New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana

Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana (A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V. destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m. ligustica and A. m. syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the first report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites.


Introduction
The mite Varroa destructor is an obligatory ectoparasite of the Eastern honeybee Apis cerana [1]. V. destructor mites spend most of their life cycle inside the colony, reproducing on the honeybee brood and feeding on the pupa. Mites transfer from one host pupa to another on nurse bees that take care of pupa [1]. During the feeding on its host this mite may transfer populations of microorganisms that it bears, and viruses in particular, as well as acquire those that belong to the host [2][3][4][5]. V. destructor made a host shift to the Western honeybee A. mellifera at the beginning of the 20 th century and spread to Europe, USA, New Zealand, Africa, and the Middle East from southern and southeastern Asia during the last century [6].

Sample Collection
The experimental colonies of A. m. ligustica (worker bees N = 48, from hives 1, 3, 5, and 23, (4 bees per hive), 14 (6 bees), 81 (9 bees), and 401 (1 bee); mites N = 606, from the same hives (85 of them from emerging bees and the rest from free falling mites), collected between October to February 2016, were described before [23]; A. m. syriaca (workers N = 15, from a subcollection of 500 workers from colonies from several apiaries; mites = 20, from tens of mites that were collected in 2013) and A. m. intermissa (workers N = 15, from a subcollection of 500 workers; V. destructor mites N = 27, from tens of mites that were collected in 2013) were described previously [24]. A. cerana drones (N = 6) and V. destructor mites (N = 20) were sampled during spring 2016 (December) in Phrae, Thailand from two colonies 3c and 4c untreated against mites (3 drones and 20 mites from each colony, respectively). The honeybees and corresponding mites were transported in RNA later™ and stored immediately at −80 • C until RNA extraction.

Samples Preparation
RNA extraction from all A. mellifera and mite samples was carried out using TRI Reagent ® (Sigma-Aldrich, Israel) according to the manufacturer's instructions as published before [24,30]. RNA from A. cerana drones and the corresponding V. destructor mites was individually extracted using a GenJet RNA purification Kit (Thermo Scientific, Burlington, Canada) according to the manufacturer's instructions.

Transcriptome and Virome Analysis
Construction and paired-end sequencing of the libraries from A. cerana and its corresponding V. destructor mites-RNA samples was performed at the Technion Genome Center on a HiSEq 2000 platform (Illumina, Haifa, Israel). Paired-end reads were assembled de novo using Trinity [31]. The obtained contigs were translated and aligned to the GenBank nonredundant (NR) database by Blastx [32]. Next-generation sequencing (NGS) of the RNA from A. m. ligustica, A. m. intermissa, and A. m. syriaca honeybees and corresponding V. destructor mites was described previously [23,24]. Metagenomic analysis of A. m. ligustica, A. m. intermissa, and A. m. syriaca subspecies and A. cerana bees, and of V. destructor mites samples were carried out as described previously and in Section 2.8 [30].

RT-PCR
cDNA was prepared using RevertAid Reverse Transcriptase (Thermo Scientific) with oligo-dT and random primers according to the manufacturer's instructions. One-hundred nanogram and 2000 ng RNA templates were used from V. destructor and honeybee samples, respectively. RT-conditions: incubation of RNA and primers at 65 • C for 5 min., followed by addition of buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 2 mM MgCl 2 , 5 mM DTT, 4 units of RNase inhibitor Ribolock ® (Thermo Scientific), and the RT enzyme (200 units) in a 25 µL volume, and further incubation at 55 • C for 30 min. The reaction was terminated by heating at 85 • C for 5 min. PCR-validations were performed with GoTaq ® (Promega Corporation, Madison, WI, USA) using 1 µL cDNA template and 0.2 µM of each forward and reverse primer in a 20 µL reaction with the following conditions; 95 • C for 4 min, 32 cycles at 94 • C for 30 s, then 56 • C (VOV-1 segments 1,2,4,5) or 57 • C (VOV-1 segments 3,6) for 50 s, 72 • C for 2 min. (VOV-1 segments 1,2,4,5) or 1 min. (VOV-1 segments 3,6), and a final extension step of 72 • C for 10 min. For VDV-4 the PCR conditions were identical to those used in segments 1, 2, 4, 5 of VOV-1 with the corresponding specific primers. Specific primers used for validations are described in the Tables S1 and S2 in the Supplementary material.

