Humoral immunity to seasonal and zoonotic influenza in the population of the Russian Federation in 2021–2025

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Introduction

The influenza A virus poses a serious threat to human health; experts currently regard this pathogen as one of the major global infectious threats. The World Health Organization (WHO) recommends vaccinating at least 50% of the population and 75% of high-risk groups against seasonal influenza. According to the regulation of Chief Public Health Officer of the Russian Federation, at least 60% of the population of each constituent entity of the Russian Federation must be vaccinated against seasonal influenza, including 75% of individuals in high-risk groups¹.

In recent decades, annual influenza epidemics have been caused by viruses of the A(H1N1pdm09) subtype, A(H3N2), and influenza B viruses of the B/Victoria and B/Yamagata genetic lineages [1]. Since 2020, B/Yamagata virus variants have almost disappeared from circulation. In the 20^th and 21^st centuries influenza pandemics were caused exclusively by influenza A virus variants [2]. A pandemic virus arises as a result of gene reassortment between human and animal influenza virus strains and the subsequent adaptation of the pathogen to humans through the accumulation of mutations.

Currently, avian influenza viruses are becoming increasingly dangerous, primarily viruses of the subtypes A(H5Nx), A(H7Nx), and A(H9N2).

Russia has established a national influenza surveillance system, an integral part of which is the animal influenza surveillance system. A WHO Collaborating Center operates at the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor, with the primary objective of identifying influenza viruses with pandemic potential in the human population.

Most serological studies of influenza viruses in Russia are limited to specific regions or short observation periods and typically do not include zoonotic viruses. This study presents the results of an analysis of 16,594 blood serum samples collected in 2021–2025 in 40 regions across 6 federal districts: the Siberian Federal District, the Far Eastern Federal District, Volga Federal District, Ural Federal District, Southern Federal District, and North Caucasus Federal District. For the first time, data are presented from a 5-year surveillance of humoral immunity against seasonal (A(H1N1), A(H3N2), influenza B) and zoonotic (A(H5Nx), A(H9N2)) influenza viruses.

The aim of the study was to investigate human blood sera for influenza—both against vaccine strains and against influenza virus strains with pandemic potential.

Materials and methods

Blood serum samples were collected by staff of the Hygienic and Epidemiological Centers in the federal subjects of the Russian Federation prior to the epidemic seasons in 2021–2025. The study was conducted with the voluntary informed consent of the patients or their legal representatives.

The blood serum samples were provided in an anonymized form, which precludes the possibility of identifying the donors. Blood for serum preparation was collected by staff of the Hygienic and Epidemiological Centers from apparently healthy individuals using standard vacuum blood collection systems. The samples were transported to the State Research Center of Virology and Biotechnology “Vector” in thermal containers with cooling elements and stored at –20°C until testing [3]. Work with human serum samples was approved by the Ethics Committee of the State Research Center of Virology and Biotechnology “Vector” of Rospotrebnadzor (Protocol No. 7 dated April 18, 2023).

The hemagglutination inhibition (HI) assay uses diagnostic reagents (PPDP) based on current vaccine strains: A/Victoria/2570/2019 (H1N1)pdm09, A/Cambodia/e0826360/2020 (H3N2), A/Darwin/9/2021 (H3N2), A/Thailand/8/2022 (H3N2), B/Washington/02/2019 (B/Victoria lineage), B/Austria/1359417/2021 (B/Victoria lineage), B/Phuket/3073/2013 (B/Yamagata lineage). When processing the results, a confidence interval for the population mean was used, which was estimated based on the sample.

When testing sera against zoonotic viruses using hemagglutination inhibition (HI) and virus neutralization (VN) assays, antigens derived from strains A/Astrakhan/3212/2020 (H5N8) and A/chicken/Khabarovsk/24-12V/2022 (H5N1) (both strains are highly pathogenic for chickens), and the low-pathogenic strain A/chicken/Primorsky Krai/03/2018 (H9N2). All strains were isolated at the Vector State Research Center of Rospotrebnadzor.

