The WEEV isolates studied here displayed wide variation in mortality and MTD by the subcutaneous route. The mouse model was used to distinguish between higher-virulence epizootic isolates of WEEV and lower-virulence enzootic viruses (Weaver et al., 1999
). The epizootic McM isolate, originally recovered from a fatal human case of encephalitis, was by far the most lethal isolate tested here. However, other isolates showed intermediate to low mortality whether recovered from mosquito vectors or horse brain. By using the intracranial and intraperitoneal routes, others have shown that enzootic isolates of WEEV from California were not uniformly of low pathogenicity and could show a range of virulence in adult mice (Hahn et al., 1988
). Nagata et al. (2006)
% mortality in each of eight isolates of WEEV by the intranasal route in adult BALB/c mice, although there was variation in MTD. One would therefore expect a greater variation between isolates in outbred strains (Hardy et al., 1974
). Forrester et al. (2008)
demonstrated that only one of ten WEEV isolates (CR46) caused >50
% mortality in subcutaneously infected Swiss–Webster mice (age of mice not given). The MTD of IMP when infected via aerosol was 7 days, considerably longer than any of the isolates tested by Nagata et al. (2006)
, and the survival rate was 90
%. Bianchi et al. (1993)
also infected outbred Swiss NIH mice (28–32 days old) with similar doses, but using the intracranial and intraperitoneal routes of infection, and showed that neuroinvasiveness was uniquely associated with epizootic strains of WEEV. This suggests that the strain and age of the mouse, the route of virus infection and virus titre are all paramount in designing a suitable animal model.
In our model, CD1 mice were infected by the subcutaneous route and were 6–8 weeks of age. The resulting range of mortality rates underlined the benefits of this mouse strain and age as a tool for investigating virus virulence. Our study also aimed to determine whether the selected WEEV isolates showed distinct phenotypes by an aerosol route of infection, as they are potential BT agents (Sidwell & Smee, 2003
). McM was 100
% lethal by aerosol, whereas IMP showed only 10
% mortality. IMP is therefore a good low-virulence strain for aerosol-infection studies to understand the molecular determinants of WEEV pathogenesis, which should lead to targets for vaccine or therapeutic design. It is of particular note that IMP caused no mortality when administered intracerebrally. McM, conversely, caused 100
% mortality within 2 days. This suggests that IMP is clearly neuroinvasive, given the titres of virus detected in the brains of subcutaneously infected mice and the presence of neuropathological lesions in the brains of one-third of IMP-infected mice. However, IMP is clearly less neurovirulent than McM, causing no intracerebellar mortality, considerably less neuropathology and having a titre at least 1 log10
lower than McM in brains. These data, combined with the types of pathology observed in the brains of infected mice (Fig. 6), suggest that McM induces more extensive damage to neurons in the central nervous system (CNS) than IMP.
In vitro growth curves in two vertebrate cell lines (BHK and Vero) and a mosquito cell line (C6/36) showed higher titres (1 log10) of McM in vertebrate cells than IMP (Fig. 2a). In contrast, IMP generated higher titres in C6/36 cells (Fig. 2b). This could be due to McM and IMP having adapted to their host types (human brain and insect cells, respectively). The growth of both isolates in Vero cells was more balanced, possibly due to both isolates having most recently been passaged in Vero cells (Fig. 2c). It is possible that additional unknown and undocumented McM passages occurred between 1941 (year of initial isolation) and 1973 (passage history first documented by the CDC). This gap in the passage history could possibly explain the high levels of in vitro and in vivo growth of McM.
Surprisingly few studies infecting adult mice subcutaneously with WEEV have been carried out, although it is a good representative route of the natural cycle of WEEV. Monath et al. (1978)
followed viraemia and organ titres after subcutaneous infection of 5–6-week-old Swiss mice with WEEV. Peak viraemia of 7.5 log10
was reached by 1 day p.i., in contrast to the present study, which found a similar length of viraemia but a lower peak viraemia of 3–5 log10
, depending on the isolate (Bowen & Calosher, 1976
). We found no association between viraemia and virulence, as no statistically significant difference was observed among isolates in the magnitude of peak viraemia. IMP, however, differed significantly from other isolates in that no viraemia was detected in mice after 2 days p.i. In cases of Venezuelan equine encephalitis virus (VEEV) and EEEV infection, it has been reported that the inapparent
apparent infection ratios are 11
1 and 3
1, respectively (Sabattini et al., 1991
McM, the most virulent isolate, grew to the highest titre in the brain, whereas the least virulent isolate (IMP) had significantly lower titres in the brain. Liu et al. (1970)
also reported that mortality in mice was associated with encephalitic signs after subcutaneous infection. It should be noted that Monath et al. (1978)
documented pathological changes in the heart during WEEV infection and observed overall mortality of 40
%, largely in the absence of neurological signs. Therefore, WEEV may be capable of causing death as a result of damage to organs other than the brain.
