the HIV infected individual. We randomly selected 10 HIV infected patients representing different geographic region of Hubei province and performed the Bayesian coalescent analysis on each set of sequences representing each patient and evaluated the BSP patterns. The estimated population size reflects the effective population size of GBV-C in each patient. Therefore, the unit of BSP should be the viral effective population size through time. To determine the putative role of positive selection (v.1) in the GBV-C viral diversity within each patient, we performed sitespecific positive selection analysis using Fixed- Effect Likelihood (FEL) via the Datamonkey web server [46]. Site with Pvalue,0.05 were considered to be under positive selection. The ML approach implemented in CODEML of PAML package version 3.15[47] was also used to detect the sites under positive selection in each patient. The codon-based substitution models (M7, M8) implemented in the CODEML allows the dN/dS to vary among sites. The likelihood ratio test (LRT) was used to compare M7 model that assume no positive selection (dN/dS,1)Table 2. Detection of recombination in complete E2 sequences by six different methods.Recombination Event Number 1 2 3aBreakpoint Positionsa 636-32 1106-493 662 – 1106 536 -Recombinant Sequence(s) ZX_M_15_014 ZX_M_15_020 JL_M_29_42 JL_M_29_RDP 6.33E-19 8.61E-10 4.95E-12 NSGENECONV 3.60E-13 1.18E-09 1.44E-08 7.35E-Maxchi 1.04E-13 1.96E-13 1.70E-09 8.92E-Chimaera 1.37E-13 3.98E-08 1.28E-09 5.79E-SiSscan 4.09E-17 6.61E-13 2.57E-11 1.14E-3Seq 6.53E-23 1.80E-05 3.38E-22 NSBreakpoint Positions Relative to U36380. NS: Not significant at p = 0.0005. doi:10.1371/journal.pone.0048417.tIntra-Host Dynamics of GBV-C in HIV PatientsFigure 2. Phylogenetic tree inferred from the complete E2 sequence data showing GBV-C variants in each HIV-infected subjects formed a unique cluster and emerged as a unique lineage with strong statistical support. Sequences representing each genotype were used as references for genotype identification. Sequences with GenBank accession numbers were the reference sequences. Isolates shaded in grey colors were the recombinant sequences (Table 2). Patients YXX_M_11 and JL_M_29 together formed a unique cluster. All the variants of JL_M_29 clustered together and appeared to emerge from a single GBV-C variant of YXX_M_11. GBV-C in patients QC_M_5, XA_M_20, and JZ_M_26 appearedIntra-Host Dynamics of GBV-C in HIV Patientsto be monophyletic and therefore shared the common ancestor. Bootstrap support 70 were shown at the base of the node. Each patient was coded with geographic region, sex, and a unique patient number. doi:10.1371/journal.pone.0048417.gwith the M8 model that assume positive selection (dN/dS.1). Sites with Bayes Empirical Bayes (BEB) posterior probabilities .95 were considered to be under positive selection.population 1662274 within JL_M_29 was emerged from a founding population (Fig. 2; Tab.