In August 2022 on a backyard farm in the Moscow region of Russia, mortality was observed among chickens, and all 45 birds of a particular farm died or were slaughtered after the onset of symptoms within a few days. Paramyxovirus was isolated from the diseased birds. Based on the nucleotide sequences of the F and NP gene fragments, it was determined that the virus belonged to subgenotype VII.1 AAvV-1 class II. The cleavage site of the F gene ₁₀₉SGGRRQKRFIG₁₁₉ and T in 546 and 555 position of the NP gene were typical for the velogenic type. The genetically closest NDV isolates were found in Iran. The mean time of death of 10-day-old chicken embryos upon infection with the minimal infectious dose was 52 h, which is typical for the velogenic pathotype. The virus caused 100% death of six-week-old chickens during oral infection as well as 100% mortality of all contact chickens, including those located in remote cages, which proves the ability of the virus to spread not only by the fecal–oral route but also by the aerosol route. That demonstrates a high level of pathogenicity and contagiousness of the isolated strain for chicken. However, mice intranasally infected with high doses of the virus did not die.

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 widespread respiratory infection, accompanied by damage to the lower respiratory tract of a person, and can lead to severe complications, up to a fatal outcome. Currently existing therapeutic agents and vaccines do not provide fully effective protection against influenza viruses. The development and creation of drugs based on the mechanism of RNA interference in the context of this problem is a promising direction. The aim of this study is to select and experimentally substantiate cellular target genes for miRNAs whose knockdown suppresses viral reproduction. A549 cells (human lung adenocarcinoma) were transfected with small interfering RNAs. After 4 hours, the transfected cells were infected with the influenza virus pri MOI = 0.1; 0.01 and 0.001. The virus-containing liquid was sampled within three days from the moment of transfection and the intensity of the dynamics of viral reproduction was assessed by the CPD titration method. The use of all small interfering RNAs at MOI = 0.1 resulted in a significantly significant decrease in the viral titer relative to non-specific control. Similar results were obtained at MOI = 0.01 and 0.001. The most effective siRNA ISSINUP98, since when using it, at MOI = 0.1, the viral titer values decreased by 1.7 lg TCD50/ml and by 3 lg TCD50/ml at MOI = 0.01 on the third day. The results showed that miRNAs directed to human cellular genes FLT4, Nup98 and Nup205, whose derivatives play an important role in the life cycle of the influenza virus, effectively reduce its reproduction in vitro. Thus, the studied genes and their products are potential targets for the development of anti-influenza drugs.

The introduction of genetic engineering methods into the production of live influenza vaccines provides significant optimization of separate stages of this process. In recent years, special attention has been paid to a method based on the direct incorporation of well-known and well-studied mutations into the genome of epidemic variants of influenza virus. Such virus obtains the ts- and att phenotypes and can be used for the production of live influenza vaccines. The article discusses the applications of this approach and problems associated with its use in medical and veterinary vaccinology.