A human isolate of bovine H5N1 is transmissible and lethal in animal models

Abstract

The outbreak of clade 2.3.4.4b highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI H5N1) in dairy cattle in the USA has so far resulted in spillover infections of at least 14 farm workers^1-3, who presented with mild respiratory symptoms or conjunctivitis, and one individual with no known animal exposure who was hospitalized but recovered^3,4. Here we characterized A/Texas/37/2024 (huTX37-H5N1), a virus isolated from the eyes of an infected farm worker who developed conjunctivitis⁵. huTX37-H5N1 replicated efficiently in primary human alveolar epithelial cells, but less efficiently in corneal epithelial cells. Despite causing mild disease in the infected worker, huTX37-H5N1 proved lethal in mice and ferrets and spread systemically, with high titres in both respiratory and non-respiratory organs. Importantly, in four independent experiments in ferrets, huTX37-H5N1 transmitted by respiratory droplets in 17-33% of transmission pairs, and five of six exposed ferrets that became infected died. PB2-631L (encoded by bovine isolates) promoted influenza polymerase activity in human cells, suggesting a role in mammalian adaptation similar to that of PB2-627K (encoded by huTX37-H5N1). In addition, bovine HPAI H5N1 virus was found to be susceptible to polymerase inhibitors both in vitro and in mice. Thus, HPAI H5N1 virus derived from dairy cattle transmits by respiratory droplets in mammals without previous adaptation and causes lethal disease in animal models.

Extended Data Figure 1.. Clinical data and virus titres associated with ferrets used to assess respiratory droplet transmission.

For infected ferrets (donors) shown in Fig. 4 (N=6 biologically independent animals per dose), daily body weights (A), body temperatures (B), and survival (C) are given. In panels (A) and (B), data points represent mean values and error bars represent the standard deviation. Survival curves in panel (C) were compared by using a log-rank Mantel-Cox test and the p-value is reported in the figure panel. Panel (D) shows virus titres in tissues of the same animals collected at the time of euthanasia or within 14 h of death. Each dot represents the titre of an individual ferret. °C, degrees Celsius; d, days; PFU, plaque forming units; PFU/g, plaque forming units per gram of tissue; PFU/ml, plaque forming units per millilitre.

Extended Data Figure 2.. huTX37-H5N1 receptor binding activity.

Four-fold serial dilutions of α2,3 and α2,6 sialylglycopolymers adhered to microtitre plates were incubated with 32 hemagglutination (HA) units of the indicated viruses or PBS (negative control). After washing, virus binding was detected by an anti-HA human monoclonal antibody (CR9114) and an HRP-conjugated secondary antibody. The absorbance values for each condition with each virus or PBS are shown. Each dot represents a single biologically independent replicate value. Three independent replicate experiments are shown.

Figure 1.. Replication in human epithelial cells.

Primary human alveolar or corneal epithelial cells grown in air-liquid interface cultures were infected with MOIs of 0.01 or 0.1 PFU per cell of huTX37-H5N1 (N=3 biological replicates per condition), NM93-H5N1 (N=2 biological replicates per condition), or VN1203-H5N1 (N=2 biological replicates per condition) and incubated at 33 °C (A) or 37 °C (B). Every 24 h for 96 h, apical surfaces were washed to collect secreted virus, which was titred using plaque assays in MDCK cells. For all conditions, all replicate data points were plotted individually, the median value is indicated by the floating bar, and variability is represented as the range. h, hours; PFU/ml, plaque-forming units per millilitre.

Figure 2.. huTX37-H5N1 is lethal in mice and spreads systemically.

