Cold-adapted SARS-CoV-2 variants with different temperature sensitivity exhibit an attenuated phenotype and confer protective immunity.

As novel SARS-CoV-2 Variants of Concern emerge, the efficacy of existing vaccines against COVID-19 is declining. A possible solution to this problem lies in the development of a live attenuated vaccine potentially able of providing cross-protective activity against a wide range of SARS-CoV-2 antigenic variants. Cold-adapted (ca) SARS-CoV-2 variants, Dubrovka-ca-B4 (D-B4) and Dubrovka-ca-D2 (D-D2), were obtained after long-term passaging of the Dubrovka (D) strain in Vero cells at reduced temperatures. Virulence, immunogenicity, and protective activity of SARS-CoV-2 variants were evaluated in experiments on intranasal infection of Syrian golden hamsters (Mesocricetus auratus). In animal model infecting with ca variants, the absence of body weight loss, the significantly lower viral titer and viral RNA concentration in animal tissues, the less pronounced inflammatory lesions in animal lungs as compared with the D strain indicated the reduced virulence of the virus variant. Single intranasal immunization with D-B4 and D-D2 variants induced the production of neutralizing antibodies in hamsters and protected them from infection with the D strain and the development of severe pneumonia. It was shown that for ca SARS-CoV-2 variants, the temperature-sensitive (ts) phenotype was not obligate for virulence reduction. Indeed, the D-B4 variant, which did not possess the ts phenotype but had lost the ability to infect human lung cells Calu-3, exhibited reduced virulence in hamsters. Consequently, the potential phenotypic markers of attenuation of ca SARS-CoV-2 variants are the ca phenotype, the ts phenotype, and the change in species specificity of the virus. This study demonstrates the great potential of SARS-CoV-2 cold adaptation as a strategy to develop a live attenuated COVID-19 vaccine.

Transcriptome Analysis of Human Glioblastoma Cells Susceptible to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Glioblastoma multiforme (GBM) accounts for almost half of all primary malignant brain tumors in adults and has a poor prognosis. Here we demonstrated the oncolytic potential of the L-16 vaccine strain of measles virus (MV) against primary human GBM cells and characterized the genetic patterns that determine the sensitivity of primary human GBM cells to oncolytic therapy. MV replicated in all GBM cells, and seven out of eight cell lines underwent complete or partial oncolysis. RNA-Seq analysis identified about 1200 differentially expressed genes (FDR < 0.05) with at least two-fold expression level change between MV-infected and uninfected cells. Among them, the most significant upregulation was observed for interferon response, apoptosis and cytokine signaling. One out of eight GBM cell lines was defective in type I interferon production and, thus, in the post-interferon response, other cells lacked expression of different cellular defense factors. Thus, none of the cell lines displayed induction of the total gene set necessary for effective inhibition of MV replication. In the resistant cells, we detected aberrant expression of metalloproteinase genes, particularly MMP3. Thus, such genes could be considered intriguing candidates for further study of factors responsible for cell sensitivity and resistance to L-16 MV infection.

New approach of genetic characterization of group A rotaviruses by the nanopore sequencing method.

Nanopore sequencing of virus genomes represented by segmented RNA (e.g. rotaviruses) requires the development of specific approaches. Due to the massive use of rotavirus vaccines, the relevance of monitoring the genetic diversity of circulating strains of group A rotaviruses (RVA) increased. The WHO recommended method of multiplex type-specific PCR does not allow genotyping of all clinically significant strains of RVA and identifying inter-strain differences within the genotype. We have described a new principle of amplification of RVA gene segments using six primers for reverse transcription and one universal primer for PCR for nanopore sequencing. The amplification of RVA genome was tested on clinical samples and three phylogenetically distant laboratory RVA strains, Wa (G1P[8]), DS-1 (G2P[4]) and 568 (G3P[3]). The developed protocol of sample preparation and nanopore sequencing allowed obtaining full-length sequences for gene segments of RVA, including the diagnostically significant segments 9 (VP7), 4 (VP4) and 6 (VP6) with high accuracy and coverage. The accuracy of sequencing of the rotavirus genome exceeded 99.5 %, and the genome coverage varied for different strains from 59.0 to 99.6 % (on average 86 %). The developed approach of nanopore sequencing of RVA genome could be a prospective tool for epidemiological studies and surveillance of rotavirus infection.

The Susceptibility of Human Melanoma Cells to Infection with the Leningrad-16 Vaccine Strain of Measles Virus.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.

Molecular-Genetic Characterization of Human Rotavirus A Strains Circulating in Moscow, Russia (2009-2014).

Enteric viruses are the most common cause of acute gastroenteritis (AGE) in young children and a significant public health problem globally. Hospital admissions of children under 5 years of age with diarrhea are primarily associated with group A rotavirus (RVA) infection. In this retrospective study, the population structure of viruses linked to AGE etiology in young children hospitalized with AGE in Moscow was evaluated, and molecular characterization of RVA strains was performed. Fecal specimens were collected from children under 5 years old hospitalized with AGE between 2009 and 2014 in Moscow, Russia. Multiplex real-time reverse transcription PCR was used to detect enteric viruses and for G/[P]-genotyping of isolated RVAs. Sequencing of RVA VP7 and VP4 cDNA fragments was used to validate the data obtained by PCR-genotyping. The main causes for hospitalization of children with AGE were RVA (40.1%), followed by noroviruses (11.4%), while adenoviruses, astroviruses, sapoviruses, enteroviruses, and orthoreoviruses were detected in 4.7%, 1.9%, 1.4%, 1.2%, and 0.2% of samples tested, respectively. Nosocomial infections, predominantly associated with RVAs and noroviruses, were detected in 24.8% of cases and occurred significantly more frequently in younger infants. The predominant RVA genotype was G4P[8], detected in 38.7% of RVA-positive cases, whereas genotypes G1P[8], G9P[8], G3P[8], and G2P[4] were found in 11.8%, 6.6%, 4.2%, and 3.3% of cases, respectively. Together, the presence of circulating RVA strains with rare VP7 and VP4 gene variants (G6 and P[9]) highlights the need to conduct continuous epidemiological monitoring of RVA infection.