Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s

The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.

Molecular Genetic Testing of Stability and Identification of Vnukovo-32 Strain Used for Production of the Cultural Concentrated Purified Inactivated Dry Rabies Vaccine

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.

IMMUNOGENESIS IN THE DESTRUCTION OF CELLS OF THE NERVOUS SYSTEM UNDER THE INFLUENCE OF THE RABIES VIRUS

The nature of rabies immunity is not well understood, the zones of deployment of protective mechanisms are unknown, and the place where antibodies meet with the virus is unclear. The issue of the mechanism of protection of the central nervous system against rabies virus is relevant. It has been hypothesized that protection (immunity) of the central nervous system to the rabies virus is induced by the distant interaction of antibodies on the nerve сells and is mediated by the energy-information field of specific antibodies circulating in the blood vessels of the central nervous system. To confirm this hypothesis, the non-contact effect of rabies antibodies on the rabies virus (strain “Shchelkovo-51”) in the culture cell (BHK-21/13) and the decrease in antigen accumulation from 1.7 to 2.9 times were experimentally shown. The obtained data confirm the main point of the hypothesis on the protection of the central nervous system against the rabies virus using an energy-information biofield.

DETERMINATION OF THE RABIES VIRUS-INFECTED VERO LINE CELL PORTION BY FLOW CYTOMETRY

Aim. Experimental substantiation of possibility to determine the rabies virus-infected Vero cell line portion in culture by flow cytometry (FC) and FITC labeled diagnostic anti-rabies immunoglobulin (DAI), manufactured in Russia. Materials and methods. Fixation and permeabilization of Vero cells, infected by rabies virus strain «Moscow 3253», was carried out by means of Суtofix/Cytoperm reagent (BD Biosciences, USA) according the Vengatesan D. et al. method (2006) and then intracellular rabies antigen was stained by DAI. Percentage of infected cells was determined by FC in 24, 48 and 72 h and as well in 48 h when the cell cultures were infected with tenfold dilutions of virus-containing fluid from 10-1 to 10-8. Results. There was a significant increase in the percentage of infected cells on average from 30 to 70% in time interval from 24 to 48 h. With 1000-fold dilution of viral-containing fluid the FC method detected the 6,9±0,21% of infected cells in Vero cultures (P<0,001, n=3). Conclusion. FC has proved to be a fast, sensitive and reliable method for determining the relative number of virus- infected Vero cells in cultures. The drug DAI had a sufficient activity for its effective use in the automated version of MFA based on the FC method. The use of FC is possible at various stages of anti-rabies drug production and control, and is also promising in terms of further improving of the rabies diagnosis.

MOLECULAR AND BIOLOGICAL CHARACTERISTICS OF VACCINARY ERA-CB 20M OF RABIES VIRUS

The molecular and biological characteristics of the vaccine against rabies virus strain ERA-CB 20M obtained by the Russian rabiologist, doctor of medical sciences S.V. Gribencha by adapting and cloning the strain ERA and SAD in a transplantable BHK-21 C13 cell culture are presented. The spectrum of the most sensitive strain of rabies ERA-CB 20M cell lines was determined and the level of glycoprotein was quantitatively determined. Primary nucleotide sequences of fragments of the genome of the strain ERA-CB 20M (genes N and G) were obtained and phylogenetic analysis was carried out. Molecular analysis showed that this strain belongs to the group of vaccine strains SAD1. When compared with the reference strain SAD1, 10% of the nucleotide differences were revealed in the gene fragment N; 15%, in the gene fragment G.