# Full genome sequencing of Corriparta virus, identifies California mosquito pool virus as a member of the Corriparta virus species

Manjunatha N Belaganahalli, Sushila Maan, Narender S Maan, Kyriaki Nomikou, Marc Guimera, Joe Brownlie, Robert Tesh, Houssam Attoui, Peter P C Mertens

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Manjunatha N Belaganahalli, Sushila Maan, Narender S Maan, Kyriaki Nomikou, Marc Guimera, et al.. Full genome sequencing of Corriparta virus, identifies California mosquito pool virus as a member of the Corriparta virus species. PLoS ONE, 2013, 8 (8), pp.e70779. 10.1371/journal.pone.0070779. hal-01606561

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# Full Genome Sequencing of Corriparta Virus, Identifies California Mosquito Pool Virus as a Member of the *Corriparta virus* Species

Manjunatha N. Belaganahalli¹, Sushila Maan¹,², Narender S. Maan², Kyriaki Nomikou¹, Marc Guimera¹, Joe Brownlie³, Robert Tesh⁴, Houssam Attoui¹, Peter P. C. Mertens¹*

¹The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom, ²College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India, ³Department of Pathology and Infectious Diseases, Royal Veterinary College, North Mymms, Hatfield, Herts, United Kingdom, ⁴Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America

## Abstract

The species *Corriparta virus* (CORV), within the genus *Orbivirus*, family *Reoviridae*, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell 'T2' and core-surface 'T13' proteins encoded by genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity (75.9%/91.6% in Seg-2/T2: 77.6%/91.7% in Seg-8/T13, respectively) were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the *Corriparta virus* species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.

**Citation:** Belaganahalli MN, Maan S, Maan NS, Nomikou K, Guimera M, et al. (2013) Full Genome Sequencing of Corriparta Virus, Identifies California Mosquito Pool Virus as a Member of the *Corriparta virus* Species. PLoS ONE 8(8): e70779. doi:10.1371/journal.pone.0070779

**Editor:** Amit Kapoor, Columbia University, United States of America

**Received:** April 6, 2013; **Accepted:** June 21, 2013; **Published:** August 27, 2013

**Copyright:** © 2013 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.

**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, partly by the Wellcome BTV Programme grant (http://www.wellcome.ac.uk/) awarded to KN and EMIDA grant (OrbiNet - K1303206) to fund MG. NSM, PPCM and HA were involved in this work, funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the European Union (EU) (SANCO/940/2002) and Defra. No additional external funding 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:** Co-author Dr. HA is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

* E-mail: Peter.Mertens@pirbright.ac.uk

## Introduction

Corriparta viruses are mosquito-borne arboviruses, classified within one of the 22 virus species currently recognised within the genus *Orbivirus*, family *Reoviridae*. Currently there are also 15 'unclassified' orbiviruses in the genus, which may represent additional species [1-6]. The orbivirus genome is composed of 10 segments of linear dsRNA, packaged as one copy of each segment within each of the non-enveloped icosahedral virus particles. The intact virion is composed of three concentric protein shells (the 'outer-capsid', 'core-surface layer and the 'subcore-shell'). Orbiviruses are transmitted by ticks or hematophagus-insect vectors (including *Culicoides*, mosquitoes or sand flies) and collectively have a wide host-range that includes both domesticated and wild ruminants, equids, camelids, marsupials, sloths, bats, birds, large canine and feline carnivores, and humans [2,7-9].

The species *Corriparta virus* currently contains six distinct viruses, that are identified as: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus; and Jacareacanga virus [2]. The structural and chemical properties of the corriparta viruses are similar to those of other orbiviruses [2]. They are sensitive to

low pH and heat, and can be modified by treatment with trypsin or chymotrypsin [10]. They have also been shown to multiply in mosquitoes after intra-thoracic inoculation [10].

Members of the *Corriparta virus* species/serogroup have been detected in Australia, Africa and South America [11]). They have been isolated from wild birds, and neutralizing antibodies were found in wild and domestic birds, cattle, marsupials, horses and man [12-15]. Corriparta virus MRM1 was isolated in 1960, from *Culex* mosquitoes, as well as from *Aedeomyia catasticta*, a rare mosquito species collected near Mitchell River in North Queensland, Australia. Subsequently, strains CS0109, V654 and V370 were also isolated in Australia [2,8,15,16]. Acado virus and Jacareacanga virus were isolated from pools of *Culex* mosquitoes collected in Ethiopia and Brazil during 1963 and 1975 respectively [8,11].

The International Committee on Taxonomy of Viruses (ICTV) has agreed 'polythetic' definitions for individual virus species [17]. The ability to exchange genome segments with other viruses belonging to the same virus species by 'reassortment' is recognised as the primary determinant of *Orbivirus* species [2,7]. However, in the absence of data concerning their compatibility for reassort-
ment, the members of individual species can be identified by other 'polythetic' parameters that include similarities in RNA and protein sequences, their RNA-segment size distribution (reflected by their migration patterns - electropherotype) during agarose gel electrophoresis [AGE], host and/or vector range, the clinical signs of infection, and serological relationships [2,7,18–20].

The members of the different *Orbivirus* species were originally identified as belonging to distinct ‘serogroups’, based on their cross-reactivity in ‘group-specific’ serological assays that include complement fixation (CF) tests, group-specific ELISA, or agar-gel-immuno-diffusion (AGID) tests, most of which target outer-core protein VP7(T13) [2,7,21]. The corriparta viruses were initially grouped primarily on the basis of CF tests [8,22]. However, a lack of neutralization assays has prevented further analysis of their intra-serogroup serological-relationships and the identification of distinct serotypes.

