First complete-genome documentation of HIV-1 intersubtype superinfection with transmissions of diverse recombinants over time to five recipients.

Human immunodeficiency virus type 1 (HIV-1) recombinants in the world are believed to be generated through recombination between distinct HIV-1 strains among coinfection or superinfection cases. However, direct evidence to support transmission of HIV-1 recombinants from a coinfected/superinfected donor to putative recipient is lacking. Here, we report on the origin and evolutionary relationship between a set of recombinants from a CRF01_AE/CRF07_BC superinfected putative donor and diverse CRF01_AE/CRF07_BC recombinants from five putative recipients. Interviews on sociodemographic characteristics and sexual behaviors for these six HIV-1-infected men who have sex with men showed that they had similar ways of partner seeking: online dating sites and social circles. Phylogenetic and recombination analyses demonstrated that the near-full-length genome sequences from six patients formed a monophyletic cluster different from known HIV-1 genotypes in maximum likelihood phylogenetic trees, were all composed of CRF01_AE and CRF07_BC fragments with two common breakpoints on env, and shared 4-7 breakpoints with each other. Moreover, 3' half-genomes of recombinant strains from five recipients had identical/similar recombinant structures with strains at longitudinal samples from the superinfected donor. Recombinants from the donor were paraphyletic, whereas five recipients were monophyletic or polyphyletic in the maximum clade credibility tree. Bayesian analyses confirmed that the estimated time to the most recent common ancestor (tMRCA) of CRF01_AE and CRF07_BC strains of the donor was 2009.2 and 2010.7, respectively, and all were earlier than the emergence of recombinants from five recipients. Our results demonstrated that the closely related unique recombinant forms of HIV-1 might be the descendent of a series of recombinants generated gradually in a superinfected patient. This finding highlights the importance of early initiation of antiretroviral therapy as well as tracing and testing of partners in patients with multiple HIV-1 infection.

Integrated analysis of lncRNA, miRNA and mRNA profiles reveals potential lncRNA functions during early HIV infection.

BACKGROUND: Long noncoding RNAs (lncRNAs) can regulate gene expression in a cis-regulatory fashion or as "microRNA sponges". However, the expression and functions of lncRNAs during early human immunodeficiency virus (HIV) infection (EHI) remain unclear. METHODS: 3 HAART-naive EHI patients and 3 healthy controls (HCs) were recruited in this study to perform RNA sequencing and microRNA (miRNA) sequencing. The expression profiles of lncRNAs, mRNAs and miRNAs were obtained, and the potential roles of lncRNAs were analysed based on discovering lncRNA cis-regulatory target mRNAs and constructing lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) networks. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on 175 lncRNA-associated differentially expressed (DE) mRNAs to investigate the potential functions of DE lncRNAs in ceRNA networks. RESULTS: A total of 242 lncRNAs, 1240 mRNAs and 21 mature known miRNAs were determined as differentially expressed genes in HAART-naive EHI patients compared to HCs. Among DE lncRNAs, 44 lncRNAs were predicted to overlap with 41 target mRNAs, and 107 lncRNAs might regulate their nearby DE mRNAs. Two DE lncRNAs might regulate their cis-regulatory target mRNAs BTLA and ZAP70, respectively, which were associated with immune activation. In addition, the ceRNA networks comprised 160 DE lncRNAs, 21 DE miRNAs and 175 DE mRNAs. Seventeen DE lncRNAs were predicted to regulate HIF1A and TCF7L2, which are involved in the process of HIV-1 replication. Twenty DE lncRNAs might share miRNA response elements (MREs) with FOS, FOSB and JUN, which are associated with both immune activation and HIV-1 replication. CONCLUSIONS: This study revealed that lncRNAs might play a critical role in HIV-1 replication and immune activation during EHI. These novel findings are helpful for understanding of the pathogenesis of HIV infection and provide new insights into antiviral therapy.

Transmitted drug resistance to Tenofovir/Emtricitabine among persons with newly diagnosed HIV infection in Shenyang city, Northeast China from 2016 to 2018.

