Investigation Of Possible Cases Of HIV Transmission By Using Miseq Platform

BackgroundRisk for occupational and nosocomial transmission of HIV attracts intensive attention of health workers, policemen and the public. Traditional ways to identify postexposure HIV infection mainly depend on epidemiologic investigation and relative document recordings. In these procedures, the management of occupational exposure to HIV is relatively normative. To confirm postexposure infections, employees should be monitored for seroconversion by testing HIV antibodies during the follow-up period. However, in some complex cases this recommendation would be inadequate, especially when the employee has been infected shortly before the exposure and is in window period at the moment, or he keeps other high risk behavior in the subsequent follow-up period. In such situations, molecular approaches for analyzing HIV genotypes and quasispecies are needed.By accessing interpersonal HIV genetic similarity, laboratory methods, including direct sequencing of PCR products, sequencing of cloned PCR products and end-point limiting-dilution PCR, are used to investigate HIV infectious events. The former one is simple and convenient, but only one dominated quasispecy can be detected. The later two approaches can obtain more major and minor quasispecies, but the disadvantages of being sophisticated and time-consuming limit their applications. By comparison, Miseq high-throughput sequencing platform (Miseq platform for short) allows to acquire massively sequencing information in one run with simply processed PCR products. This approach has been applied in several fields such as the mutation detection of HCV drug resistance. But it has not yet been reported to use Miseq platform in analysis of HIV quasispecies or identification of postexposure infection.Objective1. To develop an approach for analyzing HIV quasispecies basing on the Miseq platform and explore the requirements in tracing postexposure HIV infection;2. To investigate a possible HIV transmission chain by using the newly developed approach.Objects and Methods1. Subjects:(1) Plasma or whole blood specimens (stored in our laboratory) from3HIV infections in a possible HIV transmission chain and11controls were analyzed. The epidemiologic information were as follows, T1perhaps infected HIV by transfusing the donated blood from T2, and T2was transmitted by HIV infection T3through homosexual behavior. Among the11controls (C3to C13), C3to C6lived in a same city with T1and so did C7to C13with T2and T3.(2) Considering the possible heterosexual transmission from C5to C6, the plasma specimens from them and2epidemically unrelated controls (C3and C4) were initially used in the research of methodology.2. Experiment procedure:(1) RNA or total nucleotide acids was extracted from plasma or whole blood specimens, respectively, and then reverse transcribed the RNA or total nucleotide acids into cDNA.(2) Direct sequencing of PCR products:the specific gene regions of env, gag, and pol were amplified and then directly sequenced after purification.(3) The targeted sequence for molecular tracing by using Miseq platform was selected in env gene region, and relative universal primers were designed and optimized.(4) The antisense primer of the second round PCR was unique "fusion primer It was synthesized specifically for each specimen. The condition of nested PCR reaction was separately optimized. After that, the targeted sequences were amplified in the optimum PCR condition.(5) PCR products were purified and evaluated. The gene libraries were built only if the quality of the PCR products were up to standard.(6) Miseq platform was used to sequence the HIV quasispecies. Then sequence reads were processed and denoised.(7) Phylogenetic analysis was applied to analyze the cleaned HIV quasispecies.Results1. Research of methodology (C3to C6)(1) Result of direct sequencing of PCR product:All4specimens were amplified and sequenced successfully. The interpersonal genetic distances of env, pol and gag region indicated a close relationship between C6and C5, but could not provide any information about the transmission direction. In these3regions, sequences of env could provide more evolutionary information.(2) Result of Miseq platform sequencing:All4specimens were sequenced successfully. An average of29045(23788to37397) cleaned reads representing1314(1229to1412) unique quasispecies was obtained from each specimen. After data cleaning, the frequency of quasispecies were counted and ranked. It was found that frequency of quasispecies from4specimens decreased dramatically. The top20quasispecies in these4specimens accounted for from41.5%to66.2%of the intrapersonal HIV sequences.(3) Interpersonal genetic distance analysis:Interpersonal genetic distances were calculated by using the top5, top20, top100, top500, and all quasispecies, respectively. When the top5quasispecies were analyzed, the average genetic distance between quasispecies from C6and C5(4.2%) was significantly lower (P<0.01) than that between C6and C3(11.6%) or C6and C4(18.2%). The results of genetic distance analysis of top20or more quasispecies were similarly,suggesting a close relationship between C5and C6. (4) Phylogenetic tree analysis:Phylogenetic trees were also built in5groups. When the top5quasispecies were analyzed, sequences from C5and C6clustered together. While sequences from C3and C4exhibited completely independent clusters. The parallel relationship between sequences from C5and C6could not indicate the direction of HIV transmission. When top20or more quasispecies were analyzed, sequences from C5showed a paraphyletic relationship with those from C6, indicating the direction of HIV transmission was from C5to C6. As more quasispecies been analyzed, more obvious the paraphyletic relationship would be.2. The application of methodology:Miseq platform was used to investigate a possible HIV transmission chain (T1to T3, C7to C13)(1) The result of Miseq platform sequencing:The data of1specimen (C10) was discarded because of the poor sequencing quality. The other9specimens (Tl to T3, C7to C9, C11to C13) were sequenced successfully. An average of22685(8637to37865) cleaned reads which representsed999(553to1660) unique quasispecies was obtained from each specimen. It was found that frequency of quasispecies from9specimens decreased dramatically. The top20quasispecies in these9specimens accounted for from41.5%to68.9%of the intrapersonal HIV sequences.(2) Interpersonal genetic distance analysis:Interpersonal genetic distances were calculated by using the top100quasispecies. The average genetic distance between quasispecies from T1and T2(2.8%) was significantly lower (P<0.01) than that between T1and controls (3.6%-14.0%). The result between T2and T3(2.9%) was significantly lower (P<0.01) than that between T2and controls (3.1%～13.6%). The average genetic distances between quasispecies from C12and Tl, T2, or T3were quite low, which indicated a close relationship among them.(3) Phylogenetic tree analysis:Phylogenetic tree was built using top100quasispecies. Sequences from T1, T2. T3and C12clustered together. While sequences from other controls exhibited completely independent clusters. T3showed a paraphyletic relationship with T1. T2, and C12. T2had a similar relationship with T1. These datd supported the epidemiologic clue that T2was transmitted by T3, then T1was transmitted by T2. Without integral epidemiologic information, the relationship between C12and T3was hard to identify. Considering the fact they were all MSMs and lived closely, it was very likely that C12was infected by T3directly or indirectly.(4) The conclusion about HIV genetic relationship and transmission direction on the possible HIV transmission chain by using Miseq platform accorded with that by using PCR product cloning and EPLD-PCR in our previous study.Conclusions1. An approach for analyzing HIV quasispecies basing on Miseq platform was developed.2. The result of Miseq platform could be used to calculate the genetic similarity of HIV quasispecies from various infections. When the number of major quasispecies was enough, the direction of HIV transmission between related infections could be idenfied. The more major quasispecies been analyzed, the clearer information on transmission direction would get.3. The molecular investigations of2HIV infectious chain though sex and sex-blood donation proved that the result of Miseq platform could obviously support the epidemiologic survey. While with incomplete epidemiologic information, the data of laboratory should be interpreted prudently.4. With the feature of convenient and cost-effective, Miseq platform has a high practical value in tracing possible HIV transmissions.