HIV-1 CRF63_02A6 models as a tool for evaluating efficacy of developing antiretroviral drugs

Highly active antiretroviral therapy (HAART) allows not only to control the infection process in certain patient, but also to reduce a risk of HIV infection spreading in general, so that one of the goals for international community fighting against HIV-spread is to maximize coverage of infected subjects with HAART. Antiretroviral therapy in HIV infection is administered lifelong, so that therapeutic efficacy may be lowered due to emergence of resistant HIV-1 variants. Currently, development of new antiretroviral drugs is currently underway throughout the world, therefore standard HIV-1 models are demanded to evaluate antiviral efficacy of promising drugs. To reliably assess drug efficiency regarding Russiawide HIV-1 variants, HIV-1 genovariants widespread in Russia should be used as a virus model. A recently emerged recombinant form of CRF63_02A6 HIV-1 is spread in Russia being currently a dominant variant detected among HIV-infected individuals in an extended region of the Siberian Federal District: in the Novosibirsk, Tomsk, Omsk, Kemerovo Regions, Krasnoyarsk and Altai Krai. We have obtained CRF63_02A6 infectious isolates of HIV-1, one of which contains mutations, reducing the sensitivity to the applied inhibitors of the virus reverse transcriptase. In addition, we constructed infectious molecular clones based on HIV-1 CRF63_02A6 variants with an affinity for CCR5 coreceptors and CXCR4. Infectious isolates and molecular clones CRF63_02A6 tested as models for assessing efficacy of antiretroviral drugs using the example of the drug “Efavirenz”. The fifty percent inhibitory concentration determined on the models of HIV-1 infectious molecular clones and HIV-1 isolate 18RU7056 ranged from 0.00027 pg/ml to 0.00046 pg/ml being in agreement with data published elsewhere. Concentrations of “Efavirenz” used in the study did not suppress the replication of HIV-1 12RU6987, which is resistant to non-nucleoside reverse transcriptase inhibitors, which confirms the decrease in the sensitivity of HIV-1 12RU6987 to “Efavirenz” by no less than 10,000 times. Thus, our data demonstrate that CRF63_02A6 HIV-1 isolated strains and infectious molecular clones are relevant and complementary tools for assessing efficacy of developing drugs aimed at suppressing HIV-1, including non-nucleoside-resistant virus reverse transcriptase inhibitors

Клинико-лабораторная характеристика острой ВИЧ-инфекции у взрослых в Новосибирской области

