Journal of General Virology (2009), 90, 1433–1439 DOI 10.1099/vir.0.010074-0 Genetic and epidemiological characterization of Stretch Lagoon orbivirus, a novel orbivirus isolated from Culex and Aedes mosquitoes in northern Australia Chris Cowled,1 Gustavo Palacios,2 Lorna Melville,3 Richard Weir,3 Susan Walsh,3 Steven Davis,3 Aneta Gubala,1 W. Ian Lipkin,2 Thomas Briese2 and David Boyle1 Correspondence 1CSIRO Livestock Industries, Australian Animal Health Laboratory, East Geelong, VIC 3220, Chris Cowled Australia [email protected] 2Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, USA 3Northern Territory Department of Primary Industries, Fisheries and Mines, Berrimah Veterinary Laboratories, Berrimah, Northern Territory 0801, Australia Stretch Lagoon orbivirus (SLOV) was isolated in 2002 from pooled Culex annulirostris mosquitoes collected at Stretch Lagoon, near the Wolfe Creek national park in the Kimberley region of Western Australia. Conventional serological tests were unable to identify the isolate, and electron microscopy indicated a virus of the genus Orbivirus, family Reoviridae. Here, a cDNA subtraction method was used to obtain approximately one-third of the viral genome, and further sequencing was performed to complete the sequences of segment 1 (viral polymerase) and segment 2 (conserved inner-core protein). Phylogenetic analysis showed that SLOV should be considered a new species within the genus Orbivirus. A real-time RT-PCR test was designed to study the epidemiology of SLOV in the field. Six additional isolates of SLOV were identified, Received 4 January 2009 including isolates from four additional locations and two additional mosquito species. Horses, Accepted 8 March 2009 donkeys and goats were implicated as potential vertebrate hosts in a serological survey. INTRODUCTION majority of unidentified isolates examined to date have had features consistent with members of the family Northern Australia has long been recognized as a hotspot Reoviridae (genus Orbivirus). One of these, K49460, was for arbovirus activity. The region is prone to regular isolated in 2002 from a pool of 25 Culex annulirostris cyclones that can carry insects over large distances, u 9 u 9 potentially providing a route for exotic arbovirus incur- mosquitoes collected at Stretch Lagoon (19 40 S, 127 35 sions (St George, 1992; St George et al., 2001). To address E) near the Wolfe Creek national park in the Kimberley the risks posed by arboviruses to human and animal health region of Western Australia. K49460 was initially examined in Australia, arbovirus surveillance is carried out by a at the Arbovirus Surveillance and Research Laboratory at network of regional diagnostic laboratories using The University of Western Australia, using a panel of approaches that include insect trapping and maintenance antibodies raised against all known Australian members of sentinel animals for virus isolation and serology of the genera Alphavirus and Flavivirus (Broom et al., (National Arbovirus Monitoring Program, 2008). Viruses 1998). It was then examined further at the Berrimah from several recognized insect-transmitted virus families Veterinary Laboratories in the Northern Territory using a are regularly isolated in northern Australia. These are large panel of antisera raised against veterinary arboviruses, members of the families Togaviridae (genus Alphavirus), including all of the recognized Australian orbiviruses, as Flaviviridae, Bunyaviridae, Rhabdoviridae and Reoviridae well as the Changuinola, Kemerovo and Japanaut orbi- (genus Orbivirus). Since arbovirus monitoring began in viruses. No identification was obtained in any of these tests. Australia, a large collection of unidentified isolates has accumulated, despite extensive diagnostic testing. The The most recent longitudinal study of arboviruses in mosquitoes in the Northern Territory covered the period The GenBank/EMBL/DDBJ accession numbers for the sequences of 1982–1992 (Weir et al., 1997). Of the nine species of SLOV segments 1 and 2 are EU718676 and EU718677. orbivirus that were known in Australia during that study, 010074 G 2009 CSIRO Printed in Great Britain 1433 C. Cowled and others members of the orbivirus species Wallal virus, Warrego Lagoon orbivirus, bringing the total number of orbivirus virus, Wongorr virus, Eubenangee virus, Corriparta virus and species described in Australia to 12. Bluetongue virus were isolated from mosquitoes on multiple occasions, and CSIRO Village virus, a strain of the species Palyam virus was isolated on one occasion. The METHODS tentative orbivirus Lake Clarendon virus has not been Virus isolation and culture. The majority of viruses examined in isolated at all since its initial discovery in 1984, when it was this study were isolated by staff at the Arbovirus Surveillance and isolated from a bird-feeding tick in southeast Queensland Research Laboratory at The University of Western Australia using (St George et al., 1984). In summary, the only orbivirus published methods (Broom et al., 1989; Johansen et al., 2000; Lindsay known to be in