Detection of honey bee viruses in Apulia region by traditional PCR and High-Throughput Sequencing

The continuous losses of honey bee colonies have encouraged further investigation on honey bee infecting viruses. In 2017 and 2018, we investigated the presence of honey bee viruses in more than 35 apiaries located in Puglia region. RNA was extracted from a pool of 10 adult bees collected in each apiary then tested by RT-PCR. Results showed the presence of different viruses commonly infecting bees of which some were found for the first time in Italy. From two apiaries where dead bees and trembling bees found in front of the hives, specimens were collected and investigated by construction of total RNAseq libraries and subsequent high-throughput sequencing (HTS). Raw reads were quality-checked by FastQC. Paired reads of 101bp were assembled by metaSPAdes version 3.9.0 49 using "only-assembler" parameter and multiple kmers (-k 71, 81, 91). Obtained contigs were searched for similarity against "viruses" database by Blastn. Preliminary HTS and bioinformatic analysis retrieved the full length (~8450 bp) sequence of the Black queen cell virus (BQCV) showing high similarity (~94.11%) with the Hungarian isolate EF517515. Ongoing analysis showed other contigs with high similarity to Apis mellifera filamentous virus, a large double stranded DNA baculovirus-like virus which we found widely distributed in Apulia (up to 91% in 2017). The present work reports the first full genome sequence of the Italian isolate BQCV-IT1. HTS technology proved to be a rapid tool for identifying viruses in honeybees. Further investigations are in progress to understand the role of these viruses on honey bee health in Puglia.