Purpose of the study. To study the characteristics of acute HIV infection in adults in the Novosibirsk region in 2017– 2018.Materials and methods. 200 patients with acute HIV infection, residents of the Novosibirsk region, aged 15 to 74 years, hospitalized in 2017–2018, 104 men and 96 women were examined. The diagnosis was verified by detecting antibodies to HIV in ELISA with a negative or doubtful result of the immunoblot, as well as by determining the quantitative content of HIV RNA in the blood by PCR. The hemogram, the content of CD4 + lymphocytes in the blood were determined. In addition, HIV-1 was studied in 71 patients for belonging to a genetic group.Results. Patients of 20–40 years old prevailed – 70.5%. The sexual way was dominant in women (69.0%), injecting psychoactive substances was more often in men (32.2%). Frequent symptoms were fever (99%), exanthema (65.5%), polylimfoadenopathy (54.0%). Less common were diarrhea (36.0%), pharyngotonsillitis (23.0%), aphthous stomatitis (6.5%), hepatomegaly or hepatosplenomegaly (8.5%). Secondary diseases were revealed in 26.5% of patients. Leukopenia was recorded in 63.0%, thrombocytopenia in 62.5%, lymphocytosis and atypical cells of the lymphoid series in 14.0%. All patients had a high “viral load” – from 23,000 to 10,000,000 or more copies / ml, a decrease in the number of CD4 + less than 350 cells – in 38.0%. From among 71 patients, 19.7% had HIV-1 subtype A, and 76.0% had recombinant HIV-1 CRF63_02A. In the latter, “viral load” of more than 10,000,000 copies / ml was more often recorded (67.8% vs. 33.4%, p = 0.004).Conclusion. A significant number of cases of acute HIV infection were reported in the Novosibirsk region in 2017– 2018. Its detection is possible by examination of all febrile patients who apply to medical institutions. Early initiation of antiretroviral therapy with the appointment of well-tolerated regimens contributes to the formation of a high commitment to therapy.Цель: изучить особенности острой ВИЧ-инфекции у взрослых в Новосибирской области в 2017–2018 гг.Материалы и методы. Обследованы 200 больных острой ВИЧ-инфекцией, жителей Новосибирской области, в возрасте от 15 до 74 лет, госпитализированных в 2017–2018 гг., 104 мужчин и 96 женщин. Диагноз верифицировали выявлением антител к ВИЧ в ИФА при отрицательном или сомнительном результате иммуноблота, а также определением количественного содержания РНК ВИЧ в крови методом ПЦР. Определялась гемограмма, содержание CD4+лимфоцитов в крови. Дополнительно у 71 пациента исследован ВИЧ-1 на принадлежность к генетической группе.Результаты. Преобладали лица 20–40 лет – 70,5%. Половой путь заражения доминировал у женщин (69,0%), инъекционное потребление психоактивных веществ чаще было у мужчин (32,2%). Частыми симптомами были лихорадка (99%), экзантема (65,5%), полилимфоаденопатия (54,0%). Реже регистрировались диарея (36,0%) фаринготонзиллит (23,0%), афтозный стоматит (6,5%), гепатомегалия или гепатоспленомегалия (8,5%). У 26,5% больных выявлены вторичные заболевания. Лейкопения регистрировалась у 63,0%, тромбоцитопении – у 62,5%, лимфоцитоз и атипичные клетки лимфоидного ряда – у 14,0%. У всех пациентов установлена высокая вирусная нагрузка – от 23 000 до 10 000 000 и более копий/мл, снижение числа CD4+ менее 350 клеток – у 38,0%. Из числа 71 больных у 19,7% выявлен ВИЧ-1 субтипа А, у 76,0% выявлен рекомбинантный CRF63_02А ВИЧ-1. У последних чаще регистрировалась вирусная нагрузка более 10 000 000 копий/мл (67,8% против 33,4%, p=0,004).Заключение. В Новосибирской области в 2017– 2018 гг. регистрируется значительное число случаев острой ВИЧ-инфекции. Ее выявление возможно при обследовании всех лихорадящих больных, обращающихся в лечебные учреждения. Необходимо раннее начало антиретровирусной терапии с назначение схем с хорошей переносимостью для формирования высокой приверженности к терапии

Выявление Вич-1, резистентных к антиретровирусным препаратам, среди жителей томской области с впервые диагностированной Вич-инфекцией

