Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil

Abstract

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.

Keywords: Bee population declines; High-throughtput sequence; Honey bee viruses; Metagenomic approach; New togotovirus isolate.

Fig. 1

Maps and location of the six municipalities in the Brazilian state of Rio Grande do Sul. The samples were taken between 2016 and 2017. The maps highlight the political demarcation of each city and the number of apiaries targeted by the study: samples from 1 apiary at Montenegro (MO); samples from 2 apiaries at Pareci Novo (PN); samples from 3 apiaries at Alto Feliz (AF); sample from 1 apiary in Linha Nova (LN); samples from 7 apiaries at Ivoti (IV); and samples from 1 apiary in Novo Hamburgo (NH)

Fig. 2

The genome organization and read coverage of the putative Apis thogotovirus 1 (ATHOV-1). The reads in the sequence library were aligned with the genome (segments) as shown in blue

Fig. 3

Detection of the thogotovirus ATHOV-1 by RT-PCR in the pooled RNA for sequencing (larvae, adults, and queen bee). The expected sizes of the polymerase subunit PB2 and GP of the ATHOV-1 were confirmed the amplified DNA fragments of 150 bp (lanes 1 and 2 of S1 segment) and 200 bp (lanes 1 and 2 of S4 segment). Additionally, the amplified DNA fragment of 250 bp (lanes 1 and 2 of MK032468) corresponding to the expected of the GP of the Varroa destructor othomyxovirus (VOV-1). PCR products (5 µl) were loaded into a 1.2% agarose gel. M (100-bp molecular marker); S1 (polymerase subunit PB2); S4 (glycoprotein); 1 (larvae); 2 (adults); and 3 (queen bee). No virus was detected in a sample or RNA from the queen bee (lane 3 of S1, S4, and MK032468)

Fig. 4

Phylogenies of deduced amino acid sequences of glycoprotein (segment 4) of ATHOV-1 in comparison to homologous sequences of selected viruses, using the maximum-likelihood method. Amino acid sequences of virus proteins used in this study were obtained from GenBank. The accession numbers of all viral sequences are presented in Supplementary Table S2

Fig. 5

Sequence alignment of ATHOV-1 segment 4 with Thogoto thogotovirus (PDB accession number 5XEA), Bourbon virus (PDB accession number 5ZKX), Dhori virus (PDB accession number 5XEB), and AcMNPV (PDB accession number 3DUZ) glycoproteins. Residues that are 100% conserved are in solid red boxes. Those with similarity > 70% are labeled in red. The secondary structures are indicated and labeled above the sequences

Fig. 6

Phylogenies of deduced amino acid sequences of glycoprotein (segment 4) of VOV-1 in comparison to best hits on BLASTx non-redundant protein database search, using the maximum-likelihood method. The accession numbers of all viral sequences are presented in Supplementary Table S2. The blue box shows segment S4 of VOV-1 clustered with Apis rabdovirus 2 (KY354234.1) and other orthomyxo-like viruses