Identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models

Abstract

Infection with monkeypox virus (MPXV) causes disease manifestations in humans that are similar, although usually less severe, than those of smallpox. Since routine vaccination for smallpox ceased more than 30 years ago, there is concern that MPXV could be used for bioterrorism. Thus, there is a need to develop animal models to study MPXV infection. Accordingly, we screened 38 inbred mouse strains for susceptibility to MPXV. Three highly susceptible wild-derived inbred strains were identified, of which CAST/EiJ was further developed as a model. Using an intranasal route of infection with an isolate of the Congo Basin clade of MPXV, CAST/EiJ mice exhibited weight loss, morbidity, and death in a dose-dependent manner with a calculated 50% lethal dose (LD(50)) of 680 PFU, whereas there were no deaths of BALB/c mice at a 10,000-fold higher dose. CAST/EiJ mice exhibited greater MPXV sensitivity when infected via the intraperitoneal route, with an LD(50) of 14 PFU. Both routes resulted in MPXV replication in the lung, spleen, and liver. Intranasal infection with an isolate of the less-pathogenic West African clade yielded an LD(50) of 7,600 PFU. The immune competence of CAST/EiJ mice was established by immunization with vaccinia virus, which induced antigen-specific T- and B-lymphocyte responses and fully protected mice from lethal doses of MPXV. The new mouse model has the following advantages for studying pathogenesis of MPXV, as well as for evaluation of potential vaccines and therapeutics: relative sensitivity to MPXV through multiple routes, genetic homogeneity, available immunological reagents, and commercial production.

FIG. 1.

In vitro and in vivo comparisons of low passage and cloned MPXV-Z79. (A and B) Growth curves. BS-C-1 cells were infected with 3 PFU per cell of the parental stock (CDC) or clonal isolate (LVD) of Congo Basin (A) or West African (B) clades of MPXV. At the indicated times after infection, cells from triplicate wells were individually harvested and lysed, and titers were determined by plaque assay on BS-C-1 cells. (C and D) Infection of African dormice. Groups of 4- to 8-month-old male dormice were infected i.n. with doses of MPXV-Z79-CB2 between 0.05 and 5,000 PFU. Animals were monitored for weight loss (C) and death (D) for 30 days. Control mice were mock infected with virus diluent. Group size was three to five animals.

FIG. 2.

i.n. infection of BALB/c and CAST/EiJ mice with MPXV-Z79-CB2. Groups of five to ten 5- to 6-week-old female mice were infected i.n. with doses of MPXV-Z79-CB2 ranging from 10² to 10⁶ PFU. Another group was mock infected. Animals were monitored daily for weight loss and death for 18 days. (A) Weight loss in BALB/c mice; (B) weight loss in CAST/EiJ mice; (C) survival of CAST/EiJ mice.

FIG. 3.

i.p. infection of CAST/EiJ mice with MPXV-Z79-CB2. Groups of five or six 9- to 11-week-old female CAST/EiJ mice were infected i.p. with doses of MPXV-Z79-CB2 ranging from 1 to 1,000 PFU. A separate group was mock infected. Animals were monitored three times per week for weight loss (A) and daily for death (B).

FIG. 4.

Virus titers in organs of CAST/EiJ mice infected with MPXV-Z79-CB2.On the day of death the lungs, livers, and spleens were removed from mice infected with 1 × 10⁴ PFU or 2 × 10⁴ PFU of MPXV-Z79-CB2 by the i.n. (n = 4) or i.p. (n = 3) route. Organs were weighed and stored frozen. After thawing and homogenization, titers were determined by plaque assay on BS-C-1 cells.

FIG. 5.

i.n. infection of CAST/EiJ mice with MPXV-USA-C1. Groups of five to eight 5- to 8-week-old female CAST/EiJ mice were infected i.n. with doses of MPXV-USA-C1 from 10² to 10⁶ PFU. Animals were monitored three times per week for weight loss (A) and daily for death (B).

FIG. 6.

Virus titer in organs of CAST/EiJ mice infected with MPXV-USA-C1. On the day of death lung, liver, spleen, and ovaries were removed from mice infected i.n. with 10⁴ (n = 4), 10⁵ (n = 6), or 10⁶ (n = 4) PFU of MPXV-USA-C1. Organs were weighed and stored frozen. After thawing and homogenization, titers were determined by plaque assay on BS-C-1 cells.

FIG. 7.

Vaccination of CAST/EiJ mice protects against lethal MPXV infection. Groups of four 6- to 11-week-old female CAST/EiJ mice were vaccinated with Dryvax or mock vaccinated with diluent by scarification on the shaved back. Four weeks later, the animals were challenged i.n. with either 7 × 10³ PFU (low dose) or 7 × 10⁴ PFU (high dose) of MPXV-Z79-CB2 and monitored for 20 days for weight loss (A) and death (B).

FIG. 8.

CD8 and CD4 T-cell responses in mice vaccinated with Dryvax. Groups of CAST/EiJ and BALB/c mice were vaccinated with Dryvax or mock vaccinated. Two weeks later splenocytes were prepared and stimulated with either MVA or VACV WR. The percentage of VACV-specific IFN-γ- and TNF-expressing CD8 and CD4 cells was determined by flow cytometry.