ABSTRACT
A new Tetraparvovirus was identified in a pooled tissue sample of a Rhinophylla pumilio bat from Santa Bárbara, Pará State, Brazil. This is the second Tetraparvovirus described in bats and has higher nucleotide and amino acid identity with a Tetraparvovirus from opossum, also described in Brazil.
KEYWORDS: parvovirus, bats, metagenomics
ANNOUNCEMENT
Tetraparvoviruses, family Parvoviridae, are linear single-stranded DNA viruses of ∼5 kb in length. They have two open reading frames: one for the NS1 replicase gene and another for the VP capsid gene (1). For Tetraparvoviruses, the NS1 amino acid identity is considered for species demarcation based on a >85% threshold (https://ictv.global/report/chapter/parvoviridae/parvoviridae/tetraparvovirus). As of February 2025, there are eight species classified in this genus, which have been identified in primates, ungulates, or bats. The one from bats, Tetraparvovirus chiropteran1, was identified in some Eidolom helvum from Ghana and has a genome of 5,065 nt. According to Canuti et al., spleen and kidney were identified as the potential replication sites of this virus (2). On 07 April 2015, tissue samples were collected from a healthy adult male Rhinophylla pumilio bat from Santa Bárbara in Pará State, Brazil (1.208333°S 48.270833°W). Fragments of its spleen, lungs, heart, and lymph nodes tissues were pooled prior to processing. This project was authorized by the Ethics Committee on Animal Use of the Evandro Chagas Institute under certificates 21/2014 and 10/2024.
RNA extraction was performed using the PureLink RNA Mini Kit (Invitrogen) associated with TRIzol Reagent (Invitrogen). The first and second strands of cDNA were synthesized via the SuperScript IV VILO MasterMix (Invitrogen) and the Second Strand cDNA Synthesis Kit (Invitrogen), respectively. A cDNA library was prepared with the Nextera XT DNA library preparation kit and sequenced on an Illumina NextSeq 500 platform with 150 bp paired-end reads. Metagenomic data analysis consisted of raw data quality control and cleaning by Fastp v0.23.4 (3), rRNA removal using SortMeRNA v.2.1 (4), de novo assembly via MEGAHIT v1.2.9 (k-mers 21, 31, 41, 51, 61, 71, 81, 91, and 99) (5), and alignment against the nr protein database by DIAMOND (6). Contig inspection, map-to-reference alignment (Geneious Mapper), and multiple sequence alignment (Clustal Omega v1.2.3) were performed using Geneious Prime 2024.0 (7). Phylogenetic analysis was performed with IQ-TREE v2.2.5 (8), using the maximum likelihood method and 1,000 bootstrap iterations. Default parameters were used except where otherwise noted.
Sequencing produced 43,168,902 reads, 468 of which were related to the Parvoviridae family. From them, a 5,258 nt contig was assembled with a genome read coverage of 11.1× and 54.8% of GC content. The putative NS1 gene extends from nucleotides 206 to 2,194, and the putative VP capsid gene from nucleotides 2,222 to 4,963. The genome showed the highest nucleotide identity with the Tetraparvovirus didelphimorph1 (MG745671), which was 73.97%. It was also the closest in amino acid identity of the NS1 gene (80.96%), below the species demarcation criteria threshold. Based on this gene, the sequence grouped within the Tetraparvovirus monophyletic clade in a subclade with the opossum sequence (Fig. 1), which is from São Paulo State, Brazil (9). These findings suggest that the obtained coding-complete genome is very likely a new Tetraparvovirus species.
Fig 1.
Maximum likelihood phylogenetic tree based on the amino acid alignment of the NS1 gene. The best amino acid substitution model defined by IQ-TREE was the Q.yeast + F + G4. Tetraparvovirus species are identified by color. The red color highlights the new sequence, while the white color represents unclassified viruses and the black color species from other genera.
ACKNOWLEDGMENTS
This research is a part of the Pro-Amazonia project, grant number 3286/2013, funded by the Coordination for the Improvement of Higher Education Personnel (CAPES).
Contributor Information
Leonardo H. Almeida Hernández, Email: leohenrique96@hotmail.com.
Simon Roux, DOE Joint Genome Institute, Berkeley, California, USA.
DATA AVAILABILITY
The virus sequence and its host mtDNA sequence are the first version, PV693370.1 and PV684749.1, respectively. The raw sequencing data has the following SRA number: SRR33632182.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The virus sequence and its host mtDNA sequence are the first version, PV693370.1 and PV684749.1, respectively. The raw sequencing data has the following SRA number: SRR33632182.