Phylodynamics of major CRF01_AE epidemic clusters circulating in mainland of China.

As the most dominant HIV-1 strain in China, CRF01_AE needs to have its evolutionary and demographic history documented. In this study, we provide phylogenetic analysis of all CRF01_AE pol sequences identified in mainland China. CRF01_AE sequences were collected from the Los Alamos HIV Sequence Database and the local Chinese provincial centers of disease control and prevention. Phylogenetic trees were constructed to identify major epidemic clusters. Bayesian coalescent-based method was used to reconstruct the time scale and demographic history. There were 2965 CRF01_AE sequences from 24 Chinese provinces that were collected, and 5 major epidemic clusters containing 85% of the total CRF01_AE sequences were identified. Every cluster contains sequences from more than 10 provinces with 1 or 2 dominant transmission routes. One cluster arose in the 1990s and 4 clusters arose in the 2000s. Cluster I is in the decline stage, while the other clusters are in the stable stage. Obvious lineage can be observed among sequences from the same transmission route but not the same area. Two large clusters in high-level prevalence were found in MSM (Men who have sex with men), which highlighted that more emphasis should be placed on MSM for HIV control in mainland China.

Multiple introductions and onward transmission of HIV-1 subtype B strains in Shanghai, China.

OBJECTIVE: To investigate the viral genetic evolution, spatial origins and patterns of transmission of HIV-1 subtype B in Shanghai, China. METHODS: A total of 242 Shanghai subtype B and 1519 reference pol sequences were subjected to phylogenetic inference and genetic transmission network analyses. RESULTS: Phylogenetic analysis revealed that subtype B strains circulating in Shanghai were genetically diverse and closely associated with viral sequence lineages in Beijing (76 of 242 [31.4%]), Central China (Henan/Hebei/Hunan/Hubei) (43 of 242 [17.8%]), Chinese Taiwan (20 of 242 [8.3%]), Japan (6 of 242 [2.5%]), and Korea (7 of 242 [2.9%]), suggesting multiple introductions into Shanghai from mainland China and Taiwan, Japan, and Korea. Interestingly, a monophyletic Shanghai lineage (SH-L) (36 of 242 [14.9%]) of HIV-1 subtype B most likely originated from an Argentine strain, transferred through Liaoning infected individuals. In-depth analyses of 195 Shanghai subtype B sequences revealed that a total of 37.9% (n = 74) sequences contributed to 35 transmission networks, whereof 33.8% (n = 25) of the sequences associated with infected individuals from other provinces. CONCLUSIONS: Our new findings reflect the evolution complexity and transmission dynamics of HIV-1 subtype B in Shanghai, which would provide critical information for the design of effective prevention measures against HIV transmission.

Evolutionary Dynamics and Complicated Genetic Transmission Network Patterns of HIV-1 CRF01_AE among MSM in Shanghai, China.

To explore the evolutionary dynamics and molecular transmission patterns of HIV-1 CRF01_AE in depth among men who have sex with men (MSM) in Shanghai, we constructed phylogenetic tree and genetic transmission networks based on 1, 152 pol sequences from MSM, 282 from other risk groups and 795 references. Phylogenetic analyses identified two distinct major CRF01_AE lineages and a Shanghai-based sub-lineage. The estimated tMRCAs for lineage 1 and 2 were 1996.0 (1992.9-1999.2) and 1997.8 (1994.3-2001.4), respectively. Of the 1, 152 MSM, 681 (59.1%) were identified as belonging to 241 separate networks. Of these 681 individuals in networks, 74.2% were linked to cases diagnosed in different years, 4.3% were linked to heterosexual women, and 0.7% were linked to persons who inject drugs. A total of 71 networks including 180 individuals diagnosed in Shanghai with the same domicile were found. Recent infection (P = 0.022) and sampling year after 2011 (P < 0.001) were significantly associated with potential transmission links among the networks. Besides, a significant transmission of viruses with drug resistant mutations at V179D/E were found in the networks. Given these findings, we propose that genetic transmission analysis is a useful tool in HIV intervention strategies to curb the spread of virus and promoting public health.

Efficacy and safety of antiretroviral regimens including raltegravir to treat HIV-infected patients with hemophilia.

