Filogenetska analiza i molekularna karakterizacija virusa humane imunodeficijencije u Srbiji

Human immunodeficiency virus (HIV) is a retrovirus, the causative agent of Acquired immunodeficiency syndrome (AIDS). Since the beginning of the epidemic over 35 years ago, more than 78 million people have been infected so far and over 30 million have died. The high genetic variability and rapid evolution of HIV have been critical to its persistence and spread throughout the world. HIV-1 and HIV-2 comprise two distinct types of HIV. HIV-1 has diversified extensively into numerous genetic forms, including four groups (M, N, O, P), of which group M is causing the pandemic of HIV infection and AIDS. Group M viruses are further classified in multiple phylogenetically distinct subtypes (A-D, F, G, H, J and K), sub-subtypes (A1, A2, F1 and F2) and numerous recombinant forms. The global distribution of HIV-1 is complex and dynamic with regional epidemics representing only a subset of the global diversity. Molecular phylogenetic analysis, a method of reconstructing evolutionary relationships between nucleotide sequences, is one of the strategies for studying viral diversity and transmission dynamics. It is estimated that around half of HIV infected people are undiagnosed, making identification of transmission networks important for targeted public health intervention programs...Virus humane imunodeficijencije (HIV) je retrovirus koji uzrokuje sindrom stečene imunodeficijencije. Od početka epidemije pre 35 godina, ovim virusom je inficirano više od 78 miliona ljudi a preko 30 miliona je umrlo. Visoka genetička varijabilnost i brza evolucija HIV-a su ključni uzroci opstanka i globalnog širenjaepidemije. HIV je filogenetski klasifikovan u dva tipa: HIV-1 i HIV-2. Visoki diverzitet HIV-1 ogleda u postojanju četiri grupe (M, N, O, P) od kojih su virusi grupe M uzročnici globalne HIV-1 pandemije. Grupa M virusa je podeljena u više filogenetski različitih podtipova (A-D, F-H, J i K), pod-podtipove (A1, A2, F1 i F2) i cirkulišuće rekombinantne forme. Distribucija podtipova u svetu je složena i dinamična sa regionalnim HIV-1 epidemijama unutar globalnog diverziteta. Molekularna filogenetska analiza, metod za rekonstrukciju evolutivnih odnosa između nukleotidnih sekvenci, je tehnika za proučavanje varijabilnosti virusa i dinamike transmisije unutar regionalnih populacija. Procenjuje se da kod blizu polovine inficiranih osoba HIV infekcija nije dijagnostikovana, zbog čega je identifikacija puteva transmisije izuzetno značajna u cilju javno zdravstvenog nadzora. U ovom istraživanju primenjene su savremene filogenetske metode u analizi HIV-1 sekvenci izolata iz Srbije u cilju karakterizacije molekularne epidemiologije i dinamike transmisije, što je ključno za bolje razumevanje karakteristika aktuelne HIV-1 epidemije u Srbiji..

Variability of the HCV core region and host genetic and epigenetic factors can predict the response to pegylated interferon/ribavirin therapy in genotype 1b hepatitis C patients from Serbia

Variations in the hepatitis C virus (HCV) core sequence have been related to disease progression and response to antiviral therapy. Previously we showed that the methylation status of RASSF1A and p16 genes, and IL28B genotypes affects the response to pegylated interferon/ribavirin (PEG-IFN/RBV) therapy. Herein we investigated whether amino acid (aa) substitutions in the HCV core region alone or in combination with IL28B genotypes and RASSF1A/p16 methylation affect the response to PEG-IFN/RBV therapy and liver disease progression. Among 29 examined patients, we found no association between single aa substitutions and response to therapy. However, we observed that patients with the HCV core aa substitution at position 75 and CT/TT IL28B genotypes were non-responders (NR), (P=0.023). Moreover, these patients had unmethylated RASSF1A. In contrast, most patients (75%) with aa substitutions at position 91 and CC IL28B genotype achieved sustained virologic response (SVR), (P=0.030), and 70% of them had methylated RASSF1A gene. Our results suggest that combined analysis of aa substitutions in the core protein, the IL28B rs12979860 polymorphism, and the methylation status of the RASSF1A gene may help in predicting treatment response to PEG-IFN/RBV in genotype 1b chronic hepatitis C patients

