Several strategies have been employed to develop new diagnostic techniques to detect infections with the henipaviruses with an emphasis on assays that do not require BSL-4 containment. Expression systems were used to produce antigens for enzyme immunoassays including the NiV N protein (Chen et al., 2006
; Yu et al., 2006
), a truncated phosphoprotein antigen (Chen et al., 2007
), or the glycoproteins of NiV (Eshaghi et al., 2005a
; Eshaghi et al., 2004
; Eshaghi et al., 2005b
). Other assays described the use of monoclonal antibodies for immunohistochemical based diagnosis (Tanimura et al., 2004
; Xiao et al., 2008
), or a modified plaque assay in which the plates are prepared in BSL-4 and inactivated by gamma irradiation before staining at BSL-2 (Crameri et al., 2002
). Soluble G proteins and/or ephrin B2 coupled with a bioplex protein array have been used to measure antibody responses to the glycoproteins of henipaviruses (Bossart et al., 2007
The pVSV-NiV-F/G was initially developed to support studies to identify the cellular receptors for the henipaviruses and has recently been used as antigen for a neutralization test (Wang et al., 2006
). A similar approach using pseudotype particles or reporter virus particles (RVP) was used in a neutralization assay for SARS (Temperton et al., 2005
) and West Nile virus (Pierson et al., 2006
). This report describes the first comparison between the neutralization assay using pVSV-NiV-F/G and PRNT. The VSV-NiV-F/G neutralization assay offers several significant advantages over PRNT. The assay can be performed entirely at BSL-2, completed in less than 48 hours, and relies on automated read-outs in a 96-well format instead of manual counting of plaques. Overall, the results obtained by the assay using pVSV-NiV-F/G were consistent and correlated well with the results from PRNT. Both methods () confirmed previous reports of the ability of NiV G to induce higher neutralizing titers than NiV-F (Bossart et al., 2005
; Guillaume et al., 2006
; Mungall et al., 2006
; Tamin et al., 2002
). Also, the pseudotype particles were immunogenic when used as an antigen in an indirect enzyme immunoassay format suggesting that this may be a promising technique for developing glycoprotein specific enzyme immunoassays for NiV and other paramyxoviruses.
There is considerable variation in all diagnostic methods, including PRNT, in detecting low levels of antibodies. In this study, the results of the PRNT were regarded as the gold standard. Fifteen of the 16 discordant results between PRNT and the pseudotype neutralization assay were recorded from serum samples collected 7, 14 and 21 dpv when antibody levels were very low. With only one exception, the PRNT titers did not exceed 40 for any of the samples that appeared to give false negative results in the pseudotype assay. Some of these low titers could have been due to toxic effects of the serum in the PRNT assay. All of the five apparent false positive results in the pseudotype assay were from the serum samples collected on day 21 and these had the titers of 40.
Henipaviruses are zoonotic viruses, and their natural reservoir is fruit bats of the genus Pteropus
(Halpin et al., 2000
; Chua et al., 2002
). The geographical distribution of Pteropus
fruit bats includes parts of West Africa, South Asia and Southeast Asia, the Pacific Islands, and the eastern coast of Australia. This vast area includes some of the most heavily populated areas in the world including many developing countries (Epstein et al., 2006
). Unlike other paramyxoviruses, these viruses have the ability to infect a wide range of host species (Bellini et al., 2005
). Therefore, there is always the potential for spillover of henipaviruses from the natural reservoir into humans or farm animals. Good laboratory tests, including both serologic and virus detection methods are needed to rapidly detect outbreaks of henipaviruses.
The standard first-line serologic test for henipaviruses has been enzyme immunoassay along with neutralization tests used for confirmation. The pseudotype neutralization assay described here could be used to replace PRNT as the confirmatory serologic test in areas lacking high containment facilities. Pseudotype particles could easily be prepared in advance and stored frozen until required. The local laboratory would need only cell culture capacity and a luminometer. This new novel, high-throughput assay could also be a valuable tool for performing serologic surveillance of various bat species and domestic animals. Finally, this assay would provide a convenient and safe way to measure protective antibodies induced by experimental vaccines against henipaviruses.