The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

RESEARCH ARTICLE The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model Chikako Kataoka1, Tadaki Suzuki2, Osamu Kotani2, Naoko Iwata-Yoshikawa2, Noriyo Nagata2, Yasushi Ami3, Takaji Wakita1, Yorihiro Nishimura1,4, Hiroyuki Shimizu1* 1 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan, 2 Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan, 3 Division of Experimental Animal Research, National Institute of Infectious Diseases, Tokyo, Japan, 4 Division of Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America * Abstract OPEN ACCESS Citation: Kataoka C, Suzuki T, Kotani O, IwataYoshikawa N, Nagata N, Ami Y, et al. (2015) The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model. PLoS Pathog 11(7): e1005033. doi:10.1371/journal.ppat.1005033 Editor: Carolyn B Coyne, University of Pittsburgh, UNITED STATES Received: April 6, 2015 Accepted: June 19, 2015 Published: July 16, 2015 Copyright: © 2015 Kataoka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals. PLOS Pathogens | DOI:10.1371/journal.ppat.1005033 July 16, 2015 1 / 26 EV71 Fitness and Pathogenesis in a Non-human Primate Model Author Summary Recently, large outbreaks of hand, foot, and mouth disease, including fatal neurological cases in young children primarily because of enterovirus 71 (EV71) have been reported, particularly in the Asia Pacific regions where the disease poses a serious threat to public health. Based on mutational and structural analyses of EV71, we identified amino acid residue 145 of the capsid protein VP1 (VP1-145) as a critical molecular determinant for the binding of EV71 to a specific cellular receptor, human P-selectin glycoprotein ligand-1 (PSGL-1). VP1-145 is highly variable among EV71 isolates and has been identified as a potential neurovirulence determinant in humans and experimental mouse models. To elucidate the in vivo involvement of PSGL-1-depentent replication and pathogenesis, we investigated viral replication, genetic stability, and the pathogenicity of the PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) strains of EV71 in a cynomolgus monkey model. After the intravenous inoculation with the PB strain, viruses found to be highly mutated at VP1-145 with resultant VP1-145E variants (non-PB) inducing viremia and neuropathogenesis, presumably in a PSGL-1-independent manner. VP1-145G variants were identified only in peripheral blood mononuclear cells from two PB-inoculated monkeys. Our study provides new insights into the interplay between virus, receptors, and host in EV71-infected individuals. Introduction Enterovirus 71 (EV71) is a non-enveloped positive-stranded RNA virus belonging to the species Enterovirus A of the genus Enterovirus in the family Picornaviridae. The EV71 RNA genome is enclosed within an icosahedral capsid comprising 60 structural protein subunits (protomers), each containing four viral structural proteins, VP1-VP4 [1, 2]. According to the molecular epidemiological analysis of the capsid VP1 sequence, EV71 was previously classified into the three genogroups A, B (subgenogroups B1–B5), and C (subgenogroups C1–C5) [3, 4]. Recently, several additional genogroups of EV71 were identified as genogroups D, E, and F [5]. EV71 is a major causative agent of hand, foot, and mouth disease (HFMD) along with coxsackievirus A16 (CVA16) and coxsackievirus A6 (CVA6) [6, 7]. EV71 usually causes mild or subclinical infection. However, in some patients, EV71 may cause severe neurological symptoms, including meningitis, brainstem encephalitis, poliomyelitis-like paralysis, pulmonary edema, and death. As recent EV71 outbreaks in the Asia-Pacific region, including Malaysia, Taiwan, China, Cambodia, and Vietnam, have involved millions of children (almost all under 5 years old) including thousands of fatal cases, EV71 poses a threat to global public health [4, 8–10]. Recently, a growing number of viral and host factors associated with EV71 infection have been reported [11–14]; however, no conclusive risk factors for neuropathogenesis have yet been identified. In the previous studies, two molecules, scavenger receptor class B, member 2 (SCARB2) [15] and P-selectin glycoprotein ligand-1 (PSGL-1) [16] were identified as functional receptors for EV71. Subsequently, other cel (...truncated)