Tipizacija, filogenetska analiza i molekularna karakterizacija virusa influence A svinja na teritoriji Republike Srbije

Swine influenza is a respiratory infectious disease of pigs caused by influenza A viruses. Given not only their global distribution in pig population and the negative economic impact on pig production but also their zoonotic potential, these viruses are a significant problem in veterinary, and, occasionally, in human medicine. In order to additionally emphasize the need of controlling and preventing the infection outbreaks on pig farms and, more significantly to determinate the pandemic potential of circulating viruses, within this doctoral dissertation we have examined the presence of the virus in sick and dead pigs from 13 commercial farms, and then typed, phylogenetically analyzed, and characterized a certain number of detected viruses. Using molecular diagnostic methods, it was analyzed 255 samples of nasal, tracheobronchial swabs, and lung tissue of pigs. The genome of the influenza A virus was detected in 24 samples, which originated from pigs from 8 farms. The pandemic origin of the hemagglutinin gene of three viruses was confirmed using molecular methods, as well. Additionally, 24 positive samples were tested by virus isolation on 9 to 11 days old chicken embryos and on the MDCK cell line. One virus was successfully isolated in both biological systems (A/swine/Serbia/2/2017). By using Next-Generation Sequencing the complete genome of seven viruses was successfully sequenced. Sequences of PB2, PB1, PA, and nucleoprotein gene of the remaining two viruses were partially read and therefore have not been included in phylogenetic and molecular analysis. Based on the hemagglutinin and neuraminidase gene sequences, seven viruses were classified to the H1N1 subtype and two to the H3N2 subtype. Three of the H1 subtype viruses in the database of porcine H1 viruses belonged to the 1A 3.3.2 genetic clade, which consists of pandemic strains of the virus. The remaining four viruses belonged to the 1C 2.1 genetic clade, along with swine Eurasian “avian-like” H1avN1 viruses. Аnalysis of all genes of influenza A viruses resulting from this study determined their affiliation to one of the three lineages - Eurasian "avian-like" H1avN1, H1N1pdm09, and A/ swine/Gent/1/1984-like H3N2 lineage. Two viruses possess the H1avN1 genotype, which is the most prevalent among viruses detected in European pig populations. The remaining 5 viruses were characterized by 4 different reassortant genotypes composed of genes that belong to Eurasian "avian-like" H1avN1, H1N1pdm09, and A/ swine/Gent/1/1984-like H3N2 lineages. In one of the eight analyzed samples, results of the sequencing showed the presence of two viruses with the different origin of the PB2, M, and NS genes. These two viruses possessed a triple reassortant genome that along with their genetic and molecular characteristics implied the high potential for spillover in the human population. Phylogenetic analysis of individual segments and of the complete genome of our viruses indicates evolution relationship with pandemic human and porcine strains, Eurasian "avian-like" H1avN1 swine strains, and A/swine/Gent/1/1984-like H3N2 European swine strains. Based on the molecular characteristics of hemagglutinin, it was found that all sequenced viruses have an affinity for the α2-6 type of receptors that are dominant in the human respiratory epithelium. The present molecular markers within polymerase B2, polymerase B1, polymerase A, and hemagglutinin indicate the ability of these viruses to replicate in human cells. Altogether, these findings imply the potential for successful transmission to humans. Amino acid changes within the antigenic sites of hemagglutinin and neuraminidase indicate a lack of crossreactivity with vaccine strains used in the preparation of commercial vaccines. Based on the amino acid sequences of neuraminidase and matrix 2 proteins, it was found that all viruses are sensitive to neuraminidase inhibitors, and resistant to drugs from the group of inhibitors of the M2 ion channel, which could be significant in case of transmission of these viruses from pigs to humans