California mice (Peromyscus californicus
) intranasally challenged with 1.6×103
PFU of VPXV in 10 µl PBS (5 µl/nostril), developed widespread lesions after a five day incubation period. The disease progression was acute, with a mortality rate of 54%; survivors began to recover by day eight and no viable virus was detectable by day 21. These data contrast with the monkeypox virus (MPXV) prairie dog model of Orthopoxvirus
infection, where disease presentation is delayed (day 9–12) and generally persists for a longer period of time (24–28 days) before resolution. The mortality rate observed in prairie dogs infected with a similar dosage of West African MPXV is 0–25% 
. Although we did not identify a febrile period in this study, it is possible that the duration of pyrexia was very short and our sampling missed it; unpublished data show that California mice infected with a lower dose of VPXV (1.2×102
PFU) did present a febrile period by day 14 pi. The weight loss and skin lesion onset may be related to anorexia due to general malaise or pain associated with oral ulceration.
The sensitivity of the NA OPXV RT-PCR is superior to viral isolation by cell culture in the detection of infection. This observation has been described previously using the E9L RT- PCR assay for detection of MPXV in prairie dog studies 
. All samples with CT values of 37 (minimum of 45 genomes of VPXV) or earlier had detectable cytopathic effect (CPE) in a single passage on BSC-40 cells, while samples with CT values ≥38 did not show evidence of viable VPXV. All samples from negative control animals were confirmed negative by both RT-PCR and cell culture.
The 14 kDa band was the immunodominant band observed in all survivors which manifest an immune response. This band size is consistent with the previously described envelope protein encoded by the A27 gene ortholog of vaccinia virus Copenhagen (VV-Cop). This gene has an important role in allowing mature virus to bind to cell surface glycosaminoglycans 
and stimulates a cellular immune response 
. Previous experiments found that mice immunized against the 14 kDa protein, and later challenged with 40 times the 50% lethal dose (LD50) of wild type VACV, did not show signs of disease and had 100% survivorship 
. Our 36 kDa band may be the previously described 37 kDa envelope protein. This would be consistent with another study in which that protein was immunodominant in animals that succumbed to orthopoxvirus infection or had to be euthanized because of the severity of the disease 
. The 62 kDa band is likely a major core protein encoded by the A10 gene (VV-Cop ortholog) derived from the P4a precursor. It is the most abundant core protein found in the virion and plays an important role in its assembly. It is also important in stimulating memory B-cells and the humoral immune response 
. The inflammatory monocytosis and neutrophilia in the early phase of infection was not an unexpected result given the severity of disease observed. Although mild thrombocytopenia was observed from day six through 21, it is unlikely to be the sole cause of the hemorrhage. Further analysis of platelet function and collection of clotting data throughout the duration of clinical disease would be required to accurately define the pathogenesis of the hemorrhage.
Histological changes attributable to active virus infection were seen in the internal organs of those animals that succumbed to disease. B- type basophilic intracytoplasmic inclusion bodies, also known as “viral factories”, are a typical histopathological feature of poxvirus infections, but the intracytoplasmic ATIs bodies observed ultrastructurally are not made by all members of the genus 
. The North American orthopoxviruses, cowpox, and ectromelia have previously been shown to form ATIs 
. Our findings confirm that VPXV makes ATIs within infected cells. However, VPXV makes all three types of ATIs, which has not been reported within the genus Orthopoxvirus
, but this could be a difference between observations made from in vitro
versus in vivo
systems. A-Type inclusions showed morphological variations other than the three classic types of inclusions described. In addition non-condensed and mature particles were seen inside and around the periphery of the inclusions (). Additional studies, involving more tissue and serial thin-sections would likely provide further insight into the overall structure and composition of ATIs.
We were not able to detect an immune response in four mice. Although two of these mice (PC 024 and PC 027) presented with external and internal hemorrhagic lesions with 1×108
PFU/mL in spleen, respectively. Both mice (PC 024 and PC 027) succumbed to disease by day seven, which may be too early for IgG detection, or may be at levels below the detection limit of our assay. The other two mice (PC 010 and PC 017) which survived the infection, and were euthanized on days 42 or 49 without the production of antibodies developed single skin or tail lesions on day 7. However the CT values (38 and 40 respectively), were above the cutoff at which samples are considered positive for virus particles. Previous data indicate that lesions caused by Orthopoxvirus
infection have very high amounts of Orthopoxvirus
DNA which would not be consistent with the values observed from these lesion samples. Thus it is probable that these two animals were not infected. This is occasionally observed in both human vaccinations with Vaccinia virus
where vaccinated individuals do not get a “take” 
, and in previous animal studies where Monkeypox virus
challenged animals do not become infected and do not produce an immune response 
. Further studies to evaluate both the humoral and cellular immune responses are needed in order to understand their roles in the resolution of OPXV infection.
This study is the first report describing the pathogenesis of a NA OPXV infection in a potential rodent reservoir. Previous field studies have shown that VPXV is endemic to California’s San Francisco Bay area and although lesions were observed on wild caught animals, little was known regarding the pathogenesis of this virus in mice. The California mouse is both geographically and ecologically sympatric with Pinyon mice and VPXV. The data from this study clearly indicate that P. californicus
is susceptible to VPXV infection via the intranasal route, and that the subsequent infection can cause extreme morbidity and high mortality. It is beyond the scope of this study to characterize the pathogenesis resulting from other routes of inoculation, it is quite possible that sub-cutaneous or intra-muscular exposure could result in different disease courses. Future efforts should consider this in order to increase our understanding of Volepox virus
pathogenesis. When comparing this model with MPXV infection in prairie dogs, we noticed that prairie dogs shed higher amounts of viable virus orally (up to 1 × 106
PFU/mL), even when inoculated with lower doses of MPXV (8×102
. Swabs of the anus, eyes, and oral cavity had lower levels of VPXV than did the solid organ tissues, but both tissues and swabs of the infected animals had consistently high viral loads, which in all cases exceed the inoculation dose used in this investigation. Lung and liver contained the most viable virus (up to 2×109
PFU/mL). This could indicate that VPXV infections may occur in wild California mice and could be transmitted between individuals in a population; however, it is noteworthy that two of the inoculated individuals that did not become infected were co-housed with two infected animals.
Several other species within the genus OPXV are rodent-borne and recognized as the causative agents of a febrile rash illness in humans. Additionally, evidence suggests that OPXV Variola virus
, the causative agent of human smallpox, was initially a rodent-borne virus before evolving into an exclusively human pathogen 
. The morbidity and mortality indices observed in this study are greater than previously reported in several models of OPXV disease, even those seen in highly pathogenic species (e.g., monkeypox virus and variola virus). The mouse and vole species in which VPXV is found, are non-commensal species that have relatively little contact with humans (as compared to species such as Peromyscus maniculatus
); thus, it is possible that this virus has had little chance for transmission between the rodent hosts and humans. Due to our limited knowledge of the natural history of VPXV additional surveillance and laboratory animal studies should be pursued to address its potential risks for other animal (small mammal) and human populations.