BACKGROUND: To assess transmitted drug resistance (TDR) to tenofovir (TDF)/emtricitabine (FTC), using as pre-exposure prophylaxis, among newly diagnosed human immunodeficiency virus-1 (HIV-1)-infected residents in Shenyang city, northeast China. METHODS: Demographic and epidemiological information of all newly diagnosed HIV-1 infected residents in Shenyang city from 2016 to 2018 were anonymously collected from the local HIV epidemic database. HIV-1 pol sequences were amplified from RNA in cryopreserved plasma samples and sequenced directly. Viral subtypes were inferred with phylogenetic analysis and drug resistance mutations (DRMs) were determined according to the Stanford HIVdb algorithm. Recent HIV infection was determined with HIV Limiting Antigen avidity electro immunoassay. RESULTS: A total of 2176 sequences (92.4%, 2176/2354) were obtained; 70.9% (1536/2167) were CRF01_AE, followed by CRF07_BC (18.0%, 391/2167), subtype B (4.7%, 102/2167), other subtypes (2.6%, 56/2167), and unique recombinant forms (3.8%, 82/2167). The prevalence of TDR was 4.9% (107/2167), among which, only 0.6% (13/2167) was resistance to TDF/FTC. Most of these subjects had CRF01_AE strains (76.9%, 10/13), were unmarried (76.9%, 10/13), infected through homosexual contact (92.3%, 12/13), and over 30 years old (median age: 33). The TDF/FTC DRMs included K65R (8/13), M184I/V (5/13), and Y115F (2/13). Recent HIV infection accounted for only 23.1% (3/13). Most cases were sporadic in the phylogenetic tree, except two CRF01_AE sequences with K65R (Bootstrap value: 99%). CONCLUSIONS: The prevalence of TDR to TDF/FTC is low among newly diagnosed HIV-infected cases in Shenyang, suggesting that TDR may have little impact on the protective effect of the ongoing CROPrEP project in Shenyang city.

Natural polymorphisms in HIV-1 CRF01_AE strain and profile of acquired drug resistance mutations in a long-term combination treatment cohort in northeastern China.

BACKGROUND: The impacts of genetic polymorphisms on drug resistance mutations (DRMs) among various HIV-1 subtypes have long been debated. In this study, we aimed to analyze the natural polymorphisms and acquired DRM profile in HIV-1 CRF01_AE-infected patients in a large first-line antiretroviral therapy (ART) cohort in northeastern China. METHODS: The natural polymorphisms of CRF01_AE were analyzed in 2034 patients from a long-term ART cohort in northeastern China. The polymorphisms in 105 treatment failure (TF) patients were compared with those in 1148 treatment success (TS) patients. The acquired DRM profile of 42 patients who experienced TF with tenofovir/lamivudine/efavirenz (TDF/3TC/EFV) treatment was analyzed by comparing the mutations at TF time point to those at baseline. The Stanford HIVdb algorithm was used to interpret the DRMs. Binomial distribution, McNemar test, Wilcoxon test and CorMut package were used to analyze the mutation rates and co-variation. Deep sequencing was used to analyze the evolutionary dynamics of co-variation. RESULTS: Before ART, there were significantly more natural polymorphisms of 31 sites on reverse transcriptase (RT) in CRF01_AE than subtype B HIV-1 (|Z value| ≥ 3), including five known drug resistance-associated sites (238, 118, 179, 103, and 40). However, only the polymorphism at site 75 was associated with TF (|Z value| ≥ 3). The mutation rate at 14 sites increased significantly at TF time point compared to baseline, with the most common DRMs comprising G190S/C, K65R, K101E/N/Q, M184 V/I, and V179D/I/A/T/E, ranging from 66.7 to 45.2%. Moreover, two unknown mutations (V75 L and L228R) increased by 19.0 and 11.9% respectively, and they were under positive selection (Ka/Ks > 1, log odds ratio [LOD] > 2) and were associated with several other DRMs (cKa/Ks > 1, LOD > 2). Deep sequencing of longitudinal plasma samples showed that L228R occurred simultaneously or followed the appearance of Y181C. CONCLUSION: The high levels of natural polymorphisms in CRF01_AE had little impact on treatment outcomes. The findings regarding potential new CRF01_AE-specific minor DRMs indicate the need for more studies on the drug resistance phenotype of CRF01_AE.

The S68G polymorphism is a compensatory mutation associated with the drug resistance mutation K65R in CRF01_AE strains.