circulation in northern Australia during the et al., 1993). DPP5368 was isolated at the Berrimah Veterinary study period that was not isolated from a mosquito species Laboratories (Northern Territory), and CSIRO1747 was isolated at was epizootic hemorrhagic disease virus (EHDV) (Weir CSIRO, Brisbane. Viruses were cultivated in vitro using BSR cells (a et al., 1997). Two other Australian orbiviruses have not subclone of the hamster cell line BHK-21), which were grown at 37 C in cell-culture flasks containing Eagle’s basal medium (pH 7.2), been isolated from mosquitoes or any other arthropod to u [ supplemented with 10 mM HEPES, 6.7 mM NaHCO3,2mML- date: Middle Point orbivirus MPOV; Cowled et al., 2007; glutamine, 80 U penicillin ml21, 137 mM streptomycin and 5 % fetal related to the tentative orbivirus Yunnan orbivirus (YUOV), calf serum. isolated in China] and Elsey virus [related to the tentative orbivirus Peruvian horse sickness virus (PHSV), isolated in Electron microscopy. Infected BSR cells were scraped from the Peru]. However, these viruses have only been described culture surface and pelleted in a bench-top centrifuge (2000 g) for recently, and YUOV and PHSV were isolated from 5 min. The supernatant was adsorbed onto Parlodion-filmed, carbon- coated copper grids for 5 min and stained with 2 % phosphotungstic mosquitoes in China and Peru, respectively (Attoui et al., acid for 1 min for negative-contrast electron microscopy. The cell 2005; GenBank accession nos NC_007748–NC_007757). pellet was processed for thin-section electron microscopy as described Culicoides species are the main vectors for many of the previously (Cowled et al., 2007). Cell pellet thin sections and above-mentioned orbiviruses [including bluetongue virus supernatants were examined under a Hitachi H7000 transmission (BTV)], but the combined evidence suggests that the electron microscope at 75 kV. majority of Australian orbiviruses can also infect mosqui- toes. In the absence of vector-competency studies, it is not RNA extraction. Total RNA was purified from tissue culture supernatant harvested during the late stage of cytopathic effect. known whether mosquitoes are true vectors of many of these Supernatants were decanted from flasks and centrifuged at 300 g for orbiviruses (i.e. capable of transmitting viruses to new 10 min at 4 uC to pellet cell debris. Clarified supernatants were vertebrate hosts) or dead-end hosts. centrifuged at 100 000 g for 90 min at 4 uC to pellet virions. RNA was then extracted using an RNeasy Mini kit (Qiagen) following the The International Committee for Virus Taxonomy (ICTV) manufacturer’s instructions and quantified using a GeneQuant II list of criteria for defining orbivirus species is polythetic, DNA/RNA Calculator (Pharmacia). meaning that some but not all of the criteria need to be fulfilled in order for an isolate to qualify as a new species PCR-Select suppression subtractive hybridization Total Stretch (Mertens et al., 2005). Attoui et al. (2001) found that all Lagoon orbivirus (SLOV) RNA from tissue culture supernatant (the orbiviruses had .30 % identity in the amino acid sequence ‘tester’) was analysed using a PCR-Select kit (Clontech) following the of the viral RNA polymerase (VP1). They also found that manufacturer’s instructions. For the ‘driver’, we used total RNA from BTV-1 (Australia) cultivated in BSR cells. Briefly, RNA from tester orbiviruses within a single species group have .91 % and driver samples was converted into double-stranded cDNA and identity in the amino acid sequence of the inner core digested with RsaI. The tester cDNA was divided into two portions protein (T2). The genetic sequences of VP1 and T2 can and each portion was 59-labelled with one of two oligonucleotide therefore be used to define a new species within the genus adaptors. A sequence of hybridizations was then performed, yielding a Orbivirus. In several orbivirus species (including mixture in which fragments of sequence unique to the tester formed Bluetongue virus), T2 is encoded on genome segment 3 double-stranded cDNA hybrids with a different adaptor at each end, and is called VP3. In other species, it is encoded on permitting PCR amplification using the Advantage II kit (Clontech) to generate a cDNA library highly enriched for fragments of the SLOV segment 2 and is called VP2. Based on the ICTV criteria, genome (Diatchenko et al., 1996). The cDNA synthesis procedure was there are 22 recognized species and ten tentative species of modified for double-stranded RNA as follows: 2 mg RNA was orbiviruses (Mertens et al., 2005). Of these, genetic combined with 0.5 ml random-hexamer oligonucleotides (1.5 mg sequence data are available from 14 species. Full genome ml21; GeneWorks) and 0.5 ml formamide in a total volume of 5 ml. sequences are available for only six of them. For the This mixture was denatured at 100 uC for 1 min in a heat block, orbiviruses that have been only partially sequenced, data chilled rapidly on ice and then used in a 10 ml reverse transcription reaction following the PCR-Select kit instructions.