Nucleoprotein and Glycoprotein Based Serological Assays for Detection of Marburg Virus Infections

ABSTRACT

Background: Marburg virus disease (MVD) is a rare but highly fatal zoonotic illness with pandemic potential. Current serological tools are limited by their reliance on glycoprotein (GP)-based antigens or Marburg virus infected cell lysates, which cannot distinguish natural infection induced immune responses from the GP based vaccines. This diagnostic gap complicates the detection of missed cases particularly in areas of high vaccination coverage. Methods: We developed and validated two complementary assays: a nucleoprotein C-terminal tail (NPct) Mix-and-Read (MR) assay and a recombinant GP1,2ΔTM ELISA. Serum samples were collected 6 to 9 months after the 2024 Rwanda MVD outbreak from RT-PCR-confirmed recovered patients (n=37) and uninfected contacts (n=46). Additional controls for the assay validation, included pre-outbreak samples, Ebola virus disease recovered patients, and experimentally infected Rousettus aegyptiacus bats. Receiver operating characteristic (ROC) curve analyses were performed to assess diagnostic performance. Findings: The NPct MR assay demonstrated near-perfect discrimination between cases and controls (AUC 0.996), with 95.7% clinical sensitivity and 99.6% specificity, and no cross-reactivity with Ebola convalescent sera. The GP ELISA achieved 94.6% clinical sensitivity and 100% specificity (AUC 0.974). Both assays produced concordant results for all samples tested. In experimentally infected bats, the NPct MR assay achieved 100% sensitivity and 100% specificity. Interpretation: The MARV NPct MR assay provides a rapid, high-throughput, species-agnostic tool capable of distinguishing natural infection from vaccine-induced immunity, addressing a critical gap in the current MVD diagnostic pipeline. Its robust validation in the largest cohort of recovered patients to date and applicability to reservoir hosts highlight its broad utility for outbreak response, follow-up care, and ecological studies.