Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Figure 1. Localisations with farm IDs (in square brackets) and numbers of collected faecal samples (n = 269) of different Hungarian animal farms used for investigation of bopiviruses. Empty markers: caprine farms, black markers: bovine farms, grey markers: ovine farms, red marker: rabbit farm, blue markers: swine farms.

Figure 2. Schematic genome maps with conserved amino acid motifs and presumed cleavage sites (P5\P2′) of ovipivirus (a), gopivirus (b) and bopivirus/Hun (c) (bottom side of the panels) and their closest relatives (top side). The gene boxes corresponding to the P1 viral capsid proteins and P2 non-structural proteins are highlighted with different shades of grey. The nucleotide (upper numbers) and amino acid (lower numbers in brackets) lengths of the corresponding genomic regions of the study viruses are shown in each gene box. The positions and sequences of conserved amino acid motifs of 2C Helicase, 3C Protease and 3DRdRp (RNA-dependent RNA polymerase) are shown under each map of the study viruses. Conserved and variable amino acids are represented by uppercase and lowercase letters, respectively. The pairwise amino acid identity values (%) of each genomic region are found between the genome maps. cre: cis-acting replication element.

Figure 3. The predicted secondary RNA structures of 5′UTR-IRES core domains (H–L) of ovipivirus (a) and bopivirus/Hun (b). Numbers indicate the nucleotide positions in the complete genome of the given virus. PTB: Polypyrimidine tract-binding protein, eIF4G: Eukaryotic translation initiation factor 4 G, pY: polypyrimidine (C or U), ATG: Start codon.

Figure 4. Phylogenetic analyses of the study ovipi-, gopi-, bopiviruses (in bold) and the representative members of family Picornaviridae based on the amino acid alignments of 3CD (a) and P1 (b) genomic regions. The trees are constructed using the Maximum likelihood method and either LG + F + I + G4 (a) or Jones Taylor Thornton (b) amino acid substitution models with 1000 iterations (bootstrap). The phylogenetic position of genus Bopivirus is marked with a grey background. The lineages of supergroup 1 (SG1) in the 3CD tree are marked with red lines. 4-3-4 indicate the genera in SG1 where picornaviruses with 4-3-4 genome organisations are present. The locations of some genera are shown in italics to help the orientation. A representative member of genera Sali- and Kobu-virus were used as outgroups in the P1 phylogenetic tree.

Figure 5. Distance plot of ovipi-, gopi-, and bopi-virus/Hun compared to the bopivirus A1 (KM589358) as a query sequence (reference) in a multiple nt alignment with a window size of 200 bp and a step size of 20 bp. Each line is a comparison between the CDS of ovipivirus (red), gopivirus (green) and bopivirus/Hun (blue) and the reference genome. Horizontal black line: the position of the RT-PCR product generated by the generic bopivirus screening oligonucleotide primers (HBG-3D-Screen-R/F). Horizontal dotted lines: positions of the RT-PCR products generated by the multiple primer sets used in various bopivirus typing RT-PCR reactions (Supplementary Table S2). Vertical dotted lines: borders of given genome regions indicated in the genome map above the plot. The most diverse (*) and most conserved (#) genome regions between ovipi-, gopi- and bopi-viruses are indicated in the bopivirus genome map.

Figure 6. Phylogenetic analysis of determined bopivirus 3DRdRp sequences. The tree was constructed with the Maximum Likelihood method and Jukes-Cantor model. The percentage of trees in which the associated taxa clustered together (bootstrap) is shown (where values are >50) next to the branches. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. The origin (farm location) and silhouettes of the samples’ hosts are found after the strain names. The locations of lineage 1 of ovine/caprine and lineage 2 of bovine bopiviruses are also indicated. Black and empty arrows indicate the phylogenetic positions of different caprine (empty arrows) and ovine (black arrows) lineages originated from the same farm. The ovipi-, gopi- and bopi-virus/Hun sequences are marked in bold.

Figure 7. (a) Phylogenetic analysis of the determined bopivirus VP1 sequences with three different genotypes of genus Erbovirus which was used as an outgroup. The tree was constructed using the Maximum Likelihood method and Kimura 2 model. The percentage of trees in which the associated taxa clustered together (bootstrap) is shown (if its values are >50) next to the branches. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. The origin (farm location) and silhouettes of the samples’ hosts are found after the strain names. The locations of lineage 1 of ovine/caprine and lineage 2 of bovine bopiviruses are also indicated. The diverse ovine strains from Hajdúszoboszló are highlighted with a grey background. Percentages are pairwise nt identity values between strains indicated by different double arrows. The ovipi-, gopi- and bopi-virus/Hun sequences are marked with bold. The branches of the strains belonging to different existing (A1) and proposed genotypes (“A2”, “B1”, “B2”) are marked with different colours. (b) Frequency distribution of pairwise distances (p-distances) between the N = 20 VP1 nucleotide sequences of all study strains and…