Olexii S. Solodiankin

Aim. To study the prevalence and identify viral agents affecting cultivated button mushrooms (Agaricus bisporus) on mushroom farms in Ukraine, using molecular biological methods, and to assess the relative viral load. Methods. Visual assessment of symptoms was conducted on samples of mushroom fruiting bodies and mycelia from farms in the Kyiv, Ternopil, Rivne, Lviv, Dnipropetrovsk, and Odesa regions. Total RNA was isolated using a GeneJET Plant RNA Purification Kit. Identification of viral agents (La France disease virus – LFDV; Mushroom bacilliform virus – MBV; Agaricus bisporus virus 6 – AbV6; Agaricus bisporus virus 16 – AbV16; Agaricus bisporus virus X – AbVX) was performed using two-step Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) on a Rotor-Gene 6000 amplifier. Glyceraldehyde-3-phosphate dehydrogenase II (GPDII) gene of A. bisporus was used as an internal control for normalization and assessment of RNA/cDNA quality. Melt curve analysis was performed, and the relative amount of viral RNA was calculated using the ΔCt method. The study used a straightforward estimation of viral load based solely on the GPDII reference gene. It was used as a reference gene. This method serves as a preliminary step, acknowledging its limitations in precision. Results. Six A. bisporus samples with various morphological changes were studied: a small cap and a strongly curved stipe (Ternopil), a thickened stipe (Kyiv), a small cap and an elongated stipe (Rivne), a dense stipe and a flat cap with pink gills (Dnipro), browning of fruiting bodies (Lviv), and an abnormal cotton-like mycelium (Odesa). The performed RT-qPCR analyses identified various combinations of LFDV, MBV, AbV6, AbV16, and AbVX. Noteworthy, mushroom virus X (MVX), associated with the "bare patch" disease, was not detected in any of the samples. The most common viruses were MBV (detected in all 6 samples) and AbVX (in 5 samples). AbV6 and AbV16 viruses were detected in 4 samples, and LFDV in 3 samples. Mixed infections by more than four viruses were detected in four samples. ΔCt calculation showed a significant relative level of MBV viral RNA in most samples (especially in Dnipro and Lviv), as well as LFDV, AbV6, and AbVX in the Lviv sample, compared to the expression level of GPDII. Melt curve analysis confirmed the specificity of the PCR products. Conclusions. RNA viruses were detected and identified in the culture of A. bisporus, and their quantitative representation was assessed. On the surveyed mushroom farms, viruses LFDV, MBV, AbV6, AbV16, and AbVX are prevalent, often as mixed infections with different levels of relative viral load. The obtained results emphasize the need for implementing systematic molecular monitoring of mushroom viral diseases in Ukraine to ensure phytosanitary safety and production stability.

The aim of the work is to determine the phenotypic and some genetic characteristics associated with the pathogenicity of the S.aureus strains isolated from patients with paratraumatic eczema that developed as a result of combat injuries. Materials and methods. Phenotypic and individual genetic characteristics of Staphylococcus aureus strains, isolated from 14 patients with paratraumatic eczema who were receiving inpatient treatment in the dermatology department of the SE «Institute of Dermatology and Venereology of the National Academy of Medical Sciences of Ukraine» were determined. Microbiological studies were conducted at the Laboratory of Microbiology, Immunology and Molecular Genetics of the SE «Institute of Dermatology and Venereology of the National Academy of Medical Sciences of Ukraine» using generally accepted methods. Molecular genetic studies on the determination of genes for the production of S. aureus enterotoxins from patient samples were conducted at the Institute of Chemistry of Functional Materials of the Scientific and Technological Complex «Institute of Single Crystals» of the NAS of Ukraine. Results. The results of the conducted studies have established the main phenotypic and individual genetic characteristics of S. aureus strains, which are associated with pathogenicity and have a significant impact on the course of the disease. Conclusions. When determining the sensitivity of clinical strains of S. aureus to antibacterial drugs, it was found that the vast majority (62.5%) had a multidrug-resistant phenotype, and 12.5% were characterized as strains with extensive resistance. It was determined that both the reference and clinical strains of S. aureus were characterized by the ability to form biofilms of predominantly high density, with strains with a high ability to form biofilms accounting for 77.8%. It has been established that the foci of lesions in patients with paratraumatic eczema resulting from combat injuries are colonized by Staphylococcus aureus, which primarily produces not only the superantigen – toxic shock syndrome toxin‑1 (TSST‑1); but also, to a significant but lesser extent, staphylococcal enterotoxins B and C.