When treating HIV-infected patients with hemophilia, adverse drug reactions and interactions and the effect of treatment on bleeding disorders must be considered. Raltegravir is the first HIV integrase inhibitor, but its use in patients with hemophilia is rarely reported. Nine HIV-positive patients with hemophilia were retrospectively studied with a focus on the virological response, changes in the CD4 count, the tendency to bleed, and the response to replacement therapy before and after raltegravir-based antiretroviral therapy (ART). The nine patients were highly treatment-experienced patients and they received raltegravir-based ART for at least nine months. The patients had their own reasons for changing to raltegravir-based ART. During treatment, the CD4 count increased progressively in four patients, with a median absolute increase of 233 cells/mm(3), while the count stabilized in the remaining five patients. Two previous recipients of lopinavir/ritonavir (LPV/r) who failed to respond to lamivudine (3TC) + zidovudine (ZDV) + efavirenz (EFV) had a viral rebound. Genotyping indicated multidrug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). A pattern of resistance to raltegravir was evident, including the primary mutation N155H and the secondary mutation T97A. In the two patients, the tendency to bleed decreased markedly and monthly usage of clotting factor VIII decreased significantly decreased. In the remaining seven patients, the viral load remained < 40 copies/mL, there was no evidence of an increased tendency to bleed, and no evidence of changes in the response to replacement therapy. All of the patients had a stable condition with no signs of disease progression and no serious adverse reactions. Results indicated that Raltegravir-based therapy offered a safe and well-tolerated option for HIV-positive patients with hemophilia.

HIV-1 Genetic Diversity and Its Impact on Baseline CD4+T Cells and Viral Loads among Recently Infected Men Who Have Sex with Men in Shanghai, China.

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient's immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P<0.01), whereas viral load was significantly higher those infected with CRF01_AE than with CRF07_BC (P<0.01). In addition, the patients with >45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with <25 years of age (P<0.05). This study reveals the presence of HIV-1 subtype diversity in Shanghai and its remarkable influence on clinical outcome. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster, patient's baseline CD4+T cell count and viral load would be of great value to monitoring of disease progression, intervention for transmission, improvement of antiretroviral therapy strategy and design of vaccines.

Evidence that HIV-1 CRF01_AE is associated with low CD4+T cell count and CXCR4 co-receptor usage in recently infected young men who have sex with men (MSM) in Shanghai, China.

Men who have sex with men (MSM) have recently accounted for an alarmingly increasing proportion of HIV-1 transmission in China. In order to investigate the immune status as a result of CRF01_AE infection and CXCR4 co-receptor usage in a young Shanghai-based HIV-1-infected MSM population in Shanghai, 364 HIV-1-infected MSM with average age of 22.7 years old, newly diagnosed between Jan 2009 and Jul 2013 were analyzed for CD4+T cell count, subtyping using phylogenetic analysis, and viral co-receptor tropism using Geno2pheno and webPSSM in combination. 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. Besides, 24 second-generation recombinant strains were identified. A lower CD4+T cell count at baseline survey was observed among CRF01_AE strain-infected individuals, compared to those who were infected with CRF07_BC (P<0.01). The frequency of baseline CD4+T cell count <200 was higher and the frequency of CD4 T counts >500 lower in CRF01_AE infection than CRF07_BC infection. It is worth noting that 32.4%-40.9% of CRF01_AE strain-infected individuals were predicted to carry CXCR4-tropic viruses whereas none of CRF07_BC and subtype B were found to be as CXCR4-tropic viruses (P<0.001). As could be expected CXCR4 tropism was associated with lower CD4 T counts. This study revealed that CRF01_AE strains with high frequency of CXCR4 tropism are prevailing in the young MSM population in China and could potentially cause a severe loss of CD4+T cell count and rapid disease progression. A regular surveillance of HIV-1 subtypes, CD4+T cell count and viral co-receptor usage would be greatly beneficial for effectively monitoring disease progression, improvement of antiretroviral therapy strategy and prompt intervention of transmission.

HIV drug resistance and its impact on antiretroviral therapy in Chinese HIV-infected patients.

BACKGROUND: Highly active antiretroviral therapy (HAART) has significantly decreased mortality among Chinese HIV patients. However, emerging HIV drug resistance (HIVDR) poses a growing threat to the long-term success and durability of HAART. METHODS: Three cross-sectional surveys were conducted across the country from 2004 to 2006, respectively. Patients completed a questionnaire and provided blood for CD4 cell count, HIV viral load (VL), and HIV resistance genotyping. Factors associated with HIVDR were identified by logistic regression. RESULTS: 3667 unique patients were included across the three surveys. Among 2826 treatment-experienced patients, median duration of treatment was 17.4 (IQR 8.6-28.4) months and HIVDR was identified in 543 (19.2%). Factors significantly associated with HIVDR included ART drug distribution location, CD4 cell count, initial HAART regimen, self-reported medication adherence, and province. CONCLUSIONS: Virologic failure increased over time on therapy but a significant proportion of patients in failure had no resistance mutations identified, suggesting that treatment adherence is suboptimal and must be emphasized. Due to the significantly higher risk of HIVDR in certain provinces, additional steps to reduce HIVDR should be taken.

HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects naïve to antiretroviral therapy.

BACKGROUND: The frequencies of regulatory T cells (Tregs) increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs. METHODS: Tregs were defined as CD4(+)CD25(+)CD127(lo/-) T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer. RESULTS: Compared to HIV-1 seronegative donors, the levels of HLA-DR on CD4(+)CD25(+)CD127(lo/-) Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads (r = 0.3163, P = 0.004) and negatively with CD4 T-cell counts (r = -0.4153, P < 0.0001). In addition, significant associations between HLA-DR expression on CD4(+)CD25(+)CD127(lo/-) Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4(+) and CD8(+) T cells were also identified. CONCLUSION: HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.

[Association of PD-1 expression on CD4+ CD25 nt/hi CD127 lo regulatory T cells with disease progression in HIV-1 infected patients].

AIM: To investigate whether Programmed death-1 (PD-1) expression on peripheral CD4(+)CD25(nt/hi)CD127(lo) regulatory T cells (Treg) was associated with disease progression in HIV-1-infected patients. METHODS: Peripheral blood from 108 HIV-1-infected patients in distinct disease progression statuses and 27 healthy individuals were collected in the present investigation. PBMCs were isolated by centrifugation on Ficoll-Hypaque, followed by staining with anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE and anti-PD-1-APC. PD-1 expression on Treg was analyzed by four-color staining flow cytometry. CD4(+) T cell absolute counts were determined using Multitest CD3/CD4/CD8/CD45 kit and plasma viral loads were detected on NucliSens EasyQ. All data were analyzed using SPSS14.0 software. RESULTS: In peripheral blood of healthy individuals, Treg expressed PD-1 at very low levels (1.72%+/-0.65%). In contrast, Treg from HIV-1-infected patients showed a significantly increased PD-1 expression (5.33%+/-2.24%, P<0.01). Moreover, AIDS patients exhibited statistically higher PD-1 expression on Treg (7.87%+/-2.23%) than newly HIV-1 infected patients (3.22%+/-1.01%, P<0.05) and patients in progression to AIDS(5.21%+/-1.72%, P<0.05). PD-1 up-regulation on Treg was closely correlated with reduced CD4(+) T cell absolute counts but elevated plasma viral load. CONCLUSION: Overall, we found that PD-1 expression on peripheral Treg was up-regulated and correlated with disease progression in HIV-1-infected patients for the first time. These findings not only extend our understanding of how Treg functions in HIV-1-infected patients but also support the notion that blocking PD-1/PD-L1 interactions may represent a potential therapeutic strategy for HIV-1-infected patients.

Genetic diversity and drug resistance of human immunodeficiency virus type 1 (HIV-1) strains circulating in Shanghai.

The HIV-1 epidemic in Shanghai is rapidly increasing. To better understand the HIV-1 genetic diversity and the mutations associated with resistance to protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), 95 antiretroviral (ARV)-treated and treatment-naive HIV-1-seropositive individuals living in Shanghai were investigated. The HIV-1 pol gene in 70 of the 95 plasma samples was successfully amplified and analyzed. The result showed that CRF01_AE predominated in Shanghai with 42.9%, followed by subtype B (10%), B' (12.9%), CRF07_BC (11.4%), CRF08_BC (10%), CRF02_AG (4.3%), G (2.9%), and K (1.4%). In addition, three new intersubtype and/or inter-CRF recombinants were detected including B'/CRF01_AE (1.4%), U/G (1.4%), and U/CRF01_AE (1.4%). The mutations conferring primary and secondary resistance to PIs were detected in 3 of 70 (4.3%) patients and the mutations conferring resistance to RTIs were identified in 12 of 70 (17.2%) patients, among whom 11 of 15 (73.3%) and 1 of 55 (1.8%) were ARV-treated and treatment-naive individuals, respectively (p < 0.01). This study reveals the emergence of genetic diversity of HIV-1 currently circulating in Shanghai. HIV-1 infection by heterosexual contact is still a major route for introduction of HIV-1 variants into this city in recent years. It is believed that this information may help to guide recommendations for diagnostic assays, vaccine design, and antiretroviral regimen strategies in China.

Genetic analysis of HIV-1 strains in rural eastern Cameroon indicates the evolution of second-generation recombinants to circulating recombinant forms.

