Genome stability of the vaccine strain VACΔ6.

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, f ive virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and f ive intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certif ied for vaccine production in accordance with the "Guidelines for Clinical Trials of Medicinal Products". PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

Increased LAMP1 Expression Enhances SARS-CoV-1 and SARS-CoV-2 Production in Vero-Derived Transgenic Cell Lines.

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002‒2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis.

[Increased LAMP1 Expression Enhances SARS-CoV-1 and SARS-CoV-2 Production in Vero-Derived Transgenic Cell Lines].

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002-2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis.

Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR.

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

[Development of the drug oncolytic immunotherapy based on vaccinia viruses (Vaccinia virus, Orthopoxvirus, Chordopoxvirinae, Poxviridae) against breast cancer.]

INTRODUCTION: Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response. The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus. MATERIAL AND METHODS: MTT test, ELISA, methods of transient dominant selection. RESULTS: The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV-1 and 4647 cell cultures, and the cytopathogenic efficacy of the virus was determined in relation to cancer cultures of cells of various genesis. In in vivo experiment, it was revealed that the polyepitopic construct in the genome L-IVP_oncoB is able to initiate a change in the profile of cytokines. DISCUSSION: The obtained data characterized L-IVP_oncoB as a promising cytopathogenic and immunostimulating agent and showed the need for further study of its properties as means of oncolytic immunotherapy. CONCLUSION: The basic experiments on the evaluation of the biological properties of the obtained L-IVP_oncoB, which are necessary for the characterization of the oncolytic virus, have been carried out.

PHYLOGENETIC ANALYSIS OF BOVINE PESTIVIRUSES DETECTED IN SIBERIA.

The results of phylogenetic analysis of three species of bovine pestiviruses circulating in six regions of Siberia, as well as those detected in fetal embryonic serum (FBS) and continuous cell cultures, are presented. The typing was made based on comparison of sequences from the 5' untranslated region (5'-UTR) of the viral genome. Among the highly productive dairy cattle, circulation of five subtypes of the BVDV1 (a, b, d, f, r) and BVDV2 was established. The predominant subtype was 1b (48% positive samples). The number of subtypes of BVDV1 was as follows: BVDV1: 1а (8%), 1b (48%), 1d (8%), 1f (16%) и 1r (8%) and BVDV2 (12%). Cell cultures revealed BVDV1a. The distribution of types and subtypes of viruses had geographical differences. BVDV1b, BVDV1d, BVDV1f и BVDV1r were detected in cattle or persistently infected (PI) animals in farms with respiratory distress. BVDV 1a revealed in the serum of PI heifer without manifestation of clinical symptoms. BVDV2 were detected in cattle with pathology of reproduction. The presence of the BVDV3 (atypical pestivirus) of the Italian group was established in seven lots of FBS obtained from two manufacturers. No evidence has been found for circulating of the atypical virus among cattle of various breeds, including imported, reindeers and red deers. Studies on the molecular epizootology of pestiviruses can be used to select and optimize the control strategy and address the issue of vaccine use in a particular region.

Comparing New-Generation Candidate Vaccines against Human Orthopoxvirus Infections.

The lack of immunity to the variola virus in the population, increasingly more frequent cases of human orthopoxvirus infection, and increased risk of the use of the variola virus (VARV) as a bioterrorism agent call for the development of modern, safe vaccines against orthopoxvirus infections. We previously developed a polyvalent DNA vaccine based on five VARV antigens and an attenuated variant of the vaccinia virus (VACV) with targeted deletion of six genes (VACΔ6). Independent experiments demonstrated that triple immunization with a DNA vaccine and double immunization with VACΔ6 provide protection to mice against a lethal dose (10 LD50) of the ectromelia virus (ECTV), which is highly pathogenic for mice. The present work was aimed at comparing the immunity to smallpox generated by various immunization protocols using the DNA vaccine and VACΔ6. It has been established that immunization of mice with a polyvalent DNA vaccine, followed by boosting with recombinant VACΔ6, as well as double immunization with VACΔ6, induces production of VACV-neutralizing antibodies and provides protection to mice against a 150 LD50 dose of ECTV. The proposed immunization protocols can be used to develop safe vaccination strategies against smallpox and other human orthopoxvirus infections.

Cowpox in a human, Russia, 2015.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

The gene therapy of collagen-induced arthritis in rats by intramuscular administration of the plasmid encoding TNF-binding domain of variola virus CrmB protein.

Wistar rats with collagen-induced arthritis were intramuscularly injected with the recombinant plasmid pcDNA/sTNF-BD encoding the sequence of the TNF-binding protein domain of variola virus CrmB protein (VARV sTNF-BD) or the pcDNA3.1 vector. Quantitative analysis showed that the histopathological changes in the hind-limb joints of rats were most severe in the animals injected with pcDNA3.1 and much less severe in the group of rats injected with pcDNA/sTNF-BD, which indicates that gene therapy of rheumatoid arthritis is promising in the case of local administration of plasmids governing the synthesis of VARV immunomodulatory proteins.

Highly immunogenic variant of attenuated vaccinia virus.

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.

Attenuation of Vaccinia Virus.

Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections.

An Outbreak of Sheep Pox in Zabajkalskij kray of Russia.

In this study, we investigated recent sheep pox outbreaks that occurred in Ononsky and Borzunsky regions of Zabajkalskij kray of Russia. The outbreaks involved in 2756 animals of which 112 were infected and 3 were slaughtered. Samples of injured skin of infected sheep were analysed by electron microscopy and CaPV-specific P32 gene amplification. Following sequence analysis of entire P32 gene showed that both specimens were identical to the sequence of several sheep poxvirus isolates from China and India. The close location of China to the last decade's Russian outbreaks suggest that possible future outbreaks in Russia could occur along the border regions with countries where sheep and goat pox are not controlled.