Amid the devastating war in Ukraine, a parallel battle rages on – that against infectious diseases. The COVID-19 pandemic starkly underlined the vital importance of global health surveillance for emerging and re-emerging human and animal pathogens. This challenge has become even more acute in war-torn regions like Ukraine, where healthcare infrastructure is under siege. For viruses with pandemic potential, such as the influenza virus, worldwide surveillance initiatives are essential in tracking the virus’s movement and spread among both animals and humans. Such monitoring serves dual purposes. Firstly, it guides the formulation of the yearly vaccine, ensuring it remains effective against prevailing strains. Secondly, it alerts livestock farmers in advance, enabling the timely rollout of mitigation measures. Moreover, discerning whether a disease outbreak is of natural origin, or an intentional release becomes critical. To achieve this, the global community must share transparent and timely information through a robust network of laboratories with cutting-edge biotechnological capabilities. The war’s ramifications have severely impacted these efforts. Yet, the stakes are too high to let these facilities fade away. This situation leads to the ultimate question: Who will step up to rebuild Ukraine’s public health and disease research laboratories?

Veterinary Medicine, Inter-departmental Subject Scientific Collection, NSC

Hepatitis E virus (HEV) infects humans and several mammals and it has eight genotypes (HEV1–HEV8). HEV1–HEV4 is the causative agent of hepatitis E in humans. HEV5–HEV6 was detected only in wild boar in Japan. HEV7–HEV8 was detected in camels. HEV3–HEV4 is characterized by zoonotic potential and main natural reservoirs for this virus are swines and wild boars. Besides, HEV3 was detected in deers, dolphins, rabbits, cattle, goats that is additional risk for virus interspecies transmission from domestic animals to humans. In this paper two primer and probe sets for HEV detection by real time PCR were characterized on the basis of computer analysis of conservative fragments of overlapping open reading frames ORF2/ORF3 of HEV genome. Availability of mismatched nucleotides in the complexes of primer/probe with viral targets was applied for estimation of primer sets. One of those primer sets from literature data was tested on the first World Health Organization International Standard for HEV RNA. The second primer set is highly cited in scientific articles on PCR HEV detection in PubMed biomedical literature database. Multiple alignment was performed on the basis of computer analysis of nucleotide sequences of overlapping open reading frames ORF2/ORF3 for 108 isolates of RNA HEV genomes from GenBank by MEGA 6.0 software. It was determined that 38 HEV isolates from 108 HEV3–HEV4 analyzed isolates for mentioned above primer and probe sets have one or two mismatched nucleotides for primer (probe) complex with single-stranded amplicon. These degeneracies may reduce sensitivity and specificity of PCR assay due to decreasing effective primer concentration, complication of estimating primer annealing temperatures

