# Full genome characterization of the Culicoides-borne marsupial orbiviruses: Wallal virus, Mudjinbarry virus and Warrego viruses

Manjunatha N. Belaganahalli, Sushila Maan, Narender S. Maan, Ian

Pritchard, Peter D Kirkland, Joe Brownlie, Houssam Attoui, Peter P. C.

Mertens

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Manjunatha N. Belaganahalli, Sushila Maan, Narender S. Maan, Ian Pritchard, Peter D Kirkland, et al.. Full genome characterization of the Culicoides-borne marsupial orbiviruses: Wallal virus, Mudjinbarry virus and Warrego viruses. PLoS ONE, 2014, 9 (10), pp.e108379. 10.1371/journal.pone.0108379. hal-01606562

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# Full Genome Characterization of the *Culicoides*-Borne Marsupial Orbiviruses: Wallal Virus, Mudjinbarry Virus and Warrego Viruses

Manjunatha N. Belaganahalli¹, Sushila Maan¹,*, Narender S. Maan¹,*, Ian Pritchard², Peter D. Kirkland³, Joe Brownlie⁴, Houssam Attoui¹, Peter P. C. Mertens¹*

1 Vector-borne Viral Diseases Programme, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom, 2 Australian Animal Health Laboratory, CSIRO, Geelong, Victoria, Australia, 3 Elizabeth Macarthur Agricultural Institute, Camden, New South Wales, Australia, 4 Department of Pathology and Infectious Diseases, Royal Veterinary College, North Mymms, Hatfield, Herts, United Kingdom

<figure><img src="image_3.png" /></figure>

## Abstract

Viruses belonging to the species *Wallal virus* and *Warrego virus* of the genus *Orbivirus* were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all *Orbivirus* species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinbararry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other *Culicoides* borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct *Orbivirus* species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - *Wallal virus*). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.

**Citation:** Belaganahalli MN, Maan S, Maan NS, Pritchard I, Kirkland PD, et al. (2014) Full Genome Characterization of the *Culicoides*-Borne Marsupial Orbiviruses: Wallal Virus, Mudjinbarry Virus and Warrego Viruses. PLoS ONE 9(10): e108379. doi:10.1371/journal.pone.0108379

**Editor:** Robert Belshaw, Plymouth University, United Kingdom

**Received** May 21, 2014; **Accepted** August 19, 2014; **Published** October 9, 2014

**Copyright:** © 2014 Belaganahalli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability:** The authors confirm that all data underlying the findings are fully available without restriction. Full length sequences of Seg-1 to Seg-10 of WALV (AUS1978/09), MUDV (AUS1982/03) and WARV (AUS1969/01) have been submitted to GenBank with accession numbers KJ495745 to KJ495754; KJ495765 to KJ495774 and KJ495755 to KJ495764 respectively. These data were compared to other orbiviruses (accession numbers for the data used are given in Table S1 in File S1).

**Funding:** This work was partly funded by the Commonwealth Scholarship Commission (http://cscuk.dfid.gov.uk/) awarded to MB, partly by the Department for Environment, Food and Rural Affairs (Defra)(SE2617) awarded to SM. NSM, PCM and HA were funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the European Union (EU) (SANCO/ 940/2002; EMIDA grant OrbiNet - K1303206) and WildTech - Grant no. 222633-2. IP was part of this work, funded by CSIRO & Dept of Agriculture, Fisheries and Forestry, Australian Government. PK was funded by NSW Department of Primary Industries. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** Houssam Attoui is a member of the PLOS ONE editorial board. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.

## Introduction

The orbiviruses are classified within the genus *Orbivirus*, family *Reoviridae*. They have icosahedral and non-enveloped virus-particles, containing a genome composed of ten linear pieces of dsRNA. The orbiviruses currently include 22 virus species (which also represent distinct 'serogroups') that have been recognised by the International Committee for the Taxonomy of Viruses (ICTV), as well as 14 unclassified isolates (some of which may represent additional species) [1-6]. The orbiviruses are primarily transmitted by ticks or haematophagous-insect-vectors (including mosquitoes, *Culicoides* [biting midges] and sand flies) and collectively have a wide host-range that includes domestic and wild ruminants, equines, marsupials, sloths, bats, birds and humans [2,7,8].

Orbiviruses, including Wallal virus (WALV), Warrego virus (WARV) and Eubenangee virus (EUBV) have previously been isolated from marsupials, and there is serological evidence for infections by Wongorr virus (WGRV) [7]. Evidence for a causal link between the orbiviruses and clinical disease in marsupials was provided by the isolation of WALV and WARV from animals affected by 'kangaroo blindness' in Australia during 1994/95 [9,10]. Both viruses were isolated from Western Grey kangaroos (*Macropus fuliginosus*), red kangaroos (*Macropus rufa*), Eastern grey kangaroos (*Macropus giganteus*) and euros or wallaroo (*Macropus robustus*). Many thousands of kangaroos were affected in the states of New South Wales, South Australia and Victoria, suffering from non-suppurative choroiditis, retinal degeneration with inflammation and secondary degeneration of the optic nerve [9,10]. A histopathology slide from a kangaroo with

<footnote>* Email: peter.mertens@pirbright.ac.uk</footnote>

<footnote>‡ Current address: Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India</footnote>
chorio-retinitis seen in the early 1970s, was found to have the same
'structure' as cases in 1994/95, suggesting periodic outbreaks of
the disease [11]. Later in 1998-99, viruses belonging to the species
*Eubenangee virus* were also isolated from wallabies affected by
'Tammar Wallaby Sudden Death Syndrome' (TSDS) [12,13].

The species *Wallal virus* contains three distinct serotypes/
strains: Wallal virus (WALV), Wallal K virus (WALKV) and
Mudjinabarry virus (MUDV) [14]. WALV was first isolated in
1969 from adult *Culicoides* collected in Charleville, Queensland,
Australia [15]. MUDV was isolated in September 1971 from
*Culicoides marksi* collected in the Alligator Rivers area of the
Northern Territory of Australia [16].

