Molecular Technique For Tracing Postexposure HIV Transmission

BackgroundHIV and other hematogenous occupational exposed infections and nosocomial infections attract increasing attention from medical workers, judicial policemen and public. Traditional techniques for tracing postexposure HIV transmission mainly depend on epidemiological investigation. Molecular technique for tracing postexposure HIV transmission have been reported abroad. The molecular technique is based on analysis for high variable region gene of different individuals’ HIV quasispecies in suspicious transmission chain and provides molecular laboratory evidences to investigate for occupational exposed infections, nosocomial infection and judicial investigation.There are three types of techniques for tracing HIV transmission, list as direct sequencing after nested PCR product, sequencing of cloned PCR products and single-genome amplification. Direct sequencing after nested PCR product can only acquire preponderant quasispecies except vulnerable quasispecies. And sequencing of cloned PCR products and single-genome amplification can acquire both preponderant quasispecies and part of vulnerable quasispecies, which can provide more quasispecies sequence information for tracing-HIV transmission.This paper focused on an investigation on whether an affection was a transfusional affection, and the spread relationship in the affection, which would provide laboratory evidences for tracing HIV transmission by using sequencing of cloned PCR products and single-genome amplification.Objective1. To build two techniques for tracing HIV transmission based on quasispecies analysis, list as sequencing of cloned PCR products and single-genome amplification.2. The two built techniques of sequencing of cloned PCR products and single-genome sequencing were applied to an investigation of a possible case of HIV transmission after blood transfusion.3. To discuss influence of different sample types (whole blood, plasma) to the detection result when single-genome amplification was applied to tracing HIV transmission.Objects and Methods1. Sixteen plasma specimens were collected from3HIV infections (T1-T3) involved in a possible HIV transmission chain and13HIV/AIDS (C1-C13) controls.2. Sequencing of cloned PCR products was applied to tracing HIV transmissionFirstly, RNA was extracted from collected plasma samples. Secondly, RNA would be reverse transcribed and nested PCR amplificated. Thirdly, cloned after choosing positive amplificated product, and quasispecies were acquired. At last, the quasispecies sequences would be obtained after sequencing and phylogenetic analysis would be used to analysis the quasispecies sequences.3. Single-genome amplification was applied to tracing HIV transmission3.1The plasma samples1) RNA were extracted from collected plasma samples.2) RNA would be reverse transcribed to cDNA.3) cDNA would be gradient dilution to explore optimum dilution.4) Nested PCR amplification of96holes would be done according to the optimum dilution.5) Sequencing the positive amplificated product, and phylogenetic analysis would be used to analysis the quasispecy sequence.3.2The whole blood samples1) All nucleic acids were extracted from collected whole blood samples.2) All nucleic acids would be reverse transcribed to cDNA and provirusDNA.3) cDNA and provirusDNA would be gradient dilution to explore optimum dilution.4) The following steps were same as the plasma samples.Results1. Results of sequencing of sequencing of cloned PCR products1) The sequences of59clones from13specimens were successfully obtained, and the sequences among most clones were different.2) To calculate gene dispersion ratio of the clones sequences. The results were as follows. The mean gene dispersion ratio of different clones in one specimen were less-than3%in most of13specimens. Compared with the clone sequences from T1, the mean gene dispersion ratio of T2,T3,C12were less than others. Hence, we inferred that there were HIV transmission in T1,T2,T3and C12.3) Phylogenetic analysis of the clones sequences and the phylogenetic tree showed as follows. All clones from T1, T2, T3and C12could cluster together, and the bootstrap on the branch was100%. Part of T3clones and clones from T1, T2and C12could cluster together. T3sequences were paraphyletic with respect to T1, T2and C12sequences. From these analyses, we inferred that the direction of transmission occurred from T3to T1, T2and C12in molecular evidence.2. Results of single-genome amplification2.1The plasma samples1) In plasma samples, the sequences of153sequences from15specimens were successfully obtained. The sequences among most quasispecies sequences were different.2) To calculate gene dispersion ratio of the quasispecies sequences. The results were as follows. The mean gene dispersion ratio of different quasispecies in one specimen were less-than4%in most of specimens. Compared with the quasispecies sequences from T1, the mean gene dispersion ratio of T2, T3, C12was less than others. Hence, we inferred that there were HIV transmission in T1, T2, T3and C12.3) Adopted phylogenetic analysis to the quasispecies sequences and the phylogenetic tree showed as follows. All quasispecies from T1, T2, T3and C12could cluster together, and the bootstrap on the branch was94%. Part of T3quasispecies and quasispecies from T1, T2and C12could cluster together. T3sequences were paraphyletic with respect to T1, T2and C12sequences. From these analyses, we inferred that the direction of transmission occurred from T3to T1, T2and C12in molecular evidence.4) Adopted phylogenetic analysis to the quasispecies sequences of control samples C5and C6. Combined with epidemiological data, we inferred that the direction of transmission occurred from C5to C6.2.2The whole blood samples1) In whole blood samples, the sequences of153sequences from14specimens were successfully obtained. The sequences among most quasispecies sequences were different.2) When plasma samples and whole blood samples were applied to tracing C2and C4transmission, the direction of transmission occurred from T2to T4in molecular evidence could be obtained in whole blood samples quasispecies except plasma samples quasispecies. Apart from this, the results of plasma samples and whole blood samples were consistent. 3. The comparison of two methodsThe analysis results of two techniques for tracing postexposure HIV transmission were consistent. When two techniques for subtype classification, the results showed that the results of sequencing of cloned PCR products only predicated C8to be BC subtype, and the results of single-genome amplification predicated C8to07_BC subtype.Conclusions1. Two techniques were built for tracing HIV transmission, list as sequencing of cloned PCR products and single-genome amplification.2. The two built techniques of sequencing of cloned PCR products and single-genome amplification were applied to an investigation of a possible case of HIV transmission after blood transfusion. The results supported the possible epidemiological clue that HIV was transmitted from T3to T2, and then to T1, indicating that molecular epidemiological investigation could provide more direct evidence for tracing postexposure HIV transmission. The results of the two techniques were consistent.3. When tracing postexposure HIV transmission, just laboratory detection couldn’t predicate HIV transmission, which should combine with epidemiological data.4. When single-genome amplification was applied to tracing HIV transmission of subjects infected for a long time, the whole blood samples could obtain more quasispecies sequences than plasma samples.