Genome sequence diversity of SARS-CoV-2 in Serbia: insights gained from a 3-year pandemic study

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19-positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19- positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide

Genetic Diversity Analysis of Mitochondrial <i>Cytb</i> Gene, Phylogeny and Phylogeography of Protected Griffon Vulture (<i>Gyps fulvus</i>) from Serbia

Once a widespread and common species across the region of southeast Europe, the Griffon vulture is now confined to small and isolated populations across the Balkan Peninsula. The population from Serbia with 290 couples represents its biggest and most viable population that can serve as an important reservoir of genetic diversity from which the birds can be used for the region’s reintroduction or recolonization programs. To estimate the level of genetic diversity, the mitochondrial Cytb gene from 58 unrelated birds sampled during the marking in the nests was sequenced and compared to the homologous Griffon vulture sequences available in publicly accessible online databases. Phylogeographic analysis based on Cytb sequences showed that the most frequent haplotype is found in all Griffon vulture populations and that each population possesses private haplotypes. Our data suggest that the Griffon vulture population from Serbia should be used as a source population for restocking and reintroduction programs in the region. The observed genetic differentiation between the populations from the Iberian and Balkan Peninsulas suggest that the introduction of foreign birds from remote populations should be avoided and that birds from indigenous or neighboring populations, if available, should be used instead

Identification of a broad spectrum of mammalian and avian species using the short fragment of the mitochondrially encoded cytochrome b gene.

Mitochondrial DNA (mtDNA), especially the gene for cytochrome b (MT-CYB), has been found to be highly informative for species identification. In this study, we present the results of the analysis of a 127 bp long fragment of MT-CYB, amplified using universal primers, variable enough to be used for species identification and discrimination, even in highly degraded animal samples. The total number of analyzed species in this study was 30, including 17 mammalian and 13 bird species. Using a newly created primer pair, we successfully amplified and sequenced the target sequence in almost all tested species. The amplification was incomplete in just two species, and as a result, partial, but still variable sequences, were obtained. Using the target fragment we successfully identified all tested samples. Initial results suggested that the intraspecies genetic diversity of the target region, in all tested species, was low - from 0 to 4.72%. The interspecies genetic diversity of the target region, crucial for successful discrimination, showed relatively high diversity, ranging from 8.36% to 42.52%. Given its short length, the target region should be used for species determination, particularly in samples that are degraded or are low in DNA quantity

Further Evidence of Population Admixture in the Serbian Honey Bee Population

Socioeconomic interests and beekeeper preferences have often taken precedence over the conservation of locally native honey bee subspecies, leading to the predominance of admixture populations in human-dominated areas. To assess the genetic diversity of contemporary managed Serbian honey bee colonies, we used 14 microsatellite loci and analyzed 237 worker bees from 46 apiaries in eight localities of northern and southern Serbia. Furthermore, we compared data for nine microsatellite loci with 338 individuals from Italy, Hungary, Poland, and Spain. The standard parameters of genetic diversity in Serbian honey bee populations were in line with other analyses, although somewhat smaller. STRUCTURE analysis showed the existence of two equally distributed genetic clusters and Analysis of molecular variances could not confirm the presence of a geographically discrete population but showed local differences. Discriminant analysis of principal components showed overlapping of worker bees from different parts of Serbia. Clear genetic differentiation can be observed when comparing all populations between geographical regions and their corresponding subspecies. The absence of the A. m. macedonica subspecies from its historical distribution range in southern Serbia as well as the lack of distinctive geographical groups suggest that selective breeding, queen import, and migratory beekeeping practices strongly influence the genetic structure and diversity of honey bees, leading to the genetic uniformization and creation of the admixture population