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Project titleThe study of phenomenon of HIV-1 low genetic diversity and detection of drug resistance in circulating viral strains in Russia

Annotation
AIDS epidemic has expanded for more than 40 years, starting from late 1970th. Every year about 2,5 million new cases of HIV infection occur worldwide. The study of molecular and genetic features of HIV transmission is one of the important steps for HIV vaccine development. It was found that during sexual HIV transmission only one viral variant is transmitted in 80% of cases. This variant is selected from the heterogeneous donor viral population [Keele et al., 2008; Haaland et al., 2009; Abrahams et al., 2009]. In the case of sexual HIV infection this effect (so called genetic bottleneck) is explained by low efficiency of viral transmission through the mucosa, and probably by selective immune pressure. Another route of transmission is associated with needle or syringes sharing among injection drug users. Currently there are very few studied cases of parenteral HIV transmission in the world and data from different scientific groups are contradictory. Several years ago our group together with colleagues from the University of North Carolina (USA) studied HIV-1 variants isolated from the blood of IDU patients with acute or early infection stage. We found the transmission of single viral variant in 70% of cases. We demonstrated that low genetic diversity of HIV-1 in IDU patients with chronic infection couldn’t be the reason for low genetic diversity of viral population in acute patients. So we suggested that some unknown selecting factor can take place during parenteral HIV transmission [Masharsky et al., 2010]. We are planning to continue the study of parenteral HIV transmission features using the ultra-deep RNA sequencing, and also detect the drug resistance mutations prevalent among recently and chronically infected subjects. 1. Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, Salazar MG, Sun C, Grayson T, Wang S, Li H, Wei X, Jiang C, Kirchherr JL, Gao F, Anderson JA, Ping LH, Swanstrom R, Tomaras GD, Blattner WA, Goepfert PA, Kilby JM, Saag MS, Delwart EL, Busch MP, Cohen MS, Montefiori DC, Haynes BF, Gaschen B, Athreya GS, Lee HY, Wood N, Seoighe C, Perelson AS, Bhattacharya T, Korber BT, Hahn BH, Shaw GM. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A. 2008 May 27;105(21):7552-7

Expected results
We plan to receive data by deep RNA sequencing about the number of genomes transmitted during parenteral HIV infection and study these viral variants more thoroughly. Using the deep sequencing we are planning to study thousands of viral variants for each sample. These results can help to explain the biological mechanisms of high rate of HIV-1 distribution among injection drug users. The following results, that will be used for development of the Russian regional HIV vaccine, will be obtained: 1. The extended collection of the blood samples isolated from patients with acute HIV infection and their contacts. This collection will be supplemented with epidemiological information. 2. The nucleotide sequences of env genes of HIV-1 from acute phase of HIV-1 infection. These sequences will be downloaded to the GenBank. 3. The results of analysis of these sequences that will add to fundamental understanding of the mechanisms of genetic bottleneck. 4. The nucleotide sequences of pol genes of HIV-1 from acute and chronic phase of HIV-1 infection. These sequences will be downloaded to the GenBank. 5. The results of analysis of these sequences that will reveal the prevailing drug resistance mutations in the population. These data will be relevant for the health care system and will help to guide the HAART regimen in the region. We plan to publish at least 8 articles in Russian and international scientific magazines.

Annotation of the results obtained in 2017
1. We finished the previously created pipeline for analyzing the massive parallel sequencing (next-generation sequencing) data obtained from PCR amplification of HIV-1 genomic fragments with the use of PrimerID approach. In particular, pipeline steps for merging forward (R1) and reverse (R2) Illumina reads and detecting hybrid consensus sequences were changed and improved. 2. We benchmarked the created pipeline against synthetic sequencing data and proved its effectiveness in finding rare viral variants in comparison with alternative pipeline. 3. We used processed data of sequencing HIV-1 env gene fragments for testing “the relaxed selection” hypothesis. Our results showed selective strength intensification during acute infection, which supports the presence of “genetic bottleneck”, during HIV transmission. 4. The final version of automated pipeline was used to process the results of PrimerID deep sequencing/ HIV RNA from patients in acute phase of HIV infection was analyzed/ The obtained data confirm the “genetic bottleneck”, during HIV transmission in IDUs. 5. In analyzed sequences mutations corresponding to primary resistance to ART have not been detected.

Publications

1. Akulova E.B., Murashev B.V., Nazarenko O.V., Verevochkin S.V., Masharsky A.E., Krasnoselskikh T.V., Lioznov D.A., Sokolovsky Y.V., Kozlov A.P. Immune Responses Induced by Candidate Optimized HIV DNA Vaccine in Phase I Clinical Trial Madridge Journal of Vaccines, Vol 1, Issue 2, Pp. 31-40 (year - 2017) https://doi.org/10.18689/mjv.2017-107

2. Akulova E.B., Nazarenko O.V., Kazennova E.N., Ketlinsky S.A., Murashev B.V., Verevochkin S.V., Masharsky A.E., Toussova O.V., Kozlov A.P. The study of immune cell populations in injection drug users from St. Petersburg cohort Addiction, - (year - 2017)

