We previously showed that inbred CAST/EiJ mice derived from wild mice, in contrast to classical inbred strains, are highly susceptible to lethal MPXV infection. In order to investigate the host responses that underlie sensitivity and resistance, we first compared kinetics of virus replication in CAST/EiJ and BALB/c mice. The intranasal route was used to partially mimic upper respiratory transmission of MPXV and VARV. High levels of virus replication were detected within 2 days in the lungs of both strains of mice. In CAST/EiJ mice, virus spread quickly to other organs (liver, spleen, kidney, and brain), with high titers accumulating on days 6 to 8, the time of death. Surprisingly, kinetics of replication in the lungs of BALB/c mice paralleled that in CAST/EiJ mice. In both mouse strains, virus titers peaked on day 6, with those in BALB/c mice infected with 106 PFU similar to those in CAST/EiJ mice infected with lethal doses. However, replication in other organs differed markedly, with only minimal spread in BALB/c mice and subsequent full recovery.
CAST/EiJ mice have not been extensively used to study microbial infections. However, they have been reported to be more resistant than BALB/c mice to Bordetella pertussis
) and more sensitive to Coccidioides immitis
). We had previously shown that CAST/EiJ and BALB/c mice make similar CD8 T-cell and antibody responses following scarification with VACV, suggesting that CAST/EiJ mice are competent in their ability to mount an acquired immune response. We suspected that differences in the kinetics or magnitude of the innate immune response might underlie the sensitivity of CAST/EiJ mice to MPXV. Accordingly, we analyzed the induction of 22 different cytokines and chemokines in lung tissue following MPXV infection. Although most cytokines showed little change after infection of BALB/c mice with 106
PFU of MPXV, a sharp peak of IFN-γ was detected on day 6, coincident with the peak virus titer and preceding virus clearance. CCL5 and CXCL10, which are induced by IFN-γ, peaked on day 8 in BALB/c mice. Remarkably, neither IFN-γ nor CCL5 increased in the lungs of MPXV-infected CAST/EiJ mice after infection with either 106
PFU of MPXV, although there was some increase in CXCL10. Interestingly, CAST/EiJ mice mounted a higher IFN-γ response in the spleen following virus spread than BALB/c mice, indicating that production of this cytokine was not genetically impaired. Fierer et al. (9
) demonstrated equivalent levels of IFN-γ mRNA in the lungs of BALB/c and CAST/EiJ mice after infection with Coccidioides immitis
although they did not measure levels of the cytokine.
To determine whether the timely induction of IFN-γ in the lungs could have prevented disease in CAST/EiJ mice, we administered mouse IFN-γ intranasally just before and following infection. This treatment lowered the level of MPXV in the lungs, reduced morbidity, and completely prevented mortality, indicating that CAST/EiJ mice could effectively respond to IFN-γ. While this experiment demonstrated that exogenous IFN-γ protected CAST/EiJ mice, it remained to be determined whether the production of IFN-γ contributed to the protection of naturally resistant strains of mice. To evaluate this, IFN-γ and IFN-γ receptor gene knockout C57BL/6 mice were infected with MPXV. Both mutant strains were more sensitive to MPXV than the parent C57BL/6 mice though less sensitive than CAST/EiJ mice or STAT1-deficient C57BL/6 mice (38
). We concluded that the ability to rapidly induce IFN-γ represents an important factor, but not the sole determining factor, responsible for the different sensitivities of C57BL/6 and CAST/EiJ mice.
The participation of IFN-γ in the host response to other poxvirus infections is well known (5
), and this cytokine likely has multiple roles, as it promotes a Th1 response in addition to more direct antiviral activity (8
). Previous studies have shown that the susceptibility of inbred mouse strains to ectromelia virus (ECTV) correlates with their ability to produce IFN-γ in the spleen following ECTV infection (38
). Thus, BALB/c is a low IFN-γ producer and is highly susceptible to ECTV whereas C57BL/6 is a high IFN-γ producer and is relatively resistant. However, both low- and high-IFN-γ-producer classical inbred strains of mice are resistant to MPXV as shown here and previously (2
). Therefore, we did not anticipate that a deficiency in IFN-γ production in the lungs would be a distinguishing feature of the sensitivity of CAST/EiJ mice.
Since IFN-γ is produced mainly by NK and T lymphocytes, the deficiency of this cytokine could be related to a failure to activate or recruit such cells into the lungs. Interestingly, the percentages of NK cells in the peripheral blood of CAST/EiJ and BALB/c are 1.25% and 7.28%, respectively, whereas the percentages of CD4 T cells are similar and those of CD8 T cells are 2-fold higher in BALB/c mice than in CAST/EiJ mice (http://phenome.jax.org
). Investigation into the function and trafficking of lymphocytes may help to illuminate the basis for the defect in IFN-γ production in the lungs of CAST/EiJ mice.
To counteract IFN-γ, many poxviruses encode a soluble IFN-γ binding protein with homology to the extracellular part of the IFN-γ receptor (1
). However, there is considerable sequence diversity of IFN-γ from different species, e.g., the mouse and human proteins are only 40% identical in amino acid sequence (8
). Although the VACV IFN-γ binding protein inhibits IFN-γ from humans, cows, rabbits, rats, and chickens, it has a low affinity for mouse IFN-γ. This can explain the finding that deletion of the B8 gene, encoding the VACV IFN-γ binding protein, did not affect VACV virulence in mice (43
), although a reduction had been reported by another group (45
). In contrast, the ECTV homolog binds mouse IFN-γ and deletion of the gene reduces virulence for mice (38
). MPXV encodes a homolog of the B8 protein with 95% and 91% identities to VACV and ECTV, respectively, but its binding properties have not yet been determined.
In conclusion, intranasal infection of CAST/EiJ mice with MPXV leads to seeding and replication of the virus in the lungs followed by rapid and uncontrolled dissemination to other major organs. To achieve equivalent levels of MPXV replication in the lungs, BALB/c mice were infected with 100 times the lethal dose used for CAST/EiJ mice. However, even then spread of MPXV to other organs was minimal in BALB/c mice. The resistance of BALB/c mice correlated with the induction of IFN-γ in the lungs, which did not occur in CAST/EiJ mice. Moreover, the resistance of CAST/EiJ mice was enhanced by administration of exogenous IFN-γ and the resistance of C57BL/6 mice was reduced by knockout of the gene encoding IFN-γ or the gene encoding the IFN-γ receptor. To follow up these observations, we plan to determine the cellular response to MPXV infection of sensitive and resistant mice and to determine the role of the MPXV IFN-γ binding protein.