Molecular characterisation of influenza B virus from the 2017/18 season in primary models of the human lung reveals improved adaptation to the lower respiratory tract.

The 2017/18 influenza season was characterized by unusual high numbers of severe infections and hospitalizations. Instead of influenza A viruses, this season was dominated by infections with influenza B viruses of the Yamagata lineage. While this IBV/Yam dominance was associated with a vaccine mismatch, a contribution of virus intrinsic features to the clinical severity of the infections was speculated. Here, we performed a molecular and phenotypic characterization of three IBV isolates from patients with severe flu symptoms in 2018 and compared it to an IBV/Yam isolate from 2016 using experimental models of increasing complexity, including human lung explants, lung organoids, and alveolar macrophages. Viral genome sequencing revealed the presence of clade but also isolate specific mutations in all viral genes, except NP, M1, and NEP. Comparative replication kinetics in different cell lines provided further evidence for improved replication fitness, tolerance towards higher temperatures, and the development of immune evasion mechanisms by the 2018 IBV isolates. Most importantly, immunohistochemistry of infected human lung explants revealed an impressively altered cell tropism, extending from AT2 to AT1 cells and macrophages. Finally, transcriptomics of infected human lung explants demonstrated significantly reduced amounts of type I and type III IFNs by the 2018 IBV isolate, supporting the existence of additional immune evasion mechanisms. Our results show that the severeness of the 2017/18 Flu season was not only the result of a vaccine mismatch but was also facilitated by improved adaptation of the circulating IBV strains to the environment of the human lower respiratory tract.

Synergistic activity of an RNA polymerase PA-PB1 interaction inhibitor with oseltamivir against human and avian influenza viruses in cell culture and in ovo.

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections.

Dissemination of influenza B virus to the lower respiratory tract of mice is restricted by the interferon response.

The global burden of disease caused by influenza B virus (IBV) is substantial; however, IBVs remain overlooked. Understanding host-pathogen interactions and establishing physiologically relevant models of infection are important for the development and assessment of therapeutics and vaccines against IBV. In this study, we assessed an upper respiratory tract (URT)-restricted model of mouse IBV infection, comparing it to the conventional administration of the virus to the total respiratory tract (TRT). We found that URT infections caused by different strains of IBV disseminate to the trachea but resulted in limited dissemination of IBV to the lungs. Infection of the URT did not result in weight loss or systemic inflammation even at high inoculum doses and despite robust viral replication in the nose. Dissemination of IBV to the lungs was enhanced in mice lacking functional type I IFN receptor (IFNAR2), but not IFNγ. Conversely, in mice expressing the IFN-inducible gene Mx1, we found reduced IBV replication in the lungs and reduced dissemination of IBV from the URT to the lungs. Inoculation of IBV in both the URT and TRT resulted in seroconversion against IBV. However, priming at the TRT conferred superior protection from a heterologous lethal IBV challenge compared to URT priming, as determined by improved survival rates and reduced viral replication throughout the respiratory tract. Overall, our study establishes a URT-restricted IBV infection model, highlights the critical role of IFNs in limiting dissemination of IBV to the lungs, and also demonstrates that the lack of viral replication in the lungs may impact protection from subsequent infections. IMPORTANCE: Our study investigated how influenza B virus (IBV) spreads from the nose to the lungs of mice and the impact this has on disease and protection from re-infection. We found that when applied to the nose only, IBV does not spread very efficiently to the lungs in a process controlled by the interferon response. Priming immunity at the nose only resulted in less protection from re-infection than priming immunity at both the nose and lungs. These insights can guide the development of potential therapies targeting the interferon response as well as of intranasal vaccines against IBV.

Effect of combined infection with Salmonella and influenza virus on their respective proliferation in chicken embryonated eggs.

Background: Salmonella is a major food-borne bacterial pathogen that causes food poisoning related to the consumption of eggs, milk, and meat. Food safety in relation to Salmonella is particularly important for eggs because their shells as well as their contents can be a source of contamination. Chicken can also be infected with influenza virus, but it remains unclear how co-infection of Salmonella and influenza virus affect each other. Aim: The potential influence of co-infection of Salmonella and influenza virus was examined. Methods: Salmonella Abony and influenza virus were injected into chicken embryonated eggs. After incubation, proliferation of Salmonella and influenza virus was measured using a direct culture assay for bacteria and an enzyme-linked immunosorbent assay for influenza virus, respectively. Results: Our findings indicate that the number of colony-forming units (CFUs) of Salmonella did not vary between chicken embryonated eggs co-infected with influenza A virus and Salmonella-only infected eggs. Furthermore, we found the proliferation of influenza A or B virus was not significantly influenced by co-infection of the eggs with Salmonella. Conclusion: These results suggest that combined infection of Salmonella with influenza virus does not affect each other, at least in terms of their proliferation.