VOV-1 Prevalence
VOV-1 prevalence was determined by using RT-PCR to detect the presence of the segment 6 of the virus genome in samples of V. destructor mites and honeybees in apiaries located at the North (Haifa, Kibbutz Lehavot HaBashan, Kibbutz Dan), the Center (ARO, Nitzanei Oz, Herut, Kfar Ruth) and the South (Kibbutz Yad Mordechai) of Israel.

qRT-PCR
Viral genome copy number was quantified on a PikoReal 96 machine (Thermo Scientific) using a standard protocol (95 • C 2 min; 40 cycles of 95 • C 10 s, 60 • C 20 s, 72 • C 20 s). Each quantitative PCR analysis was performed in triplicate. Nontemplate controls (water) were included in triplicates in each assay. The KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Bio-systems) was used, in a 10 µL final volume. For each analysis 2 µL of the diluted cDNA was used (dilution factor of 4) and specific primers VOV-1-qRT-F1 and VOV-1-qRT-R1 at a concentration of 0.25 µM each (Table S1). The specificity of the amplicons synthesized during the PCR run was ascertained by performing a dissociation curve protocol from 60 • C to 95 • C. Specific primers used for quantification are provided in Table S1 in Supplementary material.

Replication Assay
Testing for viral replication (presence of the positive strand-sense RNA) was performed by synthetizing the negative-strand cDNA of fragment 6 from the RNA samples using the tagged primer VOV6-46F-TAG, as we described before to analyze replication of BRV-1 [30]. Subsequently the residual VOV6-46F-TAG primer was inactivated by adding to the mixture exonuclease-I and incubating it for another 15 min. at 37 • C (method described in de Miranda et al, 2013 [33]). Finally the exonuclease I was inactivated by heating the mixture at 80 • C for 15 min. Subsequently, PCR was performed with primers VOV6-870R and TAG (Supplementary material 1 Table S1). cDNA produced without any primer was used as control in the same reactions followed by PCR with the same primers as above. PCR was performed at 95 • C for 4 min., 30 cycles at 94 • C for 30 s, then 58 • C for 50 s, 72 • C for 1 min. and a final extension step of 72 • C for 10 min. The identity of the amplified fragment was confirmed by Sanger sequencing (performed at the Biological Services Unit of the Weizmann Institute of Science, Israel).

Bioinformatic Identification of Contigs
Each RNAseq library was de novo-assembled using Trinity assembler version 2.2.0 [31]. The assembled contigs were then searched with BLASTX [33] against the NCBI nonredundant protein database (NR) [34]. After the assembled viruses were identified in each library, each library's raw data reads were mapped using bowtie2 [35] to evaluate the virus quantity in the transcriptome. We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present. Then we analyzed data that we downloaded from recently published transcriptomes of viruses of A. m. intermissa and A. m. syriaca and their varroa from the MENA region [24] and were able to assemble the complete genome of the virus (Tables 1 and 2). Table 1. Libraries used in this study.

NGS Libraries
Library Code Accession Number * * Libraries uploaded NCBI short read archive database, raw data.

Molecular Phylogenetic Analysis
Phylogenetic analysis was done using MEGA 6 [34]. Alignment of the proteins was done using MAFFT [36], and then Maximum likelihood Phyml 3.0 was used for creating the tree [37] with a 100 bootstrap.   Interestingly, ARV-1/BRV-1 and VOV-1 showed differences in their distribution across bee and V. destructor libraries; while ARV-1/BRV-1 was present in all V. destructor libraries and in two of the  Table 3).