The purpose of this paper is to evaluate the spreading of HIV-1 resistant to antiretroviral drugs among Tomsk Oblast population with newly diagnosed HIV-infection.Materials and methods. It was collected 122 clinical samples of  peripheral blood of HIV-infected patients from Tomsk Oblast who did not take antiretroviral drugs. In HIV-1 isolated from clinical samples we studied nucleotide sequence of genome fragments encoding virus protease and reverse transcriptase. Complex analyses of epidemiologic data from patients and the presence in genome HIV-1 mutations associated with resistance development to protease inhibitors and virus reverse transcriptase were carried out.Results. Analysis of HIV-1 isolated from Tomsk Oblast naïve HIV-infected population made it possible to detect HIV-1 mutations associated with a decrease of virus sensitivity to antiretroviral drugs in 9,8% of cases. Among described mutations 50% were associated with resistance to virus protease inhibitors; 33,3% were resistance mutations to nonnuclease inhibitors of reverse transcriptase, and 16.7% were resistance mutations to nucleoside inhibitors of virus reverse transcriptase. Out of 9,8% of resistant viruses 7,3% of casesincluded mutations associated with the development of potentially low level of reduction of HIV-1 sensitivity to drugs. Main HIV-1 resistance mutations of high and average levels were registered only in 2,5% of genotyped HIV-1 isolated from people who inject drugs.Conclusion. Current study detected considerably low sampling rate of HIV-1 carrying mutations associated with resistance to antiretroviral drugs among Tomsk Oblast naïve HIV-infected population. It is believed to be caused by a relatively short period of extensive application of antiretroviral therapy in that territory. Analysis of epidemiologic data resulted in detection of factors negatively affecting prediction of further development of HIV-infection epidemic in the region including prevalence of risk behavior practice contributing to resistant HIV-1 transmission both among patients via heterosexual contacts and among people who inject drugs.Цель: оценка распространения ВИЧ-1, резистентных к антиретровирусным препаратам, среди жителей Томской области с впервые выявленной ВИЧ-инфекцией.Материалы и методы. Собрано 122 клинических образца периферической крови ВИЧ-инфицированных жителей Томской области, не принимавших антиретровирусные препараты. Для выделенных из клинических образцов ВИЧ-1 исследована нуклеотидная последовательность фрагментов генома, кодирующих протеазу и обратную транскриптазу вируса. Выполнен комплексный анализ эпидемиологических данных пациентов и наличия в геноме ВИЧ-1 мутаций, связанных с развитием резистентности к ингибиторам протеазы и обратной транскриптазы вируса.Результаты. Анализ ВИЧ-1, выделенных от наивных ВИЧ-инфицированных лиц Томской области, в 9,8% случаев выявил мутации ВИЧ-1, ассоциированные со снижением чувствительности вируса к антиретровирусным препаратам. Среди описанных мутаций 50%составляли мутации, связанные с резистентностью к ингибиторам протеазы вируса; в 33,3% – мутации резистентности к ненуклеозидным ингибиторам обратной транскриптазы и в 16,7% – к нуклеозидным ингибиторам обратной транскриптазы вируса. Из 9,8% резистентных вирусов в 7,3% случаев были выявлены мутации, ассоциированные с развитием потенциального/потенциально-низкого/низкого уровня снижения чувствительности ВИЧ-1 к препаратам. Основные мутации резистентности ВИЧ-1 высокого и среднего уровня были зарегистрированы лишь в 2,5% генотипированных ВИЧ-1, выделенных от лиц, потребляющих инъекционные наркотические препараты.Заключение. Выполненное исследование выявило среди наивных ВИЧ-инфицированных жителей Томской области достаточно низкую частоту регистрации ВИЧ-1, несущих мутации, ассоциированные с резистентностью к антиретровирусным препаратам. Вероятно это связано с относительно коротким периодом широкого применения в области  антиретровирусной терапии. Анализ эпидемиологических данных выявил факторы, негативно влияющие на прогноз дальнейшего развития эпидемии ВИЧ-инфекции в регионе – широкую распространенность практик рискованного поведения, способствующих передаче резистентных ВИЧ-1 как среди лиц, инфицированных при гетеросексуальных контактах, так и среди потребителей инъекционных наркотиков

Detection of HIV-1 resistant to antiretroviral drugs among tomsk oblast population with newly diagnosed HIV-infection

The purpose of this paper is to evaluate the spreading of HIV-1 resistant to antiretroviral drugs among Tomsk Oblast population with newly diagnosed HIV-infection.Materials and methods. It was collected 122 clinical samples of  peripheral blood of HIV-infected patients from Tomsk Oblast who did not take antiretroviral drugs. In HIV-1 isolated from clinical samples we studied nucleotide sequence of genome fragments encoding virus protease and reverse transcriptase. Complex analyses of epidemiologic data from patients and the presence in genome HIV-1 mutations associated with resistance development to protease inhibitors and virus reverse transcriptase were carried out.Results. Analysis of HIV-1 isolated from Tomsk Oblast naïve HIV-infected population made it possible to detect HIV-1 mutations associated with a decrease of virus sensitivity to antiretroviral drugs in 9,8% of cases. Among described mutations 50% were associated with resistance to virus protease inhibitors; 33,3% were resistance mutations to nonnuclease inhibitors of reverse transcriptase, and 16.7% were resistance mutations to nucleoside inhibitors of virus reverse transcriptase. Out of 9,8% of resistant viruses 7,3% of casesincluded mutations associated with the development of potentially low level of reduction of HIV-1 sensitivity to drugs. Main HIV-1 resistance mutations of high and average levels were registered only in 2,5% of genotyped HIV-1 isolated from people who inject drugs.Conclusion. Current study detected considerably low sampling rate of HIV-1 carrying mutations associated with resistance to antiretroviral drugs among Tomsk Oblast naïve HIV-infected population. It is believed to be caused by a relatively short period of extensive application of antiretroviral therapy in that territory. Analysis of epidemiologic data resulted in detection of factors negatively affecting prediction of further development of HIV-infection epidemic in the region including prevalence of risk behavior practice contributing to resistant HIV-1 transmission both among patients via heterosexual contacts and among people who inject drugs

