Genotype A3 influenza A(H5N1) isolated from fur seals shows high virulence in mammals, but not airborne transmission

Abstract

The global spread of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses has recently extended to include diverse mammalian species, raising new concerns about pandemic risk. In 2023, this clade was first detected in Russian marine mammals during a mass mortality event among northern fur seals in the Far East. Genetic analyses revealed the causative viruses to belong to genotype A3 of European origin, which is known to have circulated in wild birds across the Far East since 2022. Notably, these isolates harbor the mammalian-adaptive substitutions PB2-K482R and NP-N319K-mutations previously linked to enhanced virulence in non-H5 avian influenza viruses, but whose impact on A(H5N1) clade 2.3.4.4b viruses remained to be characterized. The heightened virulence of A3 genotype viruses is confirmed by data obtained via a mouse model. However, despite these adaptive changes, ferret transmission models showed no evidence of airborne transmission of the fur seal-derived virus. Our findings indicate that while PB2-K482R and NP-N319K may contribute to increased mammalian pathogenicity, they do not significantly increase the efficiency of respiratory transmission-a key prerequisite for human pandemic potential. Although suggesting a limited immediate pandemic threat from this A3 genotype, these results underscore the critical need for continued surveillance and functional assessment of emerging mammalian-adaptive mutations in circulating A(H5N1) viruses.

Fig. 1

Phylogenetic tree of the HA gene of A(H5Nx) viruses. Studied strains A(H5N1) are indicated in blue. Reference strains used in the virulence analysis are indicated by black squares. HA sequences from reference strains used in the analysis were obtained from the EpiFlu database of GISAID.

Fig. 2

Phylogenetic tree of the NP gene of A(H5Nx) viruses. Studied strains A(H5N1) are indicated in blue. Reference strains used in the virulence analysis are indicated by black squares. NP sequences from reference strains used in the analysis were obtained from the EpiFlu database of GISAID.

Fig. 3

Phylogenetic tree of the PB2 gene of A(H5Nx) viruses. Studied strains A(H5N1) are indicated in blue. Reference strains used in the virulence analysis are indicated by black squares. PB2 sequences from reference strains used in the analysis were obtained from the EpiFlu database of GISAID.

Fig. 4

Survival rates of BALB/c mice following intranasal infection with influenza A(H5N1) clade 2.3.4.4b virus at varying doses. Groups of mice (n = 6 per group) were intranasally inoculated with increasing doses of clade 2.3.4.4b A(H5N1) influenza virus. Survival was monitored daily over a 14-day observation period. The data is presented for four viruses: (A) A/fur seal/Sakhalin/399-1V/2023 A(H5N1), (B) A/chicken/Magadan/14-7V/2022, (C) A/turkey/Tyumen/81-96V/2021, (D) A/gull/Mari El/345-7V/2023.

Fig. 5

Airborne transmission experiment in ferrets infected with the clade 2.3.4.4b influenza A(H5N1) virus A/fur seal/Sakhalin/399-1V/2023. Donor ferrets (n = 3 per dose group) were intranasally inoculated with 5.7, 4.7, 3.7, or 2.7 log₁₀ EID₅₀/mL of virus. On day 1 post-inoculation (dpi), naïve recipient ferrets (n = 3 per group) were placed in separate cages within a TIEGEL ELC 04–60 dynamic aerobiology chamber, with unidirectional airflow from donor to recipient cages. Cages were arranged at a distance sufficient to prevent direct contact while allowing for airborne transmission. Animals were co-housed in this setup for 5 h daily over 5 consecutive days (dpi 1–5). Clinical signs were monitored throughout the study, and nasal wash samples were collected at defined time points from both inoculated and recipient ferrets. Blood samples were collected at 21 dpi from surviving donor ferrets and at 21 days post-contact (dpc) from all recipient ferrets to assess seroconversion.

Fig. 6

Results of airborne transmission experiment in ferrets. (A) Survival rates of donor ferrets following intranasal inoculation with clade 2.3.4.4b influenza A(H5N1) virus A/fur seal/Sakhalin/399-1V/2023 in the airborne transmission experiment. Four groups of three ferrets were inoculated with increasing doses of virus (2.7 – 5.7 log₁₀ EID₅₀/mL) and monitored for survival over the course of the study. (B) Infectious viral titers in nasal washes from donor ferrets following intranasal inoculation with varying doses of virus. Four groups of ferrets (n = 3 per group) were inoculated with 2.7, 3.7, 4.7, or 5.7 log₁₀ EID₅₀/mL, and nasal washes were collected daily to quantify viral shedding. Infectious virus titers are expressed as log₁₀ FFU/mL (mean ± SD for each group of ferrets). The limit of detection is indicated by the dashed line. (C) Weight loss of donor ferrets in the airborne transmission experiment following infection with 2.7, 3.7, 4.7, 5.7 log₁₀ EID₅₀/mL, respectively (mean ± SD for each ferret group). Triangles indicate animal deaths. (D) Weight loss of recipient ferrets in the airborne transmission experiment following contact with donor ferrets. (E) Change in body temperature of donor ferrets in the airborne transmission experiment following infection with 2.7, 3.7, 4.7, 5.7 log₁₀ EID₅₀/mL, respectively (mean ± SD for each ferret group). Triangles indicate animal deaths. (F) Change in body temperature of recipient ferrets in the airborne transmission experiment.