The western honey bee (Apis mellifera) is one of the most important pollinators of agricultural crops (Khalifa et al., 2021) as well as wild floral species (Hung et al., 2018). The honey bees pollination service is therefore highly valuable, both economically and ecologically. However, during the last few decades, massive losses have been reported worldwide for managed colonies derived from Apis mellifera subspecies of European origin (Neumann & Carreck, 2015).These colony losses are caused by multiple factors including an extensive way of agriculture, insufficient food supply during critical parts of the season, and the spread of parasites and diseases (Hristov et al., 2020). The parasitic mite Varroa destructor along with associated viruses have been identified as the strong driver of colony losses (Guzmán-Novoa et al., 2010). Also gut pathogenssuch as the microsporidium Nosema ceranae and the protozoa Lotmaria passim may harm honey bee health (Marín-García et al., 2022, Gómez-Moracho et al., 2020), contributing to the death of colonies (Ravoet et al., 2013).

Honey bee viruses, particularly Deformed wing virus (DWV), have been shown to be strongly associated with colony losses (Highfield et al., 2009). DWV is a non-enveloped positive single-strand (ss+) RNA virus of the Iflaviridae family (Lanzi et al., 2006) that can replicate in honey bee brood as well as in adult tissues (Gusachenko et al, 2020). As the Varroa mite serves as an efficient vector for DWV transmission, the virus is the most widespread honey bee virus in Varroa-infested colonies, severely affecting honey bee health (Ryabov et al., 2014). Although honey bees have adopted several antiviral defense strategies, including cellular and humoral immune mechanisms (Brutscher et al., 2015, Feng et al., 2020), Varroa mite feeding modifies the host immune system, allowing rapid viral replication (Yang and Cox-Foster, 2005, Nazzi et al., 2012; Kunc et al., 2022). This usually results in the manifestation of clinical symptoms in honey bees that have emerged from Varroa-infested brood, such as wing and abdomen deformities, impaired cognitive functions, shortened lifespan and death (Lanzi et al., 2006).

Beekeepers prevent the development of DWV infection indirectly by reducing the Varroa mite vector population. According to our knowledge, no commercially available antiviral treatment for honey bees is currently available on the market. However, a few experimental approaches to cope with honey bee viruses have been reported in recent years. The first involves the addition of dsRNA into honey bee royal jelly to stimulate activation of the RNAi pathway (Desai et al., 2012). This strategy, however, might not always be effective (Yang et al., 2018) and it can also have unintended consequences, such as off-target effects (Nunes et al., 2013). Reduced viral titers have also been observed after exposing bees to a heat-shock regime (McMenamin et al., 2020). Another approach to reduce honey bee viruses is by feeding honey bees with natural supplements that have been shown to possess antiviral properties in other biological systems. For example, honey bees fed a diet supplemented with thyme oil, a commonly used anti-varroa substance, showed enhanced activity of their immune system and reduced DWV loads (Parekh et al., 2021). A similar effect was shown in honey bees after feeding the grape pomace powder-supplemented diet (Pascual et al., 2023). Also, feeding honey bees with extracts from polypore mushrooms Ganoderma lucidum and Fomes fomentarius significantly reduced the levels of DWV and LSV viruses (Stamets et al., 2018). The antiviral potential of mushrooms was further confirmed by the observation that DWV levels in honey bees were significantly reduced by feeding ß glucans (Felicioli et al., 2020), common secondary metabolites present in the cell walls of mushrooms.

Secondary metabolites from higher fungi have consistently been demonstrated to be highly potent antiviral agents. Antiviral properties have been described mainly through the actions of polysaccharides, proteins, peptides, polyphenols and triterpenoids. These substances can suppress viral entrance (Pan et al., 2013), replication (Xu et al., 2012), the activity of viral enzymes (El-Mekkawy et al., 1998), cellular and viral protein production (Zhao et al., 2016, Okamoto et al., 2004), and boost host immunity (Wang et al., 2014) against a wide spectrum of viruses, including non-enveloped ss+ RNA viruses (Seo & Choi, 2021). For instance, a polysaccharide from Agaricus brasiliensis inhibited Poliovirus (Faccin et al., 2007), and a polysaccharide from Grifola frondosa suppressed the expression of the capsid protein VP-1 of Enterovirus 71 (Zhao et al., 2016). However, the underlying mechanisms of fungal antiviral actions are much better understood for enveloped viruses. For example, polyphenols from Phellinus linteus and Glaziella splendens have an inhibitory effect on neuraminidase of Influenza A (Hwang et al., 2018; Kim et al., 2019), triterpenes from Ganoderma lucidum inhibit HIV-1 protease (El-Mekkawy et al ., 1998) and lactase isolated from Lentinus tigrinus inhibits reverse transcriptase of HIV (Xu et al., 2012). Strong antiviral properties were also reported for Cortinarius caperatus, as the protein RC28 isolated from this fungus was shown to inhibit HSV-1 and 2, varicella-zoster virus, influenza A virus, and respiratory syncytial virus (Yan et al., 2015, Piraino and Brandt, 1999).

The objective of this study was to evaluate the potential of Cortinarius caperatus as the treatment against viral infection of honey bees. Thus, we examine the ability of the alcohol extract and the dry powder of the mushroom to reduce DWV titers in cage experiments as well as in field experiments using honey bee colonies in hives. Supplementary feeding of the mushroom alcohol extract was also tested for its effect on honey bee lifespan. Moreover, ossible presence of mushroom residues in honey was evaluated in field experiments. Our results demonstrated that the alcohol extract of C. caperatus can be used as a safe and effective treatment to reduce DWV infection.