Genotypic Prediction Of Co-receptor Utilization For HIV-1 CRF01_AE And CRF07_BC Isolates In China And Near Full-Length Sequence Analysis

BackgroundThe mechanism of action of most anti-HIV therapeutic drugs is to inhibit the replication of HIV in host cells,and CCR5 antagonists are a new field of anti-HIV drug development by blocking the binding of gp120 to CCR5 and preventing HIV from entering host cells.CCR5 antagonists do not work in patients with X4 tropic virus infection.Therefore,testing for HIV tropism is recommended before the treatment of co-receptor antagonists.HIV tropism is identified with phenotypic tropism testing or predicted by genotypic tropism testing.The phenotypic testing is to use a recombinant virus,pseudovirus or live virus to infect a cell line that expresses CCR5 or CXCR4 to detect the utilization of the co-receptor.The phenotypic testing is recognized as the gold standard,but the presence of high operating requirements,expensive,time consuming drawback.The genotypic testing mainly uses the amino acid or nucleotide sequence of the V3 region to predict the utilization of co-receptor.The commonly used online prediction tools are WebPSSM and Geno2pheno.The genotypic testing are cheaper,yield results more quickly and are easier to standardize.However,most genotypic testing are based on the V3 region sequences of subtype B and subtype C.Genotypic prediction feasibility of co-receptor utilization for HIV-1 CRF01_AE and CRF07_BC remains to be further studied.CRF01_AE、CRF07_BC、CRF08_BC and B have become mainly circulating subtypes in china.In recent years,a number of CRF01_AE/CRF07_BC,CRF01_AE/B,CRF01_AE/CRF07_BC/B unique recombinant forms have been found in MSM,among which CRF5501_B,CRF5901_B,CRF6701_B,CRF79₀107 and CRF80 0107 have been developed into circulating recombinant forms.The timely discovery of new recombinant viruses and analysis of the source of their parental strains will help to understand the spread of HIV-1 and provide a scientific evidence for the prevention and control of HIV-1 in China.Objective1.To understand the utilization of CRF01_AE and CRF07 BC recombinant virus co-receptors in China.To analyze genotypic prediction feasibility of co-receptor utilization for HIV-1 CRF01_AE and CRF07 BC isolates in China.2.To explore new unique recombinant forms and circulating recombinant forms by analyzing HIV-1 near full-length genome.MethodsTwenty-two CRF01_AE and nineteen CRF07 BC strains were isolated from HIV-1 infected people in Beijing,Guangxi and Sichuan.The viral RNA was extracted from the strains,and then reverse transcribed into complementary DNA.Near-endpoint diluted cDNA templates were used to amplify the near full-length genome,and subtype and recombination patterns were analyzed for near-full-length sequences.GHOST cells expressing different co-receptors were infected with live virus,HIV-1SF33（X4/R5）dual tropism was used as a positive control,normal cells were used as a negative control,cells were collected after 48 hours of culture,and GFP was analyzed by flow cytometry to obtain HIV-1 co-receptor utilization.We compared the co-receptor utilization of viruses by both genotypic（11/25 rule,WebPSSM and geno2pheno）and phenotypic method based Ghost cell line.ResultsThe first section1.Four strains used the CCR5/CXCR4 co-receptor and 18 strains used the CCR5 co-receptor among 22 CRF01 AE recombinant virus as detected by the GHOST cell line.All the 19 CRF07 BC recombinant virus only used the co-receptor as detected by the GHOST cell line.2.Consistency analysis results:For CRF01_AE recombinant virus,the consistency rate between genotypic prediction of 11/25 rule and phenotypic test was 86.4%;Among geno2pheno,the consistency rates between genotypic prediction of G2p-clinical,NGS-sanger and G2p-colon models and phenotypic tests were 95.5%,86.4%,68.2%,respectively.In WebPSSM,the consistency rates between genotypic prediction of PSSM-sinsi B,PSSM-x4r5,and PSSM-sinsi_C models and phenotypic tests were 86.4%,72.7%,and 45.5%,respectively.For CRF07 BC recombinant virus,the consistency rate between genotypic prediction of 11/25 rule and phenotypic test was 94.7%;Among geno2pheno,the consistency rates between genotypic prediction of G2p-clinical,NGS-sanger and G2p-colon models and phenotypic tests were 94.7%,94.7%,100.0%,respectively.In WebPSSM,the consistency rates between genotypic prediction of PSSM-sinsi B,PSSM-x4r5,and PSSM-sinsi C models and phenotypic tests were 100.0%,94.7%,and 100.0%,respectively.3.Net charge analysis results of amino acids in V3 region:For CRF01 AE recombinant virus,V3 net charge in R5/X4 tropism was distributed from 5 to 7,and that of R5 tropism was distributed from 2 to 7.The rank sum test showed that P<0.01,and the difference was statistically significant.For CRF07 BC recombinant virus,V3 net charge of R5 tropism was distributed from 2 to 5.4.V3 loop tip motifs results:The V3 loop tip motifis of CRF01_AE recombinant virus had four types,including GPGQ（68.1%）、GPGR（22.7%）、GPGH（4.5%）、GLGR（4.5%）.The V3 loop tip motifis of CRF07_BC recombinant virus only had GPGQ（100%）.The second section1.A novel CCR5 tropic CRF01-AE/CRF07_BC unique recombinant virus（GX2016EU10）was identified,from a 24-year-old man,who was infected by homosexual sex in Guangxi.GX2016EU10 was obtained with length of 8938bp（relative to the HXB2 nucleotide numbering system:position 631-9603）.GX2016EU10 was composed of three CRF01_AE fragments and three CRF07 BC fragments.Relative to HXB2,the positions of the five recombination breakpoints were 1245,3864,5849,7895 and 8995,respectively.2.CRF01 AE fragments（V）could be clustered into a specific cluster 4a,which was mainly prevalent among MSM in northern China,with bootstrap values of 92%.CRF07 BC fragments（Ⅱ、Ⅳ、Ⅵ）were clustered into GZ070087,which was prevalent among MSM in Guizhou,with bootstrap values of 96%,97%and 71%,respectively.Conclusion1.For the HIV-1 infected individuals with CRF01_AE isolates in China,co-receptor usage should be predicted by Geno2pheno before the treatment of co-receptor antagonists.In the case of only the nucleotide or amino acid sequence of the V3 region,it is recommended to use the NGS-sanger model for prediction;in combination with clinical indicators（CD4 count,viral load,etc.）,it is recommended to use the G2p-clinical model for prediction.2.For the HIV-1 infected individuals with CRF07 BC isolates in China,co-receptor usage should be predicted by WebPSSM before the treatment of co-receptor antagonists.3.The emergence of GX2016EU10 indicates that the HIV-1 epidemic among MSM in Guangxi is increasingly more complicated.Therefore,it is necessary to strengthen the HIV-1 molecular epidemiological investigation.Timely discovery of the distribution of URF and CRF will help us to better understand the different population speard of HIV-1,providing scientific evidence for the prevention and control of HIV-1.