An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys

Abstract

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3')], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3'), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3'). Monkeys inoculated with EV71(S1-3') showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3'), two monkeys were intravenously inoculated with EV71(S1-3'), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3') was isolated only from the spinal cord. These results indicate that EV71(S1-3') acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.

FIG. 1.

Schematic diagram of the EV71(S1-3′) genome. The sequences derived from the parental EV71(BrCr-TR) genome are represented by the black regions, and the mutations derived from PV1(Sabin) are represented by the white regions with stars above them. Corresponding mutations of PV1(Mahoney), PV1(Sabin), and EV71(BrCr-TR) are also shown. The numbers in parentheses are the nucleotide positions in the genome or the amino acid positions in the 3D^pol protein. The figure was adapted from a previously published figure with the permission of the Society for General Microbiology (5).

FIG. 2.

Experimental schedule, clinical symptoms, and virus excretion of cynomolgus monkeys inoculated with EV71(S1-3′). (A) Experimental schedule. In experiment 1 (upper panel), the antigenicity of EV71(S1-3′) in cynomolgus monkeys was examined. Three monkeys were intravenously inoculated with EV71(S1-3′) on day 0 and were then challenged by lethal inoculation with EV71(BrCr-TR) on day 45 p.i. In experiment 2 (lower panel), the tissue specificity of EV71(S1-3′) in the early phase of infection was examined. Two monkeys were sacrificed on day 4 p.i., and virus distribution and histopathology in those two monkeys were examined. The assigned numbers and ages of individual monkeys are shown. Numbers in parentheses represent days after the lethal challenge with EV71(BrCr-TR). The swabs and blood were collected at the times indicated. (B) Clinical symptoms of the monkeys. Severity of tremor is represented by +, ++, and +++. (C) Virus excretion in the throat and from rectal swabs of the monkeys inoculated with EV71(S1-3′). The swabs from which EV71(S1-3′) or EV71(BrCr-TR) was isolated are shown as positive, and swabs from which EV71(S1-3′) or EV71(BrCr-TR) was not isolated are shown as negative. Swab samples from monkeys inoculated with EV71(BrCr-TR) (numbers 4507 and 4508) were collected in our previous study (5). ND, not determined.

FIG. 3.

Anti-EV71 antibody titers of monkeys inoculated with EV71(S1-3′). The time point of lethal challenge with EV71(BrCr-TR) is indicated by an arrow. (A) Anti-EV71 neutralization activity of monkey serum. The neutralization titer of 50 μl of the serum, determined by the CPE method, is shown over the course of the infection. The serum of a monkey (number 4507) inoculated with virulent EV71(BrCr-TR) was collected in a previous study (5). (B and C) Measurement of anti-EV71 IgM (B) or anti-EV71 IgG (C) in monkey serum. Histidine-tagged EV71 virions were adsorbed on a nickel-coated plate and then monkey serum was added to the plate. Monkey anti-EV71 antibodies bound to the histidine-tagged virions were detected by HRP-conjugated goat anti-monkey IgM or anti-monkey IgG antibodies. OD450, optical density at 450 nm.