Recently, full genome sequencing and phylogenetic analyses have been used to determine the genetic relatedness and taxonomic status of individual isolates belonging to different *Orbivirus* species, including *Bluetongue virus* (BTV); *African horsesickness virus* (AHSV) and *Epizootic hemorrhagic disease virus* (EHDV) [1,23–26]. These sequence data have supported development of faster and more reliable, virus-species/serogroup, and virus-serotype specific diagnostic assays, using both conventional and real-time RT-PCRs [27–31]. Comparisons of nucleotide (nt) and amino acid (aa) sequence data also provide a basis for grouping of orbivirus isolates into topotypes and for molecular epidemiology studies [23,25,32,33]. However, full-genome sequence data are currently available for representatives of only 11 of the recognized *Orbivirus* species [24] (accession numbers given in Table S1). Additional partial-sequences are available for the highly conserved genome-segment encoding the subcore ‘T2’ protein (VP3 of BTV) of some *Orbivirus* species, including *Warrego virus* (WARV), *Wallal virus* (WALV), *Wongorr virus* (WGRV) and CORV [32] (Table S1).

‘California mosquito pool virus’ (CMPV) was isolated in 1974 from pooled *Culex tarsalis* mosquitoes collected as part of an infectious agent surveillance program conducted by The California Department of Public Health [5]. Partial sequences for genome segments 2, 4, 6, 7 and 9 from CMPV (accession numbers EU789391 to EU789395) were compared to available data for other orbiviruses, suggesting that CMPV might represent a novel virus species [5]. However, the lack of reference sequences for representatives of all *Orbivirus* species, made it impossible to confirm the taxonomic status and species identity of CMPV at that time.

We report the full genome sequence of CORV-MRM1 (AUS1960/01). Comparisons of nucleotide (nt) and deduced amino acid (aa) sequences for the conserved polymerase ‘VP1(Pol)’, subcore-shell ‘T2-protein’, and outer-core ‘T13-protein’, to data published for other orbiviruses, indicate that CORV and CMPV belong to the same species - *Corriparta virus*.

## Results

### Virus propagation and dsRNA 'electropherotype'

CORV-MRM1 induced severe cytopathic effect (CPE) in BHK cell monolayers, by 48–72 hours post infection (pi). The viral dsRNA was purified from infected cell-cultures and analysed by 1% agarose gel electrophoresis (AGE) (Figure 1). CORV Seg-3 (encoding the larger outer capsid protein VP3(OC2)) migrates close to the medium sized genome-segments (Seg-4, Seg-5 and Seg-6) giving a 2-4-4 (2-4-3-1) migration pattern (Figure 1). This contrasts with the 3-3-4 (3-3-3-1) pattern that is more typical of the

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Agarose gel (1%) electrophoresis for dsRNAs of CORV, BTV and EHDV. The purified genomic RNA of LIB2007/07 (a western-topotype isolate of BTV serotype 1 (BTV-1w)) - lane 1: AUS1982/05 (an eastern-topotype isolate of EHDV serotype 8 (EHDV-8e)) - lane 2: and CORV-MRM1 (AUS1960/01) - lane 3, were analysed by 1% agarose gel electrophoresis (AGE), then stained with ethidium bromide, visualised and photographed under UV illumination. The genome segment numbers (encoded proteins) are indicated at the side of the gel.

*doi:10.1371/journal.pone.0070779.g001*</figcaption></figure>

*Culicoides* borne orbiviruses (as illustrated for BTV-1w [LIB2007/07] and EHDV-8e [AUS1982/05]).

### Characterisation and coding assignments of CORV genome segments

Sequences for Seg-1 to Seg-10 CORV-MRM1 (AUS1960/01) have been deposited in the GenBank with accession numbers KC853042 to KC853051, respectively. They range from 3,925 bp to 790 bp (encoding proteins of 1,290 aa to 108 aa) with a total length of 19,093 bp, (Table 1). The different genome segments of CORV-MRM1 all share six fully conserved nucleotides at their 5' ends, and 10 at their 3' ends (+ve: 5'-GUAUAG.........CAAAGGAUAC-3'). Two terminal nucleotides at the 5' end and three nucleotides at the 3' end (5'-GU......UAC-3') 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).

Collectively the terminal non-coding regions (NCR) represent 4.99% of the CORV-MRM1 genome (Table 2). Comparisons with data from GenBank for other mosquito-borne-orbiviruses (MBOs) showed similar or higher percentages, while the tick-borne orbiviruses (TBOs) and *Culicoides*-borne orbiviruses (CBOs) show similar or lower values (Table 2).

Like other orbiviruses, most genome segments of CORV-MRM1 (AUS1960/01), have shorter 5' than 3' NCRs, except for Seg-6 (encoding the smaller outer capsid protein VP5(OC2)) which has a longer 5' NCR and Seg-9 (encoding VP6) which has 3' and 5' NCRs of equal length (Table 1). Exceptions also occur in Umatilla virus (UMAV) and Great Island virus (GIV) although the significance of these variations is unclear.

