Article Figures & Data
Figures
- FIG 1
Pathogenic lagovirus detections in Australia between May 2015 and October 2016. Sites where GI.1 (red triangles), GI.2 (blue circles), and GI.1a-Aus (green squares) were detected are indicated on the map separated into 3-month periods. Filled points indicate detections that were within the respective 3-month period, while hollow points indicate previous detections.
- FIG 2
Proportional detections of GI.1, GI.2, GI.1a-Aus, and mixed infections. Detections of each virus are presented as a proportion (y axis) of total cases per month (x axis) between May 2015 and October 2016.
- FIG 3
Phylogenies of the nonstructural and structural genes of Australian and global lagoviruses. Maximum likelihood phylogenies of the nonstructural genes (n = 184) (A) and structural genes (VP60 and VP10; n = 184) (B) were inferred using the newly sequenced Australian lagovirus strains (shown in boldface) and representative published sequences. The Australian GI.2 clades are collapsed due to their large size. The accession numbers of published sequences are indicated in the taxon names. The species from which the virus was collected is indicated in the taxon names of newly sequenced samples (O. cun., Oryctolagus cuniculus), and collections from wild animals are indicated by an asterisk next to the species name. The genotype of each cluster is indicated by colored boxes: GI.1, blue; GI.2, red; GI.4, green. To illustrate recombinant strains, the taxon labels are colored according to their structural gene genotype. Taxon label coloring that does not match the colored boxes indicates recombination. Phylogenies were rooted using an early European EBHSV isolate (not shown), and the scale bar is proportional to the number of nucleotide substitutions per site. Bootstrap support values are shown for the major nodes.
- FIG 4
Inference of the evolutionary history of Australian GI.1bP-GI.2 samples. A Bayesian MCMC time-scaled phylogenetic tree was constructed from 133 GI.1bP-GI.2 nonstructural gene sequences using a relaxed molecular clock (UCLD) and the GMRF Bayesian skyride model of population growth. Circles at tips are color coded to indicate the Australian state from which the virus was collected. ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; SA, South Australia; TAS, Tasmania; VIC, Victoria; WA, Western Australia. A selection of representative European GI.1bP-GI.2 sequences (EUR) were also included. Circles at the nodes are colored according to their most likely location, as estimated by ancestral state reconstruction. The size of the circles at the node represents the posterior probability that the ancestor occurred in that state, where larger circles represent a higher probability. The taxon name of BlMt-1, the first GI.2 detected in Australia, is highlighted in pale red. The taxon name of viruses isolated from hares are boldfaced. The time to most recent common ancestor is indicated at major nodes (year/month). The accession numbers of sequences obtained from GenBank are included in the taxon names. The species from which the virus was collected is indicated in the taxon name of newly sequenced samples (O. cun, Oryctolagus cuniculus; L. eur, Lepus europaeus). Samples from wild animals are indicated by an asterisk next to the species name. The scale bar is proportional to time in years. Clades and subclades have been defined arbitrarily for reference to Table 3.
- FIG 5
Linear regressions of GI.1bP-GI.2 nonstructural and structural genes. Linear regressions of root-to-tip genetic distances (y axis) against sampling time (x axis) were inferred for GI.1bP-GI.2 Australian (n = 128) and European (n = 5) nonstructural and structural genes.
Tables
- TABLE 1
Known genotypes and variants of lagoviruses infecting Oryctolagus cuniculus