The species *Warrego virus* contains three distinct serotypes/
strains: Warrego virus (WARV), Warrego K virus (WARKV) and
Mitchell River virus (MRV). WARV virus was first isolated in 1970
from adult *Culicoides* collected in Charleville, Queensland, Australia
[7,15]. Mitchell River virus was isolated in April 1969 from *Culicoides*
*spp.* trapped in the Mitchell River region in Australia [15].

The species *Eubenangee virus* contains four distinct viruses: Eubenangee virus (EUBV), Tilligerry virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) [2]. EUBV and TILV were isolated in Australia in 1963 and 1971 respectively from pools of mosquitoes [7]. They were also isolated from *Culicoides spp.* and antibodies to these viruses have been detected in cattle and marsupials [7,17]. PATAV and NGOV were isolated in 1968 and 1974 respectively in the Central African Republic, also from pools of mosquitoes [7]. Nucleotide (nt) and amino-acid (aa) sequence comparisons indicate that PATAV represents a novel and distinct *Orbivirus* species [18].

The different species of *Culicoides* borne orbivirus (CBO)
include *Bluetongue virus* (BTV) (the type species of the genus
*Orbivirus*), *African horse sickness virus* (AHSV) and *Epizootic
haemorrhagic disease virus* (EHDV), which are thought to include
the most economically important orbiviruses. Complete genomes
have been sequenced for representative isolates of BTV, EHDV,
AHSV as well as some other CBO species including *Palyam virus*
(PALV) and *Equine encephalosis virus* (EEV) (the members of
which infect domesticated livestock) [19–23]. Although WALV,
WARV and EUBV are also transmitted by adult *Culicoides*,
[9,12,24] the full genome sequence of only one of these viruses –
EUBV – has been reported [18].

Recent advances in RT-PCR and high throughput DNA
sequencing methods have generated full-genome sequence data for
multiple orbivirus isolates, helping to clarify the genetic relatedness
and taxonomic status within and between different orbivirus
species, including BTV, AHSV and EHDV [18,19,25–28].
Comparisons of nucleotide (nt) and amino acid (aa) sequence
data now provide a basis for molecular epidemiology studies and
for grouping distinct orbivirus isolates into serotypes and topotypes
[19,26,29,30].

As part of a programme to generate representative data for all
*Orbivirus* species, we report full genome nucleotide sequences for
WALV, MUDV and WARV, and present a comparison to isolates
from other *Orbivirus* species as an aid to help identify novel
orbivirus isolates. The full-genome sequences of WALV and
MUDV indicate that they represent different serotypes within the
*Wallal virus* species and that WARV represents a distinct species
within the genus *Orbivirus*.

**Materials and Methods**

Viruses

The viruses used in this study [AUS1978/09 (WALV),
AUS1982/03 (MUDV) and AUS1969/01 (WARV)] were

obtained from the Orbivirus Reference Collection (ORC) at The
Pirbright Institute. These viruses were originally taken from
naturally infected animals by qualified veterinarians, as part of
normal diagnostic testing procedures in the respective countries.
MUDV was propagated in BHK-21 cells (clone 13 obtained from
European Collection of Animal cell Cultures (ECACC -
84100501), while WALV and WARV were grown in BSR cells
(a clone of BHK) [35] or BHK cells, in Dulbecco's minimum
essential medium (DMEM) supplemented with antibiotics (100
units/ml penicillin and 100 µg/ml streptomycin) and 2 mM
glutamine. Infected cell cultures were incubated until they showed
widespread (100%) cytopathic effects (CPE). Viruses were
harvested, aliquoted and used for the dsRNA extraction, or stored
in the orbivirus reference collection (ORC) at -80°C.

Preparation of viral dsRNA

Intact genomic dsRNA was extracted from WALV, MUDV
and WARV infected cell cultures using a guanidinium isothiocy-
anate extraction procedure as described by Attoui et al [36].
Briefly, the infected cell pellet was lysed in 1 ml of commercially
available TRIZOL reagent (Invitrogen), 0.2 volume of chloroform
was added, mixed by vortexing and the mixture incubated on ice
for 10 min. The supernatant containing total RNA was separated
from cellular debris and DNA by centrifuging at 10,000 xg for
10 min at 4°C. Single stranded RNA (ssRNA) was removed by
2 M LiCl precipitation at 4°C overnight, followed by centrifuga-
tion at 10,000 xg for 5 min. An equal volume of isopropanol
containing 750 mM ammonium acetate was added to the
supernatant, then mixed and the viral dsRNA was allowed to
precipitate for a minimum of 2 hours at -20°C. The dsRNA was
pelleted by centrifugation at 10,000 xg for 10 min, washed with
70% ethanol, air dried and suspended in nuclease free water
(NFW). The RNA was either used immediately or stored at
-20°C.

Reverse transcription of dsRNA and PCR amplification of cDNAs

The genome segments of WALV, MUDV and WARV were
reverse-transcribed using a 'full-length amplification of cDNA'
(FLAC) technique described by Maan et al [37]. Briefly, a 35 base
self-priming oligonucleotide 'anchor-primer' with a phosphorylat-
ed 5' terminus was ligated to the 3' ends of the viral dsRNAs using
the T4 RNA ligase, followed by reverse transcription using RT
system (Promega). The resulting cDNAs were amplified using
complementary primers to the anchor primer. The resulting
cDNA amplicons were analyzed by agarose gel electrophoresis.
For cloning purposes, a high fidelity KOD polymerase enzyme
(Novagen) was used in the PCR.