Evolutionary dynamics of Usutu virus: Worldwide dispersal patterns and transmission dynamics in Europe

Background: Usutu virus (USUV) is an emerging mosquito-borne Flavivirus, with birds as the main zoonotic reservoir. Humans are accidental hosts and mostly develop mild or even asymptomatic infections, although severe complications such as encephalitis can also arise. Detailed characterization of the pathogen's phylogenetics may offer valuable insights into the prediction and prevention of potential epidemics; however, lack of uniformity and the number of available USUV sequences worldwide hamper comprehensive investigation. Aim: The study aimed to investigate USUV spatio-temporal dispersal inter- and intracontinentally and to estimate the dynamics of viral spread within Europe. Methods: Phylogeographic and phylodynamic analyses were done using advanced phylogenetic methods implemented in Beast 1.10.4 and Beast 2.6.4 software packages. Results: Herein, we report on a new USUV isolate from Culex pipiens collected in 2019 from Serbia. The results of this research revealed two newly described intercontinental migration events of USUV from Africa to Germany in the 1970s and from Africa to the Middle East (Israel) in the late 90s. Finally, phylodynamic analysis substantiated the ongoing active expansion of USUV in Europe. Conclusion: The data would imply a high potential for further USUV expansion in Europe. Detailed phylogenetic characterization of the pathogen may offer valuable insights into prediction and prevention of potential epidemics; however, lack of uniformity and number of available USUV sequences worldwide hampers comprehensive investigation. This study draws attention to the need for upscaling USUV surveillance

Detection of the Xanthi Chryso-like Virus in New Geographical Area and a Novel Arthropod Carrier

Here, we report on a serendipitous finding of a chryso-like virus associated with Culex pipiens mosquitos in the course of study aimed to detect and characterize West Nile virus (WNV) circulating in mosquitos in Serbia, Southern Europe. Upon initial detection of unexpected product in a PCR protocol for partial WNV NS5 gene amplification, further confirmation and identification was obtained through additional PCR and Sanger sequencing experiments. Bioinformatic and phylogenetic analysis identified the obtained sequences as Xanthi chryso-like virus (XCLV). The finding is particular for the fact that it associates XCLV with a new potential vector species and documents a novel geographical area of its distribution

Exploring Evolutionary and Transmission Dynamics of HIV Epidemic in Serbia: Bridging Socio-Demographic With Phylogenetic Approach

Previous molecular studies of Serbian HIV epidemic identified the dominance of subtype B and presence of clusters related HIV-1 transmission, in particular among men who have sex with men (MSM). In order to get a deeper understanding of the complexities of HIV sub-epidemics in Serbia, epidemic trends, temporal origin and phylodynamic characteristics in general population and subpopulations were analyzed by means of mathematical modeling, phylogenetic analysis and latent class analysis (LCA). Fitting of the logistic curve of trends for a cumulative annual number of new HIV cases in 1984–2016, in general population and MSM transmission group, was performed. Both datasets fitted the logistic growth model, showing the early exponential phase of the growth curve. According to the suggested model, in the year 2030, the number of newly diagnosed HIV cases in Serbia will continue to grow, in particular in the MSM transmission group. Further, a detailed phylogenetic analysis was performed on 385 sequences from the period 1997–2015. Identification of transmission clusters, estimation of population growth (Ne), of the effective reproductive number (Re) and time of the most recent common ancestor (tMRCA) were estimated employing Bayesian and maximum likelihood methods. A substantial proportion of 53% of subtype B sequences was found within transmission clusters/network. Phylodynamic analysis revealed Re over one during the whole period investigated, with the steepest slopes and a recent tMRCA for MSM transmission group subtype B clades, in line with a growing trend in the number of transmissions in years approaching the end of the study period. Contrary, heterosexual clades in both studied subtypes – B and C – showed modest growth and stagnation. LCA analysis identified five latent classes, with transmission clusters dominantly present in 2/5 classes, linked to MSM transmission living in the capital city and with the high prevalence of co-infection with HBV and/or other STIs.Presented findings imply that HIV epidemic in Serbia is still in the exponential growth phase, in particular, related to the MSM transmission, with estimated steep growth curve until 2030. The obtained results imply that an average new HIV patient in Serbia is a young man with concomitant sexually transmitted infection

