# Amplicon sequencing with Oxford nanopore technologies as a diagnostic alternative for small ruminant lentiviruses in sheep

## TL;DR

This paper explores using Oxford Nanopore sequencing as a more accurate diagnostic tool for Maedi-Visna virus in sheep compared to traditional methods like ELISA and qPCR.

## Contribution

The study introduces Oxford Nanopore amplicon sequencing as a novel diagnostic method for small ruminant lentiviruses in sheep.

## Key findings

- Nanopore sequencing reduced false negatives compared to ELISA (42%) and qPCR (77%).
- Blood DNA was found to be the most reliable source for virus detection using this method.
- Amplicon sequencing of gag, pol, and p25 genes improved SRLVs detection accuracy.

## Abstract

In Europe, Maedi-Visna disease has high prevalence rates at the individual and flock levels, respectively, and is regarded as one of the most significant infectious disease in sheep. The lack of treatment or a commercial vaccine underscores the need for accurate and reliable diagnostic tools to support control programs. Conventional methods, including ELISA and qPCR, provide useful but incomplete information due to the genetic variability of small ruminant lentiviruses (SRLVs) and the heterogeneous host immune response. In this work, third-generation sequencing was assessed as a diagnostic strategy, focusing on Oxford Nanopore Technologies amplicon sequencing of different regions of the virus genome. DNA from whole blood, PBMCs, semen, and nasal mucosa of 44 rams previously tested for Maedi-Visna virus by ELISA was used to generate amplicons of the gag, pol, and p25 genes. Sequencing showed that blood DNA was the most reliable source for SRLVs detection by Nanopore, despite the low proportion of monocytes present in this medium. Compared with conventional approaches, Nanopore sequencing reduced the proportion of false negatives observed with ELISA (42%) and qPCR (77%). These results highlight Nanopore amplicon sequencing as a promising diagnostic alternative, combining epidemiological relevance with technological innovation to enhance SRLVs detection and strengthen control strategies for sustainable disease management.

The online version contains supplementary material available at 10.1038/s41598-026-36989-y.