Characterization of Pipistrellus pygmaeus Bat Virome from Sweden

Abstract

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78-99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88-94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

Keywords: Sweden; bat; coronavirus; metagenomics; virome.

Figure 1

Schematic summary of the number of reads related to viruses classified by viral family at the protein level. Scale breaks were introduced in the x-axis for the visualization of the variable number of viral counts in each dataset. The exact number of viral counts are provided in Supplementary Table S1.

Figure 2

Simplot analysis of Swedish bat coronaviruses. Near full-length sequences of BtCoV/F-MV1/P.pyg/SWE/2020 (A) and BtCoV/F-MV2/P.pyg/SWE/2020 (B) were used as queries and compared with closest reference sequences in the SimPlot similarity analysis. All the analyses were performed with a Kimura model, a window size of 600 base pairs, and a step size of 100 base pairs.

Figure 3

Phylogenetic analysis of coronaviruses from Sweden based on predicted full-length ORF1ab protein. The sequences reported in this study, BtCoV/F-MV1/P.pyg/SWE/2020 (ON457560), BtCoV/F-MV2/P.pyg/SWE/2020 (ON457561), and BtCoV/F-HS1/P.pyg/SWE/2020 (ON457562), (indicated by red square boxes and red text), and the closest sequence matches from Genbank and ICTV reference sequences from each genus within the Orthocoronavirinae subfamily are included in the analysis. The tree was generated using a maximum likelihood model with 500 bootstrap iterations on ClustalW-aligned amino acid sequences, with a total of 8037 amino acid positions in the final dataset. Only bootstrap values above 50 are displayed on the nodes.

Figure 4

Phylogenetic analysis of bat picornaviruses from Sweden based on the conserved capsid protein. The sequences reported in this study, BtPp-PicoV/F-HS1/SWE/2020 (ON375332) and BtPp-PicoV/F-HS2/SWE/2020 (ON375333) (indicated by red square boxes and red text), the closest sequence matches from Genbank and ICTV reference sequences from a different genus within the Picornaviridae family (indicated in green) are included in the analysis. The tree was generated using MEGAx and a maximum likelihood model with 500 bootstrap iterations on ClustalW aligned amino acid sequences, with a total length of 256 amino acids. Only bootstrap values above 50 are displayed on the nodes.

Figure 5

Phylogenetic analysis of bat hepevirus from Sweden based on predicted conserved RdRp protein. The sequence reported in this study, BtHEV-Pp1/F-MV/P.Pyg/SWE/2020 (ON513427) (indicated by red square boxes and red text), and the closest sequence matches from Genbank and ICTV reference sequences from within the Hepeviridae family are included in the analysis. The tree was generated using MEGAx and a maximum likelihood model with 500 bootstrap iterations on ClustalW aligned amino acid sequences, with a total length of 351 amino acids. Only bootstrap values above 50 are displayed on the nodes.