# First Detection and Genetic Characterization of Influenza D Virus in Cattle in Spain

## TL;DR

Influenza D virus was first detected in Spanish cattle, with two genetic lineages identified and a high rate of co-infections observed.

## Contribution

This is the first report of Influenza D virus in Spanish cattle, identifying two genetic lineages and their co-infection patterns.

## Key findings

- Influenza D virus was detected in 12% of samples from 30 farms across 15 Spanish provinces.
- IDV-positive samples showed co-infections with three to seven other pathogens in 94.7% of cases.
- The D/660 lineage of IDV exhibited higher genetic diversity compared to the D/OK lineage.

## Abstract

We analyzed 316 samples from 210 Spanish cattle farms suffering respiratory diseases between July 2023 and September 2024 in order to detect Influenza D virus (IDV) and other respiratory pathogens. For IDV, 38 samples (12%) from 30 farms (11.1%) were positive across 15 provinces throughout Spain. IDV was more frequently detected in bronchoalveolar lavage samples (22.1%) and nasal swabs (13.5%) compared to lung tissues (5%) and other sample mixtures (5%). All IDV-positive specimens exhibited other infectious agents too; furthermore, 94.7% harbored three to seven pathogens, demonstrating the complex nature of bovine respiratory diseases. Statistical analysis revealed moderate IDV-Mycoplasma bovis and IDV-Pasteurella multocida associations, weak IDV-bovine coronavirus and IDV-Histophilus somni association and negligible association with bovine herpesvirus 1. In a subset of 13 samples showing a high concentration of IDV, we obtained partial sequences of the hemagglutinin-esterase (HEF) gene: sequencing confirmed the presence of the two major genetic IDV lineages previously detected among cattle in Europe, D/OK and D/660. The samples belonging to the D/660 lineage exhibited higher genetic diversity. This is the first report of IDV infection in Spanish cattle, confirming the circulation of both the D/OK and D/660 lineages in the cattle population.

Influenza D virus (IDV) and co-circulating respiratory pathogens were investigated using RT-qPCR in 316 samples collected from 210 Spanish cattle farms with bovine respiratory disease (BRD) outbreaks between July 2023 and September 2024. Thirty-eight IDV-positive samples, from 30 farms across 15 provinces throughout Spain, were identified. IDV was significantly more frequent in bronchoalveolar lavage samples (22.1%) and nasal swabs (13.5%) compared to lung tissues (5%) and other sample mixtures (5%). All IDV-positive specimens exhibited co-infections, with most (94.7%) harboring three to seven viral and/or bacterial pathogens, highlighting the complexity of BRD outbreaks. Cramer’s V analysis revealed moderate IDV association with Mycoplasma bovis (V = 0.255) and Pasteurella multocida (V = 0.223), and a weaker IDV-bovine coronavirus association (V = 0.202), while IDV association with Histophilus somni (V = 0.025) and bovine herpesvirus 1 (V = 0.000) was negligible. Partial sequences of the hemagglutinin-esterase (HEF) gene from a subset of 13 IDV-positive samples (Ct < 32) were obtained. This confirmed the presence of the two major genetic lineages detected among cattle in Europe, D/OK and D/660, with D/660 exhibiting higher genetic diversity, as determined by DNAsp 6.12 software. This is the first report of IDV infection in Spanish cattle, confirming the circulation of the D/OK and D/660 lineages within the cattle population.