The present study assesses copper metabolism of the host organism as a target of antiviral strategy, basing on the “virocell” concept. Silver nanoparticles (AgNPs) were used as a specific active agent because they reduce the level of holo-ceruloplasmin, the main extracellular cuproenzyme. The mouse model of influenza virus A infection was used with two doses: 1 LD₅₀ and 10 LD₅₀. Three treatment regimens were used: Scheme 1—mice were pretreated 4 days before infection and then every day during infection development; Scheme 2—mice were pretreated four days before infection and on the day of virus infection; Scheme 3—virus infection and AgNP treatment started simultaneously, and mice were injected with AgNPs until the end of the experiment. The mice treated by Scheme 1 demonstrated significantly lower mortality, the protection index reached 60–70% at the end of the experiment, and mean lifespan was prolonged. In addition, the treatment of the animals with AgNPs resulted in normalization of the weight dynamics. Despite the amelioration of the infection, AgNP treatment did not influence influenza virus replication. The possibility of using nanosilver as an effective indirectly-acting antiviral drug is discussed.

The influenza virus continually evolves because of the high mutation rate, resulting in dramatic changes in its pathogenicity and other biological properties. This study aimed to evaluate the evolution of certain essential properties, understand the connections between them, and find the molecular basis for the manifestation of these properties. To that end, 21 A(H1N1)pdm09 influenza viruses were tested for their pathogenicity and toxicity in a mouse model with a ts/non-ts phenotype manifestation and HA thermal stability. The results demonstrated that, for a strain to have high pathogenicity, it must express a toxic effect, have a non-ts phenotype, and have a thermally stable HA. The ancestor A/California/07/2009 (H1N1)pdm influenza virus expressed the non-ts phenotype, after which the cycling trend of the ts/non-ts phenotype was observed in new strains of A(H1N1)pdm09 influenza viruses, indicating that the ratio of the ts phenotype will increase in the coming years. Of the 21 tested viruses, A/South Africa/3626/2013 had the high pathogenicity in the mouse model. Sequence alignment analysis showed that this virus has three unique mutations in the polymerase complex, two of which are in the PB2 gene and one that is in the PB1 gene. Further study of these mutations might explain the distinguishing pathogenicity.

The number of lung-adapted influenza viruses is limited. Most of them are not antigenically related to current circulating viruses. Viruses similar to recent strains are required for screening modern antiviral compounds and studying new vaccine candidates against novel influenza viruses. The process by which an influenza virus adapts to a new host is rather difficult. The aim of this study was to select a non-adapted current virus whose major biological properties correspond to those of classical lab-adapted viruses. Mice were inoculated intranasally with non-lung-adapted influenza viruses of subtype H1N1pdm09. They were monitored closely for body weight loss, mortality outcomes and gross pathology for 14 days following inoculation, as well as viral replication in lung tissue. Lung-adapted PR8 virus was used as a control. The tested viruses multiplied equally well in the lower respiratory tract of mice without prior adaptation but dramatically differed in lethality; the differences in their toxicity and pathogenicity in mice were established. A/South Africa/3626/2013 (H1N1)pdm09 virus was found to be an appropriate candidate to replace PR8 as a model virus for influenza research. No prior adaptation to the animal model is needed to reach the pathogenicity level of the classical mouse-adapted PR8 virus.

Background/Objectives: Influenza viruses and SARS-CoV-2 are currently cocirculating with similar seasonality, and both pathogens are characterized by a high mutational rate which results in reduced vaccine effectiveness and thus requires regular updating of vaccine compositions. Vaccine formulations combining seasonal influenza and SARS-CoV-2 strains can be considered promising and cost-effective tools for protection against both infections. Methods: We used a licensed seasonal trivalent live attenuated influenza vaccine (3×LAIV) as a basis for the development of a modified 3×LAIV/CoV-2 vaccine, where H1N1 and H3N2 LAIV strains encoded an immunogenic cassette enriched with conserved T-cell epitopes of SARS-CoV-2, whereas a B/Victoria lineage LAIV strain was unmodified. The trivalent LAIV/CoV-2 composition was compared to the classical 3×LAIV in the golden Syrian hamster model. Animals were intranasally immunized with the mixtures of the vaccine viruses, twice, with a 3-week interval. Immunogenicity was assessed on day 42 of the study, and the protective effect was established by infecting vaccinated hamsters with either influenza H1N1, H3N2 or B viruses or with SARS-CoV-2 strains of the Wuhan, Delta and Omicron lineages. Results: Both the classical 3×LAIV and 3×LAIV/CoV-2 vaccine compositions induced similar levels of serum antibodies specific to all three influenza strains, which resulted in comparable levels of protection against challenge from either influenza strain. Protection against SARS-CoV-2 challenge was more pronounced in the 3×LAIV/CoV-2-immunized hamsters compared to the classical 3×LAIV group. These data were accompanied by the higher magnitude of virus-specific cellular responses detected by ELISPOT in the modified trivalent LAIV group. Conclusions: The modified trivalent live attenuated influenza vaccine encoding the T-cell epitopes of SARS-CoV-2 can be considered a promising tool for combined protection against seasonal influenza and COVID-19.

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44⁺CD62L⁻) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP₃₆₆); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.