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.

Using the Ground Squirrel (Marmota bobak) as an Animal Model to Assess Monkeypox Drug Efficacy.

In experiments to study the sensitivity of ground squirrels (Marmota bobak) to monkeypox virus (MPXV) at intranasal challenge, expressed pox-like clinical symptoms (hyperthermia, lymphadenitis, skin rash all over the body and mucous membranes and others) were observed 7-9 days post-infection. The 50% infective dose (ID50 ) of MPXV for these marmots determined by the presence of clinical signs of the disease was 2.2 log10 PFU. Some diseased marmots (about 40%) died 13-22 days post-infection, and the mortality rate was weakly dependent on MPXV infective dose. Lungs with trachea were primary target organs of marmots challenged intranasally (with ~30 ID50 ). The pathogen got to secondary target organs of the animals mainly via the lymphatic way (with replication in bifurcation lymph nodes). Lungs with trachea, nasal mucosa and skin were the organs where the maximum MPXV amounts accumulated in these animals. Evidences of the pathogen presence and replication were revealed in these and subcutaneously infected marmots in the traditional primary target cells for MPXV (macrophages and respiratory tract epitheliocytes), as well as in some other cells (endotheliocytes, plasmocytes, fibroblasts, reticular and smooth muscle cells). Our use of this animal species to assess the antiviral efficacy of some drugs demonstrated the agreement of the obtained results with those described in scientific literature, which opens up the prospects of using marmots as animal models for monkeypox to develop therapeutic and preventive anti-smallpox drugs.

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 Possibility of Using the ICR Mouse as an Animal Model to Assess Antimonkeypox Drug Efficacy.

As a result of the conducted experimental studies on intranasal challenge of ICR mice, rabbits and miniature pigs (even in the maximum variant) with the doses of 4.0-5.5 lg PFU of monkeypox virus (MPXV), some clinical signs such as purulent conjunctivitis, blepharitis and ruffled fur were found only in mice. The 50% infective dose (C ID50 ) of MPXV for these animals estimated by the presence of external clinical signs was 4.8 lg PFU, and L ID50 estimated by the virus presence in the lungs of mice 7 days post-infection taking into account its 10% application in the animal respiratory tract was 1.4 lg PFU. When studying the dynamics of MPXV propagation in mice challenged intranasally with 25 L ID50 of MPXV, the maximum pathogen accumulation was revealed in nasal cavity, lungs and brain: 5.7 ± 0.1, 5.5 ± 0.1 and 5.3 ± 0.3 lg PFU/ml, respectively. The pathomorphological examination of these animals revealed the presence and replication of the pathogen in the traditional primary target cells for MPXV (mononuclear phagocyte system cells and respiratory tract epitheliocytes) as well as in some other types of cells (endothelial cells, reticular cells, connective tissue cells). Our use of these animals to assess the antiviral efficacy of some drugs demonstrated the agreement of the results (a significant positive effect of NIOCH-14 and ST-246) with those described in scientific literature, which opens up the prospects of using ICR mice as animal models for monkeypox to develop preventive antismallpox drugs.

[Development of the disease in marmot at the intranasal infection with the monkeypox virus].

In experimental study the sensitivity of the Marmota bobak species to the monkeypox virus (MPXV) with the intranasal (i/n) infection was tested. It was demonstrated that 50% of the infective dose (ID50) of the MPXV on external clinical signs of the disease was 2.2 Ig plaque forming units (PFU). The percentage of the marmot mortality is slightly dependent on the infecting dose of the MPXV, therefore it is not possible to correctly determine the value of 50 % fatal dose (FD50) for these animals. The most pronounced external clinical signs of the disease were obtained in the marmots: pox-like skin rash throughout the surface of the body and mucous membranes, purulent discharge from the nose, lymphadenitis, discoordination, tremor of the extremities, fever, increased aggression, and ruffled fur. In the course of experiments intended to determine the dynamics of the accumulation of the MPXV in various organs, tissues, and blood serum of marmot infected i/n with dose of 3.7 Ig PFU, it was found that the trachea, lungs, and the bifurcation lymph nodes are the primary target organs. The trachea, lungs, nasal mucosa membrane, and skin are the organs with maximal virus replication recorded at 5, 7, 9, and 12 days after the infection. The transfer of the MPXV into the secondary target organs (nasal mucosa membrane, brain, spleen, duodenum, adrenal glands, and skin) was carried out in marmots with lymphogenic and hematogenic ways of the dissemination of the infection.

