Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2014

Recombinant ELISA antigens derived from domain III of the flavivirus envelope protein differentiate infections with West Nile virus and Murray Valley encephalitis virus in horses. (#137)

Thisun BH Piyasena 1 , Yin X Setoh 1 , Alexander A Khromykh 1 , Jody Hobson-Peters 1 , Natalie A Prow 1 , Helle Bielefeldt-Ohmann 1 , Peter D Kirkland 2 , David Perera 3 , Mary J Cardosa 3 , Roy A Hall 1
  1. Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University Of Queensland, Brisbane, QLD, Australia
  2. NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, Australia
  3. Institute of Health & Community Medicine, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia

In 2011, the mosquito-borne viruses West Nile virus (Kunjin strain - WNVKUN) and Murray Valley encephalitis virus (MVEV) were implicated in an outbreak of equine encephalitis in south-eastern Australia involving more than 1,000 animals with a case-fatality rate between 10-15%1 . Current serological tests to detect antibody against MVEV or WNVKUN in horses are compromised by cross-reactive antibodies, which reduce the ability to distinguish between infections with these closely related flaviviruses2 . This study utilised recombinant proteins derived from domain-III of the envelope protein (rE-DIII) of MVEV and WNVKUN as diagnostic antigens in ELISA to serologically differentiate between infections by these two viruses in horses.

rE-DIII proteins were bacterially expressed and purified using affinity and size-exclusion chromatography. These antigens were used in ELISA to test the reactivity of horse sera previously characterized for flavivirus-specific antibodies by neutralization assays3 4 5 . Serum samples were deemed positive if the OD was 3 standard deviations above that produced by naive sera, and results indicative of a specific infection when the OD was significantly (P<0.05) higher to one antigen compared to the other in ELISA.

Testing of a panel of 45 horse serum samples with a range of known reactivity to flaviviral antigens revealed that 8/9 horses, previously deemed positive for MVEV infection by virus neutralisation, were also positive for MVEV in the rE-DIII ELISA. Similarly, 9/15 horses previously diagnosed with WNV infection tested positive for WNVKUN in the rE-DIII ELISA. Four of the remaining WNV-positive sera did not meet the OD threshold for a positive result in the ELISA, but still produced higher OD readings to the WNVKUN rE-DIII antigen.  These results indicate that rE-DIII antigens are highly specific in ELISA and capable of differentiating between MVEV and WNVKUN infections in horses. These preliminary findings support their use in routine diagnosis of equine viral infections in the Australian context and further assessment on high throughput platforms such as the microsphere immunoassay.

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