HIV Genotyping And Drug Resistance Analysis And Co-infection Study Among Unpaid Blood Donors In Some Areas Of China

Background and Objectives Human immunodeficiency virus（HIV）is a single-stranded RNA virus.It causes human acquired immunodeficiency syndrome（AIDS）which leads to a gradual loss of immune function and lack of resistance to external pathogens.Eventually,leads to the co-infection with a variety of opportunistic pathogens resulting in multiple clinical symptoms until death.The high rate of gene evolution is easy to produce new epidemic subtypes and drug-resistant types,which make the treatment of HIV-infected people clinically greatly hindered.At present,most of the genotyping,evolutionary analysis and drug resistance studies on HIV are concentrated in hospitalized AIDS patients.However,there are few studies on blood donors.Therefore,HIV genotype,subtype distribution and phylogeny among blood donors were analyzed to monitor evolutionary trend of HIV gene molecules among blood donor.Analysis of drug resistance can guide the future treatment and drug use trends of HIV-infected patients,play an early role in preparation and research,support prevalence and evolutionary trend data.In addition,the infection of HIV is a very long process.During this period,the immune function of the host gradually declines.The cause of death from HIV infection is the co-infection with other pathogens.At present,the research on HIV co-infection is concentrated in hospitals,that is,patients who have developed or are about to develop AIDS,pathogens were mostly related with acute symptoms or high mortality,however,asymptomatic HIV infected individuals in blood donors are rarely mentioned in the research.Therefore,this study investigated the co-infection of pathogens by metagenomic detection methods in different stages of HIV infection,in samples between recent infections,long-term infections and AIDS,to analyze the differences at different stages of infection,the comparison can be used to guide the treatment and protection of HIV-infected people at different stages.Methods The first part of the experiment:Samples of HIV-infected blood donors from 5 regions（Chongqing,Urumqi,Luoyang,Mianyang,and Liuzhou）from August 2015 to February 2017 were collected and confirmed.The results showed that there were 176 anti-HIV positive plasma samples..The RNA of the specimen was extracted and reverse transcribed,followed by nested PCR to amplify and sequence the HIV POL gene fragment.The sequencing results were analyzed by MEGA 7.0 to construct the HIV molecular phylogenetic tree.The drug resistalce was analyzed in the HIV resistance gene analysis library of Stanford University.The second part of the experiment:30 HIV samples were collected:long-term infection samples,recent infection samples,AIDS samples and healthy samples（control trials）.After mixing 10 samples,the DNA and RNA were enriched by ultracentrifugation,and they were divided into three groups.After extracting the total DNA and total RNA of each group,RNA was reverse transcribed,DNA and cDNA were used as templates for random primer PCR,then the products were subject to high-throughput sequencing,and the sequencing results were classified by Kraken Pyl.0.The Hemll.0 software was used to perform clustering heat map analysis and Past3.0 software for principal component analysis.Results The proportion of successful amplification of HIV POL gene fragments in anti-HIV positive plasma samples from 5 regional blood donors was 44.3%（78/176）,including 37.8%（31/82）in Chongqing and 48.5%（16/33）in Urumqi,71.43%（10/14）in Mianyang,40%（4/10）in Luoyang,and 45.9%（18/37）in Guangxi.The gene subtypes were:B 1.3%（1/78）,CRF07_BC 74.4%（58/78）,CRF01_AE 16.7%（13/78）,CRF08_BC 5.1%（4/78）,CRF55₀1B 2.47%（2/78）.Seven（9.0%）drug resistance mutations were detected in all positive PCR product sequences,including four PI-assisted（three Q58E and one I84M mutations）drug resistance mutations,two NRTI（D67N）drug resistance mutations and one NNRTI（V179E）drug resistance mutation.There were 154 long-term infections（87.5%）and 22 recent infections（12.5%）.In the genotype distribution and drug resistance mutations,the two did not show obvious differences.The number and diversity of pathogens in the long-term infected patients were significantly richer than those in the other three groups.The overall trend was HIV long-term infection group>recent infection group>AIDS group>control group.The main co-infected pathogens are attributed in families as Pseudomonadaceae and Enterobacteriaceae.Virus are mainly:Retroviridae,Parvoviridae,Herpesviridae,Hepadnaviridae,Anelloviridae.Conclusion The diversity of HIV genotypes among blood donors in the five regions in China is high,with CRF07_BC being dominant.At the same time,the newly emerging CRF55₀1B subtype within the survey revealed that HIV infection is spreading from the high-risk group to the general population.At different stages of HIV infection,the number and diversity of co-infected pathogens increase with the development of the infection process;due to the birth of dominant species,AIDS patients resulting in a low diversity of co-infected pathogens.