HI assay was conducted in accordance with WHO guidelines². In the HI assay against vaccine virus strains, a serum dilution of 1 : 40 or higher was considered a diagnostic antibody titer. When testing sera against zoonotic strains, a serum dilution of 1 : 10 or higher was considered a diagnostic titer.

The titer of neutralizing serum antibodies was determined as the maximum serum dilution at which the number of focus-forming units (FFUs — foci of infected cells in a monolayer of MDCK cells stained with fluorescent antibodies) decreased by 50% compared to the mean FFU value in the control of the working dilution of the virus. The threshold for seropositivity in the VN assay against zoonotic strains was a serum dilution of 1:10.

For each group, the proportion of seropositive samples (titer ≥ 1 : 40) with 95% confidence intervals (CI) using Wilson’s method and the geometric mean titer with 95% CI using a parametric method based on the Student’s t-distribution for logarithmically transformed data.

The Kruskal–Wallis test was used to assess overall differences between seasons; where significant differences were found, pairwise comparisons of seasons were performed using the Mann–Whitney test. Within each season, separate analyses were performed: comparison of age groups and comparison of regions (Kruskal–Wallis test followed by pairwise comparisons). For the proportions of seropositive individuals in these same subgroups, Pearson’s χ² test was applied, followed by pairwise comparisons. In all cases, the Holm correction was used for multiple comparisons to control for Type I error. The significance level for all statistical tests was set at p < 0.05.

Results

In October–November 2021, during the COVID-19 pandemic, 1,344 blood serum samples were collected from healthy donors in five federal districts: the Siberian Federal District — 415 samples, the Southern Federal District — 267, the Far Eastern Federal District — 250, Volga Federal District — 156, and Ural Federal District — 256. Only 8–31% of the samples tested positive in HI with the vaccine strain A/Victoria/2570/2019 (H1N1)pdm09, 24–37% with A/Cambodia/e0826360/2020 (H3N2), 2–10% with B/Washington/02/2019 (B/Victoria lineage), and up to 25% with B/Phuket/3073/2013 (B/Yamagata lineage) (Fig. 1).

 

Fig. 1. The proportion of individuals tested seropositive for the current influenza vaccine strains on the eve of the 2021–2022 flu season.

 

Statistical analysis revealed significant regional differences in the prevalence of seropositivity to the A(H1N1)pdm09 (χ² = 38.2; p < 0.001) and B/Yamagata (χ² = 45.1; p < 0.001) viruses. For the A(H1N1)pdm09 virus, the highest prevalence was observed in the Urals Federal District (31%), and the lowest in the Far Eastern Federal District (8%). For the B/Yamagata virus, the highest rates were recorded in the Southern Federal District (31%) and the Siberian Federal District (25%), and the lowest in the Far Eastern Federal District (8%). Paired comparisons using the Holm correction confirmed the significance of differences between these extreme groups (p < 0.05). In all regions, the level of antibodies against the hemagglutinin of the B/Victoria virus remained low (2–10%), which likely reflects a general decline in herd immunity following the COVID-19 pandemic. For the A(H3N2) virus, the differences between regions did not reach statistical significance.

Thus, humoral immunity to seasonal influenza at the end of 2021 had significantly decreased compared with the seasons preceding the COVID-19 pandemic [4–6]. For example, during the 2019/2020 season, we detected antibodies to influenza A viruses in 40–68% of samples collected from all regions of the Russian Federation, and to influenza B/Victoria virus in 12–46% of samples [7]. During the COVID-19 pandemic, weekly monitoring revealed a change in the etiological profile of pathogens causing severe acute respiratory infections, with a sharp decline in the detection rate of influenza viruses [8].

In October–November 2022 (on the eve of the 2022/2023 flu season), 2,216 blood serum samples were collected in five federal districts: the Southern Federal District, the Volga Federal District, the North Caucasus Federal District, the Far Eastern Federal District, and the Siberian Federal District. Using the HI assay, the proportion of seropositive individuals was determined as follows: 44% for the A(H1N1)pdm09 virus, 37% for A(H3N2), and 19% for B/Victoria (Fig. 2).

