First molecular detection of Apis mellifera filamentous virus in Apis mellifera in southern Italy

The global and continuous increasing decline of honey bees over the last decade has driven several research programs to tackle biotic and abiotic causes threatening this important insect species. Among the biotic factors, several positive-sense RNA viruses have been recently associated with colony decline, and a widespread occurrence of the DNA virus "Apis mellifera filamentous virus (AmFV)" has also been reported. However, the pathogenicity of this virus on the infected colonies is still questioned, being rarely associated to acute infections or colony losses. In this work we investigated the occurrence of AmFV in the apiaries in the Apulia region (southern Italy) using conventional PCR and high-throughput sequencing (HTS). More than 35 apiaries were visited between 2017 and 2018, and about 380 honey bee specimens consisting of adults, pupae, larvae of the worker bees and in some cases queen pupae were collected and tested by PCR as groups using two diagnostic primers that amplify two partial region of the Thymidylate synthase gene and the BRO-N gene, respectively. From two apiaries where trembling and weak bees were found, specimens were collected and extracted total RNA subjected to HTS. PCR results showed the occurrence of AmFV in all sampled colonies with only two negative samples: (i) one sample consisting of 20 adult bees collected in Copertino (LE) and (ii) one sample consisting of two died pupae collected from queen cell in an apiary located in Lecce. The overall AmFV-infection rate in the Apulian colonies was approximately 91%. The nucleotide sequence determined for a selected representative AmFV positive sample, showed that based on the phylogenetic analysis of the thymidylate synthase gene and Bro gene sequences, the Apulian isolate shared more than 98% of nucleotide identity with isolates previously characterized in Belgium, Switzerland and China. The presence of this virus in the diseased-colonies was also confirmed by HTS: two large contigs (3604bp and 2112bp) were assembled from the sequence dataset and shared up to 99% similarity- at nucleotide level- with sequences of AmFV previously reported from Switzerland. This is the first detection of AmFV infections on A. mellifera populations in Italy by HTS and PCR. HTS technology proved to be a rapid tool for identifying viruses in honeybees. Analyses are still ongoing on the recovered sequence dataset to identify other putative viral agents. Although limited information is available on the potential impact of AmFV, the wide distribution of this virus in the Apulian colonies should be of concern for the healthy status of honey bee in the region.