Modeling the effects of cigarette smoke extract on influenza B virus infections in mice.

Influenza B virus (IBV) is a major respiratory viral pathogen. Due to a lack of pandemic potential for IBV, there is a lag in research on IBV pathology and immunological responses compared to IAV. Therefore, the impact of various lifestyle and environmental factors on IBV infections, such as cigarette smoking (CS), remains elusive. Despite the increased risk and severity of IAV infections with CS, limited information exists on the impact of CS on IBV infections due to the absence of suitable animal models. To this end, we developed an animal model system by pre-treating mice for two weeks with cigarette smoke extract (CSE), then infected them with IBV and monitored the resulting pathological, immunological, and virological effects. Our results reveal that the CSE treatment decreased IBV specific IgG levels yet did not change viral replication in the upper airway/the lung, and weight recovery post infection. However, higher concentrations of CSE did result in higher mortality post infection. Together, this suggests that CS induced inflammation coupled with IBV infection resulted in exacerbated disease outcome.

Sequential infections with rhinovirus and influenza modulate the replicative capacity of SARS-CoV-2 in the upper respiratory tract.

Although frequently reported since the beginning of the pandemic, questions remain regarding the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interaction with circulating respiratory viruses in coinfected patients. We here investigated dual infections involving early-pandemic SARS-CoV-2 and the Alpha variant and three of the most prevalent respiratory viruses, rhinovirus (RV) and Influenza A and B viruses (IAV and IBV), in reconstituted respiratory airway epithelial cells cultured at air-liquid interface. We found that SARS-CoV-2 replication was impaired by primary, but not secondary, rhino- and influenza virus infection. In contrast, SARS-CoV-2 had no effect on the replication of these seasonal respiratory viruses. Inhibition of SARS-CoV-2 correlated better with immune response triggered by RV, IAV and IBV than the virus entry. Using neutralizing antibody against type I and III interferons, SARS-CoV-2 blockade in dual infections could be partly prevented. Altogether, these data suggested that SARS-CoV-2 interaction with seasonal respiratory viruses would be modulated by interferon induction and could impact SARS-CoV-2 epidemiology when circulation of other respiratory viruses is restored.

Synthesis and structure-activity relationship of L-methionine-coupled 1,3,4-thiadiazole derivatives with activity against influenza virus.

In previous investigations, we identified a class of 1,3,4-thiadiazole derivatives with antiviral activity. N-{3-(Methylsulfanyl)-1-[5-(phenylamino)-1,3,4-thiadiazole-2-yl]propyl}benzamide emerged as a relevant lead compound for designing novel influenza A virus inhibitors. In the present study, we elaborated on this initial lead by performing chemical synthesis and antiviral evaluation of a series of structural analogues. During this research, thirteen novel 1,3,4-thiadiazole derivatives were synthesized by the cyclization of the corresponding thiosemicarbazides as synthetic precursors. The structures and the purities of the synthesized compounds were confirmed through chromatographic and spectral data. Four L-methionine-based 1,3,4-thiadiazole derivatives displayed activity against influenza A virus, the two best compounds being 24 carrying a 5-(4-chlorophenylamino)-1,3,4-thiadiazole moiety and 30 possessing a 5-(benzoylamino)-1,3,4-thiadiazole structure [antiviral EC50 against influenza A/H3N2 virus: 4.8 and 7.4 µM, respectively]. The 1,3,4-thiadiazole derivatives were inactive against influenza B virus and a wide panel of unrelated DNA and RNA viruses. Compound 24 represents a new class of selective influenza A virus inhibitors acting during the virus entry process, as evidenced by our findings in a time-of-addition assay. Molecular descriptors and in silico prediction of ADMET properties of the active compounds were calculated. According to in silico ADMET and drug similarity studies, active compounds have been estimated to be good candidates for oral administration with no apparent toxicity considerations.

Limbic encephalitis in a child with ovarian teratoma and influenza B. Case report and critical review of the history of autoimmune anti-N-methyl-d-aspartate receptor encephalitis.

We report the appearance of clinical symptoms and signs of N-methyl-d-Aspartate (NMDA) receptor encephalitis in a patient presenting just days after contraction of influenza B. The offending mature ovarian teratoma was identified and removed on the 10th day after the appearance of symptoms, with subsequent nearly complete resolution of symptoms over the subsequent 6 months. We provide a focused literature review of the clinical and pathophysiologic literature of anti-NMDA receptor encephalitis pertaining to influenza B virus and the pediatric population. Taken together, this study contributes to the pathophysiological understanding of anti-NMDA receptor encephalitis and aids clinicians in its early recognition and management.

Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.

CD8+ T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550-558-specific CD8+ T cells being cross-reactive between IAV and IBV. Memory CD8+ T cells towards these specificities are present in blood (CD27+CD45RA- phenotype) and tissues (CD103+CD69+ phenotype) of healthy individuals, and effector CD27-CD45RA-PD-1+CD38+CD8+ T cells in IAV/IBV patients. Our data show influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.

G-Protein-Coupled Receptor and Ion Channel Genes Used by Influenza Virus for Replication.

Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza virus for replication. Targeting these proinfluenza genes can provide therapeutic strategies to reduce virus replication. Nineteen proinfluenza G-protein-coupled receptor (GPCR) and 13 proinfluenza ion channel genes were identified in human lung (A549) cells by use of small interfering RNAs (siRNAs). These proinfluenza genes were authenticated by testing influenza virus A/WSN/33-, A/CA/04/09-, and B/Yamagata/16/1988-infected A549 cells, resulting in the validation of 16 proinfluenza GPCR and 5 proinfluenza ion channel genes. These findings showed that several GPCR and ion channel genes are needed for the production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza virus productive cycle so as to limit infection.IMPORTANCE Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation, since a mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza virus during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. The targeting of host genes is refractory to drug resistance generated by viral mutations, as well as providing a platform for the development of broad-spectrum antiviral drugs.

Anomalous influenza seasonality in the United States and the emergence of novel influenza B viruses.

The 2019/2020 influenza season in the United States began earlier than any season since the 2009 H1N1 pandemic, with an increase in influenza-like illnesses observed as early as August. Also noteworthy was the numerical domination of influenza B cases early in this influenza season, in contrast to their typically later peak in the past. Here, we dissect the 2019/2020 influenza season not only with regard to its unusually early activity, but also with regard to the relative dynamics of type A and type B cases. We propose that the recent expansion of a novel influenza B/Victoria clade may be associated with this shift in the composition and kinetics of the influenza season in the United States. We use epidemiological transmission models to explore whether changes in the effective reproduction number or short-term cross-immunity between these viruses can explain the dynamics of influenza A and B seasonality. We find support for an increase in the effective reproduction number of influenza B, rather than support for cross-type immunity-driven dynamics. Our findings have clear implications for optimal vaccination strategies.

Comparison of influenza type A and B with COVID-19: A global systematic review and meta-analysis on clinical, laboratory and radiographic findings.

We compared clinical symptoms, laboratory findings, radiographic signs and outcomes of COVID-19 and influenza to identify unique features. Depending on the heterogeneity test, we used either random or fixed-effect models to analyse the appropriateness of the pooled results. Overall, 540 articles included in this study; 75,164 cases of COVID-19 (157 studies), 113,818 influenza type A (251 studies) and 9266 influenza type B patients (47 studies) were included. Runny nose, dyspnoea, sore throat and rhinorrhoea were less frequent symptoms in COVID-19 cases (14%, 15%, 11.5% and 9.5%, respectively) in comparison to influenza type A (70%, 45.5%, 49% and 44.5%, respectively) and type B (74%, 33%, 38% and 49%, respectively). Most of the patients with COVID-19 had abnormal chest radiology (84%, p < 0.001) in comparison to influenza type A (57%, p < 0.001) and B (33%, p < 0.001). The incubation period in COVID-19 (6.4 days estimated) was longer than influenza type A (3.4 days). Likewise, the duration of hospitalization in COVID-19 patients (14 days) was longer than influenza type A (6.5 days) and influenza type B (6.7 days). Case fatality rate of hospitalized patients in COVID-19 (6.5%, p < 0.001), influenza type A (6%, p < 0.001) and influenza type B was 3%(p < 0.001). The results showed that COVID-19 and influenza had many differences in clinical manifestations and radiographic findings. Due to the lack of effective medication or vaccine for COVID-19, timely detection of this viral infection and distinguishing from influenza are very important.

Altered NKp46 Recognition and Elimination of Influenza B Viruses.

Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a binding affinity approximately seven times lower than NKp46 binding of influenza A's HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that NKp46 is not important for influenza B elimination. These findings enable us to better understand the interactions between the different influenza viruses and NK cells that are known to be crucial for viral elimination.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

Hsa-miR-30e-3p inhibits influenza B virus replication by targeting viral NA and NP genes.

