COVID-19 has killed more than 4 million people to date and is the most significant global health problem. The first recorded case of COVID-19 had been noted in Wuhan, China in December 2019, and already on March 11, 2020, World Health Organization declared a pandemic due to the rapid spread of this infection. In addition to the damage to the respiratory system, SARS-CoV-2 is capable of causing severe complications that can affect almost all organ systems. Due to the insufficient effectiveness of the COVID-19 therapy, there is an urgent need to develop effective specific medicines. Among the known approaches to the creation of antiviral drugs, a very promising direction is the development of drugs whose action is mediated by the mechanism of RNA interference (RNAi). A small interfering RNA (siRNA) molecule suppresses the expression of a target gene in this regulatory pathway. The phenomenon of RNAi makes it possible to quickly create a whole series of highly effective antiviral drugs, if the matrix RNA (mRNA) sequence of the target viral protein is known. This review examines the possibility of clinical application of siRNAs aimed at suppressing reproduction of the SARS-CoV-2, taking into account the experience of similar studies using SARS-CoV and MERS-CoV infection models. It is important to remember that the effectiveness of siRNA molecules targeting viral genes may decrease due to the formation of viral resistance. In this regard, the design of siRNAs targeting the cellular factors necessary for the reproduction of SARS-CoV-2 deserves special attention.

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

Background: Influenza is one of the most common infectious diseases, which affect the lower respiratory tract, and can lead to serious complications including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of influenza A virus in human lung cell culture. Materials and methods: Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL-185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting of the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann-Whitney U test was used for statistical analysis. Results: In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day; 26 and 29 times on the second day; 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has decreased 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times. Conclutions: We identified a number of genes such as FLT4, Nup98, and Nup205, the decrease in the expression of which can effectively suppress viral reproduction. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.

Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.

Objectives. Evaluation of changes in the viral activity of influenza A/WSN/33 after complex knockdown of combinations of cellular genes FLT4, Nup98 and Nup205 in human lung cell culture A549. Methods. The work was carried out using the equipment of the Center for Collective Use of the I. Mechnikov Research Institute of Vaccines and Sera, Russia. The authors performed transfection of combinations of small interfering ribonucleic acid (siRNA) complexes that cause simultaneous disruption of the expression of cellular genes FLT4, Nup98, and Nup205. Within three days from the moment of transfection and infection, the supernatant fluid and cell lysate were taken for subsequent viral reproduction intensity determination using the titration method for cytopathic action. The dynamics of changes in the concentration of viral ribonucleic acid (vRNA) was determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The nonparametric Mann–Whitney test was used to calculate statistically significant differences between groups.Results. Using all of the combinations of siRNA complexes, cell viability did not decrease below the threshold level of 70%. In cells treated with complex FLT4.2 + Nup98.1 + Nup205 at the multiplicity of infection (MOI) equal to 0.1, a significant decrease in viral reproduction by 1.5 lg was noted on the first day in relation to nonspecific and viral controls. The use of siRNA complexes at MOI 0.01 resulted in a more pronounced antiviral effect. The viral titer in cells treated with siRNA complexes FLT4.2 + Nup98.1 and Nup98.1 + Nup205 decreased by 1.5 lg on the first day. In cells treated with complexes FLT4.2 + Nup205 and FLT4.2 + Nup98.1 + Nup205, it decreased by 1.8 and 2.0 lg on the first day and by 1.8 and 2.5 lg on the second day, respectively, in relation to nonspecific and viral controls. When conducting real-time RT-PCR, a significant decrease in the concentration of vRNA was noted. At MOI 0.1, a 295, 55, and 63-fold decrease in the viral load was observed with the use of siRNA complexes FLT4.2 + Nup98.1, Nup98.1 + Nup205, and FLT4.2 + Nup98.1 + Nup205, respectively. On the second day, a decrease in vRNA was also observed in cells treated with complex A. A 415-fold decrease in vRNA on the third day was noted in cells treated with complex FLT4.2 + Nup205. At MOI 0.01, the concentration of vRNA decreased 9.5 times when using complex B relative to nonspecific and viral control.Conclusions. The study showed a pronounced antiviral effect of siRNA combinations while simultaneously suppressing the activity of cellular genes (FLT4, Nup98, and Nup205), whose expression products are playing important role in the viral reproduction process, and obtained original designs of siRNA complexes. The results obtained are of great importance for the creation of emergence prophylactic and therapeutic drugs, whose action is based on the mechanism of RNA interference.

Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.