Seg-5 (1,936 bp) of CORV-MRM1 which encodes the ‘tubule’ protein NS1(TuP) (one of the most abundantly expressed orbivirus
<table><caption>**Table 1.** Characteristics of Corriparta virus (CORV-MRM1) (AUS1960/01) genome segments (dsRNA) and their encoded proteins.</caption><thead><tr><th>Seg No.</th><th>Seg size (bp)</th><th>Gr-C content (%)</th><th>Protein encoded (structure/putative function)</th><th>ORFs bp (including stop codon)</th><th>5' NCR</th><th>3' NCR</th><th>No. of amino acids</th><th>Predicted protein molecular mass (kDa)</th><th>5' Termini</th><th>3' Termini</th><th>Accession numbers</th></tr></thead><tbody><tr><td>1</td><td>3,925</td><td>41.66</td><td>VP1 (Pol)</td><td>12-3884</td><td>11</td><td>44</td><td>1290</td><td>147.07</td><td>5'-GUAUAGG....</td><td>....CCAAAGGAUAC-3'</td><td>KC853042</td></tr><tr><td>2</td><td>2,952</td><td>43.43</td><td>VP2 (T2)</td><td>33-2894</td><td>32</td><td>61</td><td>953</td><td>108.75</td><td>5'-GUAUAGG....</td><td>....CCAAAGGAUAC-3'</td><td>KC853043</td></tr><tr><td>3</td><td>2,284</td><td>42.43</td><td>VP3 (OC1)</td><td>20-2236</td><td>19</td><td>51</td><td>738</td><td>84.81</td><td>5'-GUAUAGG....</td><td>....CCAAAGGAUAC-3'</td><td>KC853044</td></tr><tr><td>4</td><td>2,032</td><td>43.11</td><td>VP4 (Cap)</td><td>12-1943</td><td>11</td><td>92</td><td>643</td><td>74.83</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853045</td></tr><tr><td>5</td><td>1,936</td><td>46.33</td><td>NS1 (TuP)</td><td>20-1789</td><td>19</td><td>150</td><td>589</td><td>68.61</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853046</td></tr><tr><td>6</td><td>1,683</td><td>46.11</td><td>VP5(OC2)</td><td>56-1639</td><td>55</td><td>47</td><td>527</td><td>57.7</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853047</td></tr><tr><td>7</td><td>1,187</td><td>46.5</td><td>NS2 (VIP)</td><td>26-1141</td><td>25</td><td>49</td><td>371</td><td>41.86</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853048</td></tr><tr><td>8</td><td>1,176</td><td>47.62</td><td>VP7 (T13)</td><td>17-1081</td><td>16</td><td>98</td><td>354</td><td>38.96</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853049</td></tr><tr><td>9</td><td>1,128</td><td>47.16</td><td>VP6 (Hel)</td><td>49-1083</td><td>48</td><td>48</td><td>344</td><td>36.85</td><td>5'-GUAUAGA....</td><td>....UCAAAGGAUAC-3'</td><td>KC853050</td></tr><tr><td>10</td><td>790</td><td>47.22</td><td>NS3</td><td>143-601</td><td>142</td><td>530</td><td>152</td><td>18.37</td><td>5'-GUAUAGG....</td><td>....CCAAAGGAUAC-3'</td><td>KC853051</td></tr><tr><td></td><td></td><td>—</td><td>NS3a</td><td>17-733</td><td>16</td><td>60</td><td>238</td><td>25.92</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td>—</td><td></td><td>407-733</td><td>406</td><td>60</td><td>108</td><td>11.848</td><td></td><td></td><td></td></tr></tbody></table>

Pol = RNA polymerase; OC = outer capsid protein; Cap = capping enzyme (guanylyltransferase); Hel = helicase enzyme; T2 = protein with T = 2 symmetry; T13 = Protein with T = 13 symmetry; VIP = viral inclusion body matrix protein; TuP = tubule protein. OC1 and OC2 refer to the larger and smaller outer capsid proteins respectively.

doi:10.1371/journal.pone.0070779.t001

proteins) is longer than the homologous Seg-5 of the CBOs or TBOs (Table 2) with a long (150 bp) 3' NCR, but is smaller than that of some other MBOs [1]. Long 3' NCRs were also observed in the NS1 genome segment of some other MBOs (e.g. UMAV-278 bp; Yunnan orbivirus [YUOV]-205 bp), but not in all other orbiviruses (<118 bp).

The 'highly conserved' subcore-shell VP2(T2) protein and the 'highly variable' outer-capsid/cell-attachment protein VP3(OC1) of CORV-MRM1, are encoded by Seg-2 and Seg-3, respectively. A similar coding pattern is seen in other MBOs, but the presence of a larger OC1 in the CBOs (including bluetongue virus, the orbivirus 'type' species) results in a reversed coding assignment for these two genome-segments (Figure 1 and Table 2).

The size of the highly conserved T13 core-surface protein (VP7 of BTV) is also relatively consistent in most orbiviruses, while the viral inclusion-body matrix-protein, (non-structural protein 2 [NS2(ViP)]) is more variable in size (Table 2). As a result, NS2 of CORV-MRM1 (AUS1960/01) is encoded by Seg-7, while Seg-8 encodes the core-surface protein VP7(T13) (Table 1), in a manner similar to the other MBOs (Table 2). However, this coding-assignment is again reversed in BTV and in some CBOs (EUBV and CHUV), due to variability in the size of NS2 between different viruses [34,35]. The capping enzyme 'VP4(CaP)' of CORV-MRM1 (at 643aa) is smaller than that of the other MBOs and some of the CBOs (Table 2). However, it is encoded by the largest Seg-4 (at 2,032 bp) so far identified in any orbivirus.

Most of the genome segments of CORV-MRM1 (AUS1960/01) except Seg-9 and Seg-10 are monocistronic, encoding a protein from a single large ORF, starting from an initiation codon with a strong Kozak sequence (RNN<u>AUGG</u>) [36]. However, like BTV, CORV Seg-10 has two in-frame AUG initiation sites encoding the NS3 and NS3a proteins of 238 aa and 108 aa (starting at 17 bp and 407 bp) respectively (Table 1). The first of these (coding for NS3) has a 'weak Kozak context' (GU<u>AUGU</u>), possibly enhancing read-through and initiation of translation from the second 'in frame' initiation site (at 407 bp). This has a strong Kozak context (GU<u>AUGG</u>), but would express the smallest NS3a in any of the orbiviruses characterized to date.