Metagenomic Analysis of Viruses in
The presence of viruses varied across A. mellifera and A. cerana bees and their corresponding V. destructor libraries. From the A. mellifera libraries analyzed, IB1 and IV4 (A. m. ligustica) showed a similar percentage of viral reads of DWV, as did SB2 and SV5 (A. m. syriaca) and A. m. intermissa, and their V. destructor libraries-AB3 and AV6-showed large differences ( Figure 1). Also, A. cerana libraries-BCER and VCER-were distinct ( Figure 1 and see below). For instance, AV6 showed a smaller percentage of DWV reads compared to AB3 and to the other A. mellifera-IB1 and SB2-and their corresponding V. destructor libraries-IV4 and SV5, respectively ( Figure 1). The cDNA libraries of A. cerana (BCER) and its varroa (VCER) displayed extremely low percentage of viral reads for DWV (0.7601% and 0.6402%, accordingly). As can be seen, our samples BCER and VCER differed in their load of other viruses as well; while the BCER main viral component was ARV-2 [38], the main virus present in VCER was VDV-2 ( Figure 1). In addition, VCER displayed two viruses present in varroa parasites of A. cerana only with 38% homology to Hubei picorna-like virus 29 [39] and 36% homology to Hubei virga-like virus 14 [40] (Table 3 and see below).
The presence of viruses varied across A. mellifera and A. cerana bees and their corresponding V. destructor libraries. From the A. mellifera libraries analyzed, IB1 and IV4 (A. m. ligustica) showed a similar percentage of viral reads of DWV, as did SB2 and SV5 (A. m. syriaca) and A. m. intermissa, and their V. destructor libraries-AB3 and AV6-showed large differences ( Figure 1). Also, A. cerana libraries-BCER and VCER-were distinct ( Figure 1 and see below). For instance, AV6 showed a smaller percentage of DWV reads compared to AB3 and to the other A. mellifera-IB1 and SB2-and their corresponding V. destructor libraries-IV4 and SV5, respectively ( Figure 1). The cDNA libraries of A. cerana (BCER) and its varroa (VCER) displayed extremely low percentage of viral reads for DWV (0.7601% and 0.6402%, accordingly). As can be seen, our samples BCER and VCER differed in their load of other viruses as well; while the BCER main viral component was ARV-2 [38], the main virus present in VCER was VDV-2 ( Figure 1). In addition, VCER displayed two viruses present in varroa parasites of A. cerana only with 38% homology to Hubei picorna-like virus 29 [39] and 36% homology to Hubei virga-like virus 14 [40] (Table 3 and see below).  Table 1), cutoff at 0.0001%.

Varroa Orthomyxovirus-1 and the Hubei Virga-like 14 Homolog Virus
We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present in the data. To complete the picture we downloaded and analyzed data from the transcriptome of viruses of A. m. intermissa and A. m. syriaca and their varroa mites from the Middle East and North African (MENA) honeybees and varroa mites that were published recently but did not focus on finding new viruses [24]. This additional sequence data facilitated complete genome assembly of this new virus. According to BLASTX analysis we identified VOV-1 contigs in two cDNA libraries of bees (IB1 and SB2) and two cDNA libraries of their corresponding varroa mites (IV4 and SV5). VOV-1 showed 23-58% homology to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [25][26][27][28]. Contigs of 2198, 1899, and 358 nucleotides in length from the SV5 library showed homology of 58%, 29%, and 46% to polymerase subunits PB2, PB1, and PA encoded in segments 1, 2, and 3 of the DHOV genome, respectively (Table 4). A contig of 232 nucleotides length from the IV4 library showed homology of 41% to the glycoprotein subunit (GP) encoded in segment 4 of THOV; a contig of 1442 nucleotides length from IB1 library showed homology of 39% to the nucleoprotein subunit  Table 1), cutoff at 0.0001%.

Varroa Orthomyxovirus-1 and the Hubei Virga-like 14 Homolog Virus
We reanalyzed the data obtained before in A. m. ligustica [23] and realized that contigs of a new Orthomyxovirus were present in the data. To complete the picture we downloaded and analyzed data from the transcriptome of viruses of A. m. intermissa and A. m. syriaca and their varroa mites from the Middle East and North African (MENA) honeybees and varroa mites that were published recently but did not focus on finding new viruses [24]. This additional sequence data facilitated complete genome assembly of this new virus. According to BLASTX analysis we identified VOV-1 contigs in two cDNA libraries of bees (IB1 and SB2) and two cDNA libraries of their corresponding varroa mites (IV4 and SV5). VOV-1 showed 23-58% homology to the Orthomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [25][26][27][28]. Contigs of 2198, 1899, and 358 nucleotides in length from the SV5 library showed homology of 58%, 29%, and 46% to polymerase subunits PB2, PB1, and PA encoded in segments 1, 2, and 3 of the DHOV genome, respectively (Table 4). A contig of 232 nucleotides length from the IV4 library showed homology of 41% to the glycoprotein subunit (GP) encoded in segment 4 of THOV; a contig of 1442 nucleotides length from IB1 library showed homology of 39% to the nucleoprotein subunit (NP) encoded in segment 5 of DHOV; and a contig of 983 nucleotides from the SB2 library showed homology of 23% to matrix protein (M) encoded in segment 6 of THOV (Table 4). Phylogenetic analysis of open reading frames (ORFs) coding for polymerase subunits PB2, PB1, and PA showed that the polymerase was closely related to negative-sense ssRNA viruses belonging to the Orthomyxoviridae viral family: Thogoto virus (THOV); Aransas Bay virus (ABV); Upolu virus (UPOV) (Figure 2A (Table 4).    Based on the above contigs' sequences we designed specific primers to validate the presence of each segment of VOV-1 in the viromes of Israeli A. mellifera ligustica and their counterpart V. destructor parasites (Figure 3 and see Materials and Methods). All six segments were identified in the V. destructor virome ( Figure 3, lanes 1, 4, 7, 10, 13, and 16) but they were absent in the virome of honeybees (Figure 3,  lanes 2, 5, 8, 11, 14, and 17).
Viruses 2018, 10, x FOR PEER REVIEW 8 of 16 Based on the above contigs' sequences we designed specific primers to validate the presence of each segment of VOV-1 in the viromes of Israeli A. mellifera ligustica and their counterpart V. destructor parasites (Figure 3 and see Materials and Methods). All six segments were identified in the V. destructor virome (Figure 3, lanes 1, 4, 7, 10, 13, and 16) but they were absent in the virome of honeybees ( Figure 3, lanes 2, 5, 8, 11, 14, and 17). Furthermore, we tested the presence of segment 6 of the viral genome to estimate the prevalence of VOV-1 by RT-PCR in individual mites and honeybees. We detected VOV-1 in 35.56% of V. destructor mites from Israeli colonies located in ARO, Beit Dagan (16 of 45), and in none of 32 individual honeybees sampled from the same colonies. We also analyzed its prevalence in colonies located in the Center, North, and South of Israel by testing pools of the honeybees and V. destructor mites with the same PCR method. The virus was identified in 78.57% of V. destructor pools (11 of 14 pools) and only in 8.33% of honeybee pools (5 of 60 pools).
The number of genomic copies of VOV-1 estimated by qRT-PCR was similar in individual mites collected from honeybee colonies in ARO, Beit Dagan, in pools of mites, and in honeybee pools sampled from colonies located in the Center, North, and South of Israel: 5.11×10 2 -1.22×10 6 , 2.33×10 3 -4.88×10 5 , and 4.91×10 2 -1.38×10 5 , respectively (Table 5).  Furthermore, we tested the presence of segment 6 of the viral genome to estimate the prevalence of VOV-1 by RT-PCR in individual mites and honeybees. We detected VOV-1 in 35.56% of V. destructor mites from Israeli colonies located in ARO, Beit Dagan (16 of 45), and in none of 32 individual honeybees sampled from the same colonies. We also analyzed its prevalence in colonies located in the Center, North, and South of Israel by testing pools of the honeybees and V. destructor mites with the same PCR method. The virus was identified in 78.57% of V. destructor pools (11 of 14 pools) and only in 8.33% of honeybee pools (5 of 60 pools).
We confirmed by Sanger DNA sequencing that the above specific-primer-tagged amplicons were identical to the VOV-1 sequence comprising nucleotides 46 and 870 of segment 6 of the viral genome.
From the two undescribed viruses that we found in VCER we further investigated VDV-4. Phylogenetic analysis using the putative large ORF protein of the virus showed that it is 36% homologous to the hypothetical protein gene of spider viruses Hubei virga-like virus 14 and Hubei virga-like virus 13 as well as to the spider putative protein of the virus Nephila clavipes virus 4 ( Figure 5). Quantitation of VOV-1 genomic copy number was carried out by amplifying segment 6 using specific primers (see Materials and Methods). N = North, C = Center, and S = South of Israel, respectively. MIX, group of mites from various colonies. (p), pool.

