Susceptibility and shedding in Mx1+ and Mx1- female mice experimentally infected with dairy cattle A(H5N1) influenza viruses

Abstract

Background: Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses have spread prolifically in dairy cattle in the US, resulting in dozens of human infections, some without well-established links to animal contacts. Many wild mammals have also been affected, including peridomestic house mice.

Methods: Here, we evaluated susceptibility, tissue tropism, and shedding in female PWK/PhJ and BALB/cJ mice, two laboratory strains derived from house mice that differ in expression of the antiviral restriction factor Mx1. PWK/PhJ mice, which were selected for their natural expression of Mx1, better reflect the antiviral capacity of most wild house mice, whereas BALB/cJ mice lack functional Mx1.

Findings: We found that, regardless of Mx1 expression status, mice are susceptible to infection by dairy cattle HPAI H5N1 viruses, that infection leads to systemic spread to non-respiratory sites, and that infected animals shed virus into the environment via urine. Shed virus remained infectious in urine for at least 24 h at room temperature.

Interpretation: These findings suggest that wild house mice could contribute to HPAI H5N1 environmental contamination and may play a role in transmission to other hosts.

Funding: This work was supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Response (contract 75N93021C00014) and by grants from the Japan Agency for Medical Research and Development (JP25wm0125002, JP253fa627001, and JP24fk0108626, to Y.K.).

Keywords: H5N1; Influenza; Mouse; Mx1; Shedding.

Fig. 1

Dairy cattle HPAI H5N1 viruses induce clinical disease and spread systemically in Mx1-expressing PWK/PhJ mice. Female BALB/cJ or PWK/PhJ mice (7 weeks old) were deeply anaesthetized and intranasally inoculated with 1000 PFU of huTX37-H5N1 (N = 15 biologically independent animals per mouse strain; three BALB/cJ mice and one PWK/PhJ mouse succumbed unexpectedly within 2 days of inoculation and were excluded from further analysis), NM93-H5N1 (N = 17 or N = 15 biologically independent BALB/cJ or PWK/PhJ mice, respectively), or Isumi-H1N1 (N = 15 biologically independent animals per mouse strain). Groups of mice were monitored for body weight loss (a) and survival (b) over 14 days or were euthanized at day 3 and day 5 post-infection and tissues were collected for plaque assays in MDCK cells (c). All mouse strain/virus infection groups comprised N = 5 biologically independent mice except the following: BALB/cJ mice infected with huTX37-H5N1 and monitored for 14 days (N = 3 mice), BALB/cJ or PWK/PhJ mice infected with huTX37-H5N1 and euthanized at day 5 post-infection for tissue collection (N = 4 animals per mouse strain), and BALB/cJ mice infected with NM93-H5N1 and monitored for 14 days (N = 7 mice). In panel (a), each animal is represented by an individual line, and the asterisks indicate time points at which significantly different p-values were obtained when comparing body weights of BALB/cJ mice infected with huTX37-H5N1 or NM93-H5N1 (∗, p = 0.0318 at day 2 post-infection; ∗∗∗, p = 0.0003 at day 4 post-infection; as determined by two-way ANOVA with a Geisser-Greenhouse correction and Sidak’s multiple comparisons test). In panel (b), the asterisks indicate a significant difference in the survival curves of huTX37-H5N1- and NM93-H5N1-infected BALB/cJ mice (∗∗, p = 0.0027, log-rank Mantel–Cox test). 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; MG, mammary gland; LD, latissimus dorsi; NT, nasal turbinate.

Fig. 2

Dairy cattle HPAI H5N1 viruses are shed in the urine of Mx1-expressing PWK/PhJ mice. Female PWK/PhJ mice (7 weeks old) were deeply anaesthetized and intranasally inoculated with 1000 PFU of huTX37-H5N1 or NM93-H5N1 (N = 5 biologically independent animals for each virus isolate). Body weights (a) and survival (b) were monitored for 14 days, and urine and faeces collected on days 1–12 post-infection were tested by performing plaque assays in MDCK cells (c, huTX37-H5N1) and (d, NM93-H5N1). In panels (a), (c), and (d), each animal is represented by an individual line. In panels (c) and (d), M1 refers to mouse 1, M2 refers to mouse 2, etc. In panel (c), the asterisk indicates the huTX-37-H5N1-infected animal that succumbed to its infection. PFU/ml, plaque forming units per millilitre; d, days.

Fig. 3

Dairy cattle HPAI H5N1 viruses are shed in the urine and faeces of Mx1-deficient BALB/cJ mice. Female BALB/cJ mice (7 weeks old) were deeply anaesthetized and intranasally inoculated with 10 PFU of huTX37-H5N1 (a) or 100 PFU of NM93-H5N1 (b) (N = 5 biologically independent animals for each virus isolate). Body weights and survival were monitored for 14 days, and urine and faeces collected on days 1–14 post-infection were tested by performing plaque assays in MDCK cells. In panels showing body weights and virus titres, each animal is represented by an individual line. In both (a) and (b), M1 refers to mouse 1, M2 refers to mouse 2, etc. PFU/ml, plaque forming units per millilitre; d, days.

Fig. 4

Dairy cattle HPAI H5N1 virus is stable for at least 24 h in mouse urine. (a) NM93-H5N1 stock virus (5 μl) was spiked into fresh urine from female BALB/cJ mice or MEM-BSA (195 μl) and aliquots were used for plaque assays after incubating at room temperature for 0, 6, 12, 24, and 72 h. Titres of each replicate sample at each time point are shown separately. (b) Female BALB/cJ mice (7 weeks old) were deeply anaesthetized and intranasally inoculated with 100 PFU of NM93-H5N1 (N = 10 biologically independent animals). Body weights and survival were monitored until all animals succumbed to their infections. Urine was collected from all surviving animals daily and pooled, and then urine was incubated at room temperature and aliquots were used for plaque assays as described for panel (a). Titres of pooled urine samples that were positive for infectious virus (from days 5, 6, and 7 post-infection) are shown separately. In panels showing body weights and virus titres, each animal is represented by an individual line. PFU/ml, plaque forming units per millilitre; d, days; h, hours.