Transmission And Evolution Of HIV Quasispecies In HIV Concordant Heterosexual Couples

BackgroundResearch on tracing postexposure HIV infection, is important for understanding the spread and evolution of HIV. It is also important for carrying out the investigation of HIV occupational exposed infections, iatrogenic infection investigation and judicial investigation. Laboratory methods reported abroad mainly included direct sequencing of nest PCR products, sequencing of cloned PCR products and end-point limiting-dilution PCR.These methods were used to infer the transmission relationship as well as the transmission direction on HIV gene subtype or quasispecies level.But these methods had some disadvantages, such as,receiving a small amount of information, operating complicatedly. Our research group had successfully used Miseq high-throughput sequencing to trace postexposure HIV infection suspected by the sexually transmission. Extracte the RNAs in plasma specimens, reverse-transcribe RNAs into cDNAs, and do nest-PCR. We can infer the transmission relationship as well as the transmission direction by Miseq high-throughput sequencing.This method supported a technology of tracing postexposure HIV infection which is more simplely, cheaper and more useful.This study had optimized the HIV Miseq high-throughput sequencing technology established early. HIV couples were enrolled as the research subjects, in to study the transmission and evolution of HIV quasispecies in heterosexual transmission.Objective1. To study the transmission of HIV quasispecies in heterosexual transmission.2. To explore the potential influence of the specimen types (whole blood and plasma) and ART.3. To explore the intraspecimen and interspouse evolution of HIV concordant heterosexual couples over time.Subjects and Methods1. Subjects(1) Seven couples were selected as the research subjects, in which one person of each couple was recently infected with HIV from his or her spouse within 1.5 years (Six couples were infected within half year). The epidemiologic information were shown as follows, the former infectors were infected by non-spouse heterosexual transmission (six couples) and drug injection (one couple).The transmission direction of six couples was from husband to wife, which of the other one was from wife to husband. The 14 subjects were not received ART, apart from F3, F5, M5, F7 and M7. The plasma and whole bloodspecimens of the seven couples were collected and preserved in-80℃.(2) The specimens of one couple (F1, M1) were collected continuously, the follow up time was from 3 months to 23 months. Nine plasma and seven whole blood specimens were collected from F1 who was infected for 54 months. Six plasma and five whole blood specimens were collected from M1 who was infected after 37 months and died after being infected for 42 months.2. Experiment procedure:(1) RNAs and total nucleotide acids were extracted from plasma or whole blood specimens, respectively.(2) Reverse-transcribed the RNA into cDNA.(3) For cDNA and the total nucleic acids, the specific gene regionswere amplified by nest PCR, and then directly sequenced after purification.The gene regions and primers for subtyping and Miseq sequencing were different.(4) Agarose gel electrophoresis and ultraviolet imagingdetection.(5)Direct sequencing for PCR products and confirm HIV subtype.(6)Miseq sequencing: Optimized the previous method,including building library for single specimen in place of mixing specimens,using PE300 program in place of PE250 program. Phylogenetic analysis was applied to analyzing the cleaned quasispecies.Results1. Analysis results of seven couples(1)The subtype of HIV: The subtype of HIV from each individual was same with his or her spouse, respectively. The HIV subtype of F1/M1, F3/M3 was 01_BC. The subtype of F4/M4 was CRF07_BC. The subtype of F5/M5 was CRF08_BC. The subtype of F2/M2, F6/M6, F7/M7 was CRF01_AE.The HIV subtype results supported the result of epidemiological investigations of marital sexual transmission.(2)The distribution of HIV quasispecies: Miseq sequencing was successfully conducted on 24 specimens, including all whole blood specimens and 10 plasma specimens. Miseq sequencing was failed on four plasma specimens (two couples).83290 to 209120 (average 154176) cleaned sequences representing 29 to 455 (average 139) HIV quasispecies were obtained for each plasma specimen.The frequency of top one quasispecies in total sequences was from 10.0% to 98.9% (average 51.4%).67831 to 193202 (average 134219) cleaned sequences representing 12 to 284 (average 97) HIV quasispecies were obtained for each whole blood specimen. The frequency of top one quasispecies in total sequences was from 25.2% to 99.2%(average 75.0%). There was no significant difference (p>0.05) in the No. of sequence and quasispecies between plasma and whole blood specimens.The percentage of top one quasispecies in whole blood was obviously higher than that in plasma specimens (p<0.05).Among most of the specimens, the frequency of total top 20 quasispecies sequences was more than 80%, which was lower(about 45%) in one plasma and was more than 80% in the other 3 specimens. for 9 out of 14 subjects, the frequency of top one quasispecies sequence from whole blood specimens was more than 85%.