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. Author manuscript; available in PMC: 2009 Oct 25.
Published in final edited form as: Virology. 2008 Sep 17;380(2):170–172. doi: 10.1016/j.virol.2008.08.012

Western equine encephalitis submergence: lack of evidence for a decline in virus virulence

Naomi L Forrester 1, Joan L Kenney 1, Eleanor Deardorff 1, Eryu Wang 1, Scott C Weaver 1,*
PMCID: PMC2574696  NIHMSID: NIHMS74445  PMID: 18801549

Abstract

The incidence of western equine encephalitis (WEE) in humans and equids peaked during the mid-20th century and has declined to fewer than 1–2 human cases annually during the past 20 years. Using the mouse model, changes in WEE virus (WEEV) virulence were investigated as a potential explanation for the decline in the number of cases. Evaluation of 10 WEEV strains representing a variety of isolation locations, hosts, and all decades from the 1940’s to the 1990’s yielded no evidence of a decline in virulence. These results suggest that ecological factors affecting human and equine exposure should be investigated to explain the decline in WEE.

Introduction

Western Equine Encephalitis Virus (WEEV) is a mosquito-borne Alphavirus (family Togaviridae) that causes severe neurological disease in humans and equids. Infection can result in mild to severe neurologic sequelae in human survivors, and has an economic impact ranging from $21,000 to $3 million dollars per case (CDC, 1995). WEEV is found in western North America and South America, and within western North America utilizes a mosquito-bird cycle with Culex tarsalis as the primary vector, although some refractory populations of this species have been described. Since the discovery of WEEV in 1930, it has caused epidemics in equids and humans across the western half the U.S. and Canada, with the attack rate in humans reaching as high as 50/100,000 in Kern county, California (Reeves and Hammon, 1958). Overall, the estimated case fatality rate in humans is 3–4% (Reisen and Monath, 1988). The number of human and equine cases peaked in the 1940’s and 1950’s and has subsequently declined. Specifically, from 1964 to 1985 there was a total of 587 human WEE cases, while from 1986 to 2006 there were only 67 human cases documented (2005). This decrease in cases is in stark contrast to the numerous emerging infectious diseases identified during the latter half of the 20th century, including several arthropod-borne viral diseases of North America (Fauci, 2001), raising the question of why WEE appears to be declining. In spite of the decline in cases, WEE is still a notifiable disease and under the right circumstances it is possible that the virus may cause further epidemics. Understanding the reasons for the decline in WEE could elucidate factors involved in the decline of this and emergence of other arboviruses.

One hypothesis for the decrease in WEE is that the extant viruses are less virulent than the now-extinct virus lineages isolated during the period of peak epidemicity, perhaps as a result of unidentified selective pressures acting on WEEV or founder effects. However, a recent study identified no difference among California WEEV strains from different decades of the 20th century in avian viremia or virulence, or in infectivity for Cx. tarsalis (Reisen, Fang, and Brault, 2008). There are other possible enzootic hosts for WEEV such as the blacktail jackrabbit (Lepus californicus), and these have not yet been tested for altered responses to WEEV isolated during different time periods. Other possible ecological explanations for the reduction in WEE cases include the decline in uses of equids in agriculture, declining populations of the primary vectors (Cx. tarsalis and Aedes dorsalis) and/or changing vertebrate host populations. Another hypothesis for the WEE decline is that, due to stochastic genetic changes in WEEV or selection by hosts not yet examined, virulence for equids and humans has decreased. To test this hypothesis and to define a robust animal model for vaccine testing, we compared the virulence of 10 strains of WEEV isolated from the 1940’s through to the 1990’s using a mouse model.

Materials and Methods

Virus Strains

The WEEV strains used for this study were chosen because of their low passage histories and their isolation from a variety of hosts and locations. All 10 strains came from the group B WEEV clade (Weaver et al., 1997), the only North American clade known to persist since the 1970’s. The strain details are found in Table 1. Because the host cell of origin can affect alphavirus pathogenesis (Shabman et al., 2007), all virus stocks were prepared in C6/36 cells with 3 days of incubation at 32°C. Viruses were harvested and stored with a final concentration of 20% fetal bovine serum (FBS) at −80 degrees.

Table 1.

WEEV strains used in this study, the date and place of collection, the source of isolation and the passage history.