BACKGROUND: The rate of S68G mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has increased and is closely related to the K65R mutation among CRF01_AE-infected patients who failed treatment. We aimed to explore the temporal association of S68G and K65R mutations and disclose the role of the former on susceptibility to nucleotide/nucleoside reverse transcriptase inhibitor (NRTI) and viral replication with the K65R double mutations among CRF01_AE-infected patients who failed treatment. METHODS: The occurrence of S68G and K65R mutations was evaluated among HIV-1 of various subtypes in the global HIV Drug Resistance Database. The temporal association of S68G and K65R mutations was analyzed through next-generation sequencing in four CRF01_AE-infected patients who failed treatment with tenofovir/lamivudine/efavirenz. The impact of the S68G mutation on susceptibility to NRTI and replication fitness was analyzed using pseudovirus phenotypic resistance assays and growth competition assays, respectively. RESULTS: The frequency of the S68G mutation increased by 1.4-9.7% in almost all HIV subtypes and circulating recombinant forms in treatment-experienced patients, except subtype F. The S68G mutation often occurred in conjunction with the K65R mutation among RTI-treated patients, with frequencies ranging 21.1-61.7% in various subtypes. Next-generation sequencing revealed that the S68G mutation occurred following the K65R mutation in three of the four CRF01_AE-infected patients. In these three patients, there was no significant change detected in the half maximal inhibitory concentration for zidovudine, tenofovir, or lamivudine between the K65R and K65R/S68G mutations, as demonstrated by the phenotypic resistance assays. Virus stocks of the K65R and K65R/S68G mutations were mixed with 4:6, 1:1, and 9:1 and cultured for 13 days, the K65R/S68G mutants outgrew those of the K65R mutants irrespective of the input ratio. CONCLUSIONS: S68G may be a natural polymorphism and compensatory mutation of K65R selected by NRTIs in the CRF01_AE strain of HIV-1. This mutation does not affect susceptibility to NRTI; however, it improves the replication fitness of K65R mutants. This study deciphers the role of the S68G mutation in the HIV reverse transcriptase of the CRF01_AE strain and provides new evidence for the interpretation of drug-resistant mutations in non-B subtypes of HIV-1.

Factors associated with high-risk low-level viremia leading to virologic failure: 16-year retrospective study of a Chinese antiretroviral therapy cohort.

BACKGROUND: Low level viremia (LLV) often occurs during antiretroviral therapy (ART) against HIV-1. However, whether LLV increases the risk of virologic failure (VF) is controversial because of the non-uniform definitions of LLV and VF. METHODS: A long-term first line regimen ART cohort from 2002 to 2018 from Shenyang, northeast China, was retrospectively studied. All participants were followed up every 3 to 6 months to evaluate the treatment effect. The high-risk LLV subgroups leading to VF (with strict standards) were explored with Cox proportional hazards model and linear mixed-effect model. The association factors of high-risk LLV were further explored using multivariate logistic regression analyses. RESULTS: A total of 2155 HIV-1 infected participants were included; of these, 38.7% showed LLV. Both high level LLV (HLLV) and any other level LLV coupled with high level blip (HLB) showed higher risk of VF (hazards ratios, HRHLLV = 5.93, and HRHLB = 2.84, p <  0.05 respectively). Moreover, HR increased with prolonged duration of LLV. Independent factors associated with high-risk LLV included the zenith baseline viral load (VL) above 6 log copies/ml (aOR = 3.49, p = 0.002), nadir baseline CD4 + T cell counts below 200 cells/mm3 (aOR = 1.78, p = 0.011), Manchu (aOR = 2.03, p = 0.003), ART over 60 months (aOR = 1.81, p = 0.004), AZT + 3TC + NVP (aOR = 2.26, p <  0.001) or DDI-based regimen (aOR = 9.96, p = 0.002), and subtype B' infection (aOR = 8.22, p = 0.001). CONCLUSIONS: In case of VF with strict standards, high-risk LLV leading to VF includes VL above 400 copies/ml, occurring at least once. Serious laboratory indicators or advanced stage of infection, long term ART and subtype B' infection might also predict the occurrence of high-risk LLV.

High polymorphism rates in well-known T cell epitopes restricted by protective HLA alleles during HIV infection are associated with rapid disease progression in early-infected MSM in China.