Influenza B virus is a member of the Orthomyxoviridae family which can infect humans and causes influenza. Although it is not pandemic like influenza A virus, it nevertheless affects millions of people worldwide annually. MicroRNAs are small non-coding RNAs regulating gene expression at posttranscriptional level. They play various important roles in cellular processes including response to viral infection. MiRNA profiles from our previous study suggested that miR-30e-3p was one of the upregulated miRNAs that responded to influenza B virus infection. In this study, in silico prediction and in vitro investigation proved that this miRNA can directly target NA and NP genes of the influenza B virus and inhibit its replication. This finding might be useful for using miRNA as an alternative therapeutics for influenza virus infection.

A large effective population size for established within-host influenza virus infection.

Strains of the influenza virus form coherent global populations, yet exist at the level of single infections in individual hosts. The relationship between these scales is a critical topic for understanding viral evolution. Here we investigate the within-host relationship between selection and the stochastic effects of genetic drift, estimating an effective population size of infection Ne for influenza infection. Examining whole-genome sequence data describing a chronic case of influenza B in a severely immunocompromised child we infer an Ne of 2.5 × 107 (95% confidence range 1.0 × 107 to 9.0 × 107) suggesting that genetic drift is of minimal importance during an established influenza infection. Our result, supported by data from influenza A infection, suggests that positive selection during within-host infection is primarily limited by the typically short period of infection. Atypically long infections may have a disproportionate influence upon global patterns of viral evolution.

The association between the seasonality of pediatric pandemic influenza virus outbreak and ambient meteorological factors in Shanghai.

BACKGROUND AND OBJECTIVES: The number of pediatric patients diagnosed with influenza types A and B is increasing annually, especially in temperate regions such as Shanghai (China). The onset of pandemic influenza viruses might be attributed to various ambient meteorological factors including temperature, relative humidity (Rh), and PM1 concentrations, etc. The study aims to explore the correlation between the seasonality of pandemic influenza and these factors. METHODS: We recruited pediatric patients aged from 0 to 18 years who were diagnosed with influenza A or B from July 1st, 2017 to June 30th, 2019 in Shanghai Children's Medical Centre (SCMC). Ambient meteorological data were collected from the Shanghai Meteorological Service (SMS) over the same period. The correlation of influenza outbreak and meteorological factors were analyzed through preliminary Pearson's r correlation test and subsequent time-series Poisson regression analysis using the distributed lag non-linear model (DLNM). RESULTS: Pearson's r test showed a statistically significant correlation between the weekly number of influenza A outpatients and ambient meteorological factors including weekly mean, maximum, minimum temperature and barometric pressure (P < 0.001), and PM1 (P < 0.01). While the weekly number of influenza B outpatients was statistically significantly correlated with weekly mean, maximum and minimum temperature (P < 0.001), barometric pressure and PM1 (P < 0.01), and minimum Rh (P < 0.05). Mean temperature and PM1 were demonstrated to be the statistically significant variables in the DLNM with influenza A and B outpatients through time-series Poisson regression analysis. A U-shaped curve relationship was noted between the mean temperature and influenza A cases (below 15 °C and above 20 °C), and the risks increased for influenza B with mean temperature below 10 °C. PM1 posed a risk after a concentration of 23 ppm for both influenza A and B. High PM1, low and the high temperature had significant effects upon the number of influenza A cases, whereas low temperature and high PM1 had significant effects upon the number of influenza B cases. CONCLUSION: This study indicated that mean temperature and PM1 were the primary factors that were continually associated with the seasonality of pediatric pandemic influenza A and B and the recurrence in the transmission and spread of influenza viruses.

Next-Generation Sequencing Analysis of Cellular Response to Influenza B Virus Infection.

Influenza B virus (IBV) is a respiratory pathogen that infects humans and causes seasonal influenza epidemics. However, cellular response to IBV infection in humans and mechanisms of host-mediated restriction of IBV replication are not thoroughly understood. In this study, we used next-generation sequencing (NGS) to perform transcriptome profiling of IBV-infected human lung epithelial A549 cells at 0, 6, 12, and 24 h post infection (hpi) and characterized the cellular gene expression dynamics. We observed that more than 4000 host genes were differentially regulated during the study period, which included up regulation of genes encoding proteins, having a role in the innate antiviral immune responses, immune activation, cellular metabolism, autophagy, and apoptosis, as well as down regulation of genes involved in mitosis and cell proliferation. Further analysis of RNA-Seq data coupled with RT-qPCR validation collectively showed that double-strand RNA recognition pathways, including retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3), were substantially activated following IBV infection. Taken together, these results provide important initial insights into the intimate interaction between IBV and lung epithelial cells, which can be further explored towards elucidation of the cellular mechanisms in restriction or elimination of IBV infections in humans.

Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.