(A) and (B) BALB/cJ mice (7 weeks old, N=5 biologically independent animals per dosage) were deeply anaesthetised and intranasally inoculated with 10-fold-serial dilutions of A/Texas/37/2024 (H5N1) in 50 μl of PBS. (A) Body weight and (B) survival were monitored daily for 13 days. In panel (A), points represent mean values and error bars represent the standard deviation. (C) BALB/cJ mice (7 weeks old, N=10 biologically independent animals per virus) were deeply anaesthetised and intranasally inoculated with 10³ PFU of huTX37-H5N1 in 50 μl of PBS. At 3 and 5 days post-infection, five mice were euthanised and tissues were collected for plaque assays in MDCK cells. In panel (C), points represent titres of individual mice, the floating bars show the median titre for each tissue of each inoculation group, and variability is represented by the range. PFU, plaque forming units; d, days; PFU/g, plaque-forming units per gram of tissue; PFU/ml, plaque-forming units per millilitre.

Figure 3.. huTX37-H5N1 is lethal in ferrets and spreads systemically.

Ferrets (6–8 months old, N=8 biologically independent animals per virus) were deeply anaesthetised and intranasally inoculated with 10⁶ PFU of huTX37-H5N1 in 500 μl of PBS. Body weights (A), body temperature (B), survival (C), and clinical symptoms (D) were monitored daily; virus titres in various tissues (E) were determined by use of plaque assays in MDCK cells. In panels (A) and (B), each line represents an individual ferret. In panel (D), symptom scores were plotted by adding one point per symptom observed on each day. Some ferrets died between health checks (indicated by a ‘D’ on the figure panels), and others were euthanised after meeting humane endpoint criteria (indicated by an ‘E’ on the figure panels). A ‘D’ or ‘E’ shown in black text indicates the outcome occurred on the same day, while gray text indicates the outcome occurred previously. In panel (E), points represent titres of individual ferrets (note, the triangular points represent the ferret that died before a health check on day 3), the floating bars show the median titre for each tissue at each timepoint, and variability is represented by the range. PFU, plaque forming units; d, days; PFU/g, plaque-forming units per gram of tissue; PFU/ml, plaque-forming units per millilitre.

Figure 4.. huTX37-H5N1 transmits by respiratory droplets in ferrets.

Ferrets (6–8 months old, N=6 animals per virus) were deeply anaesthetised and intranasally inoculated with 10⁶ PFU (A), 10³ PFU (B), 10² PFU (C), or 10¹ PFU (D). One day later, naïve ferrets (N=1 animal per infected animal) were placed in adjacent cages allowing for air flow but no direct contact between animals. Nasal swab samples were collected at the indicated timepoints and tested by using plaque assays in MDCK cells. In each figure panel, from left to right are plots of survival of infected donors, survival of respiratory droplet contacts, virus titres in nasal swabs of infected donors, and virus titres in nasal swabs of respiratory droplet contacts. Dotted lines in the virus titre plots represent the limit of detection. In panel (A), the asterisk indicates a respiratory droplet contact animal from pair 3 for which a low amount of virus was detected in the nasal swab at only the day 3 post-exposure timepoint, no virus was detected in any other tissue from the same animal upon necropsy. PFU, plaque forming units; PFU/ml, plaque-forming units per millilitre; d, days; n.d., not determined. Panel (E) shows virus titres in tissues of respiratory droplet contacts collected at the time of euthanasia or within 14 h of death. Each dot represents the titre of an individual ferret.

Figure 5.. Effects of mutations on viral polymerase activity.

Panel (A) indicates the PB2 amino acids at positions 627 and 631 for each of the TX001-H5N1 polymerase complexes used in the mini-replicon assays. The amino acids encoded by the huTX37-H5N1 virus are shown for comparison. Human 293T (B) or avian DF-1 (C) cells were transfected with plasmids expressing TX001-H5N1 polymerase complex proteins and a plasmid expressing a reporter vRNA encoding the firefly luciferase gene. After incubating cells for 24 h at the temperatures indicated on the figure panels, luciferase activity was measured. Polymerase activity was calculated by standardizing firefly luciferase activity (vRNA) to Renilla luciferase activity expressed from a control plasmid and is shown as relative luciferase activity. The plots show the mean and standard deviation of 8 data points collected over 3 replicate experiments for each PB2 protein. Groups were compared by using non-parametric two-tailed Mann-Whitney tests with a Holm-Sidak adjustment for multiple comparisons. Asterisks (***) on the panels indicate that p < 0.001 for comparisons between the wild-type TX001-H5N1 polymerase complex (with PB2–627E and PB2–631L) and a given mutant. In human cells (panel B), the polymerase activity of the TX001-H5N1 PB2-E627K mutant was significantly increased at 37 °C (p = 0.000311) and at 33 °C (p = 0.000311), and the polymerase activity of the TX001-H5N1 PB2-L631M mutant was significantly decreased at 37 °C (p = 0.000311) and at 33 °C (p = 0.000311). In avian cells (panel C), the polymerase activity of the TX001-H5N1 PB2-E627K mutant was not significantly different at 39 °C (p = 0.382284) or 37 °C (p = 0.102880), and the polymerase activity of the TX001-H5N1 PB2-L631M mutant was significantly decreased at 39 °C (p = 0.000622) and at 37 °C (p = 0.000622).