T cell epitopes restricted by several protective HLA alleles, such as B*57, B*5801, B*27, B*51 and B*13, have been very well defined over the past two decades. We investigated 32 well-known T cell epitopes restricted by protective HLA molecules among 54 Chinese men who have sex with men (MSM) at the early stage of HIV-1 infection. Subjects in our cohort carrying protective HLA types did not exhibit slow CD4 T cell count decline (P = 0.489) or low viral load set points (P = 0.500). Variations occurred in 96.88% (31/32) of the known wild-type epitopes (rate 1.85-100%), and the variation rates of the strains of two CRF01_AE lineages were significantly higher than those of non-CRF01_AE strains (76.82% vs. 48.96%, P = 0.004; 71.27% vs. 8.96%, P = 0.025). Subjects infected with CRF01_AE exhibited relatively rapid disease progression (P = 0.035). Therefore, the lack of wild-type protective T cell epitopes restricted by classic protective HLA alleles in CRF01_AE HIV-1 strains may be one of the reasons why rapid disease progression is observed in Chinese MSM with HIV-1 infection.

Crosstalk in competing endogenous RNA networks reveals new circular RNAs involved in the pathogenesis of early HIV infection.

BACKGROUND: The events in early HIV infection (EHI) are important determinants of disease severity and progression rate to AIDS, but the mechanisms of pathogenesis in EHI have not been fully understood. Circular RNAs (circRNAs) have been verified as "microRNA sponges" that regulate gene expression through competing endogenous RNA (ceRNA) networks, but circRNA expression profiles and their contribution to EHI pathogenesis are still unclear. METHODS: Two different libraries were constructed with RNA from human peripheral blood mononuclear cells from 3 HARRT-naive EHI patients and 3 healthy controls (HCs). The complete transcriptomes were sequenced with RNA sequencing (RNA-Seq) and miRNA sequencing (miRNA-Seq). The differentially expressed (DE) RNAs were validated with RT-qPCR. The circRNA profile and circRNA-associated-ceRNA network in EHI were analyzed with the integrated data of RNA-Seq and miRNA-Seq. Gene ontology (GO) analysis was used to annotate the circRNAs involved in the circRNA-associated-ceRNA networks. RESULTS: A total of 1365 circRNAs, 30 miRNAs, and 2049 mRNAs were differentially expressed between HARRT-naive EHI patients and HCs. A ceRNA network was constructed with 516 DE circRNAs and 903 DE mRNAs that shared miR response elements with 21 DE miRNAs. GO analysis demonstrated the multiple roles of the circRNAs enriched in EHI with circRNA-associated-ceRNA networks, such as immune response, inflammatory response and defense responses to virus, 67 circRNAs were revealed to be potentially involved in HIV-1 replication through regulating the expression of CCNK, CDKN1A and IL-15. CONCLUSIONS: This study, for the first time, revealed a large circRNA profile and complex pathogenesis roles of circRNAs in EHI. A group of enriched circRNAs and associated circRNA-associated-ceRNA networks might contribute to HIV replication regulation and provide novel potential targets for both the pathogenesis of EHI and antiviral therapy.

Multi-layered Gag-specific immunodominant responses contribute to improved viral control in the CRF01_AE subtype of HIV-1-infected MSM subjects.

BACKGROUND: The purpose of this study was to characterize specific cytotoxic T-cell (CTL) responses in men who have sex with men (MSM) subjects infected with the human immunodeficiency virus type 1 (HIV-1) CRF01_AE subtype during the first year of infection and impacts on viral control and evolution. RESULTS: Fifteen HIV-1 primary infected cases were recruited from Liaoning MSM prospective cohort. CTL responses to Gag, Pol and Nef proteins at 3 month and 1 year post infection were detected with Gamma interferon enzyme-linked immunospot (ELISPOT) assay using optimized consensus overlapping peptides, as well as the viral quasispecies sequences from the synchronous plasma. Gag and Nef proteins were the main targets of CTL responses during the first year of HIV-1 infection, and this was evident from the data after adjusting for the length of amino acids by dividing the amino acids number of the corresponding protein and multiplying by 100. Additionally, relative magnitudes of Gag at both 3 months and 1 year post infection were significantly negatively correlated with the viral set point (p = 0.002, r = -0.726; p = 0.025, r = -0.574). While the relative magnitude of Nef at 1 year post infection were significantly positively correlated with viral set point (p = 0.004, r = 0.697). Subjects with multi-layered Gag immunodominant responses during the first year of infection had significantly lower viral set points than subjects without such responses (p = 0.002). CONCLUSION: Multi-layered Gag immunodominant responses during the first year of infection were correlated with viral control, which provides a theoretical basis for vaccine design targeting MSM subjects with the CRF01_AE subtype.