3. Dukhovlinova E., Masharsky A., Vasileva A., Porrello A., Zhou S., Verevochkin S., Toussova O., Labranche C., Montefiori D., Frishman D., Cohen M., Miller W., Hoffman I., Kozlov A., Swanstrom R. Characterization of the Transmitted Virus in an Ongoing HIV-1 Epidemic Driven by Injecting Drug Use AIDS Research and Human Retroviruses, - (year - 2017)

4. Karnaukhova Iu.K., Polev D.E., Krukovskaya L.L., Masharsky A.E., Nazarenko O.V., Makashov A.A., Kozlov A.P. A new cancer-testis long noncoding RNA, the OTP-AS1 RNA Nature Communications, - (year - 2017)

5. Löchel HF, Riemenschneider M, Frishman D, Heider D Subtype A Coreceptor Tropism Classification in HIV-1 Bioinformatics, - (year - 2017)

6. Vasileva A.A., Bizin I.V., Talalov O.V., Frishman D.I. Accurate HIV-1 population diversity analysis using deep sequencing with Primer ID approach BMC Bioinformatics, - (year - 2017)

Annotation of the results obtained in 2015
The human immunodeficiency virus (HIV-1) deep sequencing results analysis is working out on HIV-1 marker genes deep sequencing results received using PrimerID approach. At the moment we completed work on following tasks: quality control and preprocessing of raw reads (FastQC tool is used), removing low quality reads (the comparison of FastQC and Trimmomatic results was made; it was decided to use Trimmomatic tool in the following work; Trimmomatic optimal settings were chosen). Currently the methodology of processing data received using PrimerID approach is working out, including: sorting sequences amplified from the single matrix (using algorithm provided by Zhou et al., 2015; this algorithm is improving) and the following consensus sequences creation (using algorithm provided by Zhou et al.). The human immunodeficiency virus (HIV-1) deep sequencing results statistical analysis is working out on amplicons received with SGA approach and following sequencing from patients with acute HIV infection blood samples in 2011. The amplicons are aligned and converted to Nexuss-format (this format is used as an input for many bioinformatics tools including BEAST). A the moment all possible settings of BEAST (program for Bayesian MCMC analysis of molecular sequences) are tested (including: different molecular clock models; using consensus sequences in the working dataset or its absence and etc.). Currently these results have been assessing and comparing. As a result we will get the settings of BEAST program proper for characterization of HIV evolution and the required mutation rate. During the first year we collected 59 new blood plasma samples from more than 20 cases of acute and recent HIV infection, and have developed the pipeline for future analysis of these samples using the collection of acute HIV infection cases obtained in 2011. We adapted the Primer ID approach on Illumina MiSeq for subtype A virus, and currently working on optimization of bioinformatics analysis of the large-scale data. We will extend the analysis of the viral isolates from newly collected samples into several regions of env and pol genes. There was one project-related collaborative visit when the members of the State Polytechnical University visited the University of Munich for the training in bioinformatics.

Publications

Annotation of the results obtained in 2016
In 2016 we collected additional samples of blood plasma from individuals acutely infected with HIV-1 who provided the epidemiological data allowing to discriminate between sexual and parenteral routes of HIV transmission. We will analyze these sample in 2017 for nucleic acid amplification and sequencing (48 samples) From 59 samples that were collected in 2015, 24 samples were found to be suitable for nucleic acid analysis and were used for next generation sequencing. The results are the following: a) We created a software algorithm "DeePrID Analyzer" to analyze the massive parallel sequencing (next-generation sequencing) data obtained from PCR amplification of HIV-1 genomic fragments. The algorithm processes the data starting from initial fastaq file that contains the raw reads from a sequencer machine the final alignment of consensus sequences that correspond to the individual viral particles from the viral pool of a single biological sample. b) We performed comparative statistical analysis for the Ion Torrent and Illumina MiSeq deep sequencing data accounting for the strengths and limitations of both sequencing platforms. There were no significant differences observed in viral diversity among the samples studied with both platforms, however the number of high-quality reads was significantly higher in Illumina. c) The viral populations obtained from the samples of the subjects with later disease stages represent a complex mixture of viral variants with no dominant variants. The viral populations of acutely infected individuals with parenteral HIV transmission were homogeneous confirming the presence of genetic bottleneck, i.e. single variant transmission. d) We obtained pol sequences for acutely infected individuals that will be further analyzed for drug resistance mutations to estimate the rate of pre-existing drug resistance mutations in the risk group.

Publications

1. Kozlov A.P., Skochilov R.V., Toussova O.V., Verevochkin S.V., Krasnoselskikh T.V., Malov S.V., Shaboltas A.V. HIV incidence and behavioral correlates of HIV acquisition in a cohort of injection drug users in St Petersburg, Russia. MEDICINE, Medicine: November 2016 - Volume 95 - Issue 44 - p e5238 doi: 10.1097/MD.0000000000005238 (year - 2016) https://doi.org/10.1097/MD.0000000000005238

2. Toussova O.V., Kozlov A.P., Verevochkin S.V., Lancaster K., Shaboltas A.V., Masharsky A.E., Dukhovlinova E.V., Miller W.C.,d , Hoffman I.F. A Cohort Approach to Real Time Detection of Acute HIV infections among people who inject drugs in St. Petersburg, Russia AIDS Research and Human Retroviruses, - (year - 2016)