genome.
From the two undescribed viruses that we found in VCER we further investigated VDV-4. Phylogenetic analysis using the putative large ORF protein of the virus showed that it is 36% homologous to the hypothetical protein gene of spider viruses Hubei virga-like virus 14 and Hubei virga-like virus 13 as well as to the spider putative protein of the virus Nephila clavipes virus 4 ( Figure 5). RT-PCR validation of its presence in a small sample of A. cerana drones and V. destructor individuals suggested that this virus was predominant in the latter ( Figure 6). Therefore, we decided to name it VDV-4. RT-PCR validation of its presence in a small sample of A. cerana drones and V. destructor individuals suggested that this virus was predominant in the latter ( Figure 6). Therefore, we decided to name it VDV-4.

Discussion
We presumed that since Apis mellifera is a new host to Varroa destructor they might show differences both in the composition and distribution of their viral load. Our analysis of the data illustrates changes in viral composition and load among samples from A. mellifera subspecies and their V. destructor. Interestingly, a small-scale sample of A. cerana and its V. destructor showed variation in viral composition and load as well. The variation in virus composition was based on a n = 1 repetition per bee species and V. destructor transcriptomes and, consequently, the results do not necessarily reflect variations at the level of species/subspecies and could be due to other factors such as sampling region, season and/or diverse time of sampling of the different bee species and there mites, etc. A higher number of samples in a coordinated effort will be required for species/subspecies comparative purposes.

