Bead-based suspension array for simultaneous detection of antibodies against the Rift Valley fever virus nucleocapsid and Gn glycoprotein
Highlights
► A bead-based suspension array was developed for serology of Rift Valley fever virus. ► It can detect antibodies against the nucleocapsid and glycoprotein Gn simultaneously. ► The assay is capable of detecting IgG antibodies in sheep, cattle and human sera. ► The assay may be applied as DIVA test with vaccines based on surface glycoproteins.
Introduction
Rift Valley fever virus (RVFV) is a mosquito borne virus that belongs to the Phlebovirus genus of the Bunyaviridae family (Elliott, 1996). The virus was first isolated in 1930 (Daubney et al., 1931) near Lake Naivasha in Kenya and has since caused devastating outbreaks throughout Africa (Bird et al., 2009, Gerdes, 2004).
The RVFV RNA genome comprises a small (S), medium (M) and large (L) segment. The S genome segment encodes the nucleocapsid (N) protein in genomic-sense orientation and a non-structural protein (NSs) in the antigenomic-sense orientation (Elliott, 1996). The NSs protein suppresses host innate immune responses and was shown to be the major virulence determinant of the virus (Billecocq et al., 2004, Bouloy et al., 2001, Habjan et al., 2009, Ikegami et al., 2009). The M segment encodes the two structural glycoproteins Gn and Gc and two non-structural proteins, a 78-kDa protein and the NSm protein, of which the latter was shown to have an anti-apoptotic function (Won et al., 2007). The L segment encodes the RNA-dependent RNA polymerase protein.
The first commercially available ELISAs used inactivated whole virus as the antigen (Paweska et al., 2003, Paweska et al., 2005a, Paweska et al., 2005b). Although these ELISAs in general perform very well, the use of recombinant proteins can provide advantages with respect to safety, stability and cost-effectiveness. Several ELISAs based on recombinant antigen have been developed and commercialized, but all these assays detect antibodies against the N protein (Jansen van Vuren and Paweska, 2009, Jansen van Vuren et al., 2007, Paweska et al., 2007, Paweska et al., 2008). Preferably, a serological confirmation test should detect antibodies against other immunogenic proteins of the virus. The virus neutralization test is based on the detection of antibodies against the structural glycoproteins, and can therefore be used for such serological confirmation (Gerdes, 2008). However, the classical virus neutralization test requires live virus to be handled under appropriate biosafety containment facilities, and takes up to seven days for completion. A less laborious, glycoprotein-based serological assay would therefore be very advantageous.
Recently, an efficient method to produce milligram amounts of the Gn ectodomain in a highly pure, soluble form was developed (de Boer et al., 2010). In the work described in the present paper, the production of this protein was improved, and both the recombinant Gn ectodomain and purified N protein were used to develop a Luminex assay for the simultaneous detection of antibodies against both proteins.
Luminex technology uses variously coloured polystyrene beads, the surface of which is carboxylated to allow covalent coupling of antigens. Conjugated beads can be incubated with serum to capture specific antibodies, after which a fluorescent secondary antibody is added to bind to the captured serum antibodies. The Luminex system uses a red laser to determine the colour of the bead and a green laser to detect bound secondary antibodies (Krishhan et al., 2009). With this system, multiple serological components can be tested simultaneously with a single sample (Perkins et al., 2006). The work described in this paper demonstrated that a newly developed Luminex assay can detect antibodies against both the RVFV Gn and N proteins simultaneously. Moreover, the results suggest that this assay may be used to differentiate infected from vaccinated animals (DIVA) when combined with new experimental vaccines based on RVFV glycoproteins.
Section snippets
Production of the N protein
Production of recombinant RVFV nucleoprotein N has been described previously (Martin-Folgar et al., 2010). Briefly summarized, the gene encoding the N protein of RVFV strain MP-12 (GenBank accession number DQ380154.1, protein ID ABD38739.1), flanked by SalI and XhoI sites, was cloned into plasmid pET32a (Novagen, Merck KGaA, Darmstadt, Germany) to generate a fusion protein with the 109 amino acid thioredoxin protein (Trx) containing a 6× His-tag for purification purposes. The resulting plasmid,
Production of Trx-N and the Gn ectodomain
The thioredoxin fusion system was initially chosen to increase the fraction of soluble recombinant N protein in E. coli cells. After induction of expression, most of the recombinant protein was found in the lysozyme fraction, which is indicative of the accumulation in the periplasmic space. Large amounts of this protein were easily produced, facilitating further downstream nickel resin-based purification procedures. The analysis of the expression by Coomassie staining showed the presence of a
Discussion
This study reports the development of a Luminex assay that can be used for the simultaneous detection of antibodies against the RVFV N protein and the structural glycoprotein Gn. The assay was shown to be capable of detecting IgG antibodies in sheep, cattle and human sera and proof-of-concept of IgM detection was provided. Based on the limited number of sera analysed, the assay was shown to be comparable in terms of sensitivity and specificity with commercially available RVFV ELISAs. This
Acknowledgments
The authors would like to thank Dr. Catherine Cêtre-Sossah (Centre International de Recherche Agronomique pour le Développement, Montpellier, France), Dr. Philippe Marianneau and Dr. Michel Pépin (Agence Nationale de Sécurité Sanitaire, France), Dr. Martin Eiden (Friedrich Loeffler Institute, Germany) and Dr. Francesc Xavier Abad Morejón de Girón (Centre de Recerca en Sanitat Animal, Spain) for providing the ring trial sera, Dr. Christiaan A. Potgieter and Shirley J. Smith (ARC-OVI) for
References (26)
- et al.
Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge
Vaccine
(2010) - et al.
Cloning and expression of Rift Valley fever virus nucleocapsid (N) protein and evaluation of a N-protein based indirect ELISA for the detection of specific IgG and IgM antibodies in domestic ruminants
Vet. Microbiol.
(2007) - et al.
Laboratory safe detection of nucleocapsid protein of Rift Valley fever virus in human and animal specimens by a sandwich ELISA
J. Virol. Methods
(2009) - et al.
Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA
J. Virol. Methods
(2007) - et al.
Rift Valley fever virus immunity provided by a paramyxovirus vaccine vector
Vaccine
(2010) - et al.
IgG-sandwich and IgM-capture enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in domestic ruminants
J. Virol. Methods
(2003) - et al.
Validation of IgG-sandwich and IgM-capture ELISA for the detection of antibody to Rift Valley fever virus in humans
J. Virol. Methods
(2005) - et al.
Validation of an indirect ELISA based on a recombinant nucleocapsid protein of Rift Valley fever virus for the detection of IgG antibody in humans
J. Virol. Methods
(2007) - et al.
An inhibition enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants
J. Virol. Methods
(2005) - et al.
Recombinant nucleocapsid-based ELISA for detection of IgG antibody to Rift Valley fever virus in African buffalo
Vet. Microbiol.
(2008)
Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus
J. Virol. Methods
NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription
J. Virol.
Rift Valley fever virus
J. Am. Vet. Med. Assoc.
Cited by (22)
Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe
2015, VaccineCitation Excerpt :There is likely to be some variability in disease kinetics between humans and animal species due to variation in susceptibility between species, and even within species [2,51,52]. RVFV can be detected by classic virological methods which include virus isolation [53], histopathology [54], antigen detection [55,56], antibody detection [57–59] and nucleic acid based assays [60–62]. For reporting RVFV in animals, the Office International des Epizooties (OIE); World Organisation for Animal Health, require laboratory confirmation by at least two positive results from a combination of different diagnostic approaches preferably for the same specimen i.e. either positive for virus/viral RNA and antibodies or positive for IgM and IgG with demonstration of rising titres between paired serum samples collected 2–4 weeks apart [50].
A duplex recombinant viral nucleoprotein microbead immunoassay for simultaneous detection of seroresponses to human respiratory syncytial virus and metapneumovirus infections
2014, Journal of Virological MethodsCitation Excerpt :The nucleocapsid (N) protein in particular has been shown to be effective in serologic assays for the paramyxoviruses, being highly immunogenic and inducing an early and long lasting antibody response (Hummel et al., 1992; Buraphacheep et al., 1997; Liu et al., 2007). As with multiplexed molecular assays that combine multiple individual pathogen assays in a single reaction to reduce reagent and sample consumption and increase testing throughput, the Luminex bead-based suspension array technology has also been used successfully to develop multiplexed serologic assays for human and animal viruses (Anderson et al., 2011; Liao et al., 2011; Hernandez et al., 2012; van der Wal et al., 2012). In this study, the N proteins of hRSV and hMPV were expressed in a baculovirus system and their performance compared against whole virus lysate antigen in in-house serologic enzyme immunoassays (EIAs).
A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants
2013, Research in Veterinary ScienceCitation Excerpt :N protein is a highly conserved protein and considered to be the immunodominant protein of Bunyaviruses in general (Jansen van Vuren et al., 2007). Recently, a novel multiplex bead-based suspension LUMINEX assay for the simultaneous detection of antibodies to RVFV N and Gn protein was published (van der Wal et al., 2012) to facilitate the development of DIVA vaccines encoding single recombinant glycoproteins. Following these lines of thought, the aim of our study was to create a novel indirect ELISA based on bacterially expressed glycoprotein Gn which is robust enough for the use in developing countries.
A bead-based suspension array for the serological detection of Trichinella in pigs
2013, Veterinary JournalCitation Excerpt :In this context the use of serology to contemporaneously screen for this and other zoonotic pathogens such as Salmonella spp. (Wegener et al., 2003) in a multiplex format is an appealing goal. Although there are many examples illustrating bead-based assays can be used for multiplex serology in humans (Dias et al., 2005; van Gageldonk et al., 2008; Casabonne et al., 2009; Antonsson et al., 2010), the use of bead-based serology for veterinary applications is limited (Clavijo et al., 2006; Perkins et al., 2006; Go et al., 2008; Watson et al., 2009; Anderson et al., 2011; van der Wal et al., 2012). In this study, we first investigated whether a serological test for Trichinella infection in pigs could be established in a format suitable for multiplexing.
Paving the way for human vaccination against Rift Valley fever virus: A systematic literature review of RVFV epidemiology from 1999 to 2021
2022, PLoS Neglected Tropical Diseases
- 1
Present address: Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, R3E 3R2 Winnipeg, Canada.
- 2
Present address: National Institute for Public Health (RIVM), Centre for Infectious Disease Control, Laboratory for Zoonoses and Environmental Microbiology, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.