Arthropod-borne viruses (arboviruses) may be transmitted to man and other susceptible vertebrate hosts by infected arthropods such as mosquitoes. There are over 100 arboviruses which are known to cause human infections with varying degrees of morbidity and mortality . Included in the arbovirus group are single-stranded, positive sense RNA viruses belonging to the Alphavirus and Flavivirus genera of the families Togaviridae and Flaviviridae respectively . In Australia, the alphaviruses that have been implicated in human disease include Ross River virus (RRV), Barmah Forest virus (BFV) and Sindbis virus, whilst flaviviruses include Dengue virus serotypes 1-4 (DENV 1-4), Murray Valley encephalitis virus (MVEV), Kunjin virus (KUNV), Japanese encephalitis virus (JEV) and Kokobera virus. Additionally, antibodies which react with 2 other flaviviruses namely, Alfuy virus (ALFV) and Stratford virus, have also been found in human sera.
The serological diagnosis of arbovirus infections usually involves the testing of paired acute and convalescent sera in parallel, and requires stable, reliable antigens that are reproducible and provide consistent results. The development of standardised protocols to ensure the availability of diagnostic antigens is an essential requirement in any arboviral laboratory, and allows the testing of human and animal sentinel surveillance sera for the diagnosis and control of arboviral diseases. In addition, production of antigens that are suitable for multiple serological assays such as the enzyme linked immunosorbent assay (ELISA) and the haemagglutination inhibition (HAI) assay, improves the efficiency of the diagnostic laboratory and increases the accuracy, reproducibility and reliability of the results produced.
The production of antigens requires the propagation of large quantities of high titre arboviral suspensions in cell culture. Parameters such as choice of cell line, multiplicity of infection (m.o.i.) and length of incubation period, should be considered to obtain optimal viral growth. The formation of nonviable deletion mutants or defective interfering (DI) particles, a well known phenomenon resulting from serial, high multiplicity passaging of many viruses in cell culture, can interfere with the replication of infectious virus and reduce yields [3–5]. To reduce the effects of DI particle formation during the propagation of virus, it may be useful to infect a particular cell line with a stock virus previously grown in a different cell line.
Methods to produce antigens may vary depending upon the amount and purity of antigen required and protocols usually involve four steps: clarification by centrifugation, inactivation, concentration and purification. Inactivation of viruses during antigen production is not only essential to prevent cross-contamination, but is also implemented to minimise the exposure of personnel to biological hazards and maintain a high standard of safe laboratory practices and procedures.
Binary ethylenimine (BEI) has been used to successfully inactivate a number of RNA and DNA animal viruses for antigen and vaccine production, including foot-and-mouth disease virus , Rabies virus  and Newcastle disease virus . Although potentially carcinogenic, use of a stock solution of 0.1 M BEI is far less toxic  and as a chemical inactivant, may be more effective than other means of inactivation such as ultraviolet radiation . BEI is known to act on nucleic acid, with minimal effect on the immunogenic properties of the viral protein coat. In addition, we have chosen BEI inactivation as the reagents required are inexpensive and the antigens produced are highly stable .
Two methods for concentrating and purifying inactivated arboviral antigens, namely, the sucrose density gradient centrifugation (SDGC) method and the cross-flow filtration (CFF) method, were investigated. Viruses have been purified previously by layering preparations on an isopycnic (equilibrium) sucrose density gradient . A similar method using a potassium tartrate gradient has been used to prepare a purified RRV antigen for use in an IgM ELISA .
Cross-flow filtration technology has been utilized for many applications including the purification of water  and immunoglobulins . In comparison to conventional filtration methods, the use of CFF to concentrate viral particles has several advantages. During CFF, culture fluid is re-circulated in tangential flow, parallel to the filter membrane. Build-up of viral particles on the membrane is minimised by the recirculation of fluid over the surface, which also facilitates the concentration of particles present in the retentate fluid. In this way, alphavirus and flavivirus particles may be partially concentrated and purified from culture fluids with the use of an appropriately sized filter membrane.
The production of substantial quantities of good quality, adequately concentrated antigens is crucial for the reliable performance of diagnostic, serological assays. We describe standardised, reproducible methods to produce safe, inactivated arboviral antigens for use in serological tests using either SDGC or CFF protocols. The methods described in this paper produce inactivated reagents that can be used immediately in a variety of ELISA and HAI assays, decreasing hazardous risks to laboratory personnel and the requirement of multiple protocols to produce different antigens. In Australia, the application of these methods for the production of antigens to exotic and emerging arboviruses such as Chikungunya virus (CHIKV), West Nile virus (WNV) and Yellow fever virus (YFV), enables the development of diagnostic assays for the future identification, management and control of outbreaks caused by these pathogens.