Cloning and sequencing of cDNAs

Amplified cDNAs were purified and cloned. MUDV genes were
cloned into the Strataclone blunt-end PCR cloning vector 'pSC-B-
amp/kan' supplied in the StrataClone Blunt PCR cloning kit
(Stratagene). WALV (only Seg-2) and WARV amplicons were
cloned into the 'pCR-Blunt' vector supplied with the Zero Blunt
PCR Cloning Kit (Invitrogen). Recombinant plasmid-vectors
containing MUDV inserts were transformed into Solopack
competent cells (Agilent Technologies), while WALV and WARV
plasmids were transformed into One Shot TOP10 competent cells
supplied with the respective cloning kits. Clones containing
relevant inserts were identified by touch PCR using M13 universal
primers. Plasmids were extracted from the clones identified using
the QIAprep Spin MiniPrep Kit (Qiagen). The plasmids and PCR
products were sequenced using an automated ABI 3730 DNA
sequencer (Applied Biosystems). Some of the MUDV sequencing
primers detected other segments of WALV and using the
sequences generated, running primers were designed to obtain
full sequence for the remaining nine segments of WALV.

Sequence analysis and phylogenetic tree construction

'Raw' ABI sequence data were assembled into 'contigs' using
the SeqManII sequence analysis package (DNΑstar version 5.0).
The ORFs of WALV, MUDV and WARV were identified and
translated into aa sequences for further analysis using EditSeq
(DNAstar version 5.0). The putative function of each protein was
identified by BlastX comparisons to homologous orbivirus (e.g.
BTV) proteins in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.
cgi?CMD=Web&PAGE_TYPE=BlastHome). Multiple align-
ments of consensus sequences were performed using ClustalX
(Version 2.0) [38], Clustal Omega (http://www.ebi.ac.uk/Tools/
msa/clustalo/) and MAFFT [39] to ensure proper alignment.
Aligned protein sequences were back translated to nucleotide
sequences using DAMBE [40]) or RevTrans 1.4 server available
online (http://www.cbs.dtu.dk/services/RevTrans/) for further
nucleotide analysis. The best fit amino acid (aa) and nucleotide (nt)
models for Maximum likelihood (ML) analysis were determined
using ProtTest 3.0 and jModeltest respectively [41,42]. The
models were also determined using MEGA 5 software. The
consensus or simplest model given by Akaike Information
Criterion (AIC) and Bayesian Information Criterion (BIC) was
selected for ML tree construction. The aa model rtREV (G+F) was
selected for T2 tree and rtREV (I+G+F) model was selected for
both VP1 and T13 proteins. One nt model, GTR (+G) was used to
construct ML trees for all three genes. All phylogenetic trees
construction and pairwise distance calculations using p-distance
parameter were performed using MEGA 5 [43,44]. GenBank
nucleotide accession numbers for the sequences used for analysis
and phylogenetic studies are listed in the Table S1 in File S1.

Results

Virus propagation and genomic-segment
migration-pattern (electropherotype)

WALV (isolate - AUS1978/09), MUDV (isolate AUS1982/03) and WARV (isolate AUS1969/01) all induced severe cytopathic effects (CPE) in BHK and BSR (a clone of BHK cells) cell monolayers at 48-72 hours post infection. When analysed by 1% agarose gel electrophoresis (AGE), the genomic dsRNA of these viruses migrated as distinct groups, consisting of 3 large segments, 3 medium size segments, 3 small segments, and 1 very small segment (giving a 3-3-3-1 pattern) (Figure 1). This pattern is similar to that observed in other CBOs (including BTV, EHDV and EUBV) [18,37]. WALV and MUDV generated identical genome segment migration patterns ('electropherotype') during AGE, as expected for viruses belonging to the same virus species (Figure 1) [2,37]. In contrast, WARV showed clear differences in the migration of Seg-3, -7, -8 and -9 (Lane 3 in Figure 1) confirming that it belongs to a different virus species.

Genome sequence analyses of WALV (AUS1978/09),
MUDV (AUS1982/03) and WARV (AUS1969/01)

Full length sequences of Seg-1 to Seg-10 of WALV (AUS1978/
09), MUDV (AUS1982/03) and WARV (AUS1969/01) have
been submitted to GenBank with accession numbers KJ495745 to
KJ495754; KJ495765 to KJ495774 and KJ495755 to KJ495764
respectively. These data were compared to other orbiviruses
(accession numbers for the data used are given in Table S1 in File

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Agarose gel (1%) electrophoretic profile of dsRNAs of Wallal virus (WALV), Mudjinabarry virus (MUDV) and Warrego viruses (WARV). 1 = WALV (AUS1978/09); 2 = MUDV (AUS1982/03); 3 = WARV (AUS1969/01).</figcaption></figure>

doi:10.1371/journal.pone.0108379.g001

S1). The total genome size of WALV, MUDV and WARV is 19,387 bp, 19,382 bp and 19,428 bp respectively, with segments ranging from 3959 bp to 862 bp (3964 bp to 838 bp in WARV), encoding proteins of 1306 aa to 87 aa (1303 aa to 78 aa in WARV) (Table 1 and 2). As suggested by the electropherotype analysis (Figure 1), most of the individual genome segments of WALV and MUDV, with the exception of Seg-2 (VP2) and Seg-8 (NS2(ViP)) have identical sizes with similar ORFs, identical start and stop positions generating proteins of the same length (Table 1).

The different genome segments of WALV and MUDV have the
same conserved termini, sharing five fully conserved nucleotides at
their 5' and 3'ends (+ve: 5'-GUA<sup>U</sup>/<sub>A</sub>A.........A<sup>C</sup>/<sub>A</sub><sup>A</sup>/<sub>G</sub>C<sup>U</sup>/<sub>A</sub>
cUAC-3'). WARV shares six fully conserved nucleotides at the 5'
ends and four at the 3' ends (+ve: 5'-GUAAAA.........A<sup>C</sup>/<sub>A</sub><sup>A</sup>/<sub>U</sub>
cUAC-3'). Two terminal nucleotides at the 5' end and three
nucleotides at the 3' end (5'-GU.....UAC-3') of all three viruses
are characteristic of the genus *Orbivirus*, and the first and last two
nucleotides represent inverted complements (http://www.
reoviridae.org/dsRNA_virus_proteins/CPV-RNA-Termin.htm).

