Characterization of H5N1 avian influenza virus isolated from bird in Russia with the E627K mutation in the PB2 protein

Abstract

Currently A(H5Nx) avian influenza viruses are globally widespread and continue to evolve. Since their emergence in 2020 novel highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b reassortant viruses have become predominant in the world and caused multiple infections in mammals. It was shown that some of A(H5N1) viruses mostly isolated from mammals contain an E627K mutation in the PB2 protein which can lead to adaptation of influenza viruses to mammalian cells. In 2023 in Russia we have isolated two highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses from birds one of which contained an E627K mutation in the PB2 protein. This virus had increased virulence in mice. Limited airborne transmission of the virus with the PB2-E627K mutation was observed between ferrets, in which infectious virus was detected in the nasal washings of the three of the twelve recipient ferrets, and clinical symptoms of the disease were observed in one case. Both viruses showed dominant binding to avian-type sialoside receptors, which was most likely the reason for the limited transmissibility. Thus, this study indicates a possible limited increase in the pandemic potential of A(H5N1) 2.3.4.4b viruses and highlights the importance of continuous avian influenza surveillance for pandemic preparedness and response.

Fig. 1

Airborne transmission experiment for ferrets infected with the clade 2.3.4.4b influenza A(H5N1) virus A/Emu372-E627K. At 0 dpi, donor ferrets (n = 3 per dose) were inoculated with 4.7, 3.7, 2.7 and 1.7 lg EID₅₀ of A(H5N1) virus. On the second dpi, groups of naïve ferrets were placed in separate cages of the TIEGEL ELC 04–60 dynamic aerobiology chamber (with directed airflow from donor to recipient ferrets) which were positioned at a distance from the cages of groups of donor ferrets to prevent direct contact. Joint exposure of the animals lasted 5 h daily for 5 days, starting on the second and ending on the sixth dpi. Clinical course of infection was monitored, and nasal wash samples were taken at indicated time points from both inoculated and contact ferrets. Blood was taken from all survived contact ferrets to check for seroconversion on 22 and 30 dpc.

Fig. 2

Survival rates of ferrets in the airborne transmission experiment with the clade 2.3.4.4b influenza A(H5N1) virus A/Emu372-E627K. (A) Survival of inoculated ferrets (n = 3 per dose). (B) Survival of contact ferrets (n = 3 per dose).

Fig. 3

Shedding of the virus A/Emu372-E627K by inoculated and airborne contact ferrets. A, С, E, G. Infectious viral titers in nasal washes of donor ferrets (n = 3 per dose) infected with 4.7, 3.7, 2.7 and 1.7 lg EID₅₀ respectively. B, D, F, H. Infectious viral titers in nasal washes of recipient ferrets (n = 3 per dose) which were in an airborne contact with donor ferrets inoculated with 4.7, 3.7, 2.7 and 1.7 lg EID₅₀ respectively. Columns represent individual animal’s titer (mean virus titer ± SD). Dashed lines indicate the lower limit of virus titer detection (1 lg FFU/mL). The asterisk sign indicates the washes from which the viral isolate was isolated in ECE.