The first start codon of CORV-MRM1 (AUS1960/01) Seg-9 also has a moderate Kozak sequence (U<u>UGAUGA</u>) and a second down-stream ORF. However, this is in the +2 reading-frame (at 143–598 bp), encoding the 152 aa NS4 protein. NS4 has previously been identified in several other orbiviruses and has been characterised in BTV and GIV [24,37,38]. The downstream Seg-9 ORF of CORV has a strong Kozak context (<u>AGGAUGG</u>) and is expected to produce a protein in infected cells. Weak or moderate Kozak sequences have also been observed in several of the genome segments of other orbiviruses, but they still appear to be translated effectively [24,39].

The G+C content of the CORV genome is 45.16%, which is considerably higher than that of other MBOs [Peruvian horse sickness virus (PHSV) and YUOV with 36.66% and 41.59% respectively] but within the overall G+C range of the insect-borne orbiviruses [36.66% in PHSV (mosquito), to 45.86% in Equine encephalosis virus (EEV) (Culicoides)]. However, it is lower than that of the tick-borne or tick-associated orbiviruses [57.29% in GIV and 51.93% in *St Croix River virus* (SCRV)].

## Phylogenetic comparisons of subcore-shell 'T2' proteins

Comparison of homologous orbivirus proteins using BlastX analysis identified VP2, encoded by Seg-2 of CORV, as the subcore shell 'T2' protein (equivalent to VP3(T2) of BTV and VP2(T2) of YUOV and SCRV). An unrooted neighbour-joining (NJ) phylogenetic tree (Figure 2) constructed for different orbivirus
<table><caption>Table 2. Full genome sequence database available for recognised species of genus *Orbivirus* and their genome coding assignments.</caption><thead><tr><th></th><th>CORV</th><th>UMAV</th><th>PHSV</th><th>YUOV</th><th>BTV-8w</th><th>AHSV-1</th><th>EHDV-1e</th><th>EEV</th><th>EUBV</th><th>CHUV</th><th>GIV</th><th>SCRV</th></tr></thead><tbody><tr><td>Protein</td><td>size in bp</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td><td>Segment</td></tr><tr><td>Pol</td><td>3925 S1 1290 3933 S1 1299 3987 S1 1311 3993 S1 1315 3944 S1 1302 3966 S1 1305 3942 S1 1302 3963 S1 1307 3962 S1 1307 3930 S1 1295 3897 S1 1285 4089 S1 1345</td></tr><tr><td>T2</td><td>2284 S2 755 2791 S2 905 2856 S2 925 2900 S2 940 2772 S3 901 2792 S3 905 2768 S3 899 3178 S3 902 2773 S3 900 2774 S3 904 2794 S2 908 2747 S2 890</td></tr><tr><td>OC1</td><td>2952 S3 953 2411 S3 797 2747 S3 881 2688 S3 873 2939 S2 961 3218 S2 1056 2968 S2 971 2775 S2 1042 2958 S2 968 3055 S2 1002 1722 S4 551 2024 S4 654</td></tr><tr><td>Cap</td><td>2032 S4 643 2021 S5 648 1996 S4 646 1993 S4 645 1981 S4 644 1978 S4 642 1983 S4 644 1986 S4 646 1984 S4 644 1967 S4 640 1936 S3 635 2017 S3 643</td></tr><tr><td>Tup</td><td>1936 S5 589 2047 S4 580 1784 S5 554 1957 S5 574 1776 S5 552 1748 S5 548 1806 S5 551 1749 S5 548 1763 S5 556 1764 S5 545 1731 S5 531 1657 S5 517</td></tr><tr><td>OC2</td><td>1683 S6 527 1681 S6 529 1695 S6 529 1683 S6 535 1637 S6 526 1564 S6 505 1640 S6 527 1607 S6 515 1658 S6 529 1610 S6 521 1666 S6 537 1664 S6 517</td></tr><tr><td>T13</td><td>1176 S8 354 1171 S8 351 1180 S8 353 1191 S8 355 1156 S7 349 1167 S8 349 1162 S8 349 1175 S8 350 1175 S7 350 1151 S7 348 1181 S7 357 1256 S7 379</td></tr><tr><td>VIP</td><td>1187 S7 371 1357 S7 407 1613 S7 435 1504 S7 435 1125 S8 354 1166 S9 365 1186 S7 373 1181 S7 362 1128 S8 351 1059 S8 333 1172 S8 359 1463 S8 462</td></tr><tr><td>Hel</td><td>1128 S9 344 1103 S9 341 1071 S9 334 1082 S9 338 1049 S9 329 1169 S7 369 1143 S9 360 1080 S9 341 1047 S9 328 877 S9 272 1056 S9 321 764 S9 232</td></tr><tr><td>NS4</td><td>— S9 152 — S9 149 — S9 111 — S9 113 — S9 77 — S7 117 — S9 76 — S9 145 — S9 84 — S9 83 — S9 190 — S9 93</td></tr><tr><td>NS3</td><td>790 S10 238 887 S10 264 819 S10 255 825 S10 253 822 S10 229 763 S10 218 810 S10 228 759 S10 240 856 S10 242 728 S10 211 703 S10 171 764 S10 224</td></tr><tr><td>Total</td><td>19093</td><td>19402</td><td>19748</td><td>19816</td><td>19201</td><td>19531</td><td>19453</td><td>19408</td><td>19304</td><td>18915</td><td>17858</td><td>18445</td></tr><tr><td>Genome (bp)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>%NCR</td><td>4.99</td><td>5.71</td><td>5.46</td><td>5.18</td><td>3.96</td><td>3.79</td><td>4.09</td><td>3.57</td><td>4.04</td><td>3.71</td><td>5</td><td>4.64</td></tr><tr><td>G+C (%)</td><td>45.16</td><td>41.55</td><td>36.66</td><td>41.59</td><td>44.98</td><td>43.46</td><td>43.02</td><td>45.86</td><td>45</td><td>39.89</td><td>58.13</td><td>51.92</td></tr><tr><td>Vectors</td><td colspan="12">Culicoides spp</td></tr><tr><td>Mosquitoes</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Tick or Tick associated</td></tr></tbody></table>