Optimization of genetic distance threshold for inferring the CRF01_AE molecular network based on next-generation sequencing.

Introduction: HIV molecular network based on genetic distance (GD) has been extensively utilized. However, the GD threshold for the non-B subtype differs from that of subtype B. This study aimed to optimize the GD threshold for inferring the CRF01_AE molecular network. Methods: Next-generation sequencing data of partial CRF01_AE pol sequences were obtained for 59 samples from 12 transmission pairs enrolled from a high-risk cohort during 2009 and 2014. The paired GD was calculated using the Tamura-Nei 93 model to infer a GD threshold range for HIV molecular networks. Results: 2,019 CRF01_AE pol sequences and information on recent HIV infection (RHI) from newly diagnosed individuals in Shenyang from 2016 to 2019 were collected to construct molecular networks to assess the ability of the inferred GD thresholds to predict recent transmission events. When HIV transmission occurs within a span of 1-4 years, the mean paired GD between the sequences of the donor and recipient within the same transmission pair were as follow: 0.008, 0.011, 0.013, and 0.023 substitutions/site. Using these four GD thresholds, it was found that 98.9%, 96.0%, 88.2%, and 40.4% of all randomly paired GD values from 12 transmission pairs were correctly identified as originating from the same transmission pairs. In the real world, as the GD threshold increased from 0.001 to 0.02 substitutions/site, the proportion of RHI within the molecular network gradually increased from 16.6% to 92.3%. Meanwhile, the proportion of links with RHI gradually decreased from 87.0% to 48.2%. The two curves intersected at a GD of 0.008 substitutions/site. Discussion: A suitable range of GD thresholds, 0.008-0.013 substitutions/site, was identified to infer the CRF01_AE molecular transmission network and identify HIV transmission events that occurred within the past three years. This finding provides valuable data for selecting an appropriate GD thresholds in constructing molecular networks for non-B subtypes.

Low-level viremia episodes appear to affect the provirus composition of the circulating cellular HIV reservoir during antiretroviral therapy.

Low-level viremia (LLV) ranging from 50 to 1,000 copies/ml is common in most HIV-1-infected patients receiving antiretroviral therapy (ART). However, the source of LLV and the impact of LLV on the HIV-1 reservoir during ART remain uncertain. We hypothesized that LLV may arise from the HIV reservoir and its occurrence affect the composition of the reservoir after LLV episodes. Accordingly, we investigated the genetic linkage of sequences obtained from plasma at LLV and pre-ART time points and from peripheral blood mononuclear cells (PBMCs) at pre-ART, pre-LLV, LLV, and post-LLV time points. We found that LLV sequences were populated with a predominant viral quasispecies that accounted for 67.29%∼100% of all sequences. Two episodes of LLV in subject 1, spaced 6 months apart, appeared to have originated from the stochastic reactivation of latently HIV-1-infected cells. Moreover, 3.77% of pre-ART plasma sequences were identical to 67.29% of LLV-3 plasma sequences in subject 1, suggesting that LLV may have arisen from a subset of cells that were infected before ART was initiated. No direct evidence of sequence linkage was found between LLV viruses and circulating cellular reservoirs in all subjects. The reservoir size, diversity, and divergence of the PBMC DNA did not differ significantly between the pre- and post-LLV sampling points (P > 0.05), but the composition of viral reservoir quasispecies shifted markedly before and after LLV episodes. Indeed, subjects with LLV had a higher total PBMC DNA level, greater viral diversity, a lower proportion of variants with identical sequences detected at two or more time points, and a shorter variant duration during ART compared with subjects without LLV. Overall, our findings suggested that LLV viruses may stem from an unidentified source other than circulating cellular reservoirs. LLV episodes may introduce great complexity into the HIV reservoir, which brings challenges to the development of treatment strategies.

