[Genetic diversity of Siberian bovine coronavirus isolates (Coronaviridae: Coronavirinae: Betacoronavirus-1: Bovine-Like coronaviruses)].

INTRODUCTION: Bovine coronaviruses (BCoVs) are causative agents of diarrhea, respiratory diseases in calves and winter cow dysentery. The study of genetic diversity of these viruses is topical issue. The purpose of the research is studying the genetic diversity of BCoV isolates circulating among dairy cattle in Siberia. MATERIALS AND METHODS: Specimens used in this study were collected from animals that died or was forcedly slaughtered before the start of the study. The target for amplification were nucleotide sequences of S and N gene regions. RESULTS: Based on the results of RT-PCR testing, virus genome was present in 16.3% of samples from calves with diarrheal syndrome and in 9.9% with respiratory syndrome. The nucleotide sequences of S gene region were determined for 18 isolates, and N gene sequences - for 12 isolates. Based on S gene, isolates were divided into two clades each containing two subclades. First subclade of first clade (European line) included 11 isolates. Second one included classic strains Quebec and Mebus, strains from Europe, USA and Korea, but none of sequences from this study belonged to this subclade. 6 isolates belonged to first subclade of second clade (American-Asian line). Second subclade (mixed line) included one isolate. N gene sequences formed two clades, one of them included two subclades. First subclade included 3 isolates (American-Asian line), and second subclade (mixed) included one isolate. Second clade (mixed) included 8 sequences. No differences in phylogenetic grouping between intestinal and respiratory isolates, as well as according to their geographic origin were identified. CONCLUSION: The studied population of BCoV isolates is heterogeneous. Nucleotide sequence analysis is a useful tool for studying molecular epidemiology of BCoV. It can be beneficial for choice of vaccines to be used in a particular geographic region.

[Genetic diversity and distribution of bovine pestiviruses (Flaviviridae: Pestivirus) in the world and in the Russian Federation].

The genus Pestivirus of the family Flaviviridae includes 11 species. Bovine pestiviruses are the causative agents of viral diarrhea/mucosal disease and include three genetically distinct species: pestivirus A (BVDV-1), B (BVDV-2), and H (BVDV-3). The number of BVDV-1 subtypes is 21, BVDV-2 - 4, and BVDV-3 - 4, which complicates the diagnosis of associated diseases, reduces the effectiveness of vaccination and control programs.We performed the search in the PubMed, Web of Science, Scopus, eLIBRARY.RU databases for articles published in 2000-2021.Pestivirus A is distributed everywhere, although the largest number of subtypes was found in cattle in Italy and China. The virus is widespread in the Central region of the Russia (subtypes 1a and 1m). In Siberia, eleven subtypes circulate among native and imported animals: 1a (5%), 1b (35%), 1c (5%), 1d (10%), 1f (20%), 1g, 1i (both 2.5%), 1j, 1k, 1p, and 1r (all for 5%). Pestivirus B subtype is more virulent, found less frequently and mainly in the North and South America, in some European countries, and in Asia. Three subtypes have been identified in Siberia: 2a (25%), 2b (10%), and 2c (5%). Pestivirus H circulates in Europe, Asia and South America. The main route of entry is contaminated biological products. In Russia, BVDV-3 of the Italian-Brazilian group (3a) was detected in 7 lots of fetal bovine serum.The role of the virus in the occurrence of respiratory diseases in calves, abortion, systemic infection and enteritis in calves and adult animals has been established. The source of the virus in such cases was a contaminated modified live vaccine.

[Detection of bovine pestiviruses by a multiplex real-time polymerase chain reaction.]

INTRODUCTION: Pestiviruses are the cause of reproductive problems, diseases of the gastrointestinal and respiratory tracts of animals. Three species are important for cattle: Pestivirus A, B, and H. Fast and reliable methods of differentiation of these pathogens are currently needed. Aims and objectives of the study: the development of multiplex real time PCR for the simultaneous detection and differentiation of three viruses. MATERIAL AND METHODS: The nucleotide sequences of the conserved regions of the 5´-UTR genes of pes tivirusesA, B, and H served as a target. RESULTS: The reaction showed high specificity, sensitivity, reproducibility and was able to detect virus RNA at a concentration of not less than 0.6-1.2 lg TCID50/cm3. Cross-reactions with other pestiviruses wer e not observed. Real time PCR confirmed the results obtained previously in RT-PCR with gel electrophoresis detection. In a parallel study of 1823 biological samples, the results of the two reactions were completely consistent. Pestivirus spp. was detectedin 76 samples, Pestivirus A was present in 73 samples, Pestivirus B - in 3 samples, and Pestivirus H was not detected. DISCUSSION: A two-step real time PCR was developed for the simultaneous detection and differentiation of three pestiviruses. Modified pan primers of S. Vilcek et al. were used for the first reaction, and primers and probes of our own design were used for virus typing, which resulted in high reaction efficiency. CONCLUSION: On the big dairy farms for livestock maintenance, there are favorable conditions for the circulation of pathogenic viruses. In this situation, rapid diagnostic methods are needed to quickly identify of several viruses. Real-time triplex analysis can be recommended as the rapid method for mass epidemiological studies, as well as for screening fetal calf serum used for virus cultivation in medicine and veterinary practice.