Pol = Polymerase, OC1 = Outer capsid protein 1 (VP2 of BTV), T2 = Inner core protein (T2 symmetry), Cap = Capping enzyme, Tup = Tubule forming protein or Tubular protein (NS1), OC2 = Outer capsid protein 2 (VP5 of BTV), T13 = Outer core protein (T13 symmetry), VIP = Viral inclusion body protein (NS2), Hel = Helicase protein. NCR and G+C content were calculated or full genome sequences.

doi:10.1371/journal.pone.0070779.t002
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Unrooted neighbour-joining tree for orbivirus subcore-shell T2 proteins. A phylogenetic tree was constructed using distance matrices, generated using the p-distance determination algorithm and pairwise deletion parameters in MEGA 5 (1000 bootstrap replicates) [65]. Since many of the available sequences are incomplete, the analysis is based on partial sequences (aa 393 to 548, numbered with reference to the aa sequence of BTV-VP3(T2)). The numbers at nodes indicate bootstrap confidence values after 1000 replications. The tree shown in Figures 3 and 4 were drawn using the same parameters. The CORV and CMPV isolates are shown in red font in the amber coloured circle. Full names of virus isolates and accession numbers of T2 protein sequences used for comparative analysis are listed in Table S1 (supplementary data). 'e' and 'w' after serotype number indicate eastern and western topotype strains, respectively. doi:10.1371/journal.pone.0070779.g002</figcaption></figure>

T2 protein sequences (listed in Table S1 supplementary data) identified two major clusters (Figure 2). The larger group in which VP3(T2) is encoded by Seg-3, comprises the *Culicoides* transmitted orbiviruses (including BTV, AHSV, EHDV, WALV, *Eubenangee virus* [EUBV], WARV and *Palyam virus* [PALV]). However, the second group in which VP2(T2) is encoded by Seg-2, includes two sub-groups transmitted by ticks (the Great Island viruses), or by mosquitoes (WGRV, UMAV, PHSV and YUOV) respectively. SCRV, which has previously been suggested as a tick-orbivirus rather than a tick-borne-orbivirus [40], branches separately from these groups and appears to be more distantly related to other orbiviruses. VP2(T2) of CORV-MRM1 (AUS1960/01) clusters very closely with partial sequence data available for CMPV (98.78%/98.99% aa/nt identity Table 3 and 4), within the mosquito-borne group, indicating that they belong to the same virus species. These two viruses show lower levels of identity with the other *Orbivirus* species analysed (<58.53%/57.98% aa/nt identity - Table 3).

## Phylogenetic comparisons of VP1(Pol) proteins

Seg-1, which encodes the viral RNA polymerase VP1(Pol), is highly conserved across the genus *Orbivirus*, and provides a target for sequencing and RT-PCR assays to identify virus isolates at both species and genus levels [1,24,31,40]. The aa sequence of VP1(Pol) from CORV-MRM1 (AUS1960/01) was compared with

representatives of other *Orbivirus* species to construct an unrooted NJ phylogenetic tree (Figure 3). Three major clusters were identified, corresponding to the different vector-groups: CBOs, MBOs and TBOs. CORV-MRM1 (AUS1960/01) again clusters as a distinct species, close to other MBOs (>51.44/54.9% aa/nt identity - Table 3), with more distant relationships to VP1 of the *Culicoides* and tick-borne groups (<50.51%/53.99% aa/nt identity - Table 3).

## Phylogenetic comparisons of outer-core 'T13' proteins

The orbivirus core-surface protein VP7(T13) is immunodominant and represents a major serogroup-specific antigen [21]. An unrooted phylogenetic tree for VP7(T13) (encoded by Seg-8 of CORV-MRM1) (Figure 4) shows a similar topology to the VP2(T2) and VP1(Pol) trees (Figures 2 and 3), grouping viruses according to their vectors. CORV-MRM1 clusters with CMPV (sharing 91.7%/77.6% aa/nt identity) as members of a distinct species (Table 4), showing lower identities with the other MBOs (<42.57%/47.62% aa/nt identities (with UMAV)), or the CBOs and TBOs (<32.95%/41.57% aa/nt identities (with GIV)) (Table 3).

