Apis mellifera Solinvivirus-1, a Novel Honey Bee Virus That Remained Undetected for over a Decade, Is Widespread in the USA

Abstract

A metagenomic analysis of the virome of honey bees (Apis mellifera) from an apiary with high rates of unexplained colony losses identified a novel RNA virus. The virus, which was named Apis mellifera solinvivirus 1 (AmSV1), contains a 10.6 kb positive-strand genomic RNA with a single ORF coding for a polyprotein with the protease, helicase, and RNA-dependent RNA polymerase domains, as well as a single jelly-roll structural protein domain, showing highest similarity with viruses in the family Solinviviridae. The injection of honey bee pupae with AmSV1 preparation showed an increase in virus titer and the accumulation of the negative-strand of AmSV1 RNA 3 days after injection, indicating the replication of AmSV1. In the infected worker bees, AmSV1 was present in heads, thoraxes, and abdomens, indicating that this virus causes systemic infection. An analysis of the geographic and historic distribution of AmSV1, using over 900 apiary samples collected across the United States, showed AmSV1 presence since at least 2010. In the year 2021, AmSV1 was detected in 10.45% of apiaries (95%CI: 8.41-12.79%), mostly sampled in June and July in Northwestern and Northeastern United States. The diagnostic methods and information on the AmSV1 distribution will be used to investigate the connection of AmSV1 to honey bee colony losses.

Keywords: Apis mellifera; RNA virus; family Solinviviridae; honey bee; honey bee colony losses; insect; pollination service.

Figure 1

The organization of Apis mellifera solinvivirus-1 genomic RNA. (a) The schematic representation of AmSV1 genomic RNA (GenBank accession number OQ540582). The position of the main ORF and putative protein domains is shown. Amino acid positions of the protein domains are indicated above the ORF. Non-structural proteins: Hel—helicase, Prot—3C protease, structural proteins: JR—jelly roll domain of structural viral protein (VP)1, VP2; (b) NGS coverage of the AmSV1 genome. Genetic diversity of AmSV1 apiary-level population used for NGS analysis: (c) Shannon’s diversity profile (sliding window average for 100 nt positions); (d) distribution of polymorphic nucleotides (n = 419) and amino acids (n = 63), showing an alternate allele exceeding 3% in frequency in the apiary-level NGS library. (e) The maximum likelihood phylogenetic tree was generated based on the full-length protein sequences of AmSV1, as well as classified (marked with asterisk) and putative solinviviruses. For SINV3 and RAAV, the sequences of -1 translational frameshift proteins (ORF1-ORF2 fusions) were used. Bootstrap values above 50%, generated from 1000 replications, are shown to the left of corresponding nodes. The bar indicates a 10% sequence difference.

Figure 2

Accumulation of AmSV1 in different body parts of individual adult honeybee workers from AmSV1 positive apiaries. (a) Sections of a frozen worker honey bee used for RNA extraction. (b) AmSV1 loads in the head, thorax, and abdomen of 16 worker bees. (c) Correlation between AmSV1 loads in head, thorax, and abdomen.

Figure 3

Pupal injection experiment. (a) The quantification of AmSV1 genome equivalents (GE) in the pupae, injected with partially purified AmSV1 preparation (AmSV1) or buffer control (PBS), which were sampled immediately after injection (Time 0) and after 3 days of incubation at +33 °C (3 dpi). Quantification was carried out by qPCR using cDNA-generated random primers, allowing the detection of AmSV1 RNA of both polarities. Dots indicate levels of AmSV1 in individual pupae. Significantly different levels of AmSV1 RNA are indicated by different red letters (ANOVA p < 0.01). (b) Specific detection of negative-strand RNA, a virus replicative intermediate, in virus preparation (lane 0) used for injection. The pupae was injected with partially purified AmSV1 preparation (AmSV1) and sampled immediately after injection, Time 0 (lanes 2 and 3, individual pupae), or after 3 day incubation at +33 °C, with 3 dpi (lanes 6 and 7, two pools of 2 pupae). Control, buffer-injected pupae—PBS—were sampled 3 days after injection (lanes 4 and 5, two pools of two pupae). M, DNA ladder, base pairs (bp). The cDNA was produced using tagged forward primer, PCR amplification was carried out with the primer that was identical to the tag and reverse primer. The arrow marks the position of the expected 141 bp RT-PCR product.

Figure 4

Spatio-temporal distribution of AmSV1 in the USA. (a) The distribution of AmSV1 in the US apiaries in 2010, 2014, and 2021. (b) Monthly loads (0 = not detected, below 2.8 log₁₀ GE/bee) and (c) monthly distribution of AmSV1 prevalence and loads for the year 2021.

Figure 5

Connections between the prevalence of AmSV1 and (a) other honey bee parasites, including (b) field apiary observations in the U.S. 2021 apiaries. In total, 794 apiary-level samples were tested. Dashed line at OR = 1 represents the null hypothesis that AmSV1 does not associate with listed measures. DWV–A—deformed wing virus, type A; DWV-B deformed wing virus type B; ABPV—acute bee paralysis virus; CBPV—chronic bee paralysis virus; IAPV—Israeli acute paralysis virus; KBV—Kashmir bee virus; LSV2—Lake Sinai virus; Varroa—ectoparasitic mite Varroa destructor; Nosema—Vairimorpha ceranae. EFB—European foulbrood (caused by bacterium Melissococcus plutonius), Sacbrood–caused by sacbrood virus, Chalkbrood–fungal disease of honey bee brood caused by fungus Ascosphaera apis, PMS—Parasitic Mite Syndrome (caused by the mite Varroa destructor), Deformed Wings–could be caused by DWV, Shiny Black—hairless bees, SHB—infestation with small hive beetle (Aethina tumida), Wax Moth—infestation with wax moth (Galleria mellonella), Queen Cells presence, Drone Layer–queen lays unfertilized drone eggs, Queenless—queen is absent in at least one of sampled colonies, Any Queen Issues—combined Queen Cells, Drone Layer, and Queenless. A significant p-value is marked with an asterisk.