Failure to detect viral RNA in bat samples collected in the Balkan region

Bats represent a known reservoir of emerging viruses, yet no molecular data are found about the occurrence of zoonotic viruses in bats in the Balkans. The aim of this study was to determine the presence of paramyxo- and hanta-viruses in bats, examined by PCR in 95 deceased bats, that were collected in Serbia and Montenegro, during the period 2002 to 2009. All samples tested positive for beta-actin mRNA, confirming successful RNA isolation and amplification. However, no sample tested positive for virus specific RNA. Our findings might reflect tissue degradation in carcass samples and do not exclude bats as potential viral reservoir in the surveyed geographic area.Tropical Biomedicine (2016), 33(4): 780-78

HIV-1 resistance profile in plasma and peripheral blood lymphocytes in a group of naive patients

Transmitted HIV-1 drug resistance (TDR) is a persisting problem, even though the prevalence of primary resistance may remain stable or start to decline. Proviral DNA detectable in peripheral blood mononuclear cells (PBMCs) is a reservoir of drug resistant viral variants and could be an alternative marker to viral RNA for the detection of drug resistance mutations. The aim of this study was to compare the HIV-1 resistance profile between plasma viral RNA and proviral DNA in a group of untreated patients. Thirty-one HIV-1 seropositive patients without prior ARV treatment were included in the study. The presence of non-polymorphic drug resistance mutations was identified in 10 cases in proviral DNA and in 11 cases in plasma according to different scoring systems. Our results show a similar resistance profile between plasma RNA and proviral DNA, but with some discordances present. The sequencing of proviral DNA could provide useful additional information with regard to primary resistance. [Projekat Ministarstva nauke Republike Srbije, br. 175024

Sequence variability of HCV core region and host genetic and epigenetic factors can predict the response to combined PEG-IFN/RBV therapy in patients with chronic hepatitis c infection genotype 1B

Variations in the hepatitis C virus (HCV) core gene are related to the progression of liver fibrosis and therapy response. However, the influence of individual amino acid (aa) substitutions at different positions of HCV Core protein on the response to combined pegylated interferon/ribavirin (PEG-IFN/RBV) therapy and disease progression is not yet fully understood. The HCV Core protein can inactivate various genes in the host genome by affecting the methylation of their promoters, leading to liver damage and carcinogenesis. Two genes whose methylation status is affected by the HCV Core protein are the tumor suppressor genes RASSF1A and p16. We have previously shown that the methylation status of these two genes, together with the single nucleotide polymorphism (SNP) rs12979860 near the interleukin-28 beta subunit (IL-28B) gene, influences the response to combined therapy. Herein, we investigated a possible association between detected aa substitutions in HCV Core protein and response to combined therapy, liver disease progression, IL28B genotype, and the methylation status of the RASSF1A and p16 genes. In 29 examined patients we found no association between individual aa substitutions and therapy response. However, we observed that patients with HCV Core aa substitutions at position 75 and CT/TT IL28B genotypes were non-responders (NR) (p=0.023), which was associated with the presence of unmethylated RASSF1A gene. In contrast, even 75% of patients with aa substitutions at position 91 and CC IL28B genotype achieved a sustained virologic response (SVR) (p=0.030), and 70% of them had methylated RASSF1A gene. There was no significant association between the methylation status of the p16 gene and aa variations in the HCV core region. Our results suggest that combined analysis of aa substitutions in HCV Core protein, IL28B genotype, and methylation status of the RASSF1A gene may help predict response to PEG-IFN/RBV therapy in patients with chronic hepatitis C genotype 1b.xx2nd B&H Symposium of Laboratory Geneticists and Molecular Biologists (with International Participation) : Book of abstracts : 10-11 May, 2024