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Volume 28, Number 9—September 2022
Research Letter

Pathogenesis and Transmissibility of North American Highly Pathogenic Avian Influenza A(H5N1) Virus in Ferrets

Joanna A. Pulit-Penaloza, Jessica A. Belser, Nicole Brock, Poulami Basu Thakur, Terrence M. Tumpey, and Taronna R. MainesComments to Author 
Author affiliation: Author affiliation: Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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Figure

Evaluating influenza A(H5N1) virus isolate aw/SC using in vivo and in vitro models. A, B) Ferrets were intranasally inoculated with 6 log10 EID50 of aw/SC virus in 1 mL of phosphate-buffered saline and direct contact (A) and respiratory droplet (B) transmission models were established with naive ferrets (1:1 ratio) the following day as described previously (9). Nasal wash samples were collected from inoculated and contact ferrets every other day, and virus titers were determined in eggs (10). As shown, infectious virus was detected in nasal wash specimens from all inoculated ferrets up to day 7 (left side of each panel); however, ferrets exposed only by direct contact (panel A, right) or through the air (panel B, right) did not show infectious virus. C, D) Replication kinetics of aw/SC virus were evaluated in human respiratory tract cells and compared with the H1N1 viruses, A/Michigan/45/2015 (MI/45), A/Idaho/7/2018 (ID/7), A/Nebraska/14/2019 (NE/14), and A/Nebraska/15/2018 (NE/15). Calu-3 cells (ATCC, https://www.atcc.org) were grown to confluence under submerged conditions in 12-mm diameter transwell inserts (Corning, https://www.corning.com). The cells were infected apically at a multiplicity of infection of 0.01 for 1 h and then washed and incubated at 33°C (C) or 37°C (D) as previously described (6). Virus titers in triplicate cell-supernatant samples were determined by standard plaque assay in MDCK cells (10). The limit of virus detection was 1.5 log10 EID50/mL or 2 log10 PFU/mL. Error bars indicate SDs. p values provided for avian H5N1 versus human seasonal H1N1 viruses were calculated by 2-way analysis of variance with a Tukey posttest. aw/SC, A/American Wigeon/SC/22-000345-001/2021; EID50, 50% egg infectious dose.

Figure. Evaluating influenza A(H5N1) virus isolate aw/SC using in vivo and in vitro models. A, B) Ferrets were intranasally inoculated with 6 log10 EID50 of aw/SC virus in 1 mL of phosphate-buffered saline and direct contact (A) and respiratory droplet (B) transmission models were established with naive ferrets (1:1 ratio) the following day as described previously (9). Nasal wash samples were collected from inoculated and contact ferrets every other day, and virus titers were determined in eggs (10). As shown, infectious virus was detected in nasal wash specimens from all inoculated ferrets up to day 7 (left side of each panel); however, ferrets exposed only by direct contact (panel A, right) or through the air (panel B, right) did not show infectious virus. C, D) Replication kinetics of aw/SC virus were evaluated in human respiratory tract cells and compared with the H1N1 viruses, A/Michigan/45/2015 (MI/45), A/Idaho/7/2018 (ID/7), A/Nebraska/14/2019 (NE/14), and A/Nebraska/15/2018 (NE/15). Calu-3 cells (ATCC, https://www.atcc.org) were grown to confluence under submerged conditions in 12-mm diameter transwell inserts (Corning, https://www.corning.com). The cells were infected apically at a multiplicity of infection of 0.01 for 1 h and then washed and incubated at 33°C (C) or 37°C (D) as previously described (6). Virus titers in triplicate cell-supernatant samples were determined by standard plaque assay in MDCK cells (10). The limit of virus detection was 1.5 log10 EID50/mL or 2 log10 PFU/mL. Error bars indicate SDs. p values provided for avian H5N1 versus human seasonal H1N1 viruses were calculated by 2-way analysis of variance with a Tukey posttest. aw/SC, A/American Wigeon/SC/22-000345-001/2021; EID50, 50% egg infectious dose.

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