Fig 1.
Field vole amdoparvovirus (FVAV) contigs derived via short read sequencing of total host lung RNA and de novo assembly.
Showing the arrangement of coding sequences within the contigs and their relationship to putative mRNA species and to genomic and protein sequences. A. The arrangement of the FVAV DNA genome based on direct sequencing for the coding regions and central intronic regions and on the conserved pattern seen in other amodoparvoviruses for the termini [89]. Coding sequences are shown below coloured according to their contribution to NS and VP proteins. B. FVAV contigs approximated to the two most highly expressed Aleutian mink disease virus (AMDV) mRNA species, R2 and R1’ [20]. FVAV coding sequences (identified by sequence and splicing site homology), coloured according to the corresponding protein region, are shown in relation to the contigs (grey) and to the putative mRNA species (based on those documented in AMDV [20]). The putative mRNAs are also coloured by protein coding region. Likely open reading frames operational in the mRNA corresponding to the contig are shown above the contig, other overlapping reading frames are shown below. A “u” postscript indicates a region that is unique to a given protein. C. The proteins that would putatively be assembled from the coding sequences based on the conserved pattern in other studied amdoparvoviruses.
Fig 2.
Frequency distributions and scatterplot for log-transformed (log10 x + 1) standardised reads mapping to the R2 mRNA-like and R1’ RNA-like field vole amdoparvovirus (FVAV) contigs.
Based on shotgun sequenced pulmonary RNA from 38 field voles. A Loess smoother (blue) is shown with shaded 95% confidence interval. The read counts for the R2 and R1’ mRNA-like contigs were highly correlated (Spearman’s rho, rs = 0.81) and the R2-like contig was typically expressed much more abundantly (up to an order of magnitude) than the R1’-like contig.
Fig 3.
Relationships of field vole amdoparvovirus (FVAV) molecular sequences to those in other amdoparvoviruses based on Maximum Likelihood phylogenetic analyses.
A. Nucleotide sequences coding for the VP1 protein in viruses for which full length or near full-length sequences were available. This analysis assumed a GTR + G substitution model and was based on 1905 positions. B. Nucleotide sequences coding for a smaller region of the VP protein allowing the inclusion of more viruses for which fragmentary genomic information was available. This analysis assumed a HKY + G substitution model and was based on 483 positions. C. Amino acid sequences for the NS protein in viruses for which full length or near full-length sequences were available. This analysis assumed an LG + G + I substitution model and was based on 562 positions. D. Amino acid sequences for a shorter region of the NS protein allowing the inclusion of more viruses for which fragmentary genomic information was available. This analysis assumed an LG + G substitution model and was based on 150 positions. A-D. AMDV1, Aleutian mink disease virus 1 (International Committee on Taxonomy of Viruses name: Amdoparvovirus carnivoran1); AMDV2, Aleutian mink disease virus 2 (Amdoparvovirus carnivoran9); AMDV3, Aleutian mink disease virus 3 (Amdoparvovirus carnivoran10); BCAV, British Columbia amdoparvovirus (Amdoparvovirus carnivoran8); BtR1-PV/FJ2012, bat parvovirus isolate; EFAV-1, European felid amdoparvovirus 1; EPV-Amdo.1-EllLut and EPV-Amdo.2-EllLut, endogenised mole vole amdoparvoviruses; GFAV, gray fox amdoparvovirus (Amdoparvovirus carnivoran2); LaAV-1, Labrador amdoparvovirus 1 (Amdoparvovirus carnivoran6); LaAV-2, Labrador amdoparvovirus 2; YRAV1, Yunnan rodent amdoparvovirus 1; RFAV, racoon dog and fox amdoparvovirus (Amdoparvovirus carnivoran3); RFFAV, red fox faecal amdoparvovirus; RpAV, red panda amdoparvovirus (Amdoparvovirus carnivoran5); RpAV-2, red panda amdoparvovirus 2 (Amdoparvovirus carnivoran7); RtRn-ParV/GZ2016, Parvovirinae sp. Isolate; SKAV, skunk amdoparvovirus (Amdoparvovirus carnivoran4); SBEHV1, Sabeidhel virus (Amdoparvovirus chiropteran1). Positions containing gaps or missing values were not considered. Trees with the maximum log likelihood are shown; scale bars indicate substitutions per site; bootstrap support [90] for nodes is indicated where this is above 60% (n = 500 replicates). GenBank sequence identifiers are included in the taxon labels as a prefix.
Fig 4.
The distribution of field vole amdoparvovirus (FVAV) variants across spatiotemporal sampling points and host mtDNA matrilineages.
