DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

THE IMPORTANCE OF ANTI-HCV SCREENING IN PATIENTS OF LARGE MULTIFIELD HOSPITALS

In the studied cohort, HCV subtypes 1b and 3a prevailed — 47,7% and 38,9%, respectively HCV genotype 2 was detected in 7,2% of cases and HCV subtype 1a — in 5,8% of cases. HCV genotype was not determined in 0,4% of patients. Difference between a frequency of detection of subtypes 1b and 3a of HCV in men and women were recorded. Subtype 3a HCV was determined in men in 44,8% of cases, and in women — in 31,7% of cases (p < 0.01). HCV subtype 1b in women was determined with a higher frequency (55,0%, р < 0, 01), than in men (41,8%).

DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses.