Experimental Everglades virus infection of cotton rats (Sigmodon hispidus)

Abstract

Everglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis (VEE) serocomplex, circulates among rodents and vector mosquitoes and infects humans, causing a febrile disease sometimes accompanied by neurologic manifestations. EVEV circulates near metropolitan Miami, which indicates the potential for substantial human disease, should outbreaks arise. We characterized EVEV infection of cotton rats in South Florida, USA, to validate their role in enzootic transmission. To evaluate whether the viremia induced in cotton rat populations regulates EVEV distribution, we also infected rats from a non-EVEV-endemic area. Viremia levels developed in rats from both localities that exceeded the threshold for infection of the vector. Most animals survived infection with no signs of illness, despite virus invasion of the brain and the development of mild encephalitis. Understanding the mechanisms by which EVEV-infected cotton rats resist clinical disease may be useful in developing VEE therapeutics for equines and humans.

Figure 1

Map of South Florida, indicating locations of Everglades virus isolation, human cases or antibody detection (stars), and our cotton rat collection site (box). Dark line delineates national park boundary.

Figure 2

Viremia and neutralizing antibody profile in F₁ Florida (panels A,B) or wild Texas cotton rats (panels C,D) injected with Everglades virus strains FE4-71k (A, C) and FE3-7c (B, D) administered subcutaneously in the left thigh. Inoculum doses were as follows: panels A and B: 2.9 log₁₀ PFU/mL, panel C: 2.3 log₁₀ PFU/mL, panel D: 3.6 log₁₀ PFU/mL. Florida animals were bled daily; viremia or 80% plaque reduction neutralization test (PRNT) antibody titers represent geometric means of data from eight rats (strain FE3-7c) or seven rats (EVEV FE4-71k). Rats from Texas were each bled every 2 days; means (geometric) represent measurements from four animals. Bars denote standard deviations.

Figure 3

A) Viremia and neutralizing antibody profiles in F₁ Florida cotton rats serially sacrificed at daily intervals after infection with 3.2 log₁₀ PFU of Everglades virus strain FE4-71k administered subcutaneously in the left thigh. Lines on each graph represent the geometric mean viremia or mean 80% plaque reduction neutralization test (PRNT) antibody titers; the number of rats bled at each time point is denoted in parentheses above each point. Error bars denote standard deviations. Everglades virus organ titers from the brain, salivary glands, lung and heart (B) and liver, kidney, and spleen (C) of EVEV strain FE471k-infected F₁ Florida cotton rats serially sacrificed at daily intervals. Two rats, denoted "a" and "b" were sacrificed daily from days 1–7 postinfection. No virus was detected in any organ on day 7.

Figure 4

Brainstem section of sham-inoculated control rat, showing the absence of an inflammatory response (A). Vascular and perivascular infiltration of mononuclear cells within the brainstem of a Florida cotton rat 7 days after infection with 3.2 log₁₀ PFU/mL EVEV strain FE4-71k; inset enlarged to show cell infiltration (B). Cortex of cotton rat 5 weeks after infection, showing absence of inflammatory response (C). Animals in panels A and B were anesthetized with pentobarbital and perfused with phosphate-buffered saline intracardially. The rat in panel C was not perfused.