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Objective:To analyze the effects of the main drug resistance mutations in the integrase (IN) region on the resistance of HIV-1 CRF01_AE strains, and compare the differences with subtype B strains.Methods:Seven IN region mutations or combined mutations (T66K, F121Y, Q148K, N155H, G118R, R263K, Q148K/N155H) were selected from the HIV drug resistance database of Stanford University in the United States, and introduced to the IN region of HIV-1 B subtype infectious clone pNL4-3 and CRF01_AE infectious clone pGX002 by seamless cloning, homologous recombination and point mutation. The mutant plasmids were transfected into 293T cells for virus packaging. The culture was expanded in MT2 cells and infectious titers were detected. Half maximal inhibitory concentrations (IC 50) of four integrase inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG) and bictegravir (BIC), against 14 mutant viruses were detected and compared with the IC 50 against the wild-type viruses. Results:B subtype and CRF01_AE plasmids carrying seven IN region mutations or combined mutations were successfully constructed, and 14 recombinant viruses were packaged with an infectious titer of 10 4-10 6 median tissue culture infective dose (TCID 50)/ml. The recombinant viruses replicated efficiently in MT2 cells. The concentrations of HIV-1 p24 antigen contained in the supernatants of cell culture reached 830-2 700 ng/ml. Five mutations or combined mutations (T66K, F121Y, Q148K, N155H, Q148K/N155H) caused CRF01_AE and B subtype strains to be highly resistant to RAL and EVG, resulting in an increase in the IC 50 by 200 times and 2 000 times or more as compared with the IC 50 against the wild-type viruses. The same mutation-caused fold changes of IC 50 of RAL and EVG against CRF01_AE were significantly lower than that of subtype B ( P<0.01). Q148K/N155H mutation caused B subtype and CRF01_AE to be highly resistant to DTG and BIC, with IC 50 increased by more than 50 times. Other mutations had little effects on the sensitivity to DTG and BIC. Conclusions:Fourteen HIV-1 strains carrying seven INSTI resistance mutations based on B subtype and CRF01_AE were constructed. Five mutations resulted in high resistance to RAL and EVG, and there was a high level of cross-resistance. Resistance to RAL and EVG caused by the same mutation was higher in B subtype than in CRF01_AE. The combined mutation of Q148K and N155H was associated with greater resistance to DTG and BIC, indicating that the genetic barrier of DTG and BIC resistance was high. DTG and BIC could effectively inhibit the strains carrying INSTI resistance mutations without obvious subtype difference.
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Objective To understand the transmission patterns and risk factors of HIV-1 strain CRF01_AE subtypes in China,and to provide guidance for the implementation of precise intervention.Methods A total of 2 094 CRF01_AE pol sequences were collected in 19 provinces in China between 1996 and 2014.Phylogenetic tree was constructed by PhyML 3.0 software to select the transmission clusters.Transmission network was constructed by Cytoscape 3.6.0,which was further used for exploring of the major risk factors.Results Of the 2 094 sequences,12.18％ (255/2 094) were in clusters.A total of 82 transmission clusters were identified.The numbers of clusters and contained sequences in intra-provincial transmission (61,173) were significantly more than those in inter-provincial transmission (21,82).The ratio of transmission clustering in MSM increased over time from 2.41％ (2/83) during 1996-2008 to 23.61％ (72/305) during 2013-2014,showing a significant upward trend (x2 =27.800,df=1,P =0.000).The proportion of MSM with inter-provincial transmission clusters were higher than those with intra-provincial transmission clusters,which increased from 0.67％ (2/297) during 1996-2008 to 6.36％(30/472) during 2013-2014,showing a significant upward trend (x2=20.276,df=1,P=0.000).The transmission rate in homosexuals of the inter-transmission clusters (86.59％,71/82) was higher than that of intra-provincial transmission clusters (56.65％,98/173),and the difference was statistically significant (x2=22.792,P=0.000).The proportion of inter-provincial transmission clusters with more than 2 transmission routes (33.33％,7/21) was higher than that of intra-provincial clusters (13.11％,8/61),and the difference was statistically significant (x2=4.273,P=0.039).Results from the transmission network analysis indicated that the proportion of high risk population (degree≥4) with inter-provincial transmission clusters (51.22％,42/82) was significantly higher than that with intra-provincial transmission clusters (26.59％,46/173),and the difference was statistically significant (x2=14.932,P=0.000).Inter-provincial clusters were mainly detected in and and MSM.Conclusions Complex transmission networks were found for HIV-1 CRF01_AE strains in the mainland of China.Inter-provincial transmission clusters increased rapidly,MSM played an important role in the wide spread of the strain.More researches in transmission networks are needed to guide the precision intervention.