Clinical and laboratory characterictic of acute HIV-infection in adult residents of Novosibirsk region

Purpose of the study. To study the characteristics of acute HIV infection in adults in the Novosibirsk region in 2017– 2018.Materials and methods. 200 patients with acute HIV infection, residents of the Novosibirsk region, aged 15 to 74 years, hospitalized in 2017–2018, 104 men and 96 women were examined. The diagnosis was verified by detecting antibodies to HIV in ELISA with a negative or doubtful result of the immunoblot, as well as by determining the quantitative content of HIV RNA in the blood by PCR. The hemogram, the content of CD4 + lymphocytes in the blood were determined. In addition, HIV-1 was studied in 71 patients for belonging to a genetic group.Results. Patients of 20–40 years old prevailed – 70.5%. The sexual way was dominant in women (69.0%), injecting psychoactive substances was more often in men (32.2%). Frequent symptoms were fever (99%), exanthema (65.5%), polylimfoadenopathy (54.0%). Less common were diarrhea (36.0%), pharyngotonsillitis (23.0%), aphthous stomatitis (6.5%), hepatomegaly or hepatosplenomegaly (8.5%). Secondary diseases were revealed in 26.5% of patients. Leukopenia was recorded in 63.0%, thrombocytopenia in 62.5%, lymphocytosis and atypical cells of the lymphoid series in 14.0%. All patients had a high “viral load” – from 23,000 to 10,000,000 or more copies / ml, a decrease in the number of CD4 + less than 350 cells – in 38.0%. From among 71 patients, 19.7% had HIV-1 subtype A, and 76.0% had recombinant HIV-1 CRF63_02A. In the latter, “viral load” of more than 10,000,000 copies / ml was more often recorded (67.8% vs. 33.4%, p = 0.004).Conclusion. A significant number of cases of acute HIV infection were reported in the Novosibirsk region in 2017– 2018. Its detection is possible by examination of all febrile patients who apply to medical institutions. Early initiation of antiretroviral therapy with the appointment of well-tolerated regimens contributes to the formation of a high commitment to therapy