Reconstituting the epidemic history of HIV strain CRF01_AE among men who have sex with men (MSM) in Liaoning, northeastern China: implications for the expanding epidemic among MSM in China.

HIV CRF01_AE accounted for 84% of the recent infections among men who have sex with men (MSM) in Liaoning Province of northeastern China. CRF01_AE strains were grouped into two distinct clusters (designated clusters 1 and 2) that were also detected in other regions in China. Phylodynamics study revealed that these two CRF01_AE strains were independently introduced into the population of MSM in China in the early and mid-1990s. Our study elucidated unique features of dynamics and interrelationships of MSM epidemics in China.

Prospective cohort study of HIV incidence and molecular characteristics of HIV among men who have sex with men(MSM) in Yunnan Province, China.

BACKGROUND: Yunnan has the largest number of reported HIV/AIDS cases among all Chinese provinces, the reported prevalence of HIV among Yunnan men who have sex with men (MSM) passed 10%, while HIV incidence epidemic and molecular characteristics of new infected Yunnan MSM were not evaluated before. METHODS: An 18 months prospective followed up with a frequency of 3 month per visit were conducted among HIV seronegative MSM in Kunming cityduring 2009-2011. Interviewer-administrated questionnaires were carried out. Blood specimens were obtained to test for syphilis and HIV, in which HIV were evaluated by standard HIV enzyme immunoassay (EIA) and HIV nucleic acid amplification testing (NAAT). Near full-length regions of the HIV-1 were evaluated for subtyping, primary drug resistance mutations. RESULTS: During the follow-up 70.1% of the recruited 378 MSM retained in the cohort. Eleven MSM seroconverted to HIV and fifteen MSM seroconverted to syphilis. The HIV incidence and syphilis incidence was 3.5 (95% CI 1.8-6.2) cases /100 person year(PY) and 5.3 (95% CI 3.0-8.7) cases/100 PY, respectively. Multivariate analysis showed that baseline syphilis infection (aHR, 17.7), occupation (students vs. others [aHR, 5.7], retirees vs. others [aHR, 4.1]), bleeding experience after receptive anal intercourse (aHR,7.6), and minority ethnic(vs. Han) [aHR, 5.7] were independent risk factors for HIV seroconversion(each P<0.05). Among the 7/11 successfully amplified near full-length sequences, 71.4% (5/7) were CRF01_AE, and 28.6% (2/7) were CRF07_BC. Two HIV transmission pairs were detected among seroconverted minority ethnic MSM. CONCLUSIONS: HIV incidence was moderately high among Yunnan MSM. Yunnan province need to strengthen both HIV and syphilis screening among MSM population. Some subpopulations of MSM, such as students, retirees and minority ethnic groups require more HIV epidemic surveillance and strengthened behavior interventions. HIV subtypes and primary drug resistance should be continually monitored to track cross-group transmission of HIV strains.

[Study on the association between genetic variation of gag gene and HLA restricted CTL response in HIV-1 B' infected Chinese people].

OBJECTIVE: To identify the genetic variation of Gag gene in HIV-1 B' subtype infected Chinese people and to evaluate the association between the positive selection sites and HLA restricted CTL response. METHODS: We downloaded all HIV-1 B' subtype gag gene sampled from China from the LANL HIV database. The sequences were grouped by years of collection and the mean intra-subtype diversity and divergence were examined by MEGA 5.0 software. The sites under positive selection were estimated by using Hyphy software. Then analyzed the association between positively selected sites and HLA restricted CTL epitopes. RESULTS: A set of 122 B' subtype HIV-1 gag sequences were collected from Henan, Hubei, Liaoning and Yunnan provinces, grouped by sampling year as < 2001, 2001 - 2003, 2004 - 2006 and 2007 - 2010. The mean intra-subtype diversity and divergence increased over time (r(1) = 0.497, P1 < 0.001; r(2) = 0.593, P2 < 0.001). This analysis revealed 34 positively selected sites and 97.1% of these sites were located within 47 known CTL epitopes. The most frequent HLA was HLA-A*02 (21.6%) followed by A*11(9.8%), B*35(9.8%) and B*57(5.9%). The percentages of HLA-A/B/C limited CTL epitopes were 43.1%, 49.0% and 7.9%, respectively. CONCLUSION: The mean intra-subtype diversity and divergence of B' subtype gag sequences increased over time. B' subtype HIV-1 virus has accumulated adaptations to HLA restricted CTL responses in HIV-1 infected Chinese.