ABSTRACT
Objective To understand the transmission patterns and risk factors of HIV-1 strain CRF01_AE subtypes in China,and to provide guidance for the implementation of precise intervention.Methods A total of 2 094 CRF01_AE pol sequences were collected in 19 provinces in China between 1996 and 2014.Phylogenetic tree was constructed by PhyML 3.0 software to select the transmission clusters.Transmission network was constructed by Cytoscape 3.6.0,which was further used for exploring of the major risk factors.Results Of the 2 094 sequences,12.18％ (255/2 094) were in clusters.A total of 82 transmission clusters were identified.The numbers of clusters and contained sequences in intra-provincial transmission (61,173) were significantly more than those in inter-provincial transmission (21,82).The ratio of transmission clustering in MSM increased over time from 2.41％ (2/83) during 1996-2008 to 23.61％ (72/305) during 2013-2014,showing a significant upward trend (x2 =27.800,df=1,P =0.000).The proportion of MSM with inter-provincial transmission clusters were higher than those with intra-provincial transmission clusters,which increased from 0.67％ (2/297) during 1996-2008 to 6.36％(30/472) during 2013-2014,showing a significant upward trend (x2=20.276,df=1,P=0.000).The transmission rate in homosexuals of the inter-transmission clusters (86.59％,71/82) was higher than that of intra-provincial transmission clusters (56.65％,98/173),and the difference was statistically significant (x2=22.792,P=0.000).The proportion of inter-provincial transmission clusters with more than 2 transmission routes (33.33％,7/21) was higher than that of intra-provincial clusters (13.11％,8/61),and the difference was statistically significant (x2=4.273,P=0.039).Results from the transmission network analysis indicated that the proportion of high risk population (degree≥4) with inter-provincial transmission clusters (51.22％,42/82) was significantly higher than that with intra-provincial transmission clusters (26.59％,46/173),and the difference was statistically significant (x2=14.932,P=0.000).Inter-provincial clusters were mainly detected in and and MSM.Conclusions Complex transmission networks were found for HIV-1 CRF01_AE strains in the mainland of China.Inter-provincial transmission clusters increased rapidly,MSM played an important role in the wide spread of the strain.More researches in transmission networks are needed to guide the precision intervention.
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Objective To analyze genetic characteristics of HIV isolated from men who have sex with men(MSM) in Beijing and predict the epidemic trend in this population.Methods All of the HIV gene sequences in our laboratory obtained from MSM in Beijing were used,which were aligned with all of the HIV gene sequences from MSM and other populations in China downloaded from Los Alamos HIV Database.Phylogenetic trees were constructed by using software PhyML 3.0,based on which the relationships of prevalent HIV strains between Beijing MSM and other populations in China were further explored.The evolution rate,the time of most recent common ancestor (tMRCA),the epidemic parameters,the reproductive number (R0) were calculated by using software BEAST to predict HIV evolution and epidemic characteristics.Results Multiple HIV subtypes,including subtype B,CRF01_AE and CRF07_BC,were found to be prevalent among MSM in Beijing.In ML tree constructed based on strains from the whole country,three clusters including B-1,CRF01_AE-1,and CRF01_AE-2 were found among the MSM in Beijing (accounting for 40％).At least three independent introduction of B 1 cluster strains into Beijing MSM were found,which were at March 1991 (July 1984-February 1997),January 1994 (January 1989-January 1998),April 1991 (August 1984-January 1996).For CRF01_AE strains,two clusters including CRF01_AE-1 and CRF01_AE-2 were introduced into the population at December 2000 (March 1998-January 2003) and December 2001 (January 2000-July 2003) respectively.The population epidemiology of HIV in Beijing MSM was reconstructed based on sequences.The CRF01_AE-1 cluster spread more quickly than the other two clusters,and the evolution rate was higher.Conclusion Multiple HIV subtypes were found prevalent among MSM in Beijing.Although subtype B strain was introduced into Beijing MSM earlier than CRF01_AE strain,CRF01_AE strain increased more quickly than subtype B strain.More research and control of the CRF01_AE prevalence will be helpful for prevention and control of HIV epidemic in MSM in Beijing.