The HIV-1 genetic diversity in most parts of Cameroon is well described and shown to be very broad. However, little is known about the composition of the HIV-1 epidemic in the rural parts of eastern Cameroon. Therefore, we investigated 25 specimens from this region for their subtypes in gag, pol, and env gene fragments. Along with genetic material of subtypes A1, C, G, CRF01_AE, CRF02_AG, and CRF11_cpx, we also identified a large number (24%, 6/25) of distinct env sequences within the subtype A radiation. CRF02_AG was the predominant genetic form in all genes studied. Half of the specimens studied were considered "pure" based on concordant subtypes in the genes studied, whereas the other half were unique recombinant forms (URFs). Except for 1 URF, all were second-generation recombinants (SGRs), 90% of which contained genetic material of CRF02_AG in at least 1 gene. Notably, we identified individuals from 3 different villages infected with CRF01_AE(gag)CRF02_AG(pol)A(env) strains, which is indicative of the evolution of this URF to a circulating recombinant form (CRF). In addition, we identified a CRF02_AG(pol)C(env) recombinant infecting a man and a woman living in the same village, suggesting horizontal transmission of this recombinant. The current study emphasizes the power of HIV-1 recombination through the generation of SGRs and the evolution of URFs into CRFs. These findings suggest that, in a region where a predominant HIV-1 strain cocirculates among several subtypes, recombination could eventually decrease the proportion of this strain over time, such as CRF02_AG in Cameroon.

Normal values for CD4 and CD8 lymphocyte subsets in healthy Chinese adults from Shanghai.

The aim of this study was to establish reference ranges for lymphocyte subsets in Chinese adults. Venous blood specimens were obtained from 614 healthy, human immunodeficiency virus (HIV)-seronegative adults in Shanghai. Flow cytometry was used to determine percentages and absolute numbers of CD4 and CD8 T lymphocytes. Mean values for CD4 and CD8 lymphocytes were 727 and 540 cells/microl, respectively, yielding a CD4/CD8 ratio of 1.49. While CD8 lymphocyte values varied with age and gender, no significant differences in CD4 lymphocyte values were observed. Shanghai adults had approximately 100 fewer CD4 lymphocytes/microl on average than Caucasians, suggesting that lower CD4 lymphocyte cutoffs for classifying and monitoring HIV infection may be needed in China.

Identification and distribution of HIV type 1 genetic diversity and protease inhibitor resistance-associated mutations in Shanghai, P. R. China.

OBJECTIVE: To investigate the genetic diversity of the HIV-1 circulating in Shanghai and to analyze the mutations in the protease (PR) gene associated with resistance to protease inhibitors (PIs). DESIGN: The genetic diversity of HIV-1 and PI resistance-associated mutations was studied in 40 Shanghai HIV-1-seropositive treatment-naive residents. The patients studied were exposed to the infection mainly through contaminated blood products (hemophiliacs) (n = 17) and sexual contacts (n = 19). Samples from 2 injecting drug users (IDUs) and 2 children born to HIV-1 infected mothers were also analyzed. METHODS: HIV-1 partial gag, pol, and env genes in infected plasma samples were amplified by reverse transcriptase polymerase chain reaction, sequenced, and phylogenetically analyzed. Analysis of PI resistance-associated amino acid substitution in PR was performed. RESULTS: HIV-1 genes in 38 of the 40 plasma samples were successfully amplified and analyzed. Polymerase chain reaction amplification was successful for 16/17 hemophilia patients and 18/19 sexually infected individuals. While all the 16 hemophilia patients infected through contaminated blood products were infected with subtype B', the 18 patients infected through sexual contact were infected with several subtypes including subtype A (n = 2), B (n = 4), B' (n = 1), C (n = 2), CRF08_BC (n = 1), CRF01_AE (n = 7), and intersubtype recombinant CRF01_AE/B (n = 1). The 2 IDUs were infected with CRF08_BC and the 2 children born to HIV-1 infected mothers were infected with subtype B' and CRF01_AE. PI resistance-associated amino acid substitutions were found at 1 codon in primary and 7 codons in secondary regions of the PR gene. Amino acid substitutions were more frequently found in the B/B' sequences (69%) than in the non-B sequences (31%). Substitutions characteristic with the subtype B/B' sequences mainly among hemophiliacs included L63P (87%), A71V/T (27%), and V77I (93%) while those that characterized the non-B sequences mainly found among heterosexuals included M36I (69%) and K20R (19%). CONCLUSION: This study reveals the presence of multiple HIV-1 subtypes and recombinants infecting Shanghai residents. The broad HIV-1 diversity is being introduced into this city through heterosexual contacts. This study also reveals that viruses infecting these treatment-naive patients have acquired both primary or secondary mutations in their PR genes. These studies should provide the basis for further epidemiologic surveys of HIV-1 subtypes and set strategies for treatment intervention and vaccine programs.