Porcine circovirus type 2 (PCV2) is responsible for a number of porcine circovirus-associated diseases (PCVAD) that can severely impact domestic pig herds. For a non-enveloped virus with a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with eight genotypes (a–h). New genotypes of PCV2 can spread through the migration of wild boar, which are thought to infect domestic pigs and spread further through the domestic pig trade. Despite a large swine population, the diversity of PCV2 genotypes in Ukraine has been under-sampled, with few PCV2 genome sequences reported in the past decade. To gain a deeper understanding of PCV2 genotype diversity in Ukraine, samples of blood serum were collected from wild boars (n = 107) that were hunted in Ukraine during the November–December 2012 hunting season. We found 34/107 (31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n = 16) were collected from a commercial market near Kharkiv in 2019, of which 6 out of 16 (37%) samples were positive for PCV2. We sequenced the genotyping locus ORF2, a gene encoding the PCV2 viral capsid (Cap), for 11 wild boar and six domestic pig samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with resolved genotypes, the PCV2 genotype b was the most common in wild boar samples (10 out of 11, 91%), while the domestic pigs were infected with genotypes b and d. We also detected genotype b/d and b/a co-infections in wild boars and domestic pigs, respectively, and for the first time in Ukraine we detected genotype f in a wild boar from Poltava. Building a maximum-likelihood phylogeny, we identified a sublineage of PCV2 genotype b infections in both wild and domestic swine, suggesting a possible epizootic cluster and an ecological interaction between wild boar and domestic pig populations in northeastern Ukraine.

Porcine circovirus type 2 (PCV2) is responsible for a number of porcine circovirus associated diseases (PCAD) that can severely impact domestic pig herds. For a non-enveloped virus with a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with 8 subtypes (a-h). New subtypes of PCV2 can spread through migration of wild boars, which are thought to infect domestic pigs and spread further through the domestic pig trade. Despite a large swine population, the diversity of PCV2 subtypes in Ukraine has been undersampled, with few PCV2 genome sequences reported in the past decade. To gain a deeper understanding of PCV2 subtype diversity in Ukraine, samples of blood serum were collected from wild boars (n = 107) that were hunted in Ukraine during the November-December 2012 hunting season. We found 34/107 (31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n = 16) were collected from a commercial market near Kharkiv in 2019, of which 6/16 (37%) were positive for PCV2. We sequenced the genotyping locus ORF2, a gene encoding the PCV2 viral capsid (Cp), for 11 wild boar and 6 domestic pig samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with resolved subtypes, PCV2 subtype b was most common in wild boar (10/11, 91%), while domestic pigs were infected with subtypes b and d. We also detected subtype b/d and b/a co-infections in wild boar and domestic pigs, respectively, and subtype f in a wild boar from Poltava for the first time in Ukraine. Building a maximum likelihood phylogeny, we identified a sublineage of PCV2 subtype b infections in both wild and domestic swine, suggesting a possible epizootic cluster and ecological interaction in northeastern Ukraine.

The purpose of this work was to adapt the method for detection of genetic material of PCV-II developed by us for use in the field conditions in the absence of laboratory equipment and proper working conditions. To develop the technique, a liver sample from a pig was used, which was characterized as positive for the presence of PCV-II genetic material. Nucleic acid extraction was performed using an express method of our own modification. The isothermal amplification reaction was carried out using reagents manufactured by Thermo Fisher Scientific (Germany) and BioLabs (Great Britain) following the manufacturers’ recommendations when using the PCV-F3, PCV-B3, PCV-FIP, PCV-BIP primer systems. A WB-4MS water bath (Biosan, Latvia) and a 380 ml thermal mug (ZIZ, Ukraine) were used for isothermal amplification. Freeze dryer ALPHA 1-2 LD plus manufactured by Christ (Germany) was used for lyophilization of the reaction mixture. The reaction temperature was monitored using a ТТЖ-М thermometer (PJSC “Skloprylad”, Ukraine), the temperature was recorded using a SterilDisk logger (Tecnosoft, Italy). Gel Doc XR+ transilluminator (Bio-Rad, USA), SYBR Green I intercalating dye produced by Invitrogen (USA) and portable ultraviolet light source (YATO, Poland) were used to visualize and control the results of isothermal amplification. The previously developed isothermal amplification method was adapted for use in field conditions on the example of the identification of PCV-II. The developed method of isothermal amplification is technically simplified and does not require the use of special laboratory equipment

