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.

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.

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.

Anxiety is one of the most frequent psychiatric disorders. Despite the fact that most studies describe an anxiolytic effect of testosterone, hyperandrogenemia in mothers is assumed to be related to an increased risk of mood disorders in their offspring. An increasing body of scientific evidence suggests that an altered expression of interneuronal markers of the hippocampus may be the cause of anxiety. The aim of this study was to examine the influence of maternal hyperandrogenemia on behavioral parameters of anxiety-like behavior, neuropeptide Y (NPY) and parvalbumin (PV) expression in the hippocampus, and the level of the brain-derived neurotrophic factor (BDNF) in the hippocampus and cerebral cortex. Pregnant female Wistar albino rats were treated with testosterone undecanoate on the 20th day of gestation. Anxiety-like behavior in adult female offspring was evaluated by the elevated plus maze test and the open field. The number of PV and NPY immunoreactive cells in the hippocampus was determined immunohistochemically. The level of BDNF expression in the hippocampus and cerebral cortex was analyzed with the Western blot test. Prenatal hyperandrogenization increased anxiety-like behavior in female offspring and decreased expression of NPY+ and PV+ in the CA1 region of the hippocampus as compared to the control group. BDNF expression in the hippocampus and cerebral cortex of prenatally androgenized female offspring was significantly increased in comparison with the controls. Prenatal hyperandrogenization may be the cause of anxiety-like behavior in female offspring. Decrease in NPY and PV expression in the hippocampus may explain the possible mechanism of hyperandrogenization induced anxiety.

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.

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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.

Orthohantaviruses are zoonotic pathogens of humans, unique among the bunyaviruses in not being transmitted by an arthropod vector. Tula orthohantavirus (TULV) is an old-world hantavirus, of yet unclear human pathogenicity, with few reported cases of clinically relevant human infection. So far, phylogeographic studies exploring global pathways of hantaviral migration are scarce and generally do not focus on a specific hantavirus species. The aim of the present study was to reconstruct the dispersal history of TULV lineages across Eurasia, based on S segment sequences sampled from different geographic areas. Maximum likelihood and Bayesian inference methods were used to perform the phylogenetic analysis and phylogeographic reconstructions. Sampling time and trapping localities were obtained for the total of 735 TULV S segment sequences available in public databases at the time of the study. The estimated substitution rate of the analyzed partial S segment alignment was 2.26 x 10-3 substitution/site/year (95% HPD interval: 1.79 x 10-3 – 2.75 x 10-3). Continuous phylogeography of TULV S segment sequences placed the potential root and origin of TULV spread in the Black Sea region. In our study, we detect a single lineage introduction of TULV to Europe, followed by local viral circulation further on.