Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America

Abstract

Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly followed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically and phenotypically diverse, with many causing severe disease with dramatic neurologic involvement in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous system warrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants.

Fig. 1. Pathogenicity of North American HPAI Influenza A(H5N1) clade 2.3.4.4b Wigeon/SC/21 and Eagle/FL/22 viruses in ferrets.

A Experimental design of ferret pathogenesis and transmission. At 0 dpi, ferrets (n = 9 per virus) were inoculated with 10⁶ EID₅₀ units of A(H5N1) virus. Three inoculated ferrets were individually co-housed with 3 naïve contact ferrets beginning 1 dpi. Clinical course of infection was monitored, and nasal wash samples were taken at indicated time points from both inoculated and contact ferrets. The remaining inoculated ferrets were euthanized at 3 dpi and 5 dpi (n = 3 per time point per virus) for viral titration in tissues. B Survival and C weight changes of inoculated ferrets (n = 3 per virus). Ferret weights every ≈48 h were used to calculate percentage of weight change from the initial mean weight at 0 dpi. Ferret weight values are the average ± SE for each group. P values for weight change were calculated using an unpaired t-test. ^**P < 0.01. D Infectious viral titers from nasal washes (n = 3–9 ferrets per virus, mean virus titer [log₁₀ TCID₅₀/mL] ±SD) and E infectious viral titers from tissues (n = 3 ferrets per virus, mean virus titer [log₁₀ TCID₅₀ per g of wet tissue]). Symbols represent each individual animal’s titer. Dashed lines indicate the lower limit of virus titer detection (1.0 log₁₀ TCID₅₀/mL). P values for viral titers were calculated using two-way ANOVA with Tukey’s multiple-comparison post hoc test. ^***P < 0.001, ^****P < 0.0001.

Fig. 2. Genotypic diversity among North American HPAI Influenza A(H5N1) clade 2.3.4.4b viruses.

A Genotypic diversity of 58 A(H5N1) viruses (some sequences generated directly from clinical material). A list of viruses used in this study and their GenBank or GISAID accession numbers are provided in Supplementary Tables 3 and 4, respectively. The colors denote common genotypes, with the orange color representing genes of Eurasian lineage and blue representing genes of NAm lineage (the different shades of blue represent phylogenetically distinct NAm genes). B Representative tangle plot showing the association of A(H5N1) virus HA genes (left side) with PB2 genes (right side) of Eurasian and NAm lineages. Nodes on the PB2 tree represent A(H5N1) viruses and representatives of other subtypes. Asterisks (*) indicate viruses that were used in subsequent experiments.

Fig. 3. Impact of different detected genotypes of North American HPAI Influenza A(H5N1) clade 2.3.4.4b viruses on pathogenicity in ferrets.

A Experimental design of ferret pathogenesis and transmission. At 0 dpi, ferrets (n = 9 per virus) were inoculated with 10⁶ EID₅₀ units of A(H5N1) virus. Clinical course of infection was monitored, and nasal wash samples were taken at the indicated time points. Ferrets (n = 3 per virus per analysis) were euthanized at 5 dpi for viral titration in tissues and pathology (Supplementary Figs. 3–5). B Survival and C weight changes of inoculated ferrets (n = 3 per virus). Ferret weights every ≈48 h were used to calculate percentage of weight change from the initial mean weight at 0 dpi. Ferret weight values are the average ± SE for each group. D Infectious viral titers from nasal washes (n = 6 ferrets per virus, except for Scaup/GA/22 at 5 dpi, for which n = 1, mean virus titer [log₁₀ TCID₅₀/mL] ±SD) and E infectious viral titers from tissues (n = 3 ferrets per virus per time point, mean virus titer [log₁₀ TCID₅₀ per g of wet tissue]). Symbols represent each individual animal’s titer. Dashed lines indicate the lower limit of virus titer detection (1.0 log₁₀ TCID₅₀/mL). P values were calculated using two-way ANOVA with Tukey’s multiple-comparison post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Fig. 4. Phenotypic properties of North American HPAI Influenza A(H5N1) clade 2.3.4.4b viruses.

A, B Solid-phase binding of A(H5N1) viruses to biotinylated sialylglycopolymers A 3’-SialLacNAc-PAA-biotin (3’-SLN) or B 6’-SialLacNAc-PAA-biotin (6’-SLN), representing galactose-linked sialic acids α2,3-SA (the avian virus preferred receptor) and α2,6-SA (the human virus preferred receptor), respectively. The data are shown as the mean ± SD from duplicate wells and representing one of two independent experiments. C Kinetics of pH inactivation of the Wigeon/SC/21, Eagle/FL/22, and CA/04 (H1N1)pdm09 viruses at 37 °C. The data are shown as the mean ± SD from triplicate wells representing one of three independent experiments. D Minireplicon polymerase activities of Wigeon/SC/21 and Eagle/FL/22 at 37 °C. The data are shown as the mean ± SD of 3-4 measurements over the hypothesized pH range for avian viruses, and 2 measurements over previously described ranges for control virus CA/04 (H1N1)pdm09, and representing one of three independent experiments. NS = not significant as determined by paired, two-tailed t-test. E Viral replication kinetics in Calu-3 cells. Cells were inoculated at an MOI of 0.001 and incubated at 37 °C. The data are shown as the mean ± SD from triplicate wells and representing one of two independent experiments. F Viral replication kinetics in primary differentiated human airway cultures. Cultures were inoculated at an MOI of 0.005 and incubated at 37 °C. The data are shown as the mean ± SD from triplicate culture inserts and representing one of two independent experiments. The number of inserts with viral replication out of the total is indicated in parentheses. Statistical significance (one-way ANOVA) was determined by comparison to rg-A/Texas/71/2017 (H3N2) at 72hpi. G, H Neutralizing antibody levels in human serum samples (n = 48) against HA protein (as measured by HI assay, dotted line indicates limit of detection of 1:10 serum dilution) or NA protein (as measured by ELLA assay). Points joined by lines represent values for the same individual for the individual antigens tested. ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001.