СОЗДАНИЕ КОЛЛЕКЦИИ СОВРЕМЕННЫХ ИЗОЛЯТОВ ВИЧ-1, ВКЛЮЧАЮЩЕЙ ОСНОВНЫЕ РОССИЙСКИЕ ГЕНЕТИЧЕСКИЕ ВАРИАНТЫ ВИРУСА

Study objectives: Fine-tuning  of techniques for HIV-1 isolation from HIV patient blood samples; developing a collection of primary HIV-1 isolates; and investigating the biological features of currently circulating genetic variants of HIV-1 subtypes A and B and of their recombinant form CRF63_02A1. These objectives are important for better knowing of HIV-1 biology and for monitoring the characteristics of the spreading HIV-1 variants. Materials and methods: PBMC were isolated by whole blood centrifuging at 325 g for 30 min in 1,077 to 1,080 g/mL  Ficoll gradient (MP Biomedicals, USA) followed by two washings with RPMI-1640. Donor PBMC were stimulated in RPMI-1640. The culture fluid was sampled, tested for viral p24 protein, and cryopreserved. HIV-1 reproduction was assessed by the accumulation of the viral protein in culture medium using an ELISA kit provided by Vector Best (Russia).  Viral DNA was isolated from HIV patient  blood plasma using RealBest DeltaMag  kits and RT-PCR MasterMix kits (Vector Brest, Russia) and PCR BioMaster HS-Tag PCI-Color kits (Bilabmix, Russia). HIV-1 isolated were characterized genetically by sequencing env and pol genes using BigDyeTerminatorv31 kits (USA). Sixty-seven infectious variants of HIV-1 subtype A and B recombinants  CRF63_02A1 and CRF02AG strain have been isolated and characterized.Целью работы являлась отработка методик выделения изолятов ВИЧ-1 из клинического материала периферической крови ВИЧ-инфицированных пациентов, создание коллекции первичных изолятов ВИЧ-1,  изучение биологических особенностей современных генетических вариантов ВИЧ-1  субтипов А, В и циркулирующей рекомбинантной формы CRF63_02A1. Выделение изолятов ВИЧ-1 и изучение их биологических свойств крайне важны для более глубокого понимания биологии вируса и мониторинга характеристик распространяющихся вариантов ВИЧ-1. Материалы и методы. Выделение МПК проводили центрифугированием цельной крови (325 g, 30 мин) на градиенте Ficoll 1,077–1,080 г/мл (MPBiomedicals, USA) с последующей двукратной отмывкой средой RPMI-1640. Стимуляцию донорских МПК проводили в среде RPMI-1640. Отобранную культуральную жидкость анализировали на содержание вирусного белка р24 и криоконсервировали. Репродукцию вируса определяли по накоплению вирусспецифического белка p24 в культуральной жидкости, концентрацию которого выявляли с использованием тест-системы «ВИЧ-1 р24-антиген-ИФА-БЕСТ». Вирусную РНК выделяли из плазмы крови ВИЧ-инфицированных пациентов. Для выделения РНК использовали наборы «РеалБест ДельтаМаг ВГВ/ВГС/ВИЧ», «Вектор-Бест», Россия. «ОТ-ПЦР» — «РеалБест  Мастер микс ОТ», «Вектор-Бест», Россия, «ПЦР»  — «БиоМастер HS-Taq ПЦР-Color (2×)»,  «Билабмикс», Россия. Генетическую характеризацию выделенных изолятов осуществляли методом определения нуклеотидных последовательностей фрагментов генома ВИЧ-1, кодирующих области гена pol и основного белка оболочки (ген env). Для определения и анализа нуклеотидной последовательности гена pol использовали наборы BigDyeTerminatorv31, USA. Выделено и описано 67 инфекционных вариантов ВИЧ-1: CRF63_02А1, субтипа А, ВиCRF02_АG.</p

Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction

Human monkeypox and smallpox viruses: genomic comparison

AbstractMonkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR

Patterns of HIV-1 drug resistance among HIV-infected patients receiving first-line antiretroviral therapy in Novosibirsk Region, Russia

ABSTRACT: Objectives: Antiretroviral (ARV) drugs have played a vital role in controlling the HIV-1 epidemic; however, some challenges remain. ARV drugs vary in their ability to control HIV infection, displaying differences in treatment-limiting factors and genetic barriers to resistance. The current report assesses the prevalence of HIV-1 drug resistance mutations (DRMs) among patients who failed first-line antiretroviral therapy (ART) and evaluates the genetic barrier of different regimens. Methods: The study cohort (n = 271) included HIV-infected individuals who visited the Novosibirsk, Russia, HIV/AIDS clinic in 2018–2022. All patients received first-line ART prior to virological failure. Sociodemographic and HIV-related data were collected from medical records and self-reported questionnaires. HIV-1 pol gene sequences were generated, and the presence of HIV-1 DRM was assessed. The genetic barrier to resistance was assessed by combining treatment regimen and adherence data. Results: Nonoptimal ART adherence was identified in 48.3% of patients and correlated with male sex, PWID, unemployment, and rural area residence. Most of the patients with high-level adherence were identified among those who were on TDF+3TC+DTG. HIV-1 DRMs were identified in 54.6% of the patients. The analysis of HIV-1 DRM, ART regimen, and adherence data classified TDF+3TC+DTG and TDF+3TC+LPV/r as treatment regimens with a high genetic barrier, whereas EFV-containing ART was classified as a regimen with a low genetic barrier. Conclusions: The current study delivers results on the efficacy of HIV-1 ART and treatment adherence in real-world practice settings. This report suggests that ART regimens with a high genetic barrier to resistance combined with improved treatment adherence may reduce the transmission of HIV-1 resistant variants