Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus

https://doi.org/10.1016/j.jviromet.2005.09.005Get rights and content

Abstract

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.

Introduction

Nipah virus is a zoonotic pathogen belonging to the family of Parmyxoviridae, genus Henipavirus and is listed as one of the potential biological agents for bio-terrorism in the Northern Atlantic Treaty Organization handbook (Lam and Chua, 2002). Nipah virus is classified as biosafety level 4 (BSL-4) agent and laboratory personnel needs to take appropriate precautions when working with this pathogen.

In humans, Nipah virus causes severe febrile encephalitis with 40% mortality (Chua et al., 1999, Paton et al., 1999, Chua et al., 2000 and Chua, 2003). In pigs, it causes mainly a respiratory disease with lesser involvement of the central nervous system (CNS) and infrequent mortality (Goh et al., 2000 and Middleton et al., 2002). The virus can be transmitted to humans through close contact with infected pigs and their body secretions (Mohd Nor et al., 2000, Lam and Chua, 2002 and Middleton et al., 2002).

Nipah virus is related antigenically and genetically to another zoonotic pathogen from the genus Henipavirus called Hendra virus (Murray et al., 1995, Harcourt et al., 2000, Daniels et al., 2001 and Wang et al., 2001). Hendra virus is the etiological agent of a fatal respiratory and CNS disease of horses and humans (Hooper et al., 1997a, Hooper et al., 1997b, Barclay and Paton, 2000 and Hooper et al., 2001). The genome of Henipaviruses consists of a single-stranded, linear molecule of RNA of negative sense that contains an attachment glycoprotein (G), fusion (F), matrix (M), polymerase (L), nucleocapsid (N), and P/V/C genes. Nipah virus has the largest genome among paramyxoviruses (18,246 nucleotides), and has a high degree of nucleotide homology in open reading frames (ORF) of various genes with Hendra virus (Harcourt et al., 2000).

The development of mAbs to Nipah virus is critical for studying the pathogenesis of the disease and for the development of diagnostic techniques. Production of quality Nipah virus antigen for mAb development requires propagation of high titer virus in tissue culture; clarification by centrifugation, inactivation, concentration and purification of the virus. In addition, when working with BSL-4 pathogens such as Nipah virus, extensive safety testing of the inactivated virus is necessary prior to immunizing mice for hybridoma production under BSL-2 conditions.

BEI, an aziridine compound, has been used for inactivation of adventitious viruses in biological preparations. BEI reacts with viral nucleic acids while preserving conformation and accessibility of epitopes to a much greater extent than formalin and β-propriolactone (Bahnemann, 1975, Bahnemann, 1990, Kyvsgaard et al., 1997 and Blackburn and Besselaar, 1991). BEI has been used to inactivate a number of DNA viruses (African swine fever, porcine parvovirus, bovine rhinotracheitis and pseudorabies) and RNA viruses (foot and mouth disease virus, Newcastle disease virus and Rabies virus) for vaccine and antigen production (Bahnemann, 1976 and Bahnemann, 1990).

This paper describes the development and characterization of murine mAbs raised against BEI inactivated Nipah virus, identification of mAbs to N and M proteins of Nipah virus, and their application for different types of assays.

Section snippets

Virus

Human isolates of Nipah and Hendra virus were kindly provided by Drs Thomas Ksiazek and Pierre Rollin, CDC, Atlanta. Nipah and Hendra virus stocks were prepared in Vero-76 monolayers in T75 flasks (Costar, Corning Inc., Corning, NY). Confluent monolayers of Vero-76 cells were infected with Nipah/Hendra virus at 0.1 multiplicity of infection (moi). Cells were incubated at 33 °C in the presence of 5% CO2 until 80% cytopathic effect (CPE) was observed. Virus stocks were frozen at −70 °C until used.

Cell culture

Inactivation, concentration and purification of Nipah virus

Complete BEI inactivation of Nipah virus with initial stock virus titer of 2 × 105 TCID50/ml was achieved within 6 h based on virus titration assay from samples taken at different time points. During safety testing CPE was not observed in PT-K75 cells that were propagated with undiluted aliquots of BEI inactivated virus from the final time point (24 h).

The inactivated virus was concentrated and partially purified in BSL-2 using sucrose gradient centrifugation. Two distinct bands were observed

Discussion

This study describes the development of murine mAbs using nucleprotein enriched fraction of sucrose gradient from BEI inactivated Nipah virus as immunogen. These mAbs recognize Nipah virus antigen in ELISA, immunofluorescence assay, Western immunoblot and immunohistochemistry.

Target identification of the mAbs in Western immunoblot with BEI inactivated Nipah virus and recombinant Nipah virus proteins collectively indicated that five mAbs reacted with the N protein and one with M protein.

Acknowledgements

The authors are grateful to Mrs Lynn Burton for her expertise on transmission electron microscopy. The authors also would like to thank Greg Smith and Jason Gren for their laboratory support, Dr Stefanie Czub, Ms Lisa Manning and Ms Shelley Ganske for their expertise on the Nipah mAbs immunohistochemistry. This research was supported by CFIA TD funds and the CBRN Research and Technology Initiative (CRTI) under projects 0196 (HW) and 0091 (JDB).

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