Multiple Introductions of Reassorted Highly Pathogenic Avian Influenza H5Nx Viruses Clade 2.3.4.4b Causing Outbreaks in Wild Birds and Poultry in The Netherlands, 2020-2021

Abstract

Highly pathogenic avian influenza (HPAI) viruses of subtype H5Nx caused outbreaks in poultry, captive birds, and wild birds in the Netherlands between October 2020 and June 2021. The full genome sequences of 143 viruses were analyzed. HPAI viruses were mainly of subtype H5N8, followed by H5N1, but also viruses of subtypes H5N3, H5N4, and H5N5 were detected. At least seven distinct genotypes were found, carrying closely related H5 segments belonging to clade 2.3.4.4b. Molecular clock analysis suggests that the reassortments of the NA gene segments likely occurred before the introduction of these viruses into the Netherlands. Genetic analysis suggested that multiple independent introductions of HPAI H5N8 viruses occurred in the Netherlands, likely followed by local spread resulting in at least two clusters of related viruses. The analysis provided evidence for independent introductions from wild birds at 10 poultry farms, whereas for two outbreaks transmission between farms could not be excluded. HPAI H5Nx viruses were detected in dead wild birds of 33 species, but mostly infected geese and swans were found. The pathogenicity of the H5N8 virus was determined for chickens and Pekin ducks, showing reduced mortality for ducks. This study provides more insight into the genetic diversity of HPAI H5Nx viruses generated by reassortment and evolution, and the spread of these viruses between wild birds and poultry. The fast and continuing evolution of H5 clade 2.3.4.4b may provide opportunities for these viruses to adapt to specific bird species, or possibly mammalian species and humans. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses are spread by migratory wild birds. Viruses causing outbreaks in wild birds and poultry in the Netherlands in 2020-2021 were genetically analyzed, which suggested that multiple virus incursions occurred. The outbreaks in poultry were likely caused by independent introductions from wild birds; only in one case virus spread between farms could not be excluded. Viruses of subtype H5N8 were mainly observed, but also other subtypes were detected that likely evolved by exchange of genetic information before these viruses were introduced into the Netherlands. Viruses were detected in many species of dead wild birds, but mostly in geese and swans. We showed that the H5N8 virus causes a higher mortality in chickens compared to ducks. This is consistent with the fact that not many wild ducks were found dead. This study provides more insight in the evolution and spread of HPAI viruses in wild birds and poultry.

Keywords: H5N8; HPAI; avian influenza virus; introduction routes; outbreak; reassortment; virus evolution.

FIG 1

The number of dead wild birds of different species testing positive in the H5-PCR between October 2020 and June 2021. Shown is the number of viruses that were sequenced in this period (black line). The number of H5-positive birds and the total number of dead wild birds tested in this period are listed above the columns (H5+/total number of birds tested).

FIG 2

Geographical locations of HPAI H5-viruses detected and sequenced in the Netherlands (A) at commercial poultry farms (squares) and captive bird facilities (triangles) and (B) in dead wild birds (circles). The subtypes of viruses detected are marked: H5N8 (blue), H5N1 (green), H5N3 (orange), H5N4 (purple), and H5N5 (brown).

FIG 3

The intravenous pathogenicity index (IVPI) scores in (A) chickens and (B) Pekin ducks. Ten 6-weeks old birds were infected with the HPAI H5N8-2020 virus isolated from the index farm, and were monitored for clinical signs and death for 10 days. The number of normal (green), sick (orange), very sick (red), and dead (dark gray) birds is shown for 10 days after infection.

FIG 4

Phylogenetic tree of the HA segment of the poultry, captive, and wild bird viruses sequenced. HPAI H5N8 viruses sequenced in this study are shown in blue, H5N1 in green, H5N3 in orange, H5N4 in purple, and H5N5 in brown. Variants are marked by A or B. Reference viruses from other countries are shown in black. The reassortments for all gene segments are schematically shown. The closest related viruses for each gene segment are listed and are marked by color.

FIG 5

Median-joining network showing the genetic relationship between the HPAI H5N8 viruses isolated from commercial poultry farms (red), captive bird facilities (green), and dead wild birds submitted for testing. Wild birds were classified according to species: swan (light blue), goose (cobalt blue), duck (dark blue), predatory species (orange), and other species (magenta). The infected farms are marked by black numbers, and the number of nucleotide differences between viruses is shown in red numbers. Two virus clusters are present, marked A and B.