 

Fig. 2. The proportion of individuals tested seropositive for the current influenza vaccine strains on the eve of the 2022–2023 flu season.

 

In 2022, differences in seroprevalence rates were statistically significant for all three viruses: A(H1N1)pdm09 (χ² = 96.1; p < 0.001), A(H3N2) (χ² = 211.5; p < 0.001), and B/Victoria (χ² = 218.5; p < 0.001, Pearson’s χ² test). The highest seroprevalence rates for A(H1N1)pdm09 and A(H3N2) were observed in the Siberian Federal District (54% and 53%, respectively), whereas in the Volga Federal District, the level of antibodies to A(H1N1)pdm09 was significantly lower (26%). For the B/Victoria virus, the highest level was also recorded in the Siberian Federal District (38%), whereas in the other districts it did not exceed 16%. Paired comparisons using the Holm-Bonferroni correction confirmed the significance of the differences between these extreme groups (p < 0.05).

The presented data showed that herd immunity was still lower than it was prior to the COVID-19 pandemic, despite the fact that 52.8% of the country’s population received a flu vaccine in 2022 [9].

By the end of November 2023, humoral immunity to the seasonal influenza A virus among the urban population had already returned to pre-COVID-19 pandemic levels, but among the rural population it remained significantly below recommended levels, with no more than 32% of samples testing positive [10].

During October–November 2024, 1,580 blood serum samples were collected in the Siberian Federal District. All samples were tested in HI assay using vaccine strains of influenza A and B viruses: A/Victoria/4897/2022 (H1N1)pdm09, A/Thailand/8/2022 (H3N2), B/Austria/ 1359417/2021 (B/Victoria lineage) (Fig. 3).

 

Fig. 3. The proportion of individuals tested seropositive for the current influenza vaccine strains on the eve of the 2024–2025 flu season.

 

In the Siberian Federal District, statistically significant differences in seroprevalence were observed between regions for all three viruses (p < 0.05; χ² test). By the start of the epidemic season, the number of positive samples for vaccine strains of influenza A and B viruses approached or exceeded 50% of all samples collected in Khakassia, Kemerovo, Irkutsk, and Novosibirsk regions. The proportion of positive samples for all tested viruses was significantly lower than 50% in Omsk region.

Data on humoral immunity during the inter-epidemic period were obtained for two regions: samples were collected in May 2025 in the Omsk region and in August 2025 in the Kaluga region. In the Omsk region, following the epidemic season, the geometric mean titers against the influenza A/Thailand/8/2022 (H3N2) virus in the groups of children and adolescents aged 8–14 and 15–18 years were approximately 1:50 (Fig. 4). In the remaining age groups, geometric mean titers against influenza A virus strains and, in all samples, against influenza B/Austria/1359417/2021 (B/Victoria lineage) were below the protective titer of 1 : 40.

 

Fig. 4. The reciprocal of the geometric mean titer against seasonal influenza viruses during the inter-epidemic period (May 2025) in the Omsk Region.

 

An analysis of data from the Kaluga Region revealed that the proportion of samples tested positive for antibodies to vaccine strains of influenza A(H1N1)pdm09 and A(H3N2) during the inter-epidemic period, prior to the start of influenza vaccination (August 2025), exceeded or approached 50% in all age groups (Fig. 5). No significant differences between age groups were identified for these viruses. In contrast, protective titers against the influenza B/Victoria virus were 40% or lower, and differences between age groups were statistically significant (χ² = 12.9; p = 0.012). The highest level of seropositivity to B/Victoria was observed in the 19–60 age group (40%), and the lowest in the 8–14 age group (13%).

 

Fig. 5. The proportion of samples tested positive for antibodies to influenza virus vaccine strains during the inter-epidemic period (August 2025) in the Kaluga Region.