(3) The range of genetic diversity: Intraspouse genetic diversity was from 0.6% to 4.4% in plasma specimens and from 0.5% to 4.0% in whole blood specimens.There was no significantly statistical difference between the genetic diversity of all plasma and whole blood specimens (P>0.05). Interspouse genetic diversity was from 0.7% to 5.1% in plasma specimens and from 0.7% to 5.1% in whole blood specimens.(4) Phylogenetic tree analysis: HIV quasispecies of every couple clustered together separately, accompanied with higher bootstrap values (>90).Considering genetic diversity, this suggesting there were transmission relationship between each couple. We can infer right transmission direction when we analyzed plasma or whole blood only or analysed them together, apart from two plasma specimens of one couple.The longer time the couples infected, the more difficult the sequence analysis was. The sequence distinction was more obvious when the infection interval between one couple was longer. For example, the sequences clustered alone when the interval between F2 and M2 was more than 8 years.The interval between F1 and M1 was short (they were both transmited in window period), and the sequences of two subjects were clustered confusingly.(5) The potential impactof specimen types and ART on the result analysis:RNA was amplified from the plasma specimens, and 10/14 specimenswere amplified successfully. The proviral DNA was amplified from the whole blood specimens, and all specimens were amplified successfully.The quasispecies sequence information obtained by plasma and whole blood was considerable. Plasma specimens from 4/5 couples and whole blood specimens from 7/7 couples could indicate the transmission direction. Plasma specimens from 3 out of 5 snhiecls who accepted ART were failed amplified.Specimens amplified successfully can infer tbe transmission relationship and the transmission direction correctly.The analysis of last couple also infer correct transmission direction although M7 had receieved ART for 57 months.2. Analysis results of the follow-up couple(1)The distribution of HIV quasispecies:Miseq sequencing was applied successfully in 8 plasma specimens and 7 whole blood specimens of F1.Miseq sequencing was applied successfully in all specimens of M1.83290 to 180118(average 131094) and 83329 to 175811 (average 122444)cleaned sequences representing 23 to 322(average 125) and 12 to 190 (average 67)HIV quasispecies were obtained for each plasma and whole blood specimen of F1.69949 to 172087 (average 114462)and 75115 to 259369 (average 166507)cleaned sequences representing 41 to 311 (average 145)and 22 to 188 (average 98)HIV quasispecies were obtained for each plasma and whole blood specimen of M1.The quasispecies distribution of F1/M1 plasma tended to disperse gradually, which was not obvious in whole blood specimens.(2) Genetic diversity analysis: The gene diversity of HIV quasispecies in F1/M1 plasma and whole blood specimens increased gradually from being infected 1 month to 37 months. From 1 month to 37 months,the average genetic diversity of Fl had grown from 0.7% (plasma),0.6%(whole blood)to 2.3% (plasma),3.1%(whole blood),which of M1 had grown from 0.6%(plasma),0.8%(whole blood)to 0.7%(plasma),2.7%(whole blood).For each subject, diversity between different follow-up specimens and the first specimens also had a rising trend,which arrived from 0.9%(plasma),0.6%(whole blood)to 2.5%(plasma),3.4% (whole blood) for F1,and arrived from 0.8%(plasma),0.7%(whole blood) to 4.1% (plasma),3.2%(whole blood)for M1. The interspouse diversity increased over time in both plasma and whole blood specimens which arrived from 0.7%(plasma),0.7%(whole blood)to 5.4%(plasma),4.7%(whole blood).(3) Phylogenetic tree analysis: In early time of infection, the distribution of intraspouse quasispecies sequence was relatively centralized, which of interspecimen quasispecies sequence was very close.The intraspecimen quasispecies sequence distribution gradually dispersed over time.It wasthe same with the interspecimen quasispecies. All follow-up specimens received right transmission direction when we analyzed plasma or whole blood or analysed them together, apart from specimens which subjects were infected after 12 months.Conclusion1. Miseq sequencing results supported the results of epidemiological survey by analyzing the qusispecies transmission and evolution of 7 concordant heterosexual couples.The inference of transmission relationship and direction could not be influenced by ART.2. The variability and frequency of HIV quasispecies was very helpful in exploring the transmission and evolution of HIV. HIV contact tracing results from whole blood specimens were more reliable and practical than that from plasma specimens.3. Diversity between different follow-up specimens and the first specimens had a rising trend, which increased to 3% after infection for 3 years as reported aboard. The interspouse diversity increased over time, which increased to 5% after infection for 3 years as reported aboard.4. To infer the transmission direction, a variety of factors should be considered:(1) Time after being infected. It was easier to infer the transmission direction if the time after being infected was short.It need further research to calculate the time after being infected. (2) Time interval between two subjects infected. It was easier to infer the transimission direction if the interval between two subjects infected was longer. Infer the transmission direction carefully when the subjects were spread in the window period, and when the time after being infected is longer.