The G+C content of the WALV, MUDV and WARV genomes
are 39.14%, 39.41% and 38.99% respectively, well within the
range previously identified for insect-borne orbiviruses (IBOs)
(36.66% in PHSV to 45.86% in EEV) but less than tick-borne
orbiviruses (TBOs) (51.93% in St Croix River virus (SCRV) to
57.29% in Great Island virus (GIV)) [5].

Like other orbiviruses, most genome segments of WALV,
MUDV and WARV have shorter 5' than 3' terminal non-coding
regions (NCRs). However, Seg-8 (encoding the viral inclusion
body protein NS2) of WALV and MUDV, and Seg-5 (encoding
the viral tubule protein NS1) of WARV have longer 5' NCRs
(Table 1). Exceptions were also observed in Umatilla virus
(UMAV), Corriparta virus (CORV) and GIV, although the
<table><caption>**Table 2.** Characteristics of dsRNA genome segments and proteins of WARV (AUS1969/01) virus.</caption><thead><tr><th>Segment</th><th>Segment length (bp)</th><th>Protein encoded</th><th>Protein length (aa)</th><th>Protein mass (kDa)</th><th>ORFs bp (including stop codon)</th><th>5' NCRs</th><th>3' NCR</th><th>5' conserved Termini</th><th>3' conserved Termini</th><th>accession numbers</th></tr></thead><tbody><tr><td>1</td><td>3964</td><td>Pol</td><td>1308</td><td>150.69</td><td>12-3938</td><td>11</td><td>29</td><td>5'-GTAAAA</td><td>ACTTAC-3'</td><td>KJ495755</td></tr><tr><td>2</td><td>2992</td><td>OC1</td><td>979</td><td>115.48</td><td>18-2957</td><td>17</td><td>38</td><td>5'-GTAAAA</td><td>ACTTAC-3'</td><td>KJ495756</td></tr><tr><td>3</td><td>2845</td><td>T2</td><td>928</td><td>106.79</td><td>15-2801</td><td>14</td><td>47</td><td>5'-GTAAAA</td><td>ACATAC-3'</td><td>KJ495757</td></tr><tr><td>4</td><td>1965</td><td>CaP</td><td>639</td><td>74.65</td><td>9-1928</td><td>8</td><td>40</td><td>5'-GTAAAA</td><td>ACATAC-3'</td><td>KJ495758</td></tr><tr><td>5</td><td>1771</td><td>TuP</td><td>530</td><td>62.17</td><td>96-1688</td><td>95</td><td>86</td><td>5'-GTAAAA</td><td>AACTAC-3'</td><td>KJ495759</td></tr><tr><td>6</td><td>1652</td><td>OC2</td><td>528</td><td>59.45</td><td>29-1615</td><td>28</td><td>40</td><td>5'-GTAAAA</td><td>ACATAC-3'</td><td>KJ495760</td></tr><tr><td>7</td><td>1185</td><td>VIP</td><td>370</td><td>42.22</td><td>30-1142</td><td>29</td><td>46</td><td>5'-GTAAAA</td><td>ACTTAC-3'</td><td>KJ495762</td></tr><tr><td>8</td><td>1165</td><td>T13</td><td>350</td><td>38.44</td><td>19-1071</td><td>18</td><td>97</td><td>5'-GTAAAA</td><td>ACATAC-3'</td><td>KJ495761</td></tr><tr><td>9</td><td>1051</td><td>Hel</td><td>330</td><td>36.82</td><td>16-1008</td><td>15</td><td>46</td><td>5'-GTAAAA</td><td>ACATAC-3'</td><td>KJ495763</td></tr><tr><td></td><td></td><td>NS4</td><td>78</td><td>9.72</td><td>113-349</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10</td><td>838</td><td>NS3</td><td>250</td><td>27.79</td><td>21-773</td><td>20</td><td>68</td><td>5'-GTAAAAATT</td><td>ACTTAC-3'</td><td>KJ495764</td></tr><tr><td></td><td></td><td>NS3a</td><td>219</td><td>24.19</td><td>114-773</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>19428</td><td></td><td></td><td></td><td></td><td></td><td>Consensus</td><td>5'-GTAAAA</td><td>A<sup>c</sup>/k<sup>A</sup>/r<sup>c</sup>TAC-3'</td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0108379.t002
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Maximum likelihood (ML) trees showing phylogenetic comparisons of T2 protein amino acid sequences (a) and nucleotide sequences of Seg-3 (b) of WALV, MUDV and WARV, aligned with those of other *Orbivirus* species. One thousand bootstrap replicates were used for construction of the phylogenetic trees. The WALV, MUDV and WARV isolates characterised in this study are marked with green circle. BTV = Bluetongue virus; AHSV = African Horse sickness virus; EHDV = Epizootic Haemorrhagic Disease Virus; CHUV = Chuzan virus; EUBV = Eubenangee virus; TILV = Tilligerry virus; PATAV = Pata virus; EEV = Equine encephalosis virus; WARV = Warrego virus; WALV = Wallal virus; MUDV = Mudjinbarry virus; CORV = Corriparta virus; UMAV = Umatilla virus; SLOV = Stretch Lagoon orbivirus; SVIV = Sathuvachari virus; YUOV = Yunnan orbivirus; PHSV = Peruvian horse sickness virus; TRBV = Tribec virus; TRBVh = another isolate of Tribec virus; LIPV = Lipovnik virus; LIPV = Lipovnik virus isolate CzArLip 91; KEMV = Kemerovo virus; KEMVh = Kemerovo virus isolate EgAn 1169-61; GIV = Great Island virus; SCRV = St Croix river virus. Virus isolates and accession numbers of polymerase sequences used for comparative analysis are listed in Table S1 in File S1. ‘e’ and ‘w’ after serotype number indicate eastern and western strains, respectively.
doi:10.1371/journal.pone.0108379.g002</figcaption></figure>

significance of these variations is uncertain [5]. Collectively the upstream and downstream terminal NCRs represent 4.29%, 4.3% and 4.08% of the WALV, MUDV and WARV genomes respectively. Comparisons with other CBOs showed similar or higher percentages, while the mosquito-borne orbiviruses (MBOs) and TBOs show higher values [5].