Capsid genotype Polymerase genotype Original nomenclature (polymerase/capsid) First reference Location first reported Prototype/reference strain Properties Note GI.1a GI.1aP RHDVa 22 Italy DQ205345.1/CHN/JX_97/1997 Pathogenic, liver tropism Antigenic variant of GI.1b, c, d; includes GI.1a-K5 GI.1a GI.4eP RCV-A1-like/RHDVa 23 Australia KY628309/AUS/NSW/BER_2/2013 Pathogenic, liver tropism, recombinant First detected in Australia, although recombination event probably occurred elsewhere; also referred to as GI.1a-Aus GI.1b, GI.1c, GI.1d GI.1bP, GI.1cP, GI.1dP RHDV 24 China M67473.1/DEU/FRG/1988 Pathogenic, liver tropism Only known virulent genotype circulating globally prior to 1997 GI.2 GI.2P RHDV2 1, 21 Spain/Portugal KM878681.1/ESP/RHDV-N11/2011 Pathogenic, liver tropism Genetically and antigenically distinct from GI.1 (RHDV) and GI.1a (RHDVa); GI.2 (capsid) was first detected in 2010 in France (1), but the polymerase genotype was not reported. GI.2 GI.1bP RHDV/RHDV2 1, 21 Portugal KM115714.2/PRT/CBAlgarve14_1/2014 Pathogenic, liver tropism, recombinant GI.2 GI.4Pa RCV-A1-like/RHDV2 1, 21 Spain/Portugal KF442963.2/PRT/7-13_Barrancos/2013 Pathogenic, liver tropism, recombinant GI.2 GI.4eP RCV-A1-like/RHDV2 25 Australia MF598302/AUS/NSW/CAR-3/2016 Pathogenic, liver tropism, recombinant Currently only detected in Australia GI.3 NDb RCV-E1 26 France AM268419.4/FRA/06-11/2006 Mostly benign, intestinal tropism Variable pathogenicity and tissue tropism GI.4a, GI.4b, GI.4c GI.4aP, GI.4bP, GI.4cP RCV-A1 27 Australia EU871528.1/AUS/MIC-07(1-4)/2007 Benign, intestinal tropism KX357707/NZ/Southland/Gore-425A/2013 and LT708120/PLR56/08-84/2007 are also classified as GI.4 but have not been classified to the variant level GI.4d ND RCV-E2 3, 28 France LT708121.1/BO9/08-117/2008 Benign, intestinal tropism Defined in reference 3, but not characterized; characterization in reference 28 Unclassified UCc ND RCV 29 Italy X96868.1/ITA/ItalyRCV/1995 Benign, intestinal tropism First reported benign lagovirus UC UC MRCV 30 USA GQ166866.1/USA/MRCV/2001 Pathogenic, liver tropism Only detected from a single outbreak in the USA - TABLE 2
Summary of samples tested for pathogenic lagoviruses in Australia from May 2015 to October 2016
Sample and genotype No. of cases per state and territorya ACT NSW NT QLD SA TAS VIC WA Total Wild rabbit GI.2 10 18 0 2 51 0 8 18 107 GI.1 2 1 0 0 19 1 1 1 25 GI.1a-Aus 0 0 0 0 0 0 0 0 0 Mixedb 0 0 0 0 0 0 0 0 0 Negative 5 8 0 4 23 0 3 7 46 Domestic rabbit GI.2 5 22 1 0 23 1 16 13 81 GI.1 0 1 0 0 6 2 0 1 10 GI.1a-Aus 0 2 0 0 0 0 0 0 2 Mixed 0 0 0 0 0 0 1 0 1 Negative 2 25 0 0 11 5 8 0 14 Unknown GI.2 0 7 1 0 0 0 3 0 11 GI.1 0 0 0 0 5 0 0 0 5 GI.1a-Aus 0 0 0 0 0 0 0 0 0 Mixed 0 1 0 0 0 0 0 0 1 Negative 0 2 0 0 2 0 1 0 4 Hare GI.2 0 0 0 0 4 0 1 0 5 Negative 2 1 0 0 4 0 0 0 7 Total 17 52 2 2 108 4 30 33 - TABLE 3
Group-defining sites in Australian GI.2 strains from May 2015 to October 2016
Virus groupa Virus protein p16 (143)b p23/26 (224) 2C-like protein (351) p29 (275) VpGc (114) Protease (143) RdRpc (514) VP60 (579) VP10 (117) Non-P2 P2 (163) 1a S130Nd R170K I961V D1062E K1307R S2123Ne 1b 1c I1138V K1307R M1682V M87V 1d 1e V1391A 1f R2058K 2 A764E D1287E A1766V 3a E245G D262S N692Y A709S A1842S V2255I S2078N A2143Ve V62I V108I 3b V62I 4a L15S F131L K341R N928S D1287E A1360S V2132Me 4b L15S N54K F131L I1117V A2301T V2132Me A2143Ve 4c L15S F131L D1287E S2300L V2132Me A2143Ve A2126Se ↵a Groups are arbitrarily defined in Fig. 4.
↵b The length of the protein (number of amino acid residues) is given in parentheses.
↵c Protein abbreviations: VpG, viral genome-linked protein; RdRp, RNA-dependent RNA polymerase.
↵d Numbers refer to the amino acid residue site number according to numbering in GenBank accession KT280060.1.
↵e Within an extended loop region (35).
- TABLE 4
Primers and probes used for initial detection of pathogenic lagoviruses
Laboratory and name Sense Sequence (5′-3′) Strain Reference EMAI vp60-7_FOR + ACYTGACTGAACTYATTGACG GI.1 49 vp60-8_REV − TCAGACATAAGAAAAGCCATTGG 49 vp60-9_FAM Probe CCAARAGCACRCTCGTGTTCAACCT–FAM-BHQ1 Modified from reference 49 RHDVXa2010-F1 + GCACCCGGCAGTATTCTC GI.1a 23 RHDVXa2010-R1 − CCCAGCCAGCGTACATCTG 23 RHDVXa2010-P1 Probe ACTGTCCAACACTCTCCACAGAACA–FAM-BHQ1 23 RHDV2-F + CCCGGGCAACATCCTGTA GI.2 This study RHDV2-R − CCAGCCAGCGTACATTTGAC This study RHDV2-P Probe CACTGTCCAACACTCGCCACAAA–FAM-BHQ1 This study PIRSA RHDVF6793 + GGACTTTCGCTCAACAACTACTCGTCAGC 50 RHDVR7411 − ATAGCTTACTTTAAACTATAAACCCAA 50 RHDV2-For + ACCACCGAGAACGCGTCCACGTCG 17 RHDV2-Rev − GGCGGATGTCAACAAGTTCTGA 17 CSIRO GI.1a-Aus_fwd + GCGTGGCATTGTGCGCAGCATC GI.1a 25 GI.1a-Aus_rev − TGTTGGTGATAAGCCATAATCGCG 25 GI.1c_fwd + AGCAAGACTGTTGACTCAATTTCG GI.1 25 GI.1c_rev − AGGCCTGCACAGTCGTAACGTT 25 GI.2_fwd + TTTCCCTGGAAGCAGTTCGTCA GI.2 25 GI.2_rev − TGTTGTCTGGTTTATGCCATTTGC 25