A. The distribution of FVAV broken down by variant, time and locality within the Kielder Forest. Each point represents one host and is sized according to the abundance of viral mRNA reads (standardised to the mean library size of 31 million reads) and coloured according to the viral variant (see key); a small x-y jitter is applied to make points from the same site visible. DD: decimal degrees. Kielder water, excluding Bakethin Reservoir, is shown in the bottom right of each panel. The base map (https://environment.data.gov.uk/catchment-planning/WaterBody/GB30327698.geojson) contains public sector information licensed under the UK Open Government Licence v1.0.(see: https://www.nationalarchives.gov.uk/doc/open-government-licence/version/1/open-government-licence.htm). B. A host haplotype network for the cytochrome c oxidase subunit 1 (mt-co1) gene sequence with nodes (representing unique haplotypes) sized according to the number of hosts and coloured according to the frequency of occurrence of FVAV variants (see key). Nucleotide differences between haplotypes are indicated by ticks on the network edges and by edge length. C. Maximum Likelihood clustering of aligned nucleotide sequences for the R1’ mRNA-like contig and the R2 mRNA-like contig in FVAV. Analysis includes sequences from the six hosts with the highest viral expression and from which it was possible to assemble long contigs. Assuming an HKY + I substitution model and based on 1256 and 2329 positions respectively for R1’ and R2. Bootstrap node support [90] above 60% is indicated (based on 500 replicates); scale bar represents 0.01 nucleotide substitutions per site.
Fig 5.
Field vole amdoparvovirus (FVAV) was associated with the elevated relative expression of immune-associated genes in the host pulmonary transcriptome.
A. Heatmap showing the differential expression of host genes in individual voles with increasing FVAV expression. Host gene expression values (main left panel) are TMM normalised and scaled (zero mean, unit standard deviation) read counts for genes significantly differentially expressed after FDR-adjustment, almost all of which are immune-associated. FVAV expression (track at top of left panel, green) is in reads normalised to library size. B. Results from a GSEA analysis including 15 custom gene sets representing immunological and other organismal processes. Immune-associated gene sets were broadly upwardly differentially expressed, but this was not the case for other gene sets. Normalised Enrichment Scores (NES) (x-axis) for the gene sets analysed are represented by individual points that are coloured according to significance (padj) and sized according to gene set size.
Fig 6.
A machine learning algorithm (Random Forest) applied to 141 non-pulmonary host variables revealed a subtle negative association between field vole amdoparvovirus (FVAV) expression and the responsiveness of splenic T-cells.
A. Ranked plot of variable importance in the Random Forest analysis with variable sets colour-coded by group (see key). High importance variables were mostly immune gene expression measurements in cultured splenocytes stimulated with anti-CD3 and anti-CD28 antibodies intended to activate T-cells. B. Plots of Yeo-Johnson transformed relative gene expression (RQ) in the highest importance variables in the CD3/CD28-stimulated splenocyte set against FVAV expression (x-axis, reads normalised to library size). Linear mixed model analysis (LMM) indicated a significant overall negative trend in CD3/CD28-stimulated splenocyte gene expression with respect to increasing FVAV expression.
Fig 7.
Structural variation in field vole amdoparvovirus (FVAV) VP1, including characterization of highly variable loop structures that are directed towards the exterior of the viral capsid oligomer.
A. From the top, tracks show the distribution of: protein motifs and externally-directed loops; codon selection signatures; amino acid (aa) evolutionary rate derived from an Amdoparvovirus-wide alignment; consistency (see key) with an ensemble molecular model for Aleutian mink disease virus (AMDV1; International Committee on Taxonomy of Viruses name: Amdoparvovirus carnivoran1), Gray fox amdoparvovirus (GFAV; Amdoparvovirus carnivoran2) and FVAV (white indicates no data due to gaps); the distribution of amino acid polymorphisms in the Kielder Forest FVAV population. For the evolutionary rate track the y-axis indicates the relative rate in substitutions per site. B. A molecular model of FVAV VP1 colour-coded to show externally-directed loops. C. Stacked bar chart showing the relative frequency of different selection signatures (see key) across motifs, loops and other sequence regions. D. Box-and-whisker plots showing the distribution of evolutionary rate (derived from an Amdoparvovirus-wide alignment) across motifs, loops and other sequence regions. E. Box-and-whisker plots showing the distribution of FVAV consistency with an ensemble molecular model for AMDV, GFAV and FVAV across loops and other sequence regions. A-E. Loop 6 is characterised by particularly high evolutionary rate, low structural consistency and a high proportion of neutrally variable and diversified codons.
Fig 8.
The field vole amdoparvovirus (FVAV) VP1 capsid protein contains regions of low structural conservation that correspond to loops facing externally in the capsid oligomer.
