| Research backgroundHuman immunodeficiency virus(HIV)is the pathogen that causes acquired immune deficiency syndrome(AIDS).There is still no effective cure for HIV infection,and no effective vaccine to protect the uninfected at present.Antiretroviral therapy(ART)has gradually made HIV infection a controllable chronic disease,but the need for lifelong medication has led to poor adherence and the emergence of drug resistance.In addition,health resources are grossly unevenly distributed across the globe,with tens of millions of people worldwide still infected with HIV and hundreds of thousands dying from AIDS-related diseases each year.HIV is still a serious threat to global public health.The introduction of HIV-1 into China has resulted in a variety of circulating recombinant forms(CRFs)and unique recombinant forms(URFs),and the main transmission route has also undergone several transformations.The complex strain composition of HIV-1 and the concealment of the sexual transmission route have brought greater difficulties to the prevention and control of the HIV-1 epidemic in China.As an important port city and special economic zone in China,a large proportion of floating population in Shenzhen makes its HIV-1 epidemic and prevention and control situation exceptionally complex.Shenzhen is also the city where the most HIV-1subtypes are found in China.The study of HIV-1 prevalence and transmission in Shenzhen will also be an important reference for national HIV-1 prevention and control.HIV-1 CRF01＿AE,an important prevalent strain of HIV-1 in China,the strains of cluster 4 showed stronger pathogenicity than cluster 5 strains.The specific phenotypic study of the difference in pathogenicity between the two clusters will be helpful for the development of corresponding antiviral drugs and the formulation of clinical drug regimens.Research objectiveThe purpose of this study was to reveal the trend of HIV-1 prevalence in Shenzhen by the long-term and yearly longitudinal cross-sectional descriptive study of HIV-1prevalence in the city,and to identify the key populations for intervention in HIV-1prevention and control in Shenzhen to provide targets for precise intervention.By analyzing the phenotypic differences in pathogenicity of different prevalent clusters of CRF01＿AE,the important circulating recombinant strain CRF01＿AE in Shenzhen,we provide important information for the intervention of the disease process and the formulation of drug regimens.Research contentsThe subjects of the study were newly diagnosed HIV-1 infections in Shenzhen from January 2011 to June 2020.We collected the demographic information and peripheral blood samples,amplified and sequenced the sequences of the pol gene fragments of the virus.By phylogenetic analysis,we determined the viral genotypes and clusters using,described the distribution and trends of viral subtypes and epidemic clusters.The characteristics of HIV-1 high-risk transmission groups were described by construction and analysis the structure of molecular networks.And we identified the potential non-disclosed men who have sex with men population(pn MSM)of HIV-1infected men who reported heterosexual(HTS)behavior by comparing the viral evolutionary characteristics and the distribution of men and women,to provide information support for HIV-1 behavioral intervention in Shenzhen.Based on the results of the Shenzhen HIV epidemiology study,we further investigated the dominant strain CRF01＿AE and identified the characteristic difference sites of strains in epidemic clusters 4 and 5 that led to significant pathogenicity differences,and used the reverse genetics platform to introduced the mutation into the strains.We compared the biological phenotypes of virulent strains with different differential sites mutation,such as replication ability,infectivity and co-receptor binding ability,and to determine the effect of differential sites on the biological phenotypes of virulent strains.Research methods1.Plasma samples from newly diagnosed HIV-1-infected patients in Shenzhen from 2011.01 to 2020.06 and their epidemiological information were collected(by Shenzhen Center for Disease Control and Prevention).Viral RNA were extracted from the plasma of infected patients,and the gene fragments amplified by reverse transcription nested PCR and sequenced to obtain the gene fragment sequence(1.3 kb)of HIV-1 pol region.The subtype determination tools in the HIV sequences database were used to determine the viral genotypes,and the Cochran-Armitage trend test in the R package was used to analyze the distribution of epidemic viral subtypes and the change trend by year.Maximum likelihood evolutionary tree(ML tree)was constructed by using fasttree,and the Cluster Picker package was used