Highly Pathogenic Avian Influenza (HPAI) H5N1 virus in Finland in 2021-2023 - Genetic diversity of the viruses and infection kinetics in human dendritic cells

Abstract

Highly pathogenic avian influenza (HPAI) H5N1 is known for its virulence and zoonotic potential, infecting birds and mammals, thus raising public health concerns. Since 2021 its spread among birds has led to cross-species transmission causing epizootics among mammals, eventually impacting fur animal farms in Finland in 2023. To analyze the infectivity of the Finnish H5N1 isolates in human cells, representatives of diverse H5N1 isolates were selected based on the genetic differences, host animal species, and the year of occurrence. The infection kinetics of the selected H5N1 isolates from wild pheasant and fox, and fur animals blue fox and white mink were examined in human monocyte-derived dendritic cells (moDCs) with H5N1 human isolate as a control. Although the isolate from pheasant (a wild bird) showed weakly reduced replication and viral protein expression in human cells compared to mammalian isolates, no discernible differences in virus replication in moDCs was observed. This study revealed similar infectivity in human moDCs for all five H5N1 isolates, regardless of the observed genetic differences. While H5N1 human infections remain rare, the virus poses a risk for widespread epizootics in mammals such as fur animal farms and, more recently, dairy cattle.

Keywords: Finland; H5N1; genetic variation; highly pathogenic avian influenza (HPAI); human cells; immune cells; infectivity; viral kinetic.

Figure 1.

Phylogenetic tree of avian influenza H5N1 virus hemagglutinin (HA) genomes identified in Europe between the years 2020–2023. The phylogenetic relationship of highly pathogenic avian H5N1 virus HA sequences (n = 995) was analyzed using the IQ tree program. The tree was inferred by Bayesian analysis. H5 gene sequences from Europe between 2020 and 2022 are marked in purple and in 2023 labelled by country of origin. The Finnish mammalian and bird originating H5N1 virus strains selected for experimental analysis are marked in different colours and pointed with asterisk (*) in the figure.

Figure 2.

Phylogenetic network for the HA genomes of Finnish H5N1 viruses originating from mammals and wild birds from 2023 incorporated with the fox and pheasant isolates from 2021. The network is constructed employing the Median Joining (MJ) method, implemented within NETWORK 10.2.0.0 software. 147 HA genes originating from wild birds (n = 45 in blue), mammalian (n = 102, in yellow) were analyzed. The H5N1 virus strains selected for experimental analysis are marked in different colours as shown in the figure. The size of the circles represents the number of viruses of the same type, and the length of the branches refers to the nucleotide distance.

Figure 3.

Infectivity curves of avian influenza H5N1 isolates in human monocyte-derived dendritic cells (moDCs). Cells from four different blood donors were infected with five H5N1 isolates with six one to five serially diluted virus doses starting from 1/75 dilution. Cells were fixed at 6 hours and 24 hours after infection and stained with anti-H5N1 glycoprotein (GP) specific antibodies followed by flow cytometric analysis to estimate the percentage of virus-infected cells. The mock sample, the uninfected cells, was used to separate the uninfected and infected cells. The results represent the mean values + standard deviations of the means from the cells of four different donors. The multiplicity of infection (MOI) was determined as 0.5 where 50% of the cells were infected, and the MOI for each dilution was calculated based on that.

Figure 4.

H5N1 virus replication in human monocyte-derived dendritic cells. moDCs from four blood donors were infected with five H5N1 isolates with MOI 0,1 and 0,0001. Cells were collected at 1-, 3-, 6-, and 24-hours post-infection, cells from different donors were pooled, and total cellular RNA was extracted. RNA samples were analyzed with influenza A M1 RNA-specific RT-qPCR. The results are presented as the expression of viral RNA relative to mock cell RNA in MOI 0,1 (A) and MOI 0,0001 (B) infected cells, with the average of three individual experiments with standard deviation. For the statistics One-way ANOVA with Tukey’s multiple comparisons test was used. Ns refers to not significant statistical differences between the samples. The expression of viral NP and M (C) and cleavage of the hemagglutinin (HA) protein (D) in cells collected at 24 hours post-infection. Cellular protein lysates from four donors were pooled and prepared for Western blotting and stained using virus protein specific antibodies. GAPDH staining was used as an internal loading control. A representative experiment out of three is shown. NA: neuraminidase that with the H5N1 glycoprotein antibodies cross react.

Figure 5.

H5N1 virus-induced interferon lambda 1 (IFN-λ1) gene expression in human monocyte-derived dendritic cells. MoDCs were infected with five H5N1 isolates at MOI values 0.1 (A) and 0.0001 (B). Cells were collected at indicated time points after infection, pooled, and total cellular RNA was extracted. Relative IFN-λ1 gene expression was analyzed by RT-qPCR. The results are an average of three independent experiments and the data is presented as relative IFN-λ1 mRNA expression compared to uninfected cells. One-way ANOVA with Tukey’s multiple comparisons test was used for the statistics. p < 0.05 (*), not significant (ns).

Figure 6.

Productivity of H5N1 virus infections in human monocyte-derived dendritic cells. MoDCs obtained from 4 blood donors (marked with coloured A–D) were infected with H5N1 isolates at MOI 0.1 (A and C) and 0.0001 (B and D), and supernatant samples were collected at 1- and 24-hours post-infection. A and B. The infective viral titres produced from moDCs were determined by plaque assay in MDCK cells (logarithmic scale). C and D. Total RNA was extracted from supernatant and viral M1 RNA copies were quantified by RT-qPCR (linear scale). Virus titres and viral M1 RNA copy numbers were compared to the 1 h time point and are presented as fold change between 1- and 24-hours samples. Data of individual donors (A–D) are marked with indicated colours. The horizontal black lines represent the geometric means of the results from 4 blood donors. Statistics was run with One-way ANOVA with Tukey’s multiple comparisons test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), not significant (ns).