Nipah virus glycoprotein: production in baculovirus and application in diagnosis
Introduction
Nipah virus (NiV) is a negative sense nonsegmented RNA virus that possesses an RNA dependent RNA polymerase (Wang et al., 1998). Its genome encodes six proteins: nucleocapsid- (N), phospho- (P), matrix- (M), fusion- (F), glyco- (G) and large- (L) proteins (Harcourt et al., 2000, Wang et al., 2001).
NiV was first isolated by Chua et al. (1999) from the cerebrospinal fluid of an encephalitic patient, during the deadly paramyxovirus outbreak in Malaysia in 1998. The virus has been classified into a new genus, Henipaviruses, within the family Paramyxoviridae (Wang et al., 2000). It causes fatal encephalitis in humans and a respiratory syndrome in pigs. Other animals such as dogs, cats, and horses can also be infected by the virus when they come into close contact with infected pigs (Chua et al., 1999, Chua et al., 2000; Paton et al., 1999). Fruit bats are thought to be the natural reservoir of the virus and could be introduced into a pig farm through their secretions (Chua et al., 2002). Guillaume et al. (2004) supported this suggestion and reported that there is an increase contact between the fruit bats with humans and domesticated animals where the bats may transfer the viruses to new species resulting in a more virulent and even fatal disease. The outbreak of Nipah virus is currently under control, but based on the above issues, there is a pressing need for rapid detection as well as serological diagnosis of the virus for monitoring the presence of the virus and its antibodies in individuals and animals in high risk areas. Currently, production of immunological reagents for these assays require the highest level of microbiological security (biohazard level 4) which is only limited to a few laboratories in the world. Recombinant DNA technology provides an alternative means for the production of safer diagnostic reagents.
In this study, the DNA encoding the G protein of NiV was cloned and expressed in a baculovirus system. The purified product was used as reagent in an ELISA for serological screening of serum samples collected from naturally infected swine during the virus outbreak in 1998–1999. In contrast to whole virus antigen, the production of the recombinant G protein is more efficient and less time-consuming. In addition, such antigen is non-infectious and therefore no containment facilities are necessary.
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Viruses and antisera
Inactivated NiV from infected cell culture medium as well as swine antisera with known SNT were obtained from Veterinary Research Laboratory (VRI), Ipoh, Malaysia.
Viral RNA preparation and amplification of truncated G gene
Total RNA was extracted using the TRIZOL LS reagent (Life Technologies, USA) from 250 μl of NiV infected cell culture medium as recommended by the manufacturer. Extracted viral RNA was used as a template for cDNA synthesis using the Superscript II RNaseH (−) reverse transcriptase (Life Technologies, USA), which were subsequently used
Results
The cDNA encoding the truncated G protein was engineered by PCR without the leader and transmembrane regions of the protein because these regions were found to reduce the level of the full length G protein significantly in insect cells (data not shown). Therefore, truncated G protein was constructed without the transmembrane and signal regions, in order to improve its level of expression.
A recombinant baculovirus was generated by using transposon-mediated insertion of the corresponding genetic
Discussion
Here we report the cloning of a truncated G gene of the NiV and the synthesis of the protein in baculovirus. Baculovirus expression systems have been widely used for the production of antigens for diagnosis of diseases caused by viruses, such as peste des petits ruminants virus (Choi et al., 2003), pseudorabies virus (Gut-Winiarska et al., 2000), porcine parvovirus (Rueda et al., 2000), Newcastle disease virus (Errington et al., 1995, Makkay et al., 1999) and measles virus (Warnes et al., 1994).
Acknowledgements
The authors wish to thank Veterinary Research Institute (VRI, Ipoh, Malaysia) especially Dr. Sharifah Syed Hassan for generously providing the inactivated NiV swine isolate and swine anti-NiV sera. This study was supported by the grant no. 26-02-03-0128 from the Ministry of Science, Technology and the Environment of Malaysia (MOSTE). M.E. was supported by National Science Fellowship (NSF) from (MOSTE).
References (19)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Nipah virus: a recently emergent deadly paramyxovirus
Science
(2000) - et al.
Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia
Lancet
(1999) - et al.
Isolation of Nipah virus from Malaysian Island flying-foxes
Microbes Infect.
(2002) - et al.
Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies
Vet. Microbiol.
(2003) - et al.
A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus
J. Virol. Methods
(1995) - et al.
A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B
J. Virol. Methods
(2000) - et al.
Molecular characterization of Nipah virus, a newly emergent paramyxovirus
Virology
(2000) - et al.
Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens
Vet. Microbiol.
(1999)
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