Continued Evolution of H5Nx Avian Influenza Viruses in Bangladeshi Live Poultry Markets: Pathogenic Potential in Poultry and Mammalian Models

Abstract

The genesis of novel influenza viruses through reassortment poses a continuing risk to public health. This is of particular concern in Bangladesh, where highly pathogenic avian influenza viruses of the A(H5N1) subtype are endemic and cocirculate with other influenza viruses. Active surveillance of avian influenza viruses in Bangladeshi live poultry markets detected three A(H5) genotypes, designated H5N1-R1, H5N1-R2, and H5N2-R3, that arose from reassortment of A(H5N1) clade 2.3.2.1a viruses. The H5N1-R1 and H5N1-R2 viruses contained HA, NA, and M genes from the A(H5N1) clade 2.3.2.1a viruses and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. H5N2-R3 viruses contained the HA gene from circulating A(H5N1) clade 2.3.2.1a viruses, NA and M genes from concurrently circulating A(H9N2) influenza viruses, and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. Representative viruses of all three genotypes and a parental clade 2.3.2.1a strain (H5N1-R0) infected and replicated in mice without prior adaptation; the H5N2-R3 virus replicated to the highest titers in the lung. All viruses efficiently infected and killed chickens. All viruses replicated in inoculated ferrets, but no airborne transmission was detected, and only H5N2-R3 showed limited direct-contact transmission. Our findings demonstrate that although the A(H5N1) viruses circulating in Bangladesh have the capacity to infect and replicate in mammals, they show very limited capacity for transmission. However, reassortment does generate viruses of distinct phenotypes.IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have circulated continuously in Bangladesh since 2007, and active surveillance has detected viral evolution driven by mutation and reassortment. Recently, three genetically distinct A(H5N1) reassortant viruses were detected in live poultry markets in Bangladesh. Currently, we cannot assign pandemic risk by only sequencing viruses; it must be conducted empirically. We found that the H5Nx highly pathogenic avian influenza viruses exhibited high virulence in mice and chickens, and one virus had limited capacity to transmit between ferrets, a property considered consistent with a higher zoonotic risk.

Keywords: Bangladesh; influenza; influenza viruses; live poultry markets.

FIG 1

(A) Genome constellations representative of the multiple subgroups of clade 2.3.2.1a highly pathogenic (H5Nx) avian influenza A virus characterized in this study. The eight gene segments (from top to bottom) in each virus are polymerase basic 2, polymerase basic 1, polymerase acidic, hemagglutinin, nucleoprotein, neuraminidase, matrix, and nonstructural. (B) Phylogenetic trees of the HA gene. Phylogenetic analysis was done using the neighbor-joining algorithm with the Kimura two-parameter model. The reliability of phylogenetic inference at each branch node was estimated by the bootstrap method with 1,000 replications; evolutionary analyses were conducted in MEGA 7.

FIG 2

Pathogenicity and replication of the multiple subgroups of clade 2.3.2.1a highly pathogenic (H5Nx) avian influenza A viruses in mice. Groups of 5- to 6-week-old BALB/c mice (n = 5) were inoculated intranasally with the indicated doses (10², 10³, 10⁴, 10⁵, or 10⁶ EID₅₀) of A/duck/Bangladesh/26042/2015 H5N1 (subgroup H5N1-R0), A/duck/Bangladesh/25683/2015 H5N1 (subgroup H5N1-R1), A/duck/Bangladesh/33841/2017 H5N1 (subgroup H5N1-R2), or A/chicken/Bangladesh/34722/2018 H5N2 (subgroup H5N2-R3). (A and B) Body weight loss (A) and survival (B) were evaluated daily for 14 days. Groups of mice (n = 3) that were infected with 10⁶ EID₅₀ were euthanized at day 3 or 5 postinfection. (C) Lung, nasal turbinate, heart, brain, and intestine were harvested, homogenized, and used to quantify viral titers by EID₅₀ assay. Viral titers expressed as the log₁₀ EID₅₀/ml were plotted as the mean. Statistical analysis was performed using two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). A/duck/Bangladesh/26042/2015 H5N1 (subgroup H5N1-R0) is shown in red, A/duck/Bangladesh/25683/2015 H5N1 (subgroup H5N1-R1) is shown in blue, A/duck/Bangladesh/33841/2017 H5N1 (subgroup H5N1-R2) is shown in green, and A/chicken/Bangladesh/34722/2018 H5N2 (subgroup H5N2-R3) is shown in pink.

FIG 3

Pulmonary lesions and viral spread in the lungs of H5Nx-infected mice. Mice (n = 3) were infected intranasally with 10⁶ EID₅₀ of A/duck/Bangladesh/26042/2015 H5N1 (subgroup H5N1-R0), A/duck/Bangladesh/25683/2015 H5N1 (subgroup H5N1-R1), A/duck/Bangladesh/33841/2017 H5N1 (subgroup H5N1-R2), or A/chicken/Bangladesh/34722/2018 H5N2 (subgroup H5N2-R3). Mice were euthanized at 3 dpi (A) or 5 dpi (B). Lungs were harvested and fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin (HE), subjected to IHC staining with anti-NP antiserum, and analyzed by histomorphometry. Magnifications: 60× (HE), 20× (IHC), and 2× (histomorphometry). For histomorphometry, total lung areas are outlined in green, and areas with antigen-positive cells are shown in red.

FIG 4

Pathogenicity and transmission of the multiple subgroups of clade 2.3.2.1a highly pathogenic (H5Nx) avian influenza A viruses in ferrets. Groups of 3 ferrets each were intranasally inoculated with 10⁶ EID₅₀ of each virus shown. Each ferret was paired with an individual naive ferret at 24 h postinoculation. Viral titers in nasal washes from individual inoculated ferrets (D; red), direct-contact ferrets (DC; blue), and aerosol-contact ferrets (AC; green) on the days postinoculation or postcontact were determined and presented as the log₁₀ EID₅₀/ml.

FIG 5

Body weight loss (left) and changes in body temperatures (right) of individually inoculated (D), direct-contact (DC), or aerosol-contact (AC) ferrets up to 12 days postinoculation or postcontact. Ferrets were inoculated intranasally with 10⁶ EID₅₀ of A/duck/Bangladesh/26042/2015 H5N1 (subgroup H5N1-R0), A/duck/Bangladesh/25683/2015 H5N1 (subgroup H5N1-R1), A/duck/Bangladesh/33841/2017 H5N1 (subgroup H5N1-R2), or A/chicken/Bangladesh/34722/2018 H5N2 (subgroup H5N2-R3), and the body weight and temperature of the animals were monitored.

FIG 6

Viral replication of multiple subgroups of clade 2.3.2.1a highly pathogenic (H5Nx) avian influenza A viruses in ferret tissues. Three ferrets were inoculated with 10⁶ EID₅₀ of A/duck/Bangladesh/33841/2017 H5N1 (subgroup H5N1-R2) or A/chicken/Bangladesh/34722/2018 H5N2 (subgroup H5N2-R3) virus and then humanely euthanized on day 4 postinoculation to collect tissues for viral titration in eggs by EID₅₀ assay. Each bar represents a ferret, and the detection limit is shown by a dotted line.