[A comparative study of the antiviral activity of chemical compounds concerning the orthopoxviruses experiments in vivo].

In the experiments using intranasal (i/n) infection of mice with the ectromelia virus (EV) in a dose 10 LD50/head (10 x 50% lethal doselhead) or with the monkaypox virus (MPXV) in a dose 10 ID50/head (10 x 50% infective dose/ head) it was demonstrated that the antiviral efficiency of chemical compounds - the condensed derivatives of pyrrolidin-2,5-dion, as well as their predecessors and the nearest analogues, synthesized in Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences (NIOCH SB RAS) was observed. As a positive control we used the antipoxvirus chemical preparation ST-246 available from SIGA Technologies Inc. (USA), synthesized in NIOCH SB RAS by the technique suggested by the authors. It was demonstrated that the compound NIOCH-14 (7-[N'-(4-Trifluoromethylbenzoil)-hydrazidecarbonil]-tricyclo[3.2.2.02,4]non-8-en-6-carbonic acid) possessed comparable with ST-246 antiviral activity concerning EV and MPXV on all indicators used. Therefore, at infection of mice with EV (strain K-1) and peroral administration of NIOCH-14 and ST-246 in a dose 50 mkg/g of mouse weight (12-14 g) within 10 days the survival rate and average life expectancy of mice authentically exceeded the control levels. EV titers in lungs through 6 days after infection in the same groups were lower than in the control. In addition to that, after 7 days of infection of mice with MPXV (strain V79-1-005) and daily peroral administration of NIOCH-14 and ST-246 in a dose 60 mkg/g of mouse weight (9-11 g) authentic decrease in a part of infected animals and MPXV titers in lungs was observed.

[Rotaviruses in younger children in Novosibirsk in 2005-2007: detection and genotyping].

Examination of 1898 patients with acute enteric infection from March 2005 to February 2007 showed that group A rotaviruses were the most frequent cause (35%) of acute gastroenteritis among children under 3 years of age. Majority of cases of rotavirus infection was detected in infants under 1 year of age (71.8%). The peak of sporadic incidence was observed between February and May. High rate of mixed infection (45.6%) was observed - associations of rotaviruses with other viruses (noroviruses, astroviruses) and bacteria (Salmonella, Shigella, enteroinvasive Escherichia coli, Campylobacter, and opportunistic species) were detected. P- and G-genotypes of 337(50.8%) isolates of group A rotaviruses were determined by RT-PCR. The most prevalent strain was P[8]G1 (54.6%) followed by P[8]G3 (10.7%), P[8]G9 (8.6%), P[4]G2 (8.3%), and P[8]G4 (4.5%) genotypes.

[Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates].

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.

[Study of antituberculous activity of moxifloxacin conjugate with macrophage scavenger-receptor ligand].

Mycobacterium tuberculosis is an intracellular pathogen that persists in macrophages of the human host. An approach to improving the treatment of tuberculosis is target delivery of antibiotics to macrophages using ligands to macrophage receptors. The antituberculous activity of the conjugate of the antituberculous antibiotic moxifloxacin with carboxymethylglucan was studied in vitro using the J774 macrophage cell line and peritoneal macrophages. The antituberculous activity of the conjugate was higher than of the free moxifloxacin. The target antibiotic delivery to macrophage cells in tuberculosis infection was shown perspective.

[Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions].

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.