Abstract
A critical need exists for highly sensitive and specific serodiagnostic tests to discriminate infections by
pathogenic arboviruses in geographic regions, such as Brazil, where multiple flaviviruses including Zika virus
(ZIKV), four serotypes of dengue virus (DENV1-4), West Nile virus (WNV), and yellow fever (YFV), as well as
chikungunya virus (CHIKV), Mayaro virus (MAYV) (both alphaviruses) and Oropouche virus (OROV) (a
bunyavirus), are endemic. However, cross-reactivity of antibodies against different flaviviruses present major
challenges, such that even neutralization tests cannot confirm a specific flavivirus infection among individuals
who have experienced previous flavivirus infections.
The objective of the proposed research is to develop highly sensitive and specific serodiagnostic tests to
discriminate infections by pathogenic arboviruses. The central hypothesis is that a combination of fusion loop
(FL)-mutated VLPs and nonstructural protein 1 (NS1) proteins of different flaviviruses and recombinant proteins
and VLPs of CHIKV, MAYV and OROV in multiplex formats can distinguish different arbovirus infections.
The first Aim is to develop and validate multiplex IgG and IgM microsphere immunoassays (MIAs) based on
recombinant proteins to distinguish arbovirus infections. We will employ recombinant NS1 proteins of 7
flaviviruses, envelope (E) 2 protein and nucleocapsid (N) protein of CHIKV, MAYV and OROV for multiplex IgG
and IgM MIAs using Luminex 200 and test with 15 panels of convalescent-phase serum/plasma from individuals
with confirmed arbovirus infections. The second Aim is to develop and validate multiplex IgG and IgM MIAs based
on VLPs to distinguish arbovirus infections. We will use purified and FL-mutated VLPs of 7 flaviviruses and VLPs
of CHIKV, MAYV and OROV, and test with 15 panels of serum/plasma as in Aim 1. The third Aim is to compare
the multiplex IgM MIAs and IgG MIAs with currently available serodiagnostic assays to determine arbovirus
seroprevalence in Bahia, a northeastern state with multiple arbovirus transmissions in Brazil. For serodiagnosis,
we will test serum samples from patients with acute febrile illness and their follow-up at outpatient clinics of 4 study
sites (Salvador, Feira de Santana, Itabuna and Campo Formoso) in Bahia. For seroprevalence study, we will
enroll and test household members at communities of the 4 study sites.
The proposed study is innovative as it employs two promising antigens (NS1 protein and FL-mutated VLPs) to
overcome flavivirus cross-reactivity for seven flaviviruses, as opposed to traditional E protein-based tests, plus
CHIKV, MAYV and OROV in two multiplex formats to discriminate arbovirus infections in Brazil. It would
contribute to a detailed understanding of the epidemiology of 10 arbovirus infections in Bahia. The successful
employment of the multiplex platforms can serve as a new paradigm for serodiagnosis and serosurveillance in
countries where multiple arboviruses co-circulate. Most importantly, this research will facilitate the
implementation of arbovirus vaccines, in particular ZIKV, DENV and CHIKV vaccines in endemic regions.