# VLP-based indirect ELISA enables reliable sero-diagnosis and epidemiological monitoring of duck circovirus

## TL;DR

A new diagnostic test using virus-like particles improves detection of duck circovirus, aiding disease monitoring and vaccine evaluation.

## Contribution

A codon-optimized capsid gene was expressed to create virus-like particles for a more reliable iELISA for duck circovirus.

## Key findings

- VLPs assembled from codon-optimized capsid gene showed superior antigenicity compared to monomers.
- The VLP-based iELISA demonstrated high sensitivity, specificity, and repeatability for detecting duck circovirus antibodies.
- Field application revealed a 19.96% positivity rate in duck sera from Jiangsu Province with a yearly increasing trend.

## Abstract

Duck circovirus (DuCV) is an emerging immunosuppressive virus that causes growth retardation, feather loss, and increased susceptibility to secondary infections in ducks, leading to significant economic losses in the duck industry. However, existing serological assays for DuCV detection often lack accuracy and reproducibility due to antigen instability, limiting effective disease surveillance.

To develop a more reliable diagnostic tool, the full-length capsid (Cap) gene of DuCV-2 was codon-optimized, expressed in Escherichia coli, purified under denaturing conditions, and refolded to self-assemble into virus-like particles (VLPs). The morphology of the assembled VLPs (~15 nm) was confirmed by transmission electron microscopy, and their immunogenicity was evaluated in ducks. Based on these VLPs, an indirect enzyme-linked immunosorbent assay (iELISA) was established and optimized.

The VLPs elicited stronger antibody responses than Cap monomers at equivalent doses, confirming their superior antigenicity. The optimized VLP-based iELISA (250 ng/well coating concentration, 1,200 serum dilution) exhibited high sensitivity (detectable up to 1:6400 dilution), strong specificity (no cross-reactivity with other avian pathogens), and good repeatability (CV < 5%). Application of the assay to 290 field duck sera collected in Jiangsu Province (2022–2024) revealed a 19.96% positivity rate, showing a gradual yearly increase.

This study developed a highly specific, sensitive, and reproducible VLP-based iELISA for DuCV antibody detection. The method provides a practical tool for large-scale epidemiological surveillance and vaccine evaluation, and the VLP-based diagnostic strategy offers a universal framework for serological assays of other avian circoviruses.