Code Strain Place of Origin Year Host Passage History
CA46 BFS932 Bakersfield, CA 1946 Culex tarsalis sm1
MO50 Ep-6 Missouri 1950 Mosquito ce1
CA53 BFS1703 California 1953 Culex tarsalis sm1/C6/36 1
MT67 Montana 64 Montana 1967 Horse de1
CA68 S8 1-22 Paradise (Butte Co) 1968 Sciurus griseus sm1
TX71 TBT-235 Texas 1968 Gopherus berland wc1,de1,sm1
CA83 CHLV 53 Riverside Co., CA 1983 Culex tarsalis vero1
NM85 85-452-NM New Mexico 1985 Culex tarsalis sm2
CO92 CO92-1356 Larimer City, CO 1992 Culex tarsalis vero1
AZ93 93A-27 Parker, Arizona 1993 Mosquito vero1
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Virulence Assays

Swiss-Webster mice (Charles Rivers, Wilmington, MA 01887, USA) were used as a model for WEE because pilot studies (unpublished data) had determined that this strain of outbred mice was the most susceptible to severe disease. Mouse manipulations were carried out using a protocol approved by the UTMB IACUC. The route of inoculation and dose were chosen to approximate a mosquito bite and because this model was also developed for vaccine efficacy studies.

Mice were infected subcutaneously with 103 plaque forming units (pfu) of virus in a 50µl volume. Animals were bled via the retroorbital sinus on day one post infection and the serum was diluted 1:10 in phosphate buffered saline (PBS) and stored at −80°C. Mouse serum was tested for the presence of WEEV using a standard plaque assay on Vero cells (Beaty, Calisher, and Shope, 1989). Animals were monitored daily for signs of disease, and were weighed daily from day 3–14 or 17 after infection.

For 4 of the WEEV strains (TX71, MO50, CA46, CA53), surviving mice sacrificed on day 7 after infection or at the point of death were necropsied, then organs were homogenized in PBS to prepare 10% W/V suspensions and tested for WEEV using a cytopathic effect (CPE) assays on Vero cells, followed by titration using a plaque assay.

The resulting datasets were ranked using semi-quantitative levels of virulence and tested using Wilcoxon’s ranked test for correlations between the date of collection and the virulence of the WEEV strains.

Results

The murine virulence of each WEEV strain was measured as the presence and level of viremia in the serum of the blood on day 1 post infection (p.i.), the weight loss exhibited over the course of infection, and the percentage survival. The viremia levels are shown in Table 2. The mice were bled on day one p.i. because pilot studies (data not shown) had demonstrated that viremia was undetectable after this time point. There was no discernable correlation (Wilcoxon’s ranked test) between the level and presence of viremia and the year of isolation of the WEEV strains (p = 0.5).

Table 2.

Viremia levels* one day after infection with WEEV.

Virus Strain CA46 MO50 CA53 MT67 CA68 TX71 CA83 NM85 CO92 AZ93
Mouse 1 <1.7 <1.7 2.11 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7
Mouse 2 1.81 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7 <1.7
Mouse 3 <1.7 <1.7 2.52 3.60 2.18 <1.7 <1.7 <1.7 3.74 <1.7
Mouse 4 <1.7 <1.7 <1.7 3.48 <1.7 <1.7 <1.7 2.54 <1.7 <1.7
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*

Titers expressed as log10 pfu/ml. Animals were considered negative when below the detection Limit of 1.7 log10 pfu/ml.

The mice were monitored daily for weight loss and mortality, and the data are presented in Fig. 1. The survival profiles (Fig. 1b), showed little correlation between the date of WEEV collection and the percentage or mean days of survival (p=0.3). Indeed, strains MT67 and CA53, as well as MO50, showed either severe weight loss and no mortality or slight weight loss and some mortality, suggesting that the ability of the virus to induce weight loss is not necessarily predictive of mortality. In fact, the only WEEV strain that showed both consistent weight loss and high mortality was CA46, although the viremia produced by this strain was minimal.

Figure 1.

Figure 1

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(a) percentage survival and rate of progression to mortality in Swiss-Webster mice injected with 3 log10 pfu/ml s.c. in 50ul and (b) average change in weight (grams) over the course of the experiment with the date of infection set as zero. Bars indicate standard deviations of the means.

For 4 of the WEEV strains (TX71, CA46, CA53 & MO50), organs were collected on day 7 p.i. and virus titers were determined. For all 4 strains, the brain was positive with titers ranging from 7.2×108 to 1.5×1010 pfu/g. For strains CA53 and MO50, virus was only found in the brain, whereas strains CA46 and TX71 were also present in the spleen (CA46) or the lung and the liver (TX71). There was very little difference in titres among the WEEV strains.