Developing and Testing a Real-Time Polymerase Chain Reaction to Identify and Quantify Bovine Respiratory Syncytial Viruses.

The bovine respiratory syncytial virus (BRSV) known as Bovine orthopneumovirus according to the international classification is one of the most important etiological agents of respiratory diseases in calves. At present, rapid and reliable methods to detect and measure the concentrations of this pathogen are needed. The objectives of the survey are developing the real-time polymerase chain reaction (PCR) to identify and quantify the BRSV RNA and, based on it, determining the number of the virus genomes in the respiratory tract of sick animals during the disease outbreaks. The nucleocapsid (N) protein gene of the virus served as the target for amplification. Messenger RNA (mRNA) of bovine GAPDH was used as a reference gene. A panel of positive control samples at known concentrations was used to estimate the virus and GAPDH numbers. The concentration of viral RNA extracted from the biomaterial samples was quantified relative to the bovine GAPDH mRNA level. The analytical sensitivity of PCR demonstrating high specificity and reproducibility was 1 × 103 genome equivalents per 1 cm3. All 273 samples of biological material taken from the animals with the respiratory diseases were analyzed. The virus genome was detected in 19.4% of samples. The viral RNA was more frequently detected in the lungs, which comprised 10.61% of positive samples. It was less frequently found in the mucous membranes of trachea and bronchi and the lymph nodes of the lungs, which comprised 0.73% of positive samples each. Concentrations of the virus in samples varied. The highest concentration was recorded in the lungs (1.3 ± 0.5-4.8 ± 0.47 log10 copies of BRSV/GAPDH RNA). The developed test kit may be used to quantify the concentration of the bovine respiratory syncytial virus in disease pathogenesis and to estimate the efficiency of vaccine or antivirus preparations for animals.

[Detection of bovine herpesvirus 4 DNA in cattle by realtime PCR.]

INTRODUCTION: BoHV-4 is poorly understood. Data on the circulation of the virus among animals and its role in infectious diseases insufficient. Aimes and goals. Development of real-time PCR for detecting the BoHV-4 and studying the frequency of its presence in samples from sick animals. MATERIAL AND METHODS: The nucleotide sequences of the glycoprotein L gene served as a target for amplification. The sequences of reference strains published in GenBank were used to analyze and design the primers. Studies were conducted in 3 regions of Western Siberia on 5 large dairy farms. RESULTS: 27.7% of samples contained the virus. The virus was present as a monoagent in nasal cavity of calves (80.0%), lungs (46.2%) and bronchial lymph nodes (38.5%) in pneumonia. In the cases of diarrhea the virus was detected in 20%, and in cows with gynecological pathology in 10.0%. In respiratory diseases of calves the virus was detected in association with BoHV-1 (21.6%) and BoCV (20.3%), and in gynecological pathology of cows with BVDV1 (6%). DISCUSSION: According to the phylogenetic analysis of 5 identified virus isolates, four belonged to the American branch and one to the European branch. The circulation of American strains occurred in the territory of the Republic of Kazakhstan (1), Tyumen (1) and Novosibirsk (2) regions, and the European - in the Novosibirsk region. CONCLUSION: The search for viruses involved to the infectious pathology, as well as studying the genetic diversity of viruses circulating on a particular farm including imported from other countries, is relevant.

DEVELOPMENT OF THE METHOD FOR IDENTIFICATION AND GENOTYPING OF THE BACTERIA PASTEURELLA MULTOCIDA AND MANNHEIMIA HAEMOLYTICA ON THE BASIS OF THE PCR AND PHYLOGENETIC ANALYSIS OF BACTERIAL CULTURES ISOLATED FROM CATTLE.

The results of development of a method for detection and genotyping of the bacteria Pasteurella multocida capsular five groups and Mannheimia haemolytica Al based on the multiplex polymerase chain reaction (PCR) with electrophoretic detection are submitted. Diagnostic sensitivity of the developed method was 103 CFU/ml in the study of the pure cultures and 105 CFU/g in the study of biological material. A study of 260 samples of biological material from infected animals revealed Pasteurella multocida in 50.0%, and Mannheimia haemolytica in 11.2% of the investigated samples. Circulation among the tested livestock of capsular groups B and E of Pasteurella multocida was not revealed. The majority of the tested samples contained group A, in some cases, group D, and, in one case, group F. On the basis of the phylogenetic analysis circulation of two different genetic types of Pasteurella multocida of the capsular group A was revealed.