The coding assignments of the genome segments of WALV and MUDV (Table 1) are similar to those of BTV [45,46]. The coding assignments of WARV are also similar, with the exception of Seg-7 and Seg-8 (coding for ViP and VP7 respectively), which have reversed their relative order of migration/size (Table 2). Most of the genome segments are monocistronic, encoding proteins from single open reading frames (ORF). However, Seg-10 of all three viruses has two in-frame initiation sites, at or near the start of the same ORF leading to translation of NS3 and NS3a (Table 1). Recently, a novel non-structural protein ‘NS4’ (which varies between 10 kDa to 22.5 kDa, depending on *Orbivirus* species), encoded by a downstream ORF (+2 frame) on Seg-9 (which also

encodes VP6-Helicase) was identified in BTV and several other orbiviruses [33,47,48]. Similarly WALV, MUDV and WARV have a downstream ORF (+2 frame) on Seg-9 that encodes NS4.

Most of the WALV and MUDV ORF’s have a ‘strong’ Kozak sequence (RNN**AUGG**), except for the first ORFs of Seg-9 and Seg-10, which have ‘weak’ (UAG**AUGU**) or ‘moderate’ (GU**CAUGU**) contexts respectively, potentially allowing read-through of the upstream initiation codon, enhancing expression of NS4 and NS3a respectively. Similarly, most ORF’s of the WARV genome-segments have a strong (RNN**AUGG**) to moderate Kozak sequence [Seg-4 (AUC**AUGC**) and Seg-5 (UCU**AUGA**)], except Seg-9 (UGG**AUGU**) and Seg-10 (GUC**AUGC**), both of which have a weak ‘context’.

