Effect of Prior Influenza A(H1N1)pdm09 Virus Infection on Pathogenesis and Transmission of Human Influenza A(H5N1) Clade 2.3.4.4b Virus in Ferret Model

Abstract

Reports of human infections with influenza A(H5N1) clade 2.3.4.4b viruses associated with outbreaks in dairy cows in the United States underscore the need to assess the potential cross-protection conferred by existing influenza immunity. We serologically evaluated ferrets previously infected with an influenza A(H1N1)pdm09 virus for cross-reactive antibodies and then challenged 3 months later with either highly pathogenic H5N1 clade 2.3.4.4b or low pathogenicity H7N9 virus. Our results showed that prior influenza A(H1N1)pdm09 virus infection more effectively reduced the replication and transmission of the H5N1 virus than did the H7N9 virus, a finding supported by the presence of group 1 hemagglutinin stalk and N1 neuraminidase antibodies in preimmune ferrets. Our findings suggest that prior influenza A(H1N1)pdm09 virus infection may confer some level of protection against influenza A(H5N1) clade 2.3.4.4.b virus.

Keywords: A(H5N1); Influenza A virus; clade 2.3.4.4b; ferrets; influenza; pathogenesis; prior infection; respiratory infections; transmission; viruses; zoonoses.

Figure 1

Detection of cross-reactive antibodies in study of the effect of prior influenza A(H1N1)pdm09 virus infection on pathogenesis and transmission of human influenza A(H5N1) clade 2.3.4.4b virus in ferret model. After primary pH1N1 infection, we detected cross-reactive antibodies by using a high-throughput multiplex influenza antibody detection assay. Serum samples from naive and preimmune ferrets were prediluted 200-fold and added to plates containing antigen-coated microspheres, then plates were incubated with protein A–phycoerythrin conjugate. Reported values represent the mean of duplicate assays. Antibody titers were expressed as median fluorescence intensity. Full virus strain names and sources of all antigens are provided (Appendix 1 Table). HA, hemagglutinin; IAV, influenza A virus; IBV, influenza B virus; NA, neuraminidase; NP, nucleoprotein.

Figure 2

Virus shedding in study of the effect of prior influenza A(H1N1)pdm09 virus infection on pathogenesis and transmission of human influenza A(H5N1) clade 2.3.4.4b virus in ferret model. A, B) Nasal wash viral titers for influenza A(H5N1) Texas/37 virus (A) and influenza A(H7N9) Anhui/1 virus (B). C, D) Virus titers from tissues for Texas/37 H5N1 virus collected 3 days postinoculation (C) and Anhui/1 H7N9 virus collected 4 days postinoculation (D). Horizontal bars indicate median, dots indicate individual titers, whiskers indicate range of positive titers. Three naive and 3 pH1N1 preimmune ferrets were inoculated via respiratory inhalation with Texas/37 or Anhui/1 virus (Table). Nasal wash specimens (A, B) were collected daily. Virus titers were determined by standard plaque assay in MDCK cells. Tissue samples collected from nasal turbinate, ethmoid turbinate, soft palate, blood, and rectal swabs were reported in log₁₀ PFU/mL. Tissues collected from lung, brain, olfactory bulb, intestines, liver, spleen, kidney were reported in log₁₀ PFU/g. The limit of detection was 10 PFU per mL or g. Statistical analyses were performed using 2-way analysis of variance test when samples were positive for viral titers in all 3 inoculated animals; we considered p<0.05 statistically significant. When <3 inoculated ferrets had detectable virus, the detection frequency is indicated above the corresponding positions. Anhui/1, low pathogenicity influenza A(H7N9) A/Anhui/1/2013; NS, not statistically significant; Texas/37, highly pathogenic influenza A(H5N1) clade 2.3.4.4b A/Texas/37/2024.

Figure 3

Contact transmission of Texas/37 influenza A(H5N1) virus and Anhui/1 influenza A(H7N9) virus in study of the effect of prior influenza A(H1N1)pdm09 virus infection on pathogenesis and transmission of human influenza A(H5N1) clade 2.3.4.4b virus in ferret model. A, C, E) Transmission of Texas/37 H5N1 virus among ferrets; B, D, F) transmission of Anhui/1 H7N9 virus among ferrets. Different shades indicate individual animals. We inoculated 3 naive ferrets per virus (A–D) and 3 preimmune ferrets per virus (E, F) by respiratory inhalation exposure (Table). Each inoculated ferret was pair-housed with a contact recipient, with (C, D) or without preimmunity (A, B, E, F); contact was sustained for 48 hours for Texas/37 and for 72 hours for Anhui/1 before inoculated animals were humanely euthanized. Nasal wash samples were collected daily from inoculated ferrets (days 1–4 postinfection) and on alternate days from the contact animals (days 1–11 postcontact). The limit of detection was 10 PFU/mL. Two naive contact ferrets (panel A) reached humane endpoints and were euthanized on days 3 and 4 postinfection. Anhui/1, low pathogenicity influenza A(H7N9) A/Anhui/1/2013; EP, endpoint; NS, not statistically significant; Texas/37, highly pathogenic influenza A(H5N1) clade 2.3.4.4b A/Texas/37/2024.