Identification of Enteroviruses by Using Monoclonal Antibodies against a Putative Common Epitope

Abstract

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.The genus Enterovirus, belonging to the family Picornaviridae, consists of 66 different subtypes, including polioviruses (PVs), coxsackievirus group A (CVA) and CVB, echoviruses, and the numbered enteroviruses. These viruses cause a wide variety of clinical illnesses, ranging from asymptomatic to fatal, including poliomyelitis, aseptic meningitis, myopericarditis, and respiratory, hepatic, and gastrointestinal infections (15,23,24). Thus, the early diagnosis of enterovirus-related infection from various clinical isolates is important for prognostic, therapeutic, and epidemiologic purposes (1,14,18,26).The primary structure and genetic organization of enteroviruses have been demonstrated recently. Conserved sequences are located in structural and nonstructural proteins of enteroviruses (12,28,29,31,35,39). Therefore, it may be possible to use the conserved region as a common epitope for the serological diagnosis of enteroviruses. The synthetic peptides for the enterovirus group-common epitope could be used for the serological diagnosis of infections caused by a broad range of enteroviruses (3, 25). However, these approaches have not yet been proven effective for the routine laboratory diagnosis of enteroviral antigens in clinical specimens. Thus, it would be useful to develop a rapid and reliable antigen diagnostic method for enteroviral infection by using a monoclonal antibody (MAb) specific for the common epitope region.In the present study, the genetic region of the putative common epitope of enteroviruses was determined by sequenceindependent single-prim...

Monoclonal Antibodies to VP1 Recognize a Broad Range of Enteroviruses

“…EV capsid protein VP1 is one of four structural proteins of EV, and its antigenic homology among many different EV serotypes has been well documented (2,7,(16)(17)(18). The N termini of most EV VP1 proteins contain highly conserved immunogenic regions that are recognized by sera from most EV-infected patients (2).…”

“…However, the two commercially available pan-EV MAbs used in the staining assay either fail to react with multiple EV serotypes (9, 19) or cross-react with other non-EVs (8, 22). The lack of availability of highly reactive and specific pan-EV MAbs for diagnosis of EV infection could be due to the difficulties in developing specific MAbs against the extensive antigenic diversity among EVs.EV capsid protein VP1 is one of four structural proteins of EV, and its antigenic homology among many different EV serotypes has been well documented (2,7,(16)(17)(18). The N termini of most EV VP1 proteins contain highly conserved immunogenic regions that are recognized by sera from most EV-infected patients (2).…”

Virus discovery by sequence‐independent genome amplification

“…Also, sequence-independent amplification of DNA has been used for the molecular cloning of specific microdissected DNA chromosomal regions [13]. Finally, a common epitope region of enteroviruses has been identified by SISPA followed by immunoscreening [33].…”

Widespread Circulation of Echovirus Type 13 Demonstrated by Increased Seroprevalence in Toyama, Japan, between 2000 and 2003

“…3B) (9,17,18,23,25). In addition, many amino acid differences were observed between strains 2002-240-SF and Del Carmen in the VP1 region, of which the N and C termini were, respectively, common epitopes of enteroviruses (27,29).…”

“…When the amino acid sequences of strains 2002-240-SF and Del Carmen were compared, differences of 1, 7, 6, and 25 amino acids were observed in the VP4, VP2, VP3, and VP1 regions, respectively. Although the neutralization antigenic sites (N-Ags) and the crystal structure of E13 have not been determined, the N-Ags of other enteroviruses have been reported and some of them are in the same position (4,17,18,25,27,29). Therefore, the amino acid sequence differences between strains 2002-240-SF and Del Carmen were compared with the N-Ags of other enteroviruses on the three-dimen- , respectively, are projected onto the E11 structure (Protein Data Bank number 1h8t; original source of structural data, reference 30; used with permission).…”