This work aimed to obtain positive control using recombinant DNA technology for detection by PCR of a new poorly studied pathogen — porcine circovirus type 3. Recombinant positive control was designed using Clone Manager Basic. As a vector in the creation of recombinant control we used plasmid pTZ57R/T, as an insert — a fragment of the gene rep PCV-3 with the length of 418 nucleotide pairs, obtained by classical PCR. Transformation of competent cells of E. coli strain DH5a was carried out by chemical poration, followed by plating on LB-medium with the addition of ampicillin at a final concentration of 100 μg/ml. The selection of E. coli cell colonies was performed by the marker of antibiotic resistance to ampicillin. The presence of a specific insert was checked by PCR with electrophoretic visualization of the results. The developed recombinant positive control can be used for the monitoring of biological samples from pigs for the presence of genetic material PCV-3 using molecular technologies

Goal. To conduct molecular genetic studies of products of swine breeding, in particular, liver, sold in the food markets of Kharkiv, for the presence of genomic material of porcine circovirus type 2 (CVS2). Methods. Extraction of total nucleic acid, in particular CVS2 DNA, from liver homogenate, transported to the laboratory on ice was performed according to standard methods. Detection of CVS2 genetic material was performed by polymerase chain reaction (PCR) in a standard format using the PCV2 F/R primer system, which limited the rep gene fragment which encodes the virus replicase, with a length of 408–421 nucleotide pairs (n.p.), on an amplifier Biometer TAdvanced. Molecular weight markers with a resolution of 25 to 700 n.p. were used to determine the length of the amplification products. To visualize the results of amplification, the method of horizontal gel-electrophoresis was used, followed by photographing 1.5% gels using Image Lab software 5.2.1. Results. It was found that 62.5% of the studied liver samples obtained from clinically healthy pigs contain CVS2 DNA, which indicates the need to strengthen control over the spread of this virus among the pig population in Ukraine. Conclusions. Based on the results of research, it is established that the liver of pigs is potentially dangerous for consumers because of the presence of CVS2 due to the possible impact of this virus on other infectious agents when ingested, as well as human infection, which can not be excluded given the known ability of CVS2 to overcome species barrier. The necessity of monitoring pig products, in particular liver, using a standard PCR variant is shown. Мета. Провести молекулярно-генетичні дослідження продукції свинарства, зокрема, печінки, що реалізують на продуктових ринках м. Харків, щодо наявності геномного матеріалу цирковірусу свиней типу 2 (ЦВС-2). Методи. Екстракцію сумарної нуклеїнової кислоти, зокрема ДНК ЦВС-2, із гомогенату печінки, транспортованої до лабораторії на льоду, здійснювали за стандартною методикою. Детекцію генетичного матеріалу ЦВС-2 проводили способом полімеразної ланцюгової реакції (ПЛР) у стандартному форматі з використанням системи праймерів PCV-2 F/R, які обмежували фрагмент гена rep, що кодує репліказу вірусу, довжиною 408 – 421 пар нуклеотидів (п.н.), на ампліфікаторі Biometra TAdvanced. Для визначення довжини продуктів ампліфікації використовували маркери молекулярної маси з дискретністю 25 – 700 п.н. Для візуалізації результатів ампліфікації застосовували метод горизонтального гель-електрофорезу з наступним фотографуванням 1,5 %-х гелів з використанням програмного забезпечення Image Lab 5.2.1. Результати. Установлено, що 62,5 % досліджуваних зразків печінки, отриманої від клінічно здорових свиней, містять ДНК ЦВС-2, що свідчить про потребу посилення контролю за поширенням цього вірусу серед популяції свиней на території України. Висновки. На основі отриманих результатів досліджень установлено, що печінка свиней є потенційно небезпечною для споживачів через уміст ЦВС-2 внаслідок можливого впливу даного вірусу на інші збудники інфекційних захворювань при попаданні в організм, а також інфікування людини, яке не можна виключити з урахуванням відомої здатності ЦВС-2 долати видовий бар’єр. Показана необхідність проведення моніторингу продуктів свинарства, зокрема печінки, з застосуванням стандартного варіанта ПЛР.