Since 2013, the arthropod-borne Chikungunya virus (CHIKV) has cocirculated with the autochthonous Mayaro virus (MAYV) in Latin America. Both belong to the same alphavirus serocomplex, termed the Semliki Forest serocomplex. The extent of antibody cross-reactivity due to the antigenic relatedness of CHIKV and MAYV in commonly used serologic tests remains unclear. By testing 64 CHIKV- and 37 MAYV-specific sera from cohort studies conducted in Peru and Brazil, we demonstrate about 50% false-positive test results using commercially available enzyme-linked immunosorbent assays (ELISAs) based on structural antigens. In contrast, combining ELISAs for CHIKV and MAYV significantly increased positive predictive values (PPV) among all cohorts from 35.3% to 88.2% for IgM and from 61.3% to 96.8% for IgG (P < 0.0001). Testing of longitudinally collected CHIKV-specific patient sera indicated that ELISA specificity is highest for IgM testing at 5 to 9 days post-onset of symptoms (dpo) and for IgG testing at 10 to 14 dpo. IgG cross-reactivity in ELISA was asymmetric, occurring in 57.9% of MAYV-specific sera compared to 29.5% of CHIKV-specific sera. Parallel plaque reduction neutralization testing (PRNT) for CHIKV and MAYV increased the PPV from 80.0% to 100% (P = 0.0053). However, labor-intense procedures and delayed seroconversion limit PRNT for patient diagnostics. In sum, individual testing for CHIKV or MAYV only is prone to misclassifications that dramatically impact patient diagnostics and sero-epidemiologic investigation. Parallel ELISAs for both CHIKV and MAYV provide an easy and efficient solution to differentiate CHIKV from MAYV infections. This approach may provide a template globally for settings in which alphavirus coemergence imposes similar problems. IMPORTANCE Geographically overlapping transmission of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in Latin America challenges serologic diagnostics and epidemiologic surveillance, as antibodies against the antigenically related viruses can be cross-reactive, potentially causing false-positive test results. We examined whether widely used ELISAs and plaque reduction neutralization testing allow specific antibody detection in the scenario of CHIKV and MAYV coemergence. For this purpose, we used 37 patient-derived MAYV-specific sera from Peru and 64 patient-derived CHIKV-specific sera from Brazil, including longitudinally collected samples. Extensive testing of those samples revealed strong antibody cross-reactivity in ELISAs, particularly for IgM, which is commonly used for patient diagnostics. Cross-neutralization was also observed, albeit at lower frequencies. Parallel testing for both viruses and comparison of ELISA reactivities and neutralizing antibody titers significantly increased diagnostic specificity. Our data provide a convenient and practicable solution to ensure robust differentiation of CHIKV- and MAYV-specific antibodies.

Dengue is a major public health problem in tropical and sub-tropical regions worldwide. Since the Zika epidemic and the increased co-circulation of other arboviruses, the serology-based diagnosis of dengue has become more problematic due to the high antigenic resemblance, especially among the flavivirus family. Therefore, a more comprehensive understanding of the diversity, specificity and temporal evolution of the antibody response following dengue infection is needed. In order to close this knowledge gap, we used a high-density peptide microarray of 9,072 linear peptides covering the entire proteome diversity of dengue, Zika, yellow fever and chikungunya viruses. The IgM and IgG antibody responses were measured against the designed microarray in symptomatic dengue infected individuals from an arbovirus endemic area in Peru and in overseas travelers returning to Belgium, as representatives of multiple-exposed and primary infections, respectively. Serum samples were collected longitudinally across four time points over the period of six months in Peru and over two time points in travelers. We show that epitopes eliciting the strongest flavivirus cross-reactive antibodies, in both primary and secondary infections were concentrated in the capsid, E, NS1, NS3 and NS5 proteins. The IgG antibody responses against NS1 and NS3 followed a rise-and-fall pattern, with peak titers between two to four weeks after onset of illness. The response to the E and NS5 proteins increased rapidly in the acute phase and was maintained at stable levels until at least 6 months after illness. A more scattered IgM antibody reactivity across the viral proteome was observed in the acute phase of the disease and that persisted through the 6-month window. The magnitude, breadth (i.e. number of unique epitopes targeted) and depth (i.e. number of epitope variants recognized) of the IgG response was higher in secondary infections compared to primary infections. For IgM antibodies, the magnitude of the response was higher in primary infected individuals whereas the breadth and depth of the response was lower in this group compared with the endemic subjects. Finally, through this arboviral proteome-wide epitope mapping, we were able to identify IgM and IgG dengue-specific epitopes which can be useful serological markers for dengue diagnosis and serostatus determination.

Latin America is a global hot spot of the COVID-19 pandemic. Serological studies in Latin America have been mostly performed in urban settings.

In a world with an increasing population at risk of exposure to arthropod-borne flaviviruses, access to timely and accurate diagnostic tests would impact profoundly on the management of cases. Twenty peptides previously identified using a flavivirus proteome-wide microarray were evaluated to determine their discriminatory potential to detect dengue virus (DENV) infection. This included nine peptides recognized by IgM antibodies (PM peptides) and 11 peptides recognized by IgG antibodies (PG peptides). A bead-based multiplex peptide immunoassay (MPIA) using the Luminex technology was set-up to determine Ab binding levels to each of these peptides in a panel of 323 carefully selected human serum samples. Sera are derived from individuals either infected with different viruses, namely, the four DENV serotypes, Zika virus (ZIKV), yellow fever virus (YFV), chikungunya virus (CHIKV), West Nile virus (WNV) and Human immunodeficiency virus (HIV), or receiving vaccination against YFV, tick-borne encephalitis (TBEV), and Japanese encephalitis virus (JEV). Additionally, a set of healthy controls were included. We targeted a minimum specificity of 80% for all the analysis. The PG-9 peptide had the best sensitivity (73%) when testing DENV sera from acute patients (A-DENV; &lt;8 days since symptom onset). With sera from convalescent DENV patients (C-DENV; &gt;10 days since symptom onset) the FPG-1 peptide was the best seromarker with a sensitivity of 86%. When combining all A-DENV and C-DENV samples, peptides PM-22 and FPG-1 had the best-diagnostic performance with a sensitivity of 60 and 61.1%, and areas under the curve (AUC) of 0.7865 and 0.8131, respectively. A Random forest (RF) algorithm was used to select the best combination of peptides to classify DENV infection at a targeted specificity &gt;80%. The best RF model for PM peptides that included A-DENV and C-DENV samples, reached a sensitivity of 72.3%, while for PG peptides, the best RF models for A-DENV only, C-DENV only and A-DENV + C-DENV reached a sensitivity of 88.9%, 89.1%, and 88.3%, respectively. In conclusion, the combination of multiple peptides constitutes a founding set of seromarkers for the discrimination of DENV infected individuals from other flavivirus infections.

We sequenced the envelope gene of dengue virus serotype 2 (DENV-2-E) in samples from an outbreak reported in 2018, in Yurimaguas, Peru. The strain belongs to lineage 2 of the American/Asian genotype. We report a variant with two novel mutations (I379T and V484I) located in domain III of DENV2-E.