Panels show the results of comparative molecular modelling of the VP1 protein in FVAV, Aleutian mink misease virus 1 (AMDV1; International Committee on Taxonomy of Viruses name: Amdoparvovirus carnivoran1) and Gray fox amdoparvovirus (GFAV; Amdoparvovirus carnivoran2). A. Residue-level consistency of species-specific models with an ensemble model averaging the models for all three species. Low structural conservation (low consistency) is concentrated in the same regions in all three species. The locations of amino acid polymorphisms found in the Kielder Forest FVAV population are shown by dashed lines. B-E. SWISS-MODEL molecular model structures for FVAV VP1, coloured according to residue consistency (see colour scale) (B-D) or location of polymorphisms (E) and in equivalent orientations. B. “Cartoon” representation (representing the protein backbone) of monomer. C-E. “Spacefill” representations (representing atoms as spheres, with sizes proportional to their van der Waals radii). C. VP1 molecule within the context of the capsid structure (note low consistency regions are externally directed). D. VP1 monomer. E. VP1 monomer showing positions of polymorphic residues (olive).
Fig 9.
Structural variation in field vole amdoparvovirus (FVAV) NS1.
A. From the top, tracks show the distribution of: protein domains and motifs; codon selection signatures; amino acid (aa) evolutionary rate derived from an Amdoparvovirus-wide alignment; the distribution of amino acid polymorphisms in the Kielder Forest FVAV population. Domain nomenclature follows PROSITE; PV_NS1_nuc, nuclease domain; SF3_HELICASE_1, helicase domain. For the evolutionary rate track the y-axis indicates the relative rate in substitutions per site. B. Box-and-whisker plots showing the distribution of evolutionary rate (derived from an Amdoparvovirus-wide alignment) across polymorphic and invariant sites in FVAV in the Kielder Forest (left-hand panel) and across FVAV nuclease and helicase domains and other sequence regions (right-hand panel). C. Stacked bar chart showing the relative frequency of different selection signatures (see key) across polymorphic and invariant sites in FVAV in the Kielder Forest (left-hand panel) and across the FVAV nuclease and helicase domains and other sequence regions (right-hand panel).
Fig 10.
Host-specific features of the viral capsid.
The structure of VP1 in field vole amdoparvovirus (FVAV) was more consistent with a phylogenetically divergent endogenized lineage also occurring in arvicoline rodent hosts, EPV-Amdo.1-EllLut, than it was with more closely related lineages in other hosts. VP1 molecular modelling results are presented for species clustering with, but falling outside, a well-supported VP clade of carnivoran-infecting amdoparvoviruses (see Fig 3) and also for endogenized amdoparvovirus (EPV-Amdo.1-EllLut) which clusters separately. Upper panels show superimposed species-specific VP1 models; lower panels show tracks representing regions and measurements along the linear amino acid sequence of VP1. Lower panels: tracks showing the position of FVAV motifs and loops (coloured as in Fig 8) (bottom), the aligned by-residue structural model consistency of each virus with FVAV (middle) (darker colours indicate low consistency, see colour scale on left) and the aligned difference in consistency with FVAV between EPV-Amdo.1-EllLut and each other virus in the FVAV cluster (redder colours indicate regions where FVAV is more consistent with EPV-Amdo.1-EllLut than with its cluster-mates; see colour scale on left) (top). The scale at the bottom indicates amino acid residue position along the linear sequence of VP1 in FVAV. Alignment gaps for non-FVAV lineages are shown in grey in the top two tracks. Upper panels: showing three-dimensional representations of modelled VP1 structures in regions (labelled, Roman numerals) where FVAV is more consistent with EPV-Amdo.1-EllLut than with cluster-mates. Superimposed molecular structures for different viral lineages are colour-coded (see key). Regions I (Loop 4) and III (Loop 7) show local differences in loop structures in a “cartoon” representation (representing the protein backbone). For region II (Loop 6), which encompasses an extended run of residues with indels, the entire superimposed VP1 structure is shown in a “spacefill” representation with FVAV, EPV-Amdo.1-EllLut and Yunnan rodent amdoparvovirus (YRAV1, a lineage occurring in murine rodents) atoms shown in transparency. (The spacefill representation shows atoms as spheres, with sizes proportional to their van der Waals radii.) Note that for region II, all of the rodent-infecting lineages have Loop 6 structures projecting in a similar plane compared to gray fox amdoparvovirus (GFAV; International Committee on Taxonomy of Viruses name: Amdoparvovirus carnivoran2) (canid hosts) and Sabeidhel virus (SBEHV1; Amdoparvovirus chiropteran1) (bat hosts). Furthermore, the Loop 6 structure in YRAV1 is larger and differently directed compared to those in FVAV and EPV-Amdo.1-EllLut, which are smaller and more similarly situated.