Use of Adjuvant Compositions Based on Squalene Ensures Induction of Neutralizing Antibodies against SARS-CoV-2.

Squalene-based adjuvant compositions that can provide effective induction of specific humoral immune response have been developed. Recombinant receptor-binding domain (RBD) of surface S-protein of SARS-CoV-2 was used to evaluate the properties of the composition. Immunization of mice with the developed squalene-based compositions in combination with RBD allows obtaining high titers of specific antibodies: from 105 to 2×106. The blood sera from immunized mice exhibit neutralizing activity against SARS-CoV-2 Delta variant (B.1.617.2) with a titer up to 1:2000.

[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.

Immunogenicity of the DNA/Protein Combined Vaccine against COVID-19.

During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity.

[Development and assessment of a reagent kit for RNA detection of CrimeanCongo hemorrhagic fever virus with using reverse transcription loop-mediated isothermal amplification method.]

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector¼ (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.

[TULA HANTAVIRUS IN CRIMEA].

Genetic evidence of the Tula virus (TULV) in Crimea region of Russia is presented. Based on the reverse transcription PCR and subsequent sequence analysis, a total of 4 RNA isolates of the TULV were identified from the tissue samples of the Altai voles Microtus obscurus captured in the Bakhchisaray district of the Republic Crimea. Phylogenetic analysis of the S-, M-, and L-segment sequences of the Crimean TULV strains showed that they formed distinct genetic lineage, Russia IV, in the TULV variant. New sequences were most closely related to the lineage Russia I sequences obtained from common vole (M. arvalis) captured in the Tula region in Central Russia

[Intranasal vaccine against COVID-19 based on a recombinant variant of the Sendai virus (Paramyxoviridae: Respirovirus) strain Moscow].

INTRODUCTION: Intranasal vaccination using live vector vaccines based on non-pathogenic or slightly pathogenic viruses is the one of the most convenient, safe and effective ways to prevent respiratory infections, including COVID-19. Sendai virus is the best suited for this purpose, since it is respiratory virus and is capable of limited replication in human bronchial epithelial cells without causing disease. The aim of the work is to design and study the vaccine properties of recombinant Sendai virus, Moscow strain, expressing secreted receptor-binding domain of SARS-CoV-2 Delta strain S protein (RBDdelta) during a single intranasal immunization. MATERIALS AND METHODS: Recombinant Sendai virus carrying insertion of RBDdelta transgene between P and M genes was constructed using reverse genetics and synthetic biology methods. Expression of RBDdelta was analyzed by Western blot. Vaccine properties were studied in two models: Syrian hamsters and BALB/c mice. Immunogenicity was evaluated by ELISA and virus-neutralization assays. Protectiveness was assessed by quantitation of SARS-CoV-2 RNA in RT-PCR and histological analysis of the lungs. RESULTS: Based on Sendai virus Moscow strain, a recombinant Sen-RBDdelta(M) was constructed that expressed a secreted RBDdelta immunologically identical to natural SARS-CoV-2 protein. A single intranasal administration of Sen-RBDdelta(M) to hamsters and mice significantly, by 15 and 107 times, respectively, reduced replicative activity of SARS-CoV-2 in lungs of animals, preventing the development of pneumonia. An effective induction of virus-neutralizing antibodies has also been demonstrated in mice. CONCLUSION: Sen-RBDdelta(M) is a promising vaccine construct against SARS-CoV-2 infection and has a protective properties even after a single intranasal introduction.

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.

Surveillance and identification of influenza A viruses in wild aquatic birds in the Crimea, Ukraine (2006-2008).

The ecology of avian influenza (AI) viruses in wild aquatic birds of Asia is poorly understood, especially for the H5N1 high pathogenicity AI (HPAI) viruses. From March 2006 through November 2008, 20 AI viruses were isolated in the Crimea region of Ukraine with an overall frequency of virus recovery of 3.3%. All the viruses were isolated from three species of dabbling ducks: mallard (Anas platyrhynchos), wigeon (Anas penelope), and garganey (Anas querquedula), making the frequency of virus recovery for dabbling ducks 6.3%. The viruses were predominantly isolated during the fall sampling period. All viruses were genetically and antigenically characterized. No H5N1 HPAI viruses were isolated, but other HA and NA subtypes were identified including H3N1 (2), H3N6 (3), H3N8 (4), H4N6 (6), H5N2 (3), H7N8 (1), and H10N6 (1) subtypes. All isolates were of low pathogenicity, as determined by the intravenous pathogenicity index of 0.00. For H5N2 and H7N8 isolates, the HA gene was sequenced and the phylogenetic analysis revealed possible ecologic connections of the Crimea region with AI viruses from Siberia and Europe. No influenza A isolates were recovered from other Anseriformes (diving ducks [two species of pochards] and graylag geese), Columbiformes (collared doves), Gruiformes (coot), and Galliformes (gray partridges).

Pathogenesis of infectious disease of mice caused by H5N1 avian influenza virus.

The pathogenesis of a disease caused by Qinghai-like H5N1 influenza virus in BALB/c mice was studied. Clinical, morphological, and immunological characteristics of the experimental infection caused by highly pathogenic A/duck/Tuva/01/06/ (H5N1) virus are described.

Pathogenicity assessment of wild-type and mouse-adapted influenza A(H1N1) pdm09 viruses in comparison with highly pathogenic influenza A(H5N1) virus.

Here we compare the results of pathological and virological examinations of mice experimentally infected with either wild-type or mouse-adapted pandemic A(H1N1) pdm09 viruses and highly pathogenic avian influenza (HPAI) virus A(H5N1). Mice were sacrificed on days 1, 3, 6, and 10 post infection or whenever morbidity was severe enough to justify euthanasia. Morbidity rates were calculated on the basis of clinical signs (weight loss, poor hair coat, hunched posture and paresis); virus-induced disease was characterised by the histopathology of lung; virus dissemination was determined by virus isolation on organ samples of lung, brain, liver, kidney and spleen. All mice infected with mouse-adapted A(H1N1) pdm09 died in the course of the experiment, whereas 20% of animals survived the infection with A(H5N1). Echinocyte formation changed the rheological properties of blood in animals infected with either mouse-adapted A(H1N1) pdm09 or A(H5N1). To sum up, the adaptation of pandemic A(H1N1) pdm09 virus can confer an enhanced virulence similar to or even exceeding that of HPAI A(H5N1) virus.

Studying the pathogenicity of avian influenza virus subtype H5N1 strains from the Russian Federation using mouse model.

The pathogenicity of strains of avian influenza virus subtype H5N1 from the Russian Federation (A/turkey/Suzdalka/12/05, A/goose/Krasnoozerskoye/627/05, A/duck/Tuva/01/06, A/chicken/Reshoty/02/06, and A/chicken/Krasnodar/123/060) was studied in mice. Morphological and immunological characteristics of experimental viral infection with avian influenza virus subtype H5N1 were evaluated.