Cross antigenicity among enteroviruses as revealed by immunoblot technique

doi:10.1016/0042-6822(83)90181-2

Virology

Volume 129, Issue 2, September 1983, Pages 431-442

https://doi.org/10.1016/0042-6822(83)90181-2 Get rights and content

Abstract

Antigenic relationships of various human and two animal picornaviruses were investigated by the immunoblotting (“Western blot”) technique. The viruses included all coxsackievirus B types (1–6), poliovirus types 1–3, several strains of echovirus 11, EMC virus, and FMDV. Antisera included human sera and sera from rabbits hyperimmunized with either purified picornaviruses, viral structural polypeptides (VP₅), boiled or “sampleboiled” virions. Group-specific reactions of various extent were observed among the human picornaviruses, but not with EMC virus. These reactions were obtained with human sera (whole serum, IgG- and IgM-fraction) as well as with “monospecific” (neutralization test) rabbit antisera. Among cross reacting polypeptides VP₁ was predominant with the notable exception of coxsackie B4, where VP₁ (defined according to cleavage pattern) migrates in our gel system as second largest polypeptide. Antisera prepared vs VP₁ had neutralizing activity as demonstrated with five different echovirus 11 strains (titers up to 2000). Antisera vs VP₁ (and other VP₅) exhibited cross-reactivity in the immunoblots. Antisera to the three poliovirus types (and to certain echovirus 11 strains) showed a surprisingly narrow cross-reacting spectrum which—in the case of poliovirus—could not be broadened by additional hyperimmunization of the rabbits with heated poliovirus 2. The significance of these results for a diagnostic ELISA in patients with picornavirus infections is dealt with.

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HFMD can be caused by different members of the enterovirus family, such as echoviruses and coxsackievirus A (CAV) but the main culprits have been identified as enterovirus 71 (EV71) and coxsackievirus A16 (CAV16) (King et al., 2000). Besides their similar pathogenicity, these viruses also are closely related genetically (Mertens et al., 1983). Infection results in symptoms of varying severity ranging from herpangina and HFMD to neuropathogenicity, including acute flaccid paralysis, aseptic meningitis, and brainstem or cerebellar encephalitis which may occasionally cause death.
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Identification of immunodominant VP1 linear epitope of enterovirus71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in western blots
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A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)–SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.
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A-2 plaque virus (A2 virus) was originally isolated from the icteric-phase sera of US servicemen with viral hepatitis in the 1960s, but apart from a preliminary characterization little is known about the agent. We have now successfully cloned and sequenced the complete viral genome. A2 viral RNA consists of 7312 nucleotides, excluding the 62 nucleotide poly(A) tract at the 3′ end, with one large open reading frame. Although clearly a member of the Picornaviridae, there is low homology to the available sequences, suggesting it is only loosely related to the classic rhino/enterovirus genus. In addition, there was no reactivity with group specific monoclonal antibody blends against polioviruses, enteroviruses 70 and 71, coxsackievirus B, and echoviruses. Two tamarins were inoculated with A2 virus to study viral pathogenesis. Both animals that received A2 virus became transiently viremic 1 week after the infection, as determined by RT–PCR, and they developed an antibody response to A2 virus. However, no physical signs or biochemical abnormalities, including elevated liver transaminases, were observed. In addition, no liver samples from patients with fulminant hepatitis (n = 7) or controls (n = 7) were positive for A2 viral RNA nor was anti-A2 neutralizing antibody detected in sera from hepatitis patients (n = 14), healthy laboratory donors (n = 14), or US blood donors (n = 33); however, most sera contained antibodies reactive with A2 virus proteins. These results suggest that A2 virus is a new member of the Picornaviridae but that its pathogenicity in nonhuman primates and association with human disease still need to be determined.
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In order to study immunological reactivity of individual enterovirus polypeptides and evaluate their usefulness for enterovirus diagnosis, the genes coding for viral structural and nonstructural proteins were expressed using recombinant baculoviruses. A histidine-tailed coxsackievirus B3 (CBV3) VP1 capsid protein was expressed and purified by immobilized metal ion affinity chromatography for EIAs. To elucidate the usefulness of the other CBV3 capsid proteins for immunoassays, recombinant baculovirus expressing the whole CBV3 capsid polyprotein region was constructed. For the detection of a potentially broader spectrum of enteroviruses, the conserved nonstructural P3 region was expressed. The P3 region encodes four nonstructural proteins including a cysteine protease (3C) and an RNA-dependent RNA-polymerase (3D). The 3C polypeptide was shown to be proteolytically active indicating that the baculovirus system is capable of expressing biologically functional enterovirus proteins. Immunoblot analysis detected antibodies against the VP1, 3C and 3D proteins in human serum samples. When the baculovirus-expressed antigens were compared with lysates of enterovirus-infected cells and a synthetic peptide in EIA highly similar results were obtained with recombinant VP1 and the lysate antigens. Although reactive in immunoblots, the P3 encoded proteins were not satisfactory for EIA.
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Cross antigenicity among enteroviruses as revealed by immunoblot technique

Abstract

Virology

Virology

Lancet

Virology

Virology

Immune and antibody responses to an isolated capsid protein of foot-and mouth disease virus

J. Immunol.

An experimental protein vaccine for foot-and-mouth disease

Perspect Virol.

Le polypeptide structural VP1 du poliovirus type 1 induit des anticorps neutralisants

C. R. Acad. Sci. Paris

Hybridoma antibodies to poliovirus N and H antigen

Arch. Virol.

Nature of the antibody response to the foot-andmouth disease virus particle, its 12S protein subunit and the isolated immunizing polypeptide VP1

J. Gen. Virol.

Isolated poliovirus capsid protein VP1 induces a neutralizing response in rats

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay

Med, Microbiol. Immunol.

Specificity of IgM antibodies in acute human coxsackievirus B infections, analysed by indirect solid phase enzyme immunoassay and immunoblot technique

J. Gen. Virol.

A phase contrast microprecipitin test with poliovirus antigens

Archiv. Gesamte Virusforsch.

Spectrum and characteristics of the virus inhibitory action of 2(a-hydroxybenzyl)-benzimidazole

J. Exp. Med.

Relationship between poliovirus and echovirus 6 antigens. I. Immunization experiments in Guinea pigs

Archiv Gesamte Virusforsch.

The preparation of 131I labelled human growth hormone of high specific radioactivity

Biochem. J.

The preparation of ¹³¹I labelled human growth hormone of high specific radioactivity