A Non-Mouse-Adapted Enterovirus 71 (EV71) Strain Exhibits Neurotropism, Causing Neurological Manifestations in a Novel Mouse Model of EV71 Infection

Abstract

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.

Attenuation of Human Enterovirus 71 High-Replication-Fidelity Variants in AG129 Mice

“…8A and B). The results indicated that RG/B4 accumulated in the hind limb muscle and brain throughout the whole experiment; the viral titer and RNA copy number reached the highest levels before the death of infected mice, similarly to a previous description of wild-type B4 infection in AG129 mice (30). In contrast, all high-replication-fidelity variants showed lower viral titers and RNA copy numbers in the hind limb muscle and brain than RG/B4 at each time point.…”

“…Young suckling AG129 mice showed neurological symptoms and even died with wild-type EV71-B4 infection because AG129 mice lack a functional interferon system and support both the spread from the primary infection site and the persistence of EV71 (30). As IFNs play an important role in the antiviral defense against EV71 infection, the LD 50 differences between the parental RG/B4 and its high-fidelity variants described in AG129 mice might even be widened in an immunocompetent host, where fewer mutations in the population of high-fidelity variants might result in a lower likelihood of mutations capable of circumventing established innate and adaptive immune responses.…”

“…Previous studies revealed that high-replication-fidelity RNA virus variants are attenuated through restricted replication and lower fitness in an animal model (10)(11)(12)(13). To evaluate the effect of high-replication-fidelity EV71 variants on viral pathogenicity in vivo, we used the AG129 mouse model, which has been well defined for wild-type EV71-B4 infection with neurotropism (30). We also generated RG/B4 variants with mutations at the position G64 of RdRp, which was identified as participating in a fidelity checkpoint in the RdRp of poliovirus (13).…”

“…Alternatively, immunodeficient mice (29,30) and transgenic mice with a human EV71 receptor (31,32) were found to be vulnerable to EV71 infection. The AG129 mouse is immunodeficient and without alpha/beta and gamma interferon (IFN) receptors; it can be infected by the non-mouse-adapted EV71 strain 41 (EV71-B4 in this paper, where B4 indicates the subgenogroup) (30). The infected mice display progressive limb paralysis prior to death and virus accumulation in the central nervous system, indicating a clear neurotropism of EV71 and making this model relevant for EV71 pathogenicity studies and for EV71 vaccines and drug testing.…”

Impact of SCARB2 on pro-inflammatory cytokines in tissues of EV71-infected mice

“…EV71, together with coxsackievirus A16 (CVA16) infections are generally associated with hand, foot and mouth disease (HFMD), but EV71 infection occasionally progress to severe neurological disease, including aseptic meningitis, poliomyelitis-like paralysis, and possibly fatal encephalitis in neonates, especially brain stem encephalitis associated with pulmonary edema and cardiac insufficiency which are the primary manifestations in patients with neurologic involvement [11,12]. Numerous animal models have been developed to study the pathogenesis of EV71 infection using the mouse-adapted strain of EV71 in innate immunodeficient mice [13][14][15]. The EV71 BrCr strain was demonstrated to induce neurological manifestation of tremor, ataxia, and brain edema in cynomolgus monkeys [16].…”