[Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging].

INTRODUCTION: African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4-6 hours and the late 8-20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results. OBJECTIVE: Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain. MATERIALS AND METHODS: For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum. RESULTS: The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented. CONCLUSION: A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

[Comparative molecular and genetic characterization of rabies viruses (Rabies lyssavirus, Lyssavirus, Rhabdoviridae) circulated in the Russian Federation in 1985-2016.]

INTRODUCTION: Rabies caused by the neurotropic virus of the genus Lyssavirus, Rhabdoviridae family, which infects all warm-blooded vertebrates including human beings. The homology level of the amino acid sequences for Lyssaviruses nucleoprotein reaches 78-93%. Aim - study the genetic diversity and molecular epidemiology of Lyssaviruses circulated in the Russian Federation in 1985-2016. MATERIAL AND METHODS: 54 isolates of rabies virus isolated from animals, and 2 isolates from humans, 4 vaccine strains of rabies virus: RV-97, ERA, Shchelkovo 51, ERAG333 used in phylogenetic study. Phylogenetic analysis was performed using Genbank data on genome fragments of 73 rabies virus isolates and 9 EBLV-1 isolates. DNASTAR V.3.12, Bio Edit 7.0.4.1 and MEGA v.10.0.5, Primer Premier 5 programs have been used. RESULTS: Comparative molecular genetic analysis of genomes fragments of 130 Lissaviruses, isolated on the territory of the RF, Ukraine in 1985-2016, vaccine strains of rabies virus, showed their distribution by geographical feature. Comparison of the nucleoprotein fragments of the rabies virus isolates with vaccine strains revealed 4 marker mutations: V56I (Eurasian group), L/V95W (Central group), D101N/S/T, and N/G106D. Phylogenetic analysis of the isolate «Juli¼, isolated from a human bitten by a bat proved his belonging to the European Bat lyssavirus-1a. DISCUSSION: Study of the molecular epidemiology of rabies within the Russian Federation allows for the genotyping of the viruses and helps to study the hidden mechanisms of rabies infection in animal and human populations, and to characterize vaccine strains, including during oral vaccination. CONCLUSION: Further study of the molecular epidemiology of rabies within the Russian Federation and the countries bordering it is important.

[Formation of population gene pools of zoonotic viruses, potentially threatening biosafety].

The possible formation of population gene pools of zoonotic viruses with a respiratory route of transmission and a possibility of a pandemic at different stages of biosphere evolution is analyzed. Forming of Poxviruses  (Entomopoxvirinae) gene pool could be the beginning of transformation from Plants to Arthropoda (Carbon - 375 million years ago) with further evolution connected with Rodentia (Pliocene - 75-70 million years ago) and further separation of genera (500-300 thousand years ago), and respiratory transmission (epidemics) between humans (10-2 thousand years BC). Smallpox comeback would be possible. Orthomyxoviruses relicts (genus Isavirus) were possibly connected with Ichthya (Silurian - 500-410 million years ago), and then close interaction with Aves (the Cretaceous, 125-110 million years ago) with the division of genera and respiratory transmission (epidemics) between humans (10-2 thousand BC). Next pandemic of influenza A could be catastrophic in terms of the number of victims and economic damage.Coronaviruses formed a gene pool by interaction with Amphibia (subfamily Letovirinae) and then with Chiroptera in Tertiary (110-75 million years ago) with transformation to Artiodactyla (Eocene - 70-60 million years ago), and only 10-2 thousand years BC acquired the ability to a respiratory transmission and became Alphaviruses, a seasonal infection of humans. A similar situation is possible in the near future with SARS-CoV-2. Pandemics associated with zoonoses even more serious than COVID-19 are likely. Constant monitoring of  populational gene pools of zoonotic viruses is necessary.

MOLECULAR-GENETIC CHARACTERIZATION OF FIELD ISOLATES OF RABIES VIRUS IDENTIFIED IN THE TERRITORY OF VLADIMIR, MOSCOW, TVER, NIZHNY NOVGOROD AND RYAZAN REGIONS.

The article presents a molecular genetic study of genomes of field isolates of rabies virus isolated in the Vladimir, Moscow, Tver, Nizhny Novgorod and Ryazan regions, with the aim of carrying out phylogenetic analysis. We studied 20 samples of purified PCR products containing the rabies virus nucleoprotein. The samples were provided by the Vladimir veterinary service. Sequencing and phylogenetic analysis of the gene showed that 12 fragments of isolates under study were close to the Central phylogenetic group of the rabies virus; namely - 5 isolates from the Vladimir region, 2 from the Nizhny Novgorod region, 2 from the Moscow region, and 3 from the Tver region. Eight studied isolates from the Nizhny Novgorod and Ryazan regions were attributed to the Eurasian phylogenetic group.

Monitoring of rabies in wild animals in the Kirov region after oral immunization.

This work presents the results of the molecular genetic research on genomes of field isolates of the rabies virus circulating in the territory of the Kirov region in order to analyze the phylogenetic relationship between the wild isolate genomes and to determine the possible reversion of the vaccine strain of the rabies virus used in the oral vaccine to virulent variant. We studied 24 brain samples from wild carnivores shot after oral immunization of the area with Rabivak-O/333. A bait with the vaccine provided by the Veterinary Service of the Kirov was also studied. All samples were found to be positive for the presence of the rabies virus as established by FAT and RT-PCR techniques. Phylogenetic analysis of N genome fragments of the rabies virus showed that the field isolates from the Kirov regions were genetically close to the field isolates from Buryatia 2012. Analysis of G genome fragments showed that the Kirov field isolates were close to the isolates from Lipetsk (2011), as well as to the Ukrainian isolates (2006 and 2010). Molecular genetic analysis of the gene fragments N and G for the field isolates and fragments of the genome of the rabies virus vaccine did not reveal any reversion to the virulent vaccine strain.

[Two cases of hydrophobia in the Republic of Tatarstan: in vivo and postmortem laboratory diagnosis].

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in viva studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in viva and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY9563I9 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9% from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21 .0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype.