A Retrospective Investigation On The Pathogenicity And Co-receptor Usage Of HIV-1 CRF01_AE Circulating Among Recently Infected Men Who Have Sex With Men(MSM)

Objective 1. To investigate the immune status and pathogenic-related CXCR4 co-receptor usage of HIV-1 CRF01_AE among recently infected men who have sex with men(MSM) in China. 2. To investigate the relationship between immune status, genetic subtypes, and co-receptor tropism among recently CRF01_AE-infected MSM individuals in China. 3. To explore the source of X4 among CRF01_AE-infected MSM individuals in China.Methods Three-hundred and sixty-four HIV-1-infected MSM individuals between age 18 and 25 living in Shanghai and newly diagnosed between January 2009 and July 2013, were analyzed retrospectively. Reverse transcription and nested polymerase chain amplification for partial genes of pol and env were performed by a home brew PCR procedure. A molecular algorithm that a frequency of ambiguous calls in bulk sequencing of pol gene under 0.44% was applied to distinguish recent infection from long-standing infection. Viral sequences were analyzed for co-receptor usage based on V3 loop sequences, using Web PSSM and Geno2 Pheno. Fisher’s exact test was used to calculate the tropism genotypic distributions among various subtypes, years, and CD4+T cell count groups. Nonparametric Tests(Nemenyi and Man-Whitney U) was used for statistical analysis of the relationship between CD4+T cell count and genetic subtypes and viral tropisms. Phylogenetic trees were constructered with software Mr Bayes and BEAST to indentify transmission clusters.Results 1. A total of 276 individuals were identified as recently infected. Subtype assignment were as follows: 176(63.8%) CRF01_AE, 77(27.9%) CRF07_BC, and 23(8.3%) subtype B/B’. Besides, 24 second-generation recombinant strains were identified. 2. A low level of baseline CD4+T cell count was found among 176 CRF01_AE-infected persons, compared to those who infected with 77 CRF07_BC(χ2=8.066, P=0.018), but not to 23 subtype B(χ2=1.774, P=0.412). The median CD4+T cell count in three subtypes-related groups were 415.5(IQR: 274.3-537.0) cells/μL, 476.5(IQR: 339.3-617.5) cells/μL, and 475.0(IQR: 351.0-590.0) cells/μL, respectively. 3. Remarkably, all CRF07_BC and subtype B viruses were predicted to be CCR5-using and thus CXCR4-usage was selectively observed in CRF01_AE(χ2=55.348, P<0.001; χ2=52.221, P<0.001). Although there was little discrepancy in tropism prediction between these two algorithms, a high accordant result was observed in CRF01_AE-infected persons(40.9% vs 32.4%). 4. In view of the fact that all predicted CXCR4-tropic viruses were found to be present among CRF01_AE strains(n=176), we analyzed the relationship between CXCR4-tropic virus and different stratified CD4+T cell count. It was not unexpected that the frequencies of predicted CXCR4-tropic strain were higher in individuals with CD4+T cell count ≤200 cells/μL(76.2% in algorithm I and 61.9% in algorithm II), compared to the subjects with higher CD4+T cell counts(algorithm I: χ2=12.228, P=0.006; algorithm II: χ2=11.940, P=0.008). 5. Among the 176 CRF01_AE infections, 22 and 16 of 176 sequences sorted into clusters. The percentage of sorted into clusters was no difference between X4 and R5(22/72=30.6% VS 36/104=34.6%, χ2=0.317, P>0.05; 16/72=22.2% VS 23/104=22.1%, χ2<0.001, P>0.05).Conclusions This study for the first time in China revealed that CRF01_AE strain with a high frequency of CXCR4-tropism has been circulating in young MSM population. The X4 among the recently CRF01_AE-infected MSM individuals maybe obtained through transmission. These might cause a severe loss of CD4+T cell count and speed up disease progression, compared to CRF07_BC strain. A regular surveillance of HIV-1 genetic subtypes, CD4+T cell count and viral co-receptor usage would be greatly beneficial for effectively monitoring disease progression, improvement of antiretroviral strategy and prompt intervention of transmission.