## Relationships between CORV and CMPV

The sequences of CORV-MRM1 Seg-4/VP4(Cap), Seg-6/VP5(OC2), and Seg-9/VP6(Hel)/NS4 were also compared with
<table><caption>**Table 3.** Percent nucleotide (nt) and amino acid (aa) identities of Corriparta virus MRM1 (AUS1960/01) with other orbiviruses.</caption><thead><tr><th rowspan="2">SI no.</th><th rowspan="2">Orbivirus species</th><th rowspan="2">Abbreviation</th><th colspan="2">T2</th><th colspan="2">VP1</th><th colspan="2">T13</th></tr><tr><th>aa</th><th>nt</th><th>aa</th><th>nt</th><th>aa</th><th>nt</th></tr></thead><tbody><tr><td rowspan="3">1</td><td rowspan="3">Bluetongue virus</td><td>BTV</td><td>37.11</td><td>47.33</td><td>47.40</td><td>52.41</td><td>25.07</td><td>38.71</td></tr><tr><td>BTV25</td><td>36.22</td><td>47.00</td><td>46.84</td><td>52.12</td><td>23.63</td><td>38.14</td></tr><tr><td>BTV26</td><td>37.11</td><td>46.56</td><td>46.84</td><td>51.68</td><td>24.21</td><td>37.85</td></tr><tr><td>2</td><td>African horse sickness virus</td><td>AHSV</td><td>37.06</td><td>47.64</td><td>47.90</td><td>53.03</td><td>23.85</td><td>39.85</td></tr><tr><td>3</td><td>Epizootic haemorrhagic disease virus</td><td>EHDV</td><td>37.19</td><td>46.88</td><td>47.40</td><td>53.06</td><td>24.50</td><td>39.67</td></tr><tr><td>4</td><td>Eubenangee virus</td><td>EUBV</td><td>36.60</td><td>47.53</td><td>47.60</td><td>52.88</td><td>27.30</td><td>42.53</td></tr><tr><td>5</td><td>Palyam virus</td><td>PALV</td><td>35.77</td><td>47.14</td><td>50.51</td><td>53.99</td><td>27.67</td><td>41.02</td></tr><tr><td>6</td><td>Equine encephalosis virus</td><td>EEV</td><td>33.96</td><td>45.36</td><td>46.61</td><td>51.26</td><td>24.71</td><td>39.18</td></tr><tr><td>7</td><td>Warrego virus</td><td>WARV</td><td>44.72</td><td>51.78</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>8</td><td>Wlallal virus</td><td>WALV</td><td>44.64</td><td>50.83</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>9</td><td>Umatilla virus</td><td>UMAV</td><td>50.17</td><td>55.21</td><td>59.66</td><td>58.12</td><td>42.57</td><td>47.62</td></tr><tr><td></td><td></td><td>SLOV</td><td>50.39</td><td>54.29</td><td>57.94</td><td>57.23</td><td></td><td></td></tr><tr><td>10</td><td>Peruvian horse sickness virus</td><td>PHSV</td><td>46.37</td><td>53.45</td><td>52.06</td><td>55.38</td><td>33.52</td><td>43.94</td></tr><tr><td>11</td><td>Yunnan orbivirus</td><td>YUOV</td><td>44.78</td><td>53.16</td><td>51.44</td><td>54.90</td><td>34.09</td><td>45.36</td></tr><tr><td>12</td><td>Corriparta virus*</td><td>CORV</td><td>98.78</td><td>98.99</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>13</td><td>Wongorr virus</td><td>WGRV</td><td>58.53</td><td>57.98</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>14</td><td>Great Island virus</td><td>GIV</td><td>45.20</td><td>49.87</td><td>50.51</td><td>52.13</td><td>32.95</td><td>41.57</td></tr><tr><td>15</td><td>St Croix river virus</td><td>SCRV</td><td>24.15</td><td>38.46</td><td>39.01</td><td>45.23</td><td>22.03</td><td>35.94</td></tr><tr><td>16</td><td>Pata virus</td><td>PATAV</td><td>36.19</td><td>46.84</td><td>47.23</td><td>52.28</td><td>26.59</td><td>40.75</td></tr></tbody></table>

the incomplete sequence data available for CMPV (Table 4). CMPV again grouped closely with CORV-MRM1 (AUS1960/01), sharing aa/nt identities of 75/67.4% and 86.2/74.3% in VP4(Cap) and VP5(OC2) respectively, further supporting the identification of CMPV as a member of *Corriparta virus* species. Seg-9 of CORV-MRM1 (coding for NS4 and VP6(Hel)) shares only 42.6/61.6% aa/nt identities with CMPV. However, no closer matches were found in either of these proteins with members of other *Orbivirus* species. Further analysis of CMPV Seg-9 confirmed the presence of an alternate ORF, encoding a 153 aa NS4 protein, which shares 55.9%/63.2% aa/nt identity with NS4 of CORV-MRM1.

## Discussion

Collectively the orbiviruses infect a wide range of hosts and are transmitted by a diverse group of vectors, including *Culicoides*, mosquitoes, sand flies and ticks [7]. Initially, the different ‘serogroups’ of orbiviruses, which are now recognised as distinct virus species, were identified by CF, AGID, immunofluorescence (IF) tests and/or enzyme-linked immunosorbent assays (ELISA). However, low level serological cross-reactions have been detected between some of the more closely related *Orbivirus* species (for example between isolates of BTV (the *Orbivirus* type species) and EHDV [41] making it difficult to conclusively identify the species of new isolates by these techniques alone. The high quality reference-strains and antisera needed for these assays are not widely available for all of the existing serogroups/species, and may themselves represent a biosecurity risk. In addition, serological methods do not generate absolute quantitative values for the relatedness of individual virus strains.

In contrast, nucleotide sequence data for reference orbivirus strains and novel isolates can be compared and transmitted easily between laboratories, without risk, providing highly reproducible and fully quantitative numerical values for the relatedness of each genome segment/protein. These data can also be used to unambiguously identify different genome segments, proteins, virus species, topotypes and serotypes [1,25,33,39,42].

Due to their economic significance, full genomes of multiple isolates (including reference strains) of BTV, EHDV and AHSV have already been characterized [23,43–49]. These data have supported development of rapid and reliable molecular methods and diagnostic tools (RT-PCR assays) for identification of virus serogroup/species and serotype [27–31,49–51].

Sequence variations in the outermost orbivirus capsid and cell-attachment protein, VP2(OC1) of BTV, correlate with both the geographic origin of the virus (topotype) and with its serotype [42,47]. In contrast sequence variation in the core proteins VP1(Pol) and T2 (VP3 of BTV) correlate only with virus genus, species and topotype [1,32,33,39,40]. However, a lack of full-genome sequence data for all 22 recognized *Orbivirus* species has hindered identification of isolates belonging to novel *Orbivirus* species and development of nucleic acid based diagnostic tests.

