Abstract:
O’Nyong-nyong fever (ONNF) is an acute, mosquito-borne, non-fatal illness
characterized primarily by debilitating polyarthralgia and/or polyarthritis. It is caused by
O’Nyong-nyong virus (ONNV), first isolated from a febrile patient in 1959 in Northern
Uganda. O’Nyong-nyong virus has been associated with rapidly spreading epidemics in
Africa recording more than 2 million cases to date. A key element for control of
arbovirus transmission is early diagnosis of infections. The main challenges in ONNV
diagnosis, include (i) lack of commercial kits and specific assays to distinguish the
closely related group A alphaviruses, in which ONNV belongs, and (ii) lack of effective
treatments and vaccines. This study aimed to (i) determine the best cell line for ONNV
isolation and large-scale production of ONNV purified proteins, (ii) generate ONNV
monoclonal and polyclonal antibodies and (ii) assembly of an ELISA-based assay for
sensitive detection of ONNV. O’Nyong-nyong virus strain SG650 was isolated in Vero
cells, purified by sucrose gradient centrifugation, and used as an immunogen in BALB/c
mice and New Zealand White rabbit immunization. Mice that developed sufficient
antibody titers against ONNV were sacrificed and their spleen cells fused with parental
myeloma cells using hybridoma technology for generation of anti-ONN monoclonal
antibodies (mAbs). Similarly, rabbits that developed sufficient antibody titer were
sacrificed and their serum purified and used to generate polyclonal antibodies (pAbs).
The five MAbs generated were characterized and their potential for diagnostics
determined and confirmed using an indirect IgG ELISA and Focus Neutralization Assay
(FRNT50). The polyclonal antibodies were either conjugated with horseradish peroxidase
for antigen detection ELISA development, used as capture antibodies for antigen
detection ELISA or used unconjugated as primary antibodies for FRNT assays. To the
best of our knowledge, this is the first documented effort to generate mAbs and pAbs
against ONNV strain SG650. Generated antibodies can be explored and utilized for
development and evaluation of serological assays to facilitate fast and timely diagnosis
of ONNV infections in the laboratory, conduct surveillance and enhance differential
diagnostic capability of arboviral and undefined febrile illnesses.