Discussion
We presumed that since Apis mellifera is a new host to Varroa destructor they might show differences both in the composition and distribution of their viral load. Our analysis of the data illustrates changes in viral composition and load among samples from A. mellifera subspecies and their V. destructor.
Interestingly, a small-scale sample of A. cerana and its V. destructor showed variation in viral composition and load as well. The variation in virus composition was based on a n = 1 repetition per bee species and V. destructor transcriptomes and, consequently, the results do not necessarily reflect variations at the level of species/subspecies and could be due to other factors such as sampling region, season and/or diverse time of sampling of the different bee species and there mites, etc. A higher number of samples in a coordinated effort will be required for species/subspecies comparative purposes.
We found that in the libraries studied the viral reads of Deformed wing virus, one of the most important factors affecting honeybee colony health and survival, were 82.20%, 99.20%, and 97.90% in A. m. ligustica, A. m. syriaca, and A. m. intermissa, respectively, and only 0.76% in A. cerana. In addition, we observed that a few viruses of importance were present in A. mellifera libraries but not in A. cerana's. Namely, ABPV + IAPV in A. m. ligustica and its V. destructor, and in A. m. syriaca; BeeMLV/VdMLV in all A. mellifera libraries and corresponding V. destructor mites, as well as in varroa from A. cerana; BQCV in all A. mellifera and corresponding V. destructor and SBV in A. m. ligustica and all three libraries of varroa mites parasitizing A. mellifera. ABPV seems to be uncommon to A. cerana in China and South Korea [41,42], or to show low prevalence in wild colonies [43], as was in A. cerana in Northern Thailand [44]. SBV was reported with high prevalence in Southeast Asia [41,[45][46][47] as well as BQCV, that showed relatively high prevalence in viral populations of A. cerana in China, South Korea, and Vietnam [42,43,48]. These two viruses were absent in our libraries and that could be due also to the small sample size of them.
Furthermore, we identified recently characterized viruses in A. mellifera and/or in varroa mites, namely Apis rhabdovirus-1/Bee rahbdovirus-1 (ARV-1/BRV-1) [30,38], Varroa destructor virus-2 and -3 (VDV-2 and VDV-3) [23], and a new Varroa orthomyxovirus-1 (VOV-1). Some of these viruses were identified as common to honeybees and V. destructor like ARV-1/BRV-1 and VOV-1, while others appeared to be restricted to mites, such as VDV-2 and VDV-3. Interestingly, VOV-1's presence was limited to A. m. ligustica and A. m. syriaca as well as to V. destructor mites parasitizing them. VDV-2 was detected in all V. destructor libraries and VDV-3 was absent in Varroa destructor mites parasitizing A. cerana. ARV-1/BRV-1 was found previously in A. mellifera, V. destructor, and in Bombus impatiens, but our finding that it is present in A. cerana suggests that it may have a broader host range. V. destructor mite parasites of A. cerana appear to bear two novel viruses with low homology to Hubei picorna-like virus-29 [39] and to Hubei virga-like virus-14 [40], which were absent in V. destructor mites from A. mellifera. We validated the presence of a Hubei virga-like virus-14 and designed it VDV-4.
Data suggest that following V. destructor invasion there is high selection on DWV strains such that only a single strain seems to dominate, though which strain dominates varies across colonies and studies [20,[49][50][51]. This process is accompanied with increase in the collapse of V. destructor-infested colonies [15,51,52]. Moreover, laboratory experiments showed that DWV undergoes rapid selection following its injection in the honeybee hemolymph, similarly to what happens during parasitization of Varroa destructor on A. mellifera [20]. In our study, we measured differences in DWV loads between samples of A. mellifera subspecies and their V. destructor counterparts. V. destructor from A. m. intermissa showed lower DWV levels compared to its parasitized host (26.83% and 97.90%, respectively). Interestingly, it was reported that A. m. intermissa was more resistant to V. destructor parasitization than other A. mellifera subspecies [53,54]. Again, the results are subjected to the above-mentioned limitations of the analysis including the n = 1 repetitions of the transcriptomic data per subspecies.
We characterized VOV-1, a novel virus common to A. mellifera and V. destructor and VDV-4, a novel virus of V. destructor from A. cerana. VOV-1 possess a single-stranded negative-sense RNA genome and belongs to the Orthomyxoviridae family that among others includes the genus Thogotovirus. Most of the Thogotoviruses have been associated with ticks [55] and relatively few of them have been described in Acari or other types of hematophagous arthropods [56,57]. The VOV-1 genome has six segments and this is the first report of the complete genome of a single-stranded negative-sense RNA segmented virus seen in honeybees and varroa mites. We provide evidence that VOV-1 replicates in individual varroa mites, and we found positive sense-virus RNA in pools collected from A. m. ligustica. Interestingly, it showed greater prevalence in V. destructor mites compared with honeybees of 78.57% and 8.33%, respectively, in samples from apiaries located at the North (Haifa, Kibbutz Lehavot HaBashan, Kibbutz Dan), the Center (ARO, Nitzanei Oz, Herut, Kfar Ruth), and the South (Kibbutz Yad Mordechai) of Israel. This is why, taking together the above data, we decided to name it Varroa orthomyxovirus-1, VOV-1.
The discovery of these novel viruses in Apis mellifera and its recently acquired obligatory parasite Varroa destructor opens a new venue for investigation of viral interactions in honeybee colonies. A number of questions emerge concerning this new host-pathogen relationship that could interfere the preexisting balance. What is the pathology associated with VOV-1? Do varroa mites transmit VOV-1 and VDV-4 directly, e.g., transovarially, or via their host bee? Where do the viruses accumulate in varroa? Is VOV-1 infectious to A. mellifera?
Furthermore, there is another issue concerning those viruses crucial for colony health and survival-DWV, ABPV, IAPV, and CBPV-and their interaction with newly discovered ARV1/ BRV-1, VOV-1, and VDV-2 and -3: Do they affect one another on a mutual base? If they do, on what level and what are the factors that may be involved (e.g., colony location, kind of treatment against varroa, season, colony resistance and/or hygienic behavior, etc.).
Our findings and the tools that we have developed in this study pave the way to investigate these questions and extend our knowledge and understanding of the role played by viral pathogens in honeybee colonies.