The full-genome sequence data presented here for CORV-MRM1 (AUS1960/01) provides a primary reference for identification of other (novel) members of the species *Corriparta virus* (CORV). Conserved nucleotide sequences are present at both the upstream and downstream termini of the genome segments of different *Orbivirus* species [2,52]. All of the genome segments of CORV-MRM1 (AUS1960/01) contain the terminal sequences (5’-GUAUAG...CAAAGGAUAC-3’) showing several differences from those of BTV (e.g. 5’-GUUAAA...ACUUAC-3’). The significance of a longer conserved 3’ terminal region in CORV-

<footnote>*data previously published for corriparta virus MRM1 (Ac No. AF530086).</footnote>

<footnote>doi:10.1371/journal.pone.0070779.t003</footnote>
<table><caption>Table 4. Percent identities between Corriparta virus (CORV-MRM1) (AUS1960/01) and California mosquito pool virus (CMPV) proteins and genome segments.</caption><thead><tr><th>Segment (Protein)</th><th>Seg-1/VP1 (Pol)</th><th>Seg-2/VP2 (T2)</th><th>Seg-3/VP3 (OC1)</th><th>Seg-4/VP4 (CaP)</th><th>Seg-5/NS1 (TuP)</th><th>Seg-6/VP5 (OC2)</th><th>Seg-7/NS2 (ViP)</th><th>Seg-8/VP7 (T13)</th><th>Seg-9#/VP6 (Hel) &amp; NS4</th><th>Seg-10/(NS3)</th></tr></thead><tbody><tr><td>Genome segment of CORV (bp)</td><td>3,925</td><td>2,952</td><td>2,284</td><td>2,032</td><td>1,936</td><td>1,683</td><td>1,187</td><td>1,176</td><td>1,128</td><td>790</td></tr><tr><td>Partial sequence of CMPV (bp)*</td><td>—</td><td>358</td><td>—</td><td>288</td><td>—</td><td>646</td><td>—</td><td>737</td><td>596</td><td>—</td></tr><tr><td>% aa identity (CORV vs CMPV)</td><td>—</td><td>91.6</td><td>—</td><td>75</td><td>—</td><td>86.2</td><td>—</td><td>91.7</td><td>42.1/55.9<sup>§</sup></td><td>—</td></tr><tr><td>% nt identity (CORV vs CMPV)</td><td>—</td><td>75.9</td><td>—</td><td>67.4</td><td>—</td><td>74.3</td><td>—</td><td>77.6</td><td>61.6/63.2<sup>§</sup></td><td>—</td></tr></tbody><tfoot><tr><td colspan="11">*Only partial CMPV sequences are available in GenBank.<br/>#Identities calculated using reverse complement sequences of CMPV.<br/>§Indicates identities of NS4.<br/>doi:10.1371/journal.pone.0070779.t004</td></tr></tfoot></table>

MRM1 is unknown, although it has been suggested that these regions may play a role in initiation of transcription or translation of the RNA or its packaging during virus replication [53,54].

The orbivirus proteins VP1(Pol), 'T2' and 'T13', which are highly conserved have taken priority in development of molecular diagnostic assays and in phylogenetic analyses [24,29,31,32,55]. Studies with large numbers of different BTV and EHDV isolates show >73%, >83% and >73% intra-species aa identities in VP1, T2 and T13 respectively, providing useful markers for the identification and classification of existing and novel orbivirus isolates [1,40,44].

CORV-MRM1 shares less than 60% aa identity (Table 3) in VP1, T2 and T13, with members of the other recognised *Orbivirus* species, confirming the classification of *Corriparta virus* as a distinct species. CORV-MRM1 is most closely related to other MBO species, particularly *Umatilla virus* (UMAV), with 59.66% and 50.17% aa identity in VP1 and T2 proteins respectively (Table 3). However, CORV-MRM1 shows 91.6%/75.9% aa/nt identity and 91.7%/77.6% aa/nt identity to CMPV in its T2 and T13 protein/gene sequences, indicating that they belong to the same virus species and therefore that CMPV does not represent a new species as previously suggested [5].

Phylogenetic analyses show that although the size of the orbivirus sub-core-shell T2 protein is relatively constant across the genus (between 890 aa in SCRV to 953 aa in CORV), the size of the larger of the two outer coat proteins (OC1) is much more variable (between 551 aa in GIV to 1056 aa in AHSV). This results in a change in relative size, order and numbering of the genome segments encoding the T2 and OC1 proteins, between the tick-associated/transmitted orbiviruses [VP2(T2) and VP4(OC1) - encoded by Seg-2 and Seg-4], the MBOs [VP2(T2) and OC1(VP3) - encoded by Seg-2 and Seg-3 respectively], and the CBOs [VP3(T2) and VP2(OC1) - encoded Seg-3 and Seg-2 respectively] [1,23,34,39,40,56].

Previous studies have suggested that the MBOs have evolved from tick-borne ancestors, with CBOs being last to evolve [39]. The concatermerisation of orbivirus genome segments and subsequent mutations may provide a mechanism that can progressively increase the size of individual genome segments [39,56]. It may therefore be significant that the size of OC1 increases in the order: TBOs (551 aa in GIV and 654 aa in SCRV), MBOs (755 aa in CORV to 881 aa in PHSV) and CBOs (961 aa in BTV to 1056 aa in AHSV-1), (Table 2).

Previous studies also indicate that the orbiviruses have evolved through a process of 'co-speciation' with their vectors [39]. Phylogenetic analyses of the conserved Pol, T2 and T13 proteins (presented here - Fig. 2, 3 and 4), show consistent grouping of the CBOs, MBOs and TBOs. In each case CORV-MRM1 groups with the other MBOs (WGRV, UMAV, PHSV and YUOV).