to screen out the epidemic clusters with sequence gene distance <4.5% and bootstrap value >90% within the cluster.The risk factors influencing virus gene clustering were analyzed based on the epidemiological information of infected patients.2.Hy Phy was used to calculate the sequence pairwise gene distances,and Cytoscape was used to visualize the network of the main prevalent subtypes CRF07＿BC,CRF01＿AE and CRF55＿01B in Shenzhen with a 0.50% gene distance threshold.We analyzed the molecular network structure.The epidemiological characteristics of infected patients in the growing transmission cluster,emerging transmission cluster and stable cluster were analyzed to determine the epidemiological trends of each transmission cluster,and to identify the characteristics of the population in the continuing transmission cluster,so as to provide key targets for the intervention of the HIV-1 epidemic in Shenzhen.Based on the obtained gene sequences,we used the Stanford University online HIV drug resistance analysis tool to detect the transmitted drug resistance mutation(TDRM)in different HIV-1 strains,to determine the occurrence of transmitted drug resistance(TDR)in Shenzhen in different years.We compared the prevalence of TDR before and after 2016,the year of implementation of“treat-all” strategy,to determine the impact of the strategy on the prevalence of TDR in Shenzhen.3.pn MSM among males self-reported to be infected with HIV-1 through HTS behavior were identified by phylogenetic and molecular network analysis.The criteria for pn MSM identification were: the men self-reported to be infected with HIV-1through HTS behavior(1)clustered in the ML tree and the time span of diagnosis is less than 5 years;(2)the transmission clusters formed in the molecular network that do not contain a female,and contained at least one HTS male and one MSM,were considered as the pn MSM.The importance of pn MSM in HIV-1 transmission was determined by evaluating their centrality metrics in the molecular network by Cytoscape.4.Studies have shown that the CRF01＿AE epidemic cluster 4 strain is significantly more pathogenic to infected individuals than epidemic cluster 5.Phylogenetic analysis of all CRF01＿AE near full length viral sequences collected in China in the HIV sequences database identified epidemic clusters 4 and 5.The sequences of the two clusters were analyzed for differential sites and the characteristic differential sites that could change the protein structure were screened out.Characteristic sites mutations were introduced on a laboratory self-constructed CRF01＿AE full-length infectious clonal plasmid.Mutant virulent strains with the ability to replicate the infestation were rescued by transfection.The replication ability,replication fitness,single round of infestation,co-receptor binding rate and membrane fusion inhibitor sensitivity of the mutant strains were evaluated at the cellular level,to identify the key genetic sites that differ in pathogenicity of different epidemic clusters of viruses.Research results1.The dominant strains of HIV-1 in Shenzhen from 2011 to 2020(as of June)were CRF07＿BC,CRF01＿AE and CRF55＿01B,showing different trends in prevalence.In this study,a total of 13116 pol 1.3 kb sequences of HIV-1 virus newly diagnosed in Shenzhen were obtained.The infected persons were mainly male(90.55%),single(62.41%)and MSM(52.71%).The median age of infected persons was 36(Inter Quartile Range,IQR: 30-44).According to subtype identification,the main prevalent HIV-1 strain in Shenzhen is the recombinant strain CRF07＿BC,CRF01＿AE and CRF55＿01B,which accounting for 87.48% of the total.In addition,more than 20 subtypes and CRFs have been identified in Shenzhen,and there was a certain proportion(2.63%)of unique recombinant strains.When analyzing the change trend of the proportion of each subtype,it was found that the proportions of CRF07＿BC and CRF55＿01B and URFs were on the rise,while that of CRF01＿AE,CRF08＿BC and B subtypes showed a downward trend(P<0.05).The proportion of infected men(93.43%),MSM(57.34%)and single(65.34%)who clustered in the large epidemic cluster(n≥4)were significantly higher than those who did not included in a epidemic cluster(P<0.05).2.The molecular networks of CRF07＿BC,CRF01＿AE and CRF55＿01B exhibited network structures with different epidemic trends.After “treat-all”,the proportion of drug resistance transmission increased,but no high-risk drug resistance transmission cluster was formed.Molecular networks were constructed for dominant prevalent subtypes of Shenzhen identified in the first part,CRF07＿BC,CRF01＿AE and CRF55＿01B.The results showed that the rate of infected patients clustering in the network decreased by year(P<0.05),suggesting a decreasing trend of HIV-1 