IMP may replicate more slowly in the brain (lower neurovirulence) or affect the innate immune response differently, resulting in delayed pathogenesis. The data in Fig. 3(a) indicate clearly that IMP replicates or enters the brain at a much slower rate than McM when administered subcutaneously. Even when administered by aerosol, IMP titres in the brain (although significantly higher than those in mice infected by the subcutaneous route) are 2–5 log10
lower than McM titres. The high titres (108
) detected in the brains of infected mice are indicative of the efficiency of McM replication. This trend is seen in mice infected with virus by either route. The intracerebral route proved to be most neuroinvasive, with no McM-infected mice surviving beyond 48 h. These data, combined with the types of pathology observed in the brains of infected mice (Fig. 6), indicate that McM induces more extensive damage to neurons in the CNS than IMP, and the possible restriction of the ability of IMP to replicate in the neurons is responsible for the slower multiplication in the CNS, as seen with other alphaviruses such as Semliki Forest virus (Atkins et al., 1999
). Thus, it is the difference in the rate of development of neuronal damage in the brains of mice infected with McM (compared with IMP) that could result in a lethal threshold before the immune system can intervene fully (Fig. 6).
The titres of both WEEV isolates in other organs in our investigation showed that virus administered by aerosol generated higher titres in the brain, spleen, lung and salivary gland than virus administered subcutaneously. As the inguinal and popliteal lymph nodes are found peripherally, it was not surprising that virus titres measured in those tissues were higher in subcutaneously infected mice than aerosol-infected mice (Fig. 3b, c). The virus titre of IMP was considerably higher in the knee joint of mice infected by either route than was observed with McM (Fig. 3e). This observation, combined with the virus titres in the brains and the absence of mortality in mice infected intracerebrally with IMP, suggests that IMP is a more arthralgic isolate, whereas McM is more encephalitic (although still detectable in the knee-joint tissues). Future immunopathological and quantitative investigations will help to elucidate this issue.
infected 1–2-day-old suckling mice and 3-week-old mice subcutaneously with WEEV isolated in Kern County, CA, USA, in 1957. In suckling mice, damage to mesodermally derived tissues resulted in death within 48 h. Three-week-old mice showed no clinical signs of disease although, beginning at 9 days p.i., encephalitis and changes including inflammatory infiltrates and haemorrhaging in some organs, such as lungs and liver, were noted. Aguilar (1970)
used a different strain and age of mouse (Swiss Mice – Rockefeller strain), as well as a different virus isolate, which may explain this discrepancy. The change in tissue tropisms observed with ageing may continue, therefore, as mice mature past 3 weeks of age, to the point where WEEV does not replicate efficiently in many organs, but retains neuroinvasiveness. It was evident that both McM and IMP replicated in most organs; however, only the presence of virus in the brain, adrenal, inguinal lymph node, knee, liver, pancreas, skeletal muscle and spleen showed any significant difference between isolates (Fig. 4a). When analysing statistical significance between virus titres in mice inoculated by different routes (aerosol and subcutaneous), it was noted that only in the brain, lung and salivary gland were titres significantly higher via the aerosol route, and titres of both viruses in the knee, popliteal lymph node and skeletal muscle were significantly higher in mice infected subcutaneously (Fig. 4b). Given the routes of infection (respiratory tract and inner thigh) and the location of these organs with respect to the route of virus, these results are to be expected.
McM and IMP were the most genetically diverse isolates examined, having 66 amino acid changes (13 major changes in the type of amino acid observed) spanning their genomes, as illustrated in Table 2. Potential pathogenic determinants have been mapped to a region of the WEEV genome containing the carboxy-terminal 13 aa of the E2 gene, the 6K and E1 genes, and the 3′ NTR. Three of the major changes in E2 were clustered within a 68 aa stretch and could possibly play an important role in the efficiency with which WEEV binds/enters host cells. Previous studies have shown that the secondary structure of the stem–loops in the 5′ NTR of other alphavirus genomes plays a role in virus neurovirulence (Evans et al., 1985
; Kinney et al., 1993
; Logue et al., 2008
; Netolitzky et al., 2000
). The single nucleotide difference between MCM and IMP at position 34, however, did not affect the secondary structure of the sole stem–loop of the WEEV 5′ NTR. Follow-up studies to determine the role of each of these virus elements in pathogenicity are under way.
This investigation demonstrated the effectiveness of our animal model in identifying two distinct WEEV isolates, representative of high and low virulence in mice. Through a series of in vivo and in vitro analyses, we have been able to show statistically significant differences in virus growth and survival between these isolates. Having selected two distinctly different WEEV isolates and mapped their amino acid and non-coding changes, we will identify which changes play an important role in virulence and neuropathogenesis and incorporate these data into the development of antiviral therapeutics for WEEV.