Statistical Tests

The viruses were ranked by weight loss, presence/absence and level of viremia, percentage survival and date of collection. The Wilcoxon Ranked Test indicated no significant correlations between viremia, weight loss and survival (mortality rate and mean survival time). The strongest correlation between year of WEEV collection and virulence was observed for weight loss. However, this was not significant at the 95% confidence interval (p=0.3).

Discussion

WEEV typically circulates enzootically between birds and the mosquitoes Cx. tarsalis and Ae. dorsalis as well as other members of the Dorsalis complex such as Ae. melanimon and Ae. campestris. A recent study that examined recent and older WEEV isolates in these hosts indicated no consistent differences in strains isolated during different decades and from different regions of California (Reisen, Fang, and Brault, 2008). However, potential differences in hosts involved in a secondary transmission cycle between the blacktail cottontail and Ae. melanimon (Hardy, 1987), which may contribute to WEEV maintenance, have not been examined. Furthermore, the dead-end hosts that generally indicate the occurrence of WEE epizootics, equids and humans, may respond to WEEV strains in ways not indicated by avian infections. Therefore, we used mice as a model for mammalian virulence.

Previous murine model studies utilized the Argentinean strains and the McMillan strain of WEEV (Bianchi et al., 1993; Nagata et al., 2006). The Argentinean strains represented by Ag80-646 probably circulate in a distinct ecosystem from the North American strains (Bianchi, Aviles, and Sabattini, 1997; Calisher et al., 1985) and therefore they were not included in our study as they are unlikely to have much bearing on the decline in virulence of WEEV in North America. Additionally, the route of infection in the previous studies was intranasal (i.n.), intracranial (i.c.) or intraperitoneal (i.p.) (Hardy et al., 1997). In our experience, WEEV is nearly always fatal via the i.n. and i.c. routes (data not shown). We hoped to develop a system that was closer to natural mosquito-borne infection, and as such the s.c. route was utilized.

The strains selected for this study were taken from the group B clade of the WEEV as defined by Weaver et al. (1997), with the strain TX71 included as a control strain from outside the group. Strains M050, CA46 and CA53 were taken from a subclade that had limited virus isolates from the later decades, whereas the rest of the strains were from a more variable and long-lived lineage. There was no reproducible difference in virulence between the two subclades, as defined by viremia or survival. In all of the statistical tests performed, there was no obvious correlation between year of isolation and any measure of viremia that was used. Weight loss correlated well with the date of collection (Spearman’s correlation coefficient r=0.002), although the results were not significant under Wilcoxon’s ranked test (p=0.3). This indicates that more WEEV strains and/or larger mouse cohort sizes may be needed to increase the power of the study, or a different animal model.

To date, no experimentally supported hypothesis has been suggested for the decline in WEE. However possible ecological changes should also be considered. Interestingly, unlike WEEV, the closely related avian alphavirus, eastern equine encephalitis virus (EEEV) has not shown the same trend of decreasing incidence, and until the introduction of West Nile virus, neither had the ecologically similar avian flavivirus, St. Louis encephalitis virus (SLEV) (Fang and Reisen, 2006; Hachiya et al., 2007). It is possible that the ecological changes unique to the WEEV transmission cycle could explain this discrepancy. One such possibility is the decline in the use of equids on farms in the U.S. during the past century, coincident with the industrialisation of agriculture (Reisen and Monath, 1988). However, equids are not considered amplification hosts so their abundance should not affect human exposure. Other possible explanations for the decline in human exposure are social trends that limit mosquito exposure, such as the increase in air conditioning (Gahlinger et al., 1986). Possible ecological factors such as a decline in abundance or a change in the distribution of the mosquito vectors Cx. tarsalis and Ae. dorsalis should also be evaluated, as any component of the enzootic transmission cycle may limit the number of spillover cases. Also, the western part of the U.S., particularly Colorado and California, were drier in the latter part of the 20th Century (Karl et al. 1995) and this may have contributed to a decline in WEEV transmission. It is important to determine the reasons for the decline in WEEV, as the mechanisms that have contributed to this decline could change in the future, affecting the circulation and disease caused by this and/or other arboviruses.

Acknowledgments

This work was supported by a grant from the NIAID through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, NIH grant U54 AI057156. JKL was supported by the NIH T32 Pre–Doctoral Training Program in Emerging and Tropical Infectious Diseases, AI007526. ERD was supported by the NIH T32 Pre-Doctoral Biodefense Training Program, AI060549 and by the James W. McLaughlin Fellowship Fund.

Footnotes

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