Multiple CRF01_AE/CRF07_BC Recombinants Enhanced the HIV-1 Epidemic Complexity Among MSM in Shenyang City, Northeast China.

The transmission of Unique Recombinant Forms (URFs) has complicated the molecular epidemic of HIV-1. This increasing genetic diversity has implications for prevention surveillance, diagnosis, and vaccine design. In this study, we characterized the HIV-1 URFs from 135 newly diagnosed HIV-1 infected cases between 2016 and 2020 in Shenyang, northeast China and analyzed the evolutionary relationship of them by phylogenetic and recombination approaches. Among 135 URFs, we found that the CRF01_AE/CRF07_BC recombinants were the most common (81.5%, 110/135), followed by CRF01_AE/B (11.9%, 16/135), B/C (3.7%, 5/135), and others (3.0%, 4/135). 94.8% (128/135) of patients infected by URFs were through homosexual contact. Among 110 URFs_0107, 60 (54.5%) formed 11 subclusters (branch support value = 1) and shared the consistent recombination structure, respectively. Four subclusters have caused small-scale spread among different high-risk populations. Although the recombination structures of URFs_0107 are various, the hotspots of recombinants gathered between position 2,508 and 2,627 (relative to the HXB2 position). Moreover, the CRF07_BC and CRF01AE fragments of URFs_0107 were mainly derived from the MSM population. In brief, our results reveal the complex recombinant modes and the high transmission risk of URFs_0107, which calls for more attention on the new URFs_0107 monitoring and strict control in the areas led by homosexual transmission route.

Comparative miRNA transcriptomics of macaques and mice reveals MYOC is an inhibitor for Cryptococcus neoformans invasion into the brain.

Cryptococcal meningoencephalitis (CM) is emerging as an infection in HIV/AIDS patients shifted from primarily ART-naive to ART-experienced individuals, as well as patients with COVID-19 and immunocompetent hosts. This fungal infection is mainly caused by the opportunistic human pathogen Cryptococcus neoformans. Brain or central nervous system (CNS) dissemination is the deadliest process for this disease; however, mechanisms underlying this process have yet to be elucidated. Moreover, illustrations of clinically relevant responses in cryptococcosis are currently limited due to the low availability of clinical samples. In this study, to explore the clinically relevant responses during C. neoformans infection, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton, a major feature of HIV/AIDS patients, was a centric pathway regulated in both infection models. Notably, assays of clinical immune cells confirmed an enhanced macrophage "Trojan Horse" in patients with HIV/AIDS, which could be shut down by cytoskeleton inhibitors. Furthermore, myocilin, encoded by MYOC, was found to be a novel enhancer for the macrophage "Trojan Horse," and an enhanced fungal burden was achieved in the brains of MYOC-transgenic mice. Taken together, the findings from this study reveal fundamental roles of the cytoskeleton and MYOC in fungal CNS dissemination, which not only helps to understand the high prevalence of CM in HIV/AIDS but also facilitates the development of novel therapeutics for meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.

CT virtual endoscopy of the ampulla of Vater: preliminary report.

AIM: To explore multi-slice spiral CT (MSCT) virtual endoscopy (CTVE) in the detection of Vater's ampulla lesions. METHODS: In addition to 30 healthy volunteers, 18 cases of common bile duct stones, and 7 cases of ampullary carcinoma were scanned with MSCT including virtual endoscopy (VE) reconstruction. Patterns of the duodenal papilla were then observed, and its size was measured. RESULTS: Reconstructed images of CTVE in the volunteers showed that the normal type of the duodenal papilla was nodular in 16 cases, V-shaped in 8 cases, and Y-shaped in 6 cases. Its mean diameter was 0.84 ± 0.17 cm in the healthy volunteers; in patients with common bile duct stones of nodular type, mean diameter was 1.72 ± 0.32 cm. In ampullary cancer patients with an irregular protruded type, its diameter was 2.30 ± 0.85 cm, Overall the mean differences between the groups were statistically significant (P < 0.001). CONCLUSION: CTVE is a convenience, no-wound, and precise clinical examination mode utilizing which the shape of duodenal papilla can be observed, and size of the latter can be measured.

