Nipah virus, which was first recognized during an outbreak of encephalitis with high mortality in Peninsular Malaysia during 1998-1999, is most closely related to Hendra virus, another emergent paramyxovirus first recognized in Australia in 1994. We have studied the morphologic features of Nipah virus in infected Vero E6 cells and human brain by using standard and immunogold electron microscopy and ultrastructural in situ hybridization. Nipah virions are enveloped particles composed of a tangle of filamentous nucleocapsids and measured as large as 1900 nm in diameter. The nucleocapsids measured up to 1.67 microm in length and had the herringbone structure characteristic for paramyxoviruses. Cellular infection was associated with multinucleation, intracytoplasmic nucleocapsid inclusions (NCIs), and long cytoplasmic tubules. Previously undescribed for other members of the family Paramyxoviridae, infected cells also contained an inclusion formed of reticular structures. Ultrastructural ISH studies suggest these inclusions play an important role in the transcription process.
Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.
Matched MeSH terms: Paramyxovirinae/growth & development