Figure 6.. Zanamivir has minimal effects on the outcome of TX001-H5N1 infection in mice.

Female BALB/c mice (N=15 per treatment condition) were deeply anaesthetised and intranasally infected with 10 PFU of wild-type A/dairy cattle/Texas/24–008749-001/2024 (H5N1) or a mutant virus expressing neuraminidase T438I. At 1 h post-infection, mice were treated with the indicated compounds daily for 5 days. (A) Body weights and survival were monitored daily for 14 days (N=5 mice per treatment condition). In the plots showing weight loss, each point represents the mean value, and the error bars represent the standard deviation. (B) Virus titres in the lung, nasal turbinate (NT), or brain were determined by using plaque assays in MDCK cells at day 3 (N=5 mice per treatment condition) and day 5 (N=5 mice per treatment condition) post-infection. Each point represents an individual titre value, bars represent the mean titre, and the error is represented by standard deviation. Titres were compared by using a non-parametric two-tailed Mann-Whitney test without adjustments for multiple comparisons. Asterisks on the panels indicate that p < 0.05 (*) or p < 0.01 (**). In mice infected with the wild-type (WT) virus, significantly reduced titres were observed in the lung (p = 0.0079), nasal turbinate (p = 0.0238), and brain (p = 0.0159) tissues of zanamivir-treated mice at day 3 post-infection, and in the lung (p = 0.0079), nasal turbinate (p = 0.0079), and brain (p = 0.0079) tissues of zanamivir-treated mice at day 5 post-infection. In mice infected with the NA-T438I mutant virus, significantly reduced titres were observed only in the lung tissues (p = 0.0079) of zanamivir-treated mice at day 3 post-infection. d, days; PFU/g, plaque forming units per gram of tissue.

Figure 7.. Bovine H5N1 is susceptible to polymerase inhibitors.

Female BALB/c mice (N=15 per treatment condition) were deeply anaesthetised and intranasally infected with 10 PFU of wild-type A/dairy cattle/Texas/24–008749-001/2024 (H5N1) or a mutant virus expressing neuraminidase T438I. At 1 h post-infection, mice were treated with the indicated compounds daily for 5 days. (A) Body weights and survival were monitored daily for 14 days (N=5 mice per treatment condition). In the plots showing weight loss, each point represents the mean value, and the error bars represent the standard deviation. (B) Virus titres in the lung, nasal turbinate (NT), or brain were determined by use of plaque assays in MDCK cells at day 3 (N=5 mice per treatment condition) and day 5 (N=5 mice per treatment condition) post-infection. Each point represents an individual titre value, bars represent the mean titre, and the error is represented by standard deviation. Titres were compared by using a non-parametric one-sided Kruskal–Wallis test with the Dunn’s multiple comparisons procedure. Asterisks on the panels indicate that p < 0.05 (*) or p < 0.01 (**). WT, wild-type; d, days; PFU/g, plaque-forming units per gram of tissue.

Figure 8.. Schematic representation of the possible evolution of dairy cow influenza lineages.

PB2–627 and PB2–631 amino acids are shown for the avian progenitor virus, the huTX37-H5N1 virus, and a representative dairy cattle isolate (NM93-H5N1).