 

For the past 20 years, we have been conducting year-round monitoring of antibodies to zoonotic influenza viruses, primarily avian influenza viruses. Samples are collected from poultry farm workers who have had contact with infected and/or dead birds, as well as from residents of regions located near the migration routes of wild waterfowl. When testing human blood sera against avian influenza viruses, a serum dilution of 1:10 was considered a significant titer in the HI and VN assays. In our studies, serum titers in the HI assay with A(H5Nx) viruses did not exceed 1 : 20. Data we obtained in 2021–2022 showed that only a few serum samples neutralized zoonotic viruses in the VN assay in MDCK cell culture at a titer of 1 : 10 [11].

In 2023, 4,095 serum samples were collected across 40 regions of the Russian Federation; only 1 sample tested positive for the influenza A(H5N1) virus. In 2024, 4,801 human serum samples were collected from 35 regions of the Russian Federation; of these, 4 (0.1%) samples tested positive in HI assay with influenza A(H5N1) virus. In 2025, 4,138 samples were collected from 40 regions; none of them tested positive in the HI (Table). All samples positive in HI assay with A(H5Nx) virus were tested in a VN assay; none of the samples neutralized the A(H5N8) and/or A(H5N1) virus in MDCK cells even at a 1 : 10 dilution. All samples that tested positive in the HI assay (titers of 1 : 40 and higher) with A(H9N2) virus were tested in MDCK cell culture with the A/chicken/Primorsky Krai/03/2018 (H9N2); in more than 85% of the samples, virus-neutralizing antibodies with a titer of 1:20 or higher were detected.

 

Number of blood serum samples tested positive in HI assay with A/H5 and A/H9 viruses in 2023–2025

Year of sample collection	Total samples collected	Number of seropositive А(Н5Nх) samples		Number of seropositive A(H9N2) samples
		n	%	n	%
2023	4095	1	< 0.01	39	1.0
2024	4801	4	0.1	54	1.0
2025	4138	0	0	43	1.0

 

Discussion

A high level of herd immunity against currently circulating strains of the influenza virus provides, first, protection for the vaccinated population against the disease, and second, protection for the unvaccinated population and people in high-risk groups through the vaccinated individuals. The immune response to vaccination in people from at-risk groups may be lower than in healthy people aged 18–64, which can affect the effectiveness of vaccines in these population groups. The fewer infected people there are around, the lower the likelihood of people from at-risk groups becoming ill. Monitoring herd immunity to vaccine strains of the influenza virus allows for predicting the burden of an upcoming epidemic and adjusting anti-epidemic measures.

Furthermore, low population immunity to seasonal influenza increases the risk of the emergence of virus variants with pandemic potential, as it raises the likelihood of reassortment between human and animal influenza virus genomes [12]. However, reassortment cannot be the sole and final event in the emergence of a pandemic virus; further adaptation of the virus to a new host is necessary. To track the circulation of such an adapting virus, a study of serum samples from people involved in the raising and processing of poultry, as well as those living in areas where influenza outbreaks have occurred among poultry or wild waterfowl is being conducted, with the aim of detecting antibodies to zoonotic influenza. According to WHO experts, the greatest current threat is posed by the highly pathogenic avian influenza viruses A(H5Nx) and A(H7N9), as well as the low-pathogenic virus A(H9N2), which is highly adapted to domestic poultry. This is because highly pathogenic viruses are widespread in live poultry markets and readily undergo reassortment with the A(H9N2) virus, while the population lacks immunity to new variants of the virus [13, 14]. Furthermore, the A(H9N2) virus circulates among poultry worldwide, and a small number of mutations, for example, the T187P + M227L mutations in hemagglutinin and the 627K mutation in the PB2 protein, lead to the emergence of mutants that cause death in mice and transmission of the virus to guinea pigs upon direct contact [14].

Herd immunity to seasonal influenza in Russia, following a sharp decline in the fall of 2020, was not restored until 2023–2024. Insufficient herd immunity not only increases the burden of seasonal epidemics but also raises the likelihood of new influenza virus variants emerging.