[Coreceptor usage of human immunodeficiency virus-1 in primary infection through sexual contact transmission].

OBJECTIVE: To evaluate the coreceptor usage characters and evolution of human immunodeficiency virus-1 (HIV-1) primary infection. METHODS: A total of 12 HIV-1 primary infection cases were recruited from a high-risk population for this prospective cohort trial. The blood samples were collected at the earliest time after infection and after a viral setpoint respectively. RNA was extracted from plasma. The env genes were amplified through reverse transcription-PCR (RT-PCR) and nested PCR. And the env fragments were ligated to T vector and then sequenced. The pattern of coreceptor usage was predicted with five different online tools. RESULTS: Among them, 11 were predicted as CCR5 virus transmission and 1 as CXCR4 virus transmission. The viruses in 1 case were detected as a switch of coreceptor usage from CCR5 to CXCR4. The consistency of five different prediction rules was 88.3%. CONCLUSION: Although the CCR5 usage virus is predominant in primary infection through sexual transmission, CXCR4 usage virus can also be detected. A switch of coreceptor usage may happen within the first year post-infection. Adopting multiple rules may improve the efficiency and validity for the prediction of coreceptor usage based on genotypes.

Characterization of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Strain Among Men Who Have Sex with Men in Liaoning, China.

CRF01_AE and CRF07_BC are two widespread human immunodeficiency virus type 1 (HIV-1) strains among different high-risk populations, including men who have sex with men (MSM), in China. This co-epidemic of various HIV strains enables the production of second-generation recombinants. In this study, we detected a novel HIV-1 CRF01_AE/CRF07_BC recombinant from LN321945, an MSM lived in Liaoning province, northeast China. The phylogenetic and recombination analyses indicated the near full-length genome (NFLG) sequence of LN321945 had six recombination breakpoints, with three CRF07_BC fragments inserted into a CRF01_AE backbone. Further subregion trees analysis revealed that both CRF01_AE and CRF07_BC fragments were derived from two predominant HIV-1 strains among MSM. In addition, the NFLG of LN321945 was revealed to be clustered closely to another CRF01_AE/CRF07_BC recombinant previously identified in Shaanxi province, northwest China, but these two recombinants had distinct recombination structure and origin of CRF01_AE fragments. Hence, this study identified a second-generation recombinant between the main strains circulating among MSM, indicating more complicated trend of HIV-1 epidemic in China.

The Viral Founder Effect and Economic-Driven Human Mobility Shaped the Distinct Epidemic Pattern of HIV-1 CRF01_AE in Northeast China.

Background: In China, two distinct lineages shaped the epidemic of HIV-1 CRF01_AE among men who have sex with men (MSM), of which the uneven distributions were observed geographically. One lineage spread across China, while another dominated in Northeast China. Understanding the drivers of viral diffusion would provide guidelines for identifying the source and hotspots of HIV transmission among MSM to target interventions in China. Methods: We collected the pol sequences between 2002-2017 to reconstruct the spatiotemporal history of CRF01_AE lineages in Shenyang, one economic center of Northeast China, using the Bayesian phylogeographic and phylodynamic approaches. Importantly, for the datasets with the high sample density, we did the down-sampling to avoid the sampling bias. Results: Two lineages accounted for 97%, including 426 and 1516 sequences, and homosexuals and bisexuals were above 80%. One lineage appeared earlier 7 years than another (1993 vs. 2002) among homosexuals and bisexuals, whereas among heterosexuals, both lineages were observed firstly in 2002. 96% viral migrations within one lineage were from homosexuals toward bisexuals (49%) and male-heterosexuals (46%). Within another, except for homosexuals (72%), bisexuals (23%) served as the top second source, and female-heterosexuals (11%) were the third recipients following bisexuals (44%) and male-heterosexuals (39%). Although the basic reproduction number (R0) of two lineages were similar and both of the effective production number (Re) fell below 1 at the most recent sampling time, the starts of the Re declining varied. Conclusions: Our findings revealed that throughout the viral national spread chain, Shenyang is the source for the initial expanding of one lineage, where is only a sink of another, proving that the viral founder effect and regional human mobility contributed to the uneven distribution of two lineages, and emphasizing the important roles of the area where the virus originated and economy-driven migrants in HIV transmission.