prevalence in Shenzhen.A large proportion of stable clusters(53.08%)and a smaller proportion of growing transmission clusters(0.26%)and emerging transmission clusters(20.00%)were present in the CRF01＿AE network,showing an overall trend of gradually weakening transmission,consistent with the trend in the proportion of newly diagnosed infections per year for this subtype.The molecular networks of CRF07＿BC and CRF55＿01B had a greater proportion of growing transmission clusters(33.02% and27.52%,P<0.05)and emerging transmission clusters(2.22% and 4.59%,P<0.05).Infected persons under 39 years of age,males,and those with junior high school or higher education level were more likely to enter the persistent transmission cluster.After the implementation of the “treat-all” policy,the proportion of HIV-1-infected patients with transmissible drug-resistant mutations in Shenzhen increased significantly(P<0.01),but the proportion of infections with TDRM clustered in the network remained stable,and no high-risk transmission clusters were formed in the molecular networks.3.A certain proportion of pn MSM-infected patients existed in the Shenzhen HIV-1epidemic,and their network centrality was comparable to that of MSM.In this part of the study,a total of 154(4.10%,154/3753)pn MSM individuals were identified among those infected with the three main prevalent subtypes(CRF01＿AE,CRF07＿BC and CRF55＿01B)in Shenzhen from 2011-2019.Of whom 76(49.35%)were infected with CRF01＿AE,50(32.47%)with CRF07＿BC,and the remaining 28(18.18%)with CRF55＿01B infected.Regarding the marital status,a greater proportion(31.82%)of pn MSM infected individuals were married than those who self-reported as MSM(P<0.001).pn MSM and self-reported MSM infected individuals did not differ significantly in all measures of centrality in the network,indicating that their position in the network and involvement in transmission were comparable to MSM infected individuals.The men self-reported to be infected by HTS who aged 39 years and younger and infected with CRF01＿AE strain were more likely to be identified as pn MSM(P<0.05).4.The characteristic site of CRF01＿AE strains of epidemic clusters 4 and 5 was identified,which lead to significant differences in protein structure and biological phenotype.We obtained a total of 188 conformant CRF01＿AE viral near full-length sequences from HIV sequences databases,and confirmed 29 sequences were belonging to epidemic clusters 4,and 42 sequences were belonging to epidemic clusters 5,respectively.Multiple different sites were found between the two epidemic clusters of viral gene sequences,in which one of them,a characteristic difference site in the gp41 envelope glycoprotein amino acid sequence of env region gene 779-792(HXB2:8559-8600)between the two clusters of viral sequences was found to lead to the difference in protein structure.Two clusters of mutant virulent strains were transformed based on CRF01＿AE infectious clones.Virus strains with two epidemic clusters of characteristically different sites were obtained using CRF01＿AE infectious cloning and reverse genetics techniques.Experiments at the cellular level revealed that epidemic cluster 4 mutant strains were more replication-competent,replication-adapted and single round of infection than epidemic cluster 5.The epidemic cluster 5 mutant strain showed higher binding to the co-receptor CCR5 than epidemic cluster 4 and increased sensitivity to the membrane fusion inhibitor T20.Research conclusionThis study systematically analyzed the HIV-1 epidemic in Shenzhen over nearly 10 years,clarified the HIV epidemic trends and key populations for prevention and control by molecular network,providing information to support the prevention and control of the HIV epidemic in Shenzhen.We found that the implementation of health policies can significantly change the prevalence of chronic infectious diseases,and that epidemic monitoring should be strengthened after the implementation of health policies to provide timely and effective information for adjusting interventions and health strategies.The effective screening of pn MSM population would facilitate effective behavioral interventions for pn MSM and their sexual partners.We identified characteristic differential sites that have a significant impact on CRF01＿AE viral replication capacity,which provides basic information for the development of antiviral therapeutic drugs.In conclusion,this study systematically analyzed the HIV-1prevalence in Shenzhen and explored the differences in pathogenicity of different HIV-1strains,which is important for the formulation of HIV-1 prevention and control policies in Shenzhen and elucidation of the virus infection mechanism. |
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