Hematophagous arthropods are important vectors for zoonotic pathogens. To date, a huge number of viruses have been identified in these arthropods, with a considerable proportion of them being human pathogens. However, the viromes of hematophagous arthropods are still largely unresearched. In this study, a number of arthropods were collected from Belgrade, Serbia including mosquitoes, ticks and bedbugs. The viromes of these arthropods were identified and characterized using Illumina MiSeq sequencing. In total, 21 viruses belonging to 11 families were characterized, with 11 of them representing novel species. These results may contribute to our knowledge of RNA viruses in arthropods and the discovery of novel human pathogens.

Rickettsiales bacteria in arthropods play a significant role in both public health and arthropod ecology. However, the extensive genetic diversity of Rickettsiales endosymbionts of arthropods is still to be discovered. In 2016, 515 arthropods belonging to 9 species of four classes (Insecta, Chilopoda, Diplopoda and Arachnida) were collected in Serbia. The presence and genetic diversity of Rickettsiales bacteria were evaluated by characterizing the 16S rRNA (rrs), citrate synthase (gltA) and heat shock protein (groEL) genes. The presence of various Rickettsiales bacteria was identified in the majority of tested arthropod species. The results revealed co-circulation of five recognized Rickettsiales species including Rickettsia, Ehrlichia and Wolbachia, as well as four tentative novel species, including one tentative novel genus named Neowolbachia. These results suggest the remarkable genetic diversity of Rickettsiales bacteria in certain arthropod species in this region. Furthermore, the high prevalence of spotted fever group Rickettsia in Ixodes ricinus ticks highlights the potential public health risk of human Rickettsia infection.

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.

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.

The aim of this prospective cohort study was to determine the prevalence of gut colonization with multidrug-resistant (MDR) bacteria, risk factors for colonization, infection risk, and outcomes among preterm neonates hospitalized at a tertiary-care center in Serbia. During the period from December 2017 to April 2018, 103 neonates were screened for rectal carriage at admission and on the seventh day of life. Characterization of MDR strains was done by conventional microbiology and molecular methods. Out of 61 (59.2%) colonized neonates, 12 (11.6%) were found colonized at admission, while 49 (47.6%) became colonized at the study site. Among a total of 72 MDR isolates, extended-spectrum beta-lactamase (ESBL)-producing enterobacteria prevailed (56/72, 77%), followed by Acinetobacter baumannii (14/72, 19%). The majority of ESBL-producing strains carried multiple genes (blaTEM/blaCTX-M-15 or blaTEM/blaSHV). Longer previous hospitalization and delivery by cesarean section were associated with MDR colonization, while mechanical ventilation was a risk factor for colonization at the study site. Infections due to MDR bacteria were more frequent among colonized than non-colonized neonates, but not significantly, and mortality was low (1%) in the studied neonates. These results indicate that hospitalized preterm neonates in Serbia are rapidly colonized with a diversity of MDR species and resistance phenotypes/genotypes.

The aim of this study was to estimate substitution rate and imprints of natural selection on parvovirus B19 genotype 1. Studied datasets included 137 near complete coding B19 genomes (positions 665 to 4851) for phylogenetic and substitution rate analysis and 146 and 214 partial genomes for selection analyses in open reading frames ORF1 and ORF2, respectively, collected 1973–2012 and including 9 newly sequenced isolates from Serbia. Phylogenetic clustering assigned majority of studied isolates to G1A. Nucleotide substitution rate for total coding DNA was 1.03 (0.6–1.27) x 10⁻⁴ substitutions/site/year, with higher values for analyzed genome partitions. In spite of the highest evolutionary rate, VP2 codons were found to be under purifying selection with rare episodic positive selection, whereas codons under diversifying selection were found in the unique part of VP1, known to contain B19 immune epitopes important in persistent infection. Analyses of overlapping gene regions identified nucleotide positions under opposite selective pressure in different ORFs, suggesting complex evolutionary mechanisms of nucleotide changes in B19 viral genomes.