Weak or moderate Kozak sequences have previously been observed in several of the genome segments of other orbiviruses, although they still appear to be translated effectively [18,33]. The down-stream NS4 ORF of Seg-9 in WALV and MUDV has a strong Kozak context (**AAG**AUGG) encoding an 87 aa protein,
<table><caption>**Table 3.** Percent amino acid and nucleotide identities of WALV (AUS1978/09), MUDV (AUS1982/03) and WARV (AUS1969/01) with other orbiviruses in VP1(Pol), T2 and T13 protein/genes.</caption><thead><tr><th rowspan="2">Orbivirus</th><th colspan="2">WALV</th><th colspan="2">T13</th><th colspan="2">MUDV</th><th colspan="2">WARV</th><th colspan="2"></th></tr><tr><th>VP1</th><th>T2</th><th>aa</th><th>nt</th><th>VP1</th><th>T2</th><th>T13</th><th>VP1</th><th>T2</th><th>T13</th></tr></thead><tbody><tr><td colspan="11"><strong>Vector: CBOs</strong></td></tr><tr><td>BTV1e</td><td>63.28</td><td>61.83</td><td>68.63</td><td>64.81</td><td>48.71</td><td>54.35</td><td>63.36</td><td>61.83</td><td>68.74</td><td>65.15</td><td>48.71</td></tr><tr><td>BTV8w</td><td>63.59</td><td>62.38</td><td>68.97</td><td>66.37</td><td>49.00</td><td>52.34</td><td>63.74</td><td>62.07</td><td>69.08</td><td>66.07</td><td>49.00</td></tr><tr><td>BTV25</td><td>62.74</td><td>62.48</td><td>67.52</td><td>64.59</td><td>49.57</td><td>53.39</td><td>62.89</td><td>62.71</td><td>67.63</td><td>65.18</td><td>49.57</td></tr><tr><td>BTV26</td><td>63.28</td><td>61.38</td><td>67.41</td><td>65.55</td><td>50.14</td><td>53.20</td><td>63.20</td><td>61.59</td><td>67.52</td><td>65.04</td><td>50.14</td></tr><tr><td>EHDV1w</td><td>62.82</td><td>63.54</td><td>69.71</td><td>65.63</td><td>49.57</td><td>54.25</td><td>62.89</td><td>63.31</td><td>69.82</td><td>65.81</td><td>49.57</td></tr><tr><td>EHDV2e</td><td>62.51</td><td>63.28</td><td>69.82</td><td>66.07</td><td>51.00</td><td>54.15</td><td>62.59</td><td>62.84</td><td>69.93</td><td>66.78</td><td>51.00</td></tr><tr><td>PATAV</td><td>64.54</td><td>63.28</td><td>68.71</td><td>65.70</td><td>55.17</td><td>58.81</td><td>64.46</td><td>63.49</td><td>68.82</td><td>65.48</td><td>55.17</td></tr><tr><td>EUBV</td><td>70.96</td><td>66.77</td><td>72.86</td><td>66.89</td><td>57.71</td><td>60.67</td><td>70.96</td><td>66.97</td><td>72.86</td><td>67.33</td><td>57.71</td></tr><tr><td>TILV</td><td>69.73</td><td>66.21</td><td>72.41</td><td>67.89</td><td>58.57</td><td>60.00</td><td>70.04</td><td>66.39</td><td>72.41</td><td>67.93</td><td>58.57</td></tr><tr><td>WALV</td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>99.16</td><td>93.57</td><td>99.78</td><td>90.36</td><td>100.00</td></tr><tr><td>MUDV</td><td>99.16</td><td>93.57</td><td>99.78</td><td>90.36</td><td>100.00</td><td>97.71</td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td></tr><tr><td>WARV</td><td>64.37</td><td>65.29</td><td>66.30</td><td>65.55</td><td>48.29</td><td>55.24</td><td>64.60</td><td>65.36</td><td>66.41</td><td>65.04</td><td>48.29</td></tr><tr><td>AHSV1</td><td>58.80</td><td>61.59</td><td>61.40</td><td>61.92</td><td>46.70</td><td>53.39</td><td>58.57</td><td>60.80</td><td>61.29</td><td>62.48</td><td>46.70</td></tr><tr><td>CHUV</td><td>57.63</td><td>60.21</td><td>60.29</td><td>61.36</td><td>39.94</td><td>49.33</td><td>57.78</td><td>60.44</td><td>60.40</td><td>62.25</td><td>39.94</td></tr><tr><td>EEV</td><td>52.65</td><td>56.16</td><td>50.83</td><td>56.17</td><td>40.29</td><td>48.86</td><td>52.65</td><td>56.32</td><td>50.83</td><td>55.28</td><td>40.29</td></tr><tr><td colspan="11"><strong>Vector: MBOs</strong></td></tr><tr><td>PHSV</td><td>48.18</td><td>55.10</td><td>36.30</td><td>48.85</td><td>21.78</td><td>42.02</td><td>48.02</td><td>55.20</td><td>36.41</td><td>48.96</td><td>21.78</td></tr><tr><td>YUOV</td><td>46.49</td><td>54.11</td><td>38.20</td><td>49.11</td><td>23.78</td><td>40.78</td><td>46.41</td><td>53.93</td><td>37.97</td><td>50.37</td><td>23.78</td></tr><tr><td>UMAV</td><td>47.86</td><td>54.04</td><td>36.01</td><td>47.97</td><td>24.07</td><td>38.11</td><td>47.54</td><td>53.78</td><td>36.12</td><td>48.20</td><td>24.07</td></tr><tr><td>SLOV</td><td>47.08</td><td>53.78</td><td>36.68</td><td>47.79</td><td>--</td><td>--</td><td>46.92</td><td>53.73</td><td>36.79</td><td>48.46</td><td>--</td></tr><tr><td>CORV</td><td>48.16</td><td>53.87</td><td>37.86</td><td>48.52</td><td>23.78</td><td>38.78</td><td>48.16</td><td>54.21</td><td>37.75</td><td>48.29</td><td>23.78</td></tr><tr><td>SVIV</td><td>46.87</td><td>52.57</td><td>37.75</td><td>49.33</td><td>25.00</td><td>41.00</td><td>47.10</td><td>52.85</td><td>37.75</td><td>49.89</td><td>25.00</td></tr><tr><td colspan="11"><strong>Vector: TBOs</strong></td></tr><tr><td>TRBV</td><td>45.08</td><td>49.33</td><td>35.86</td><td>44.02</td><td>25.21</td><td>38.68</td><td>44.93</td><td>48.81</td><td>35.97</td><td>43.76</td><td>25.21</td></tr><tr><td>KEMV</td><td>45.91</td><td>49.74</td><td>35.08</td><td>44.39</td><td>24.93</td><td>37.25</td><td>45.83</td><td>49.74</td><td>35.19</td><td>43.65</td><td>24.93</td></tr><tr><td>GIV</td><td>45.28</td><td>48.90</td><td>36.53</td><td>45.58</td><td>24.93</td><td>37.25</td><td>45.13</td><td>49.11</td><td>36.64</td><td>44.69</td><td>24.93</td></tr><tr><td colspan="11"><strong>Vector: Tick associated</strong></td></tr><tr><td>SCRV</td><td>36.08</td><td>43.55</td><td>23.49</td><td>37.78</td><td>19.53</td><td>34.79</td><td>36.08</td><td>43.73</td><td>23.49</td><td>37.86</td><td>19.53</td></tr></tbody></table>

BTV = Bluetooth virus; BTV1e = BTV-1 eastern topology; BTV8w = BTV-8 western topology; EHDV = Epizootic haemorrhagic disease; EHDV1w = EHDV-1 western topology; EHDV2e = EHDV-2 eastern topology; PATAV = Pata virus; EUBV = Eubengee virus; TILV = Tiliigerry virus; WALV = Wallal virus; MUDV = Mudjinbarry virus; WARV = Warego virus; AHSV = African horse sickness virus; CHUV = Chuzan virus; EEV = Equine encephalosis virus; PHSV = Peruvian horse sickness virus; YUOV = Yunnan orbivirus; UMAV = Umatilla virus; SLOV = Stretch Lagoon orbivirus; CORV = Corriparta virus; SVIV = Satuthuvari virus; TRBV = Tribec virus; KEMV = Kemorovo virus; GIV = Great Island virus; SCRV = St Croix river virus. The highest identities were written in bold font.

doi:10.1371/journal.pone.0108379.t003
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Maximum likelihood (ML) trees showing phylogenetic comparisons of the VP1 protein (a) and nucleotide sequences of Seg-1 (b) of WALV, MUDV and WARV, aligned with those of other *Orbivirus* species. One thousand bootstrap replicates were used for construction of the phylogenetic trees. The WALV, MUDV and WARV isolates characterised in this study are marked with green circle. WGRV = Wongorr virus; DAGV = D'Aguilar virus; PICV = Picola virus; PARV = Paroo river virus; CMPV = California mosquito pool virus; MPOV = Middle point orbivirus; BRDV = Broadhaven virus. Further abbreviations used in this figure are same as mentioned in Figure 2. Virus isolates and accession numbers of polymerase sequences used for comparative analysis are listed in Table S1 in File S1. 'e' and 'w' after serotype number indicate eastern and western strains, respectively.

doi:10.1371/journal.pone.0108379.g003</figcaption></figure>

and in WARV has a moderate Kozak sequence (GGGAUGC) encoding 78 aa protein. In all three viruses WALV, MUDV and WARV, the second initiation site (for NS3a) in Seg-10 has the strong Kozak context (A^A/CGAUGG).