To more fully characterize population protection (the presence of herd immunity), indicators such as a geometric mean antibody titer of more than 1 : 40 in each age group and a seropositivity rate of at least 75% in at-risk groups and at least 60% in the general population are used. Our study has limitations regarding the sample characteristics, as we lack data on which samples were obtained from individuals in risk groups. Furthermore, there are no data on the geometric mean titer (except for samples obtained from the Hygienic and Epidemiological Center in the Omsk Region in May 2025).

As part of surveillance for avian influenza, it was shown that in 2021–2025, only a few samples tested positive in HI assay with A(H5Nx) influenza viruses, and about 1% of samples tested positive for antibodies to the A(H9N2) virus. However, all samples positive in HI assay with A(H5) virus were negative in the VN assay, and approximately 85% of samples positive in HI assay with A(H9N2) contained virus-neutralizing antibodies.

These varying results in the VN assay can be explained by the biological properties of influenza A viruses of different subtypes. For example, influenza A(H5Nx) virus strains, which are related in terms of hemagglutinin gene nucleotide sequences, often differ so much in their antigenic properties that they do not react in the HI assay with reference sera obtained against other strains. Apparently, the absence of viral neutralization by serum in cell culture is associated with very low titers in the HI assay, since titers in the HI assay were no higher than 1 : 20 when testing sera against zoonotic strains.

The situation is different for the influenza A(H9N2) virus. Strains that are not even closely related in terms of the nucleotide composition of the hemagglutinin gene exhibit similar antigenic properties and interact well in the HI assay with sera against other A(H9N2) strains. In our case, titers in the HI were 1 : 40 and higher.

Conclusion

Humoral immunity to seasonal influenza in Russia, which had plummeted during the COVID-19 pandemic, did not recover until 2023–2024. Insufficient herd immunity increases the likelihood of new influenza virus variants emerging. However, there is currently no stable circulation of influenza A(H5Nx) and A(H9N2) viruses in the human population in Russia.

Nevertheless, vaccination against seasonal influenza is necessary for at least 75% of workers at poultry farms and other organizations directly involved in the breeding and handling of farm poultry to reduce the risk of the emergence of an influenza virus with pandemic potential in the event of simultaneous human infection with seasonal and zoonotic influenza viruses.

 

¹ Resolution of the Chief State Sanitary Doctor of the Russian Federation dated July 8, 2025 No. 14 "On measures to prevent influenza, acute respiratory viral infections, and the new coronavirus infection (COVID-19) in the 2025–2026 epidemic season" (registered July 25, 2025 No. 83060).

² World Health Organization. Manual for the laboratory diagnosis and virological surveillance of influenza. World Health Organization, ‎2011. URL: https://iris.who.int/handle/10665/44518

About the authors

Alina R. Muratova

State Research Center of Virology and Biotechnology "Vector"

Author for correspondence.
Email: muratova_ar@vector.nsc.ru
ORCID iD: 0009-0003-5379-6452

chief specialist, Department of zoonotic infections and influenza

Russian Federation, Koltsovo

Tatyana N. Ilyicheva

State Research Center of Virology and Biotechnology "Vector"

Email: ilicheva_tn@vector.nsc.ru
ORCID iD: 0000-0002-2354-7688

Doctor of Biological Sciences, Associate Professor, leading researcher, Department of zoonotic infections and influenza

Russian Federation, Koltsovo

Ksenia I. Ivanova

State Research Center of Virology and Biotechnology "Vector"

Email: ivanova_ki@vector.nsc.ru
ORCID iD: 0009-0002-3191-1360

junior researcher, Department of zoonotic infections and influenza

Russian Federation, Koltsovo

Anastasia A. Moiseeva

State Research Center of Virology and Biotechnology "Vector"

Email: chalaya_aa@vector.nsc.ru
ORCID iD: 0000-0001-7048-2357

junior researcher, Department of zoonotic infections and influenza

Russian Federation, Koltsovo

Vasily Yu. Marchenko

State Research Center of Virology and Biotechnology "Vector"

Email: marchenko_vyu@vector.nsc.ru
ORCID iD: 0000-0002-1339-6732

Dr. Sci. (Biol.), Head, Department of zoonotic infections and influenza

Russian Federation, Koltsovo

References

Supplementary files