The dissertation is dedicated to the molecular structure studies and phylogenetic relationships of porcine circovirus type II (PCV-2) isolates, which circulates on the territory of Ukraine, and method development for pathogen detection based on the isothermal amplification assay with its adoption for using in field conditions.
The data due to the PCV-2 distribution in the world and in Ukraine for the period 2009–2019 were analyzed and summarized.
An epizootic screening was conducted and new data on the distribution of PCV-2 among wild pigs in Ukraine were obtained. Thus, PCV-2 DNA was found in 31.8% of total tested clinical samples from wild boars, indicating that virus is circulating in the wild boar population in Ukraine.
Data on the etiological structure of PCV-2-associated diseases in domestic pigs in farms in the Eastern region of Ukraine were obtained. It has been found that the structure of such diseases is caused by PCV-2 and primary infectious agents — PRRS, porcine parvovirus, porcine adenovirus, M. hyopneumoniae in various associations. As the result association of PCV-2, parvovirus and pig adenovirus was dominated in the structure of circovirus-associated diseases and consists 21.36%. The PCV-2 and parvovirus association was 5.83%. The least commonly encountered association was PCV-2, PRRS and M. hyopneumoniae — 1.94%; while the PCV-2 and PRRS association was 20.39% and the PVC-II and M. hyopneumoniae only 2.91%.
For the first time in Ukraine, an isothermal amplification assay (LAMP) has been developed for the detection of PCV-2 genetic material. The primer set (PCV-F3, PCV-B3, PCV-FIP, PCV-BIP) was designed based on the target sequence of the capsid gene and the protocol of their application was optimized. Optimal isothermal reaction parameters required for amplification products was implemented. An adaptation of the developed method for use in the field conditions was carried out in the absence of laboratory equipment and proper working conditions.
The possibility of using PCR for genotyping of PCV-2 was studied. It has been found that the conventional PCR cannot completely replace the classical approaches for PCV-2 genotyping by sequencing due to the high genetic variability of this pathogen causes the low specificity of the primers for its genotyping.
For the first time in the world, DNA sequencing of PCV-2 was performed using nanopore technology using a developed PCV-2 seqF/R primer system flanking a 798 bp region in PCV-2 genome containing the complete sequence of the cap gene. We obtained 11 complete PCV-2 capsid gene sequences (in nucleotide position from 970 to 1,768 of the PCV-2 genome) suitable for phylogenetic studies.
The peculiarities of the molecular-genetic structure of the PCV-2 circulating on the territory of Ukraine were investigated and the phylogenetic analysis of its isolates was conducted. Ten Ukrainian isolates were identified as 2B subtype and one of the sequenced isolates (Poltava 2) belong to the 2F genotype that was confirmed firstly in Ukraine. Despite the relationship of the Ukrainian isolates (except Poltava 2) to the isolates from England, Argentina, Italy, Romania, the USA, South Korea, and previously isolated in the territory of Ukraine, they were found forming a separate clade. The main group in this clade was formed by isolates Zaporizhzhia 1, Volyn 1, Chernihiv 1, Chernihiv 2, Chernihiv 3, Chernihiv 4, Luhansk 2, and Poltava 1, while the samples Chernivtsi 1 and Chernivtsi 2 formed a separate branch inside the clade, indicating a common source of origin Ukrainian PCV-2 isolates and their autochthonous nature in Ukraine.
The phylogenetic analysis of the PCV-2 amino acid sequences was performed. The differences in the structure of the capsid protein of the studied isolates were established. Six genetic subgroups within Ukrainian isolates of the five phenotypes of the PCV-2 genotype 2B were determined.
The epitope MP-Lcd in the region 75–92 of the capsid protein of Ukrainian isolates was analysed. It was found that the sequences that formed the main clade of Ukrainian isolates had no differences. Samples Chernivtsi 1 and Chernivtsi 2, and Poltava 2, which formed separate branches, differed from the main group by amino acid sequence in positions 91 (valine (V) → isoleucine (I)) and 80 (leucine (L) → valine (V) ) respectively.
The genetic distance between the nucleotide sequences of PCV-2 was evaluated and the correlation of geographical and genetic distances of the main clade of Ukrainian isolates was investigated. There is no statistically significant difference between the geographical distance and the genetic structure of the studied sequences, which testifies to the homogeneity of the PCV-2 isolates circulating on the territory of Ukraine among the wild boar population.