### Phylogenetic relationships of Seg-1/VP1(Pol)

The conserved nature of the orbivirus RNA dependent RNA polymerase (Pol) (encoded by Seg-1) has previously been used to help classify orbivirus isolates at the genus and species level based on sequence comparisons and identity levels [18,25,27,31]. Phylogenetic comparisons of Seg-1/VP1(Pol) from WALV, MUDV and WARV with other orbiviruses (Figure 2), showed three groups containing the CBOs, MBOs and TBOs. In each case WALV, MUDV and WARV grouped with the CBOs.

Seg-1/VP1(Pol) of WALV and MUDV share 99.16/93.57 aa/nt identity, confirming that they belong to the same *Orbivirus* species. In comparisons to other orbiviruses they show a maximum of 70.96%/66.97% aa/nt identity with EUBV and a minimum of 36.08%/43.55% with SCRV (Table 3). VP1(Pol) of WARV

shared up to a maximum of 65.41%/63.72% aa/nt identities with EHDV-2w, indicating its distinctiveness from other *Orbivirus* species within the CBOs.

### Phylogenetic relationships of the subcore-shell 'T2' protein

BlastX analyses confirmed that the subcore shell T2 protein - VP3 - is encoded by Seg-3 of WALV, MUDV and WARV. Phylogenetic trees constructed for the T2 protein of different orbiviruses (Figure 3 and Figure S1 in File S1) showed that the CBOs (including BTV, AHSV, EHDV, WALV, EUBV, WARV and PALV) form a single group, in which T2(VP3) is encoded by Seg-3. The TBOs (GIV) and the MBOs (CORV, WGRV, Peruvian horse sickness virus [PHSV] and Yunnan orbivirus [YUOV]), form two further groups, in both of which the subcore T2(VP2) is encoded by Seg-2. The T2 protein (encoded by Seg-2) of SCRV branches separately, representing a more distantly related orbivirus from ticks.
<figure><img src="image_7.png" /></figure>

**Figure 4. Maximum likelihood (ML) trees showing phylogenetic comparisons of the T13 protein (a) and gene (b) sequences of WALV, MUDV and WARV, aligned with those of other *Orbivirus* species.** One thousand bootstrap replicates were used for construction of the phylogenetic trees. The WALV, MUDV and WARV isolates characterised in this study are marked with green circle. Abbreviations used in this figure are same as mentioned in Figure 2 and 3. Virus isolates and accession numbers of polymerase sequences used for comparative analysis are listed in Table S1 in File S1. ‘e’ and ‘w’ after serotype number indicate eastern and western strains, respectively.
doi:10.1371/journal.pone.0108379.g004

The T2 protein/gene of WALV and MUDV clustered together, sharing 99.78%/90.36% aa/nt identities (Table 3), confirming that they belong to the same *Orbivirus* species (Figure 3). Comparisons to other orbiviruses showed a maximum of 72.86%/67.89% aa/nt identities with EUBV, down to 23.49%/37.44% with SCRV supporting classification of WALV and MUDV within a separate virus species. In contrast, WARV branched separately from other orbiviruses, sharing maximum aa/nt identities of 67.33%/65.55% with EUBV/WALV and a minimum of 24.4%/37.17% with SCRV (Table 3) confirming its classification within a distinct species.

## Phylogenetic comparisons of Seg-7/VP7(T13)

The core surface protein VP7(T13) is the most abundant of the orbivirus structural proteins and is a strongly immuno-dominant serogroup/species-specific antigen [49]. The ML trees constructed for VP7(T13) protein and gene sequences of WALV, MUDV, WARV and other orbiviruses (Figure 4) exhibited similar topology to the T2 and VP1(Pol) trees (Figures 2 and 3) with three distinct ‘vector-groups’ (CBOs, MBOs and TBOs). WALV and MUDV again grouped together, while WARV branched separately within the CBOs. When compared to other orbiviruses, the VP7(T13) proteins/genes of WALV and MUDV (which share 100%/

97.71% aa/nt identity) showed a maximum of 58.57%/60.67% aa/nt identity with EUBV and TILV. In contrast WARV again showed maximum aa/nt identity with EHDV (56.45%/59.45%). WALV and WARV share 39.94% to 58.57% aa sequence identity with the other CBOs; 20.06% to 25.21% aa identity with MBOs; 21.04% to 25.21% aa identity with the TBOs; but only 19.53% to 20.41% aa identity with the more distantly related SCRV (Table 3).

## Phylogenetic comparisons of Seg-2/VP2(OC1)

Orbivirus outer capsid protein (OC1) determines virus serotype and is highly variable, both in sequence identity levels and in size. VP2(OC1) of the CBOs is encoded by Seg-2 (VP2), by Seg-3 (VP3) of the MBOs and by Seg-4 (VP4) of the TBOs [5,18,33]. In WALV, MUDV and WARV viruses, VP2(OC1) is encoded by Seg-2, consistent with *Culicoides* transmission. An unrooted NJ phylogenetic tree constructed for OC1 of different orbiviruses (Figure 5) identified three major clusters, again corresponding to their ‘vector groups’ in a manner similar to the trees for VP1, T2 and T13 proteins (Figures 2, 3 and 4). These groupings suggest that despite a high level of aa sequence variation in response to immunological pressure from the vertebrate host (different serotypes), the orbiviruses have maintained a significant level of
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Unrooted neighbour-joining tree showing the relationships between amino acid sequences of OC1 protein of different orbiviruses. One thousand bootstrap replicates were used for construction of the phylogenetic trees. The VP2(OC1) protein of WALV, MUDV and WARV isolates characterised in this study are marked with green circle. CBOs = *Culicoides* borne orbiviruses; MBOs = Mosquito borne orbiviruses; TBOs = Tick borne orbiviruses. Abbreviations used for viruses in this figure are same as mentioned in Figure 2 and 3. Virus isolates and accession numbers of polymerase sequences used for comparative analysis are listed in Table S1 in File S1. 'e' and 'w' after serotype number indicate eastern and western strains, respectively.</figcaption></figure>

doi:10.1371/journal.pone.0108379.g005

conservation in OC1 that is related to vector group. Despite much higher identity levels in their conserved genes and proteins, WALV and MUDV share only 67.89%/69.22% aa/nt identity in Seg-2/VP2(OC1), confirming that they represent different serotypes (WALV-1 and WALV-2 respectively) within the same *Orbivirus* species.