Corriparta viruses have been isolated in Australia, Africa and South America [11]. However, the data presented here clearly identify CMPV, which was isolated in North America [5], as a member of the species *Corriparta virus*. The occurrence of these closely related viruses in the Americas and Australia indicates that there has been spread of viruses between these regions, which could be due to movement of infected hosts or vectors. Similar movements are also suggested by the detection of other orbiviruses (UMAV and PHSV, and individual serotypes of BTV and EHDV) in more than one continent, e.g, in Australia, Africa and the Americas [1,28,57–59]. Additional strains of each serogroup/ species, from different locations/origins, need to be isolated and characterised/sequenced to better understand their geographical distribution and its significance.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Unrooted neighbour-joining for orbivirus polymerase VP1(Pol) proteins. An unrooted NJ phylogenetic tree for orbivirus VP1(Pol) proteins was constructed using a p-distance algorithm and pairwise deletion parameters, as indicated in Figure 1. The CORV-MRM1 isolate characterised in this study is indicated in red font in amber coloured circle. Full names of virus isolates and accession numbers of polymerase sequences used for comparative analysis are listed in Table S1 (supplementary data). 'e' and 'w' after serotype number indicate eastern and western topotype strains, respectively.</figcaption></figure>

doi:10.1371/journal.pone.0070779.g003

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Unrooted neighbour-joining for orbivirus outer-core VP7(T13) proteins. An unrooted NJ phylogenetic tree for orbivirus VP7(T13) proteins was constructed using a p-distance algorithm and pairwise deletion parameters, as indicated in Figure 1. The CORV-MRM1 and CMPV isolates are shown in red font in amber coloured circle. Full names of virus isolates and accession numbers of T13 protein sequences used for comparative analysis are listed in Table S1 (supplementary data). 'e' and 'w' after serotype number indicate eastern and western strains, respectively.</figcaption></figure>
The sequences and relative sizes of the VP1(Pol), T2 and OC1 proteins are important evolutionary markers that can help differentiate/group orbiviruses by species, serotypes and topotype. The sequence data generated in this study will facilitate the use of phylogenetic analyses to identify other novel isolates belonging to the *Corriparta virus* species, as well as helping to identify the arthropod vectors involved in their transmission. Further studies are still needed to define the different serotype and topotypes of CORV.

## Materials and Methods

### Virus propagation

CORV-MRM1 (AUS1960/01), obtained at passage level MB6/BHK2 from the Orbivirus Reference Collection at The Pirbright Institute, was propagated in BHK-21 cell monolayers [clone 13 obtained from European Collection of Animal cell Cultures (ECACC - 84100501)], 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 at 37°C until they showed widespread (100%) cytopathic effects (CPE). Then viruses were harvested, aliquoted and used for the extraction of viral dsRNA. All virus isolates used in these studies were obtained from diagnostic samples of naturally infected animals and were taken as a part of normal diagnostic investigations by qualified veterinarians in the individual countries.

### Extraction and purification of CORV dsRNA

Cell monolayers showing 100% CPE after infection with CORV-MRM1, were harvested and pelleted at 3000 g for 5 min. The viral dsRNA was released and purified using TRIzol® reagent (Invitrogen) as described by Attoui *et al* [60]. Briefly, the infected cell pellet was lysed in 1 ml of TRIzol®, then 0.2 volume of choloroform was added, vortexed and the mixture incubated on ice for 10 min. The aqueous phase, containing total RNA, was separated from the phenol-chloroform phase by centrifugation at 10,000 g for 10 min before 900 µl of isopropanol was added prior to incubation at -20°C for 2 hours. The RNA was pelleted at 10,000 g for 10 min, washed with 70% ethanol, air dried and dissolved in 100 µl of nuclease free water (NFW). Single stranded RNA (ssRNA) was removed by precipitation with 2M LiCl (Sigma) at 4°C overnight, followed by centrifugation at 10,000 g for 5 min. An equal volume of isopropanol, containing 750 mM ammonium acetate, was mixed with the supernatant. After precipitation at -20°C for 2 hours, the viral dsRNA was pelleted at 10,000 g for 10 min, washed with 70% ethanol, air dried and suspended in 50 µl of NFW. The RNA was either used immediately or stored at -20°C.

### Reverse transcription and PCR amplification

CORV-MRM1 genome segments were reverse-transcribed into cDNA using the full-length amplification (FLAC) technique described by Maan *et al*. [61]. Briefly, a 35 base oligonucleotide 'anchor-primer', with a phosphorylated 5' terminus, was ligated to the 3' ends of the viral dsRNAs using the T4 RNA ligase overnight at 16°C. Then dsRNA segments were fractionated on 1% agarose gel and recovered from the gel using a 'silica binding' method (RNaid® kit, MP Biomedicals) as per the manufacturer's

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instructions. The dsRNA eluted in NFW, was denatured at 99°C for 5 minutes, and then snap chilled on ice before synthesising first-strand cDNA using RT system (Promega). The resulting cDNAs were amplified using primers complementary to the anchor primer and high fidelity KOD polymerase enzyme (Novagen). PCR amplicons were analyzed by agarose gel electrophoresis.

### Cloning and sequencing of cDNA segments

cDNA amplicons were purified and cloned into the 'pCR®-Blunt' vector supplied with the Zero Blunt® PCR Cloning Kit (Invitrogen). Recombinant plasmid-vectors containing CORV-MRM1 inserts were transformed into One Shot® TOP10 competent cells supplied with the cloning kit. Clones containing the desired inserts were identified by colony 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).

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## Supporting Information

<caption>**Table S1 Nucleotide accession numbers for sequences used in phylogenetic analysis.**
(DOCX)</caption>

### Acknowledgments

The authors wish to acknowledge support from international colleagues who provided virus isolates for analyses and comparisons.

### Author Contributions

Conceived and designed the experiments: MB SM NSM HA PPCM. Performed the experiments: MB SM NSM KN MG. Analyzed the data: MB SM NSM HA. Contributed reagents/materials/analysis tools: MB SM RT JB PPCM. Wrote the paper: MB SM PPCM.

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