## Discussion

The majority of the existing *Orbivirus* species were initially recognised as distinct ‘serogroups’, sharing common antigens detectable by complement-fixation (CF), agar gel immunodiffusion (AGID), fluorescent antibody (FA) tests, or enzyme-linked immune-sorbent assays (ELISA) [50,51]. However, these serological assays are relatively insensitive, can lack specificity and can show significant levels of cross-reactivity between closely related *Orbivirus* species [52–54]. Identification of new orbivirus isolates (by species and serotype) was also dependent on the availability of reference antisera for the virus species that have already been recognised.

The advent of more rapid and reliable sequencing methods for dsRNA virus genomes has generated data for reference strains of

the individual virus species, that can be easily disseminated for phylogenetic comparisons [5, 19, 34, 37]. These data sets now represent a primary tool for the identification and classification of novel orbivirus isolates [25–27,31,55].

The orbivirus sub-core-shell ‘T2’, polymerase ‘Pol’ and outer-core ‘T13’ proteins are all highly conserved, with >83%, >73% and >73% aa identity respectively within a single *Orbivirus* species. Consequently, these proteins and their comparisons have been used as ‘markers’ for identification and classification of existing and novel orbivirus isolates [5, 18, 26, 31, 32]. The highest inter-species identity levels that have been detected in these conserved proteins are between the closely related and intensively studied species: *Bluetongue virus* and *Epizootic haemorrhagic disease virus* (with maximum aa identities of 80%, 73%, and 66% in T2, Pol and T13 protein respectively). The genes encoding these proteins have also served as targets for development of orbivirus genus-specific and species-specific RT-PCR assays that can be used for diagnosis and virus discovery [18, 29, 56–58].

Nucleotide variation of up to 31.6% (equivalent to >68.4% nt identity) has been observed in the serotype controlling VP2(OC1) gene between different BTV isolates within a single serotype, with a maximum of 71.5% nt identity between different types providing
useful ‘bench marks’ for typing of isolates in other orbivirus species
[32,34]

Phylogenetic analysis of the three most conserved VP1, T2 and
T13 proteins/genes and the highly variable OC1 protein of the
orbiviruses, that are presented here show three distinct ‘clusters’
that correspond to the arthropod vectors used by each virus
(Figure 2, 3 and 4), indicating that the orbiviruses have evolved
through ‘co-speciation’ with their arthropod vectors [18,33]. The
TBOs and MBOs form distinct phylogenetic clusters originating
from a common branch but are more closely related to each other
than to the CBOs (Figure 4 and 5). The TBOs appear to provide
an ancestral ‘root’ for the insect transmitted orbiviruses. The
identification of a smaller OC1 proteins in all of the MBOs (when
compared to the CBOs) and a much smaller protein (~50%) in the
TBOs suggests that significant evolutionary changes in OC1 may
have played a significant role in the initial vector preference and
evolutionary separation of these groups of viruses. The changes in
size of the OC1 protein of the IBOs compared to the TBOs could
be related to an historic concatemerization event [59] doubling the
size of the protein during the evolution of the MBOs and CBOs.

Phylogenetic trees for the different genome segments/proteins
of WALV, MUDV and WARV confirm that members of the same
*Orbivirus* species cluster more closely together, while members of
distinct species branch separately. The trees constructed for
orbivirus Pol, T2 and T13 genes are almost entirely congruent,
also showing similarities to the OC1 tree, although with higher
levels of identity within each virus species. This suggests that
functional interactions between the different viral proteins/RNAs
may restrict the exchange of genome segments (reassortment)
between virus species.

WALV and MUDV are indistinguishable in complement
fixation tests and can reassort with high frequency [60],
demonstrating that they belong to the same *Orbivirus* species -
*Wallal virus*. The data presented here, showing that WALV and
MUDV share >99%/90% aa/nt identities in their conserved Pol,
T2 and T13 proteins support this conclusion (Table 3). However,
WALV and MUDV can be distinguished in serum neutralization
tests [60,61] and are therefore regarded as distinct serotypes -
WALV-1 and WALV-2 respectively. This is supported by the data
presented here, showing that they share <68%/70% aa/nt
identity in outer capsid protein VP2(OC1).

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The utility of sequence data for phylogenetic analysis of viral
pathogens, their classification, as well as diagnostic test and
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becoming an accepted standard for molecular epidemiological
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Full genome references sequences are now available for fourteen
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**Supporting Information**

**File S1 Table S1 and Figure S1.** Table S1. Nucleotide accession numbers for sequences used in phylogenetic analysis. Figure S1. Unrooted Neighbour-joining phylogenetic tree comparing orbivirus T2 proteins. (DOCX)

Acknowledgments

The authors would like to thank all collaborators for providing virus
isolates for tests and analyses. P. Mertens is a Jenner Investigator.

Author Contributions

Conceived and designed the experiments: MB SM NSM HA PCM.
Performed the experiments: MB SM NSM. Analyzed the data: MB SM
HA NSM PCM. Contributed reagents/materials/analysis tools: MB SM
HA PCM IP PK JB. Wrote the paper: MB SM HA NSM PCM.

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