A New Orbivirus Isolated from Mosquitoes in North-Western Australia Shows Antigenic and Genetic Similarity to Corriparta Virus but Does Not Replicate in Vertebrate Cells

The discovery and characterisation of new mosquito-borne viruses provides valuable information on the biodiversity of vector-borne viruses and important insights into their evolution. In this study, a broad-spectrum virus screening system, based on the detection of long double-stranded RNA in inoculated cell cultures, was used to investigate the presence of novel viruses in mosquito populations of northern Australia. We detected and isolated a new virus (tentatively named Parry’s Lagoon virus, PLV) from Culex annulirostris, Culex pullus, Mansonia uniformis and Aedes normanensis mosquitoes that shares genomic sequence similarities to Corriparta virus (CORV), a member of the Orbivirus genus of the family Reoviridae. Despite moderate to high (72.2% to 92.2%) amino acid identity across all proteins when compared to CORV, and demonstration of antigenic relatedness, PLV did not replicate in several vertebrate cell lines that were permissive to CORV. This striking phenotypic difference suggests that PLV has evolved to have a very restricted host range, indicative of a mosquito-only life cycle.


Introduction
Viruses of the Reoviridae family are icosahedral, non-enveloped and consist of a 9-12 segmented double-stranded RNA (dsRNA) genome [1]. The family consists of two clear subfamilies, the Spinareovirinae ("spiked" core particles) and the Sedoreovirinae ("spikes" absent on core particles). Reoviruses are separated into 16 distinct genera [1]. In addition to infecting plants, reoviruses are

Mosquito Collection and Initial Pool Screening
The study sites described in this paper have previously been described in detail [13,14]. Adult mosquitoes were collected in the Kimberley region of northern Western Australia [15] using methods already described [16]. Mosquitoes were homogenised [17] in pools of up to 25 mosquitoes. Homogenates were inoculated onto 96 well monolayers of C6/36 cells and subsequently passaged onto additional 96 well monolayers of C6/36, Vero and PS-EK (porcine stable-equine kidney) cells as described elsewhere [18]. Monolayers were examined microscopically for evidence of infection, and C6/36 cell monolayers were fixed and assayed by fixed cell ELISA using a panel of flavivirus and alphavirus generic and specific monoclonal antibodies [19].

Parry's Lagoon Virus Detection and Isolation from Mosquito Homogenates
Mosquito pools that were negative for flaviviruses and alphaviruses and did not induce cytopathic effect when inoculated onto vertebrate cell lines (see Section 2.2) were further assessed for the presence of novel viruses using the MAVRIC system developed in our lab [12]. Briefly, virus isolation was performed using mosquito homogenate inoculation onto C6/36 monolayers and incubation at 28˝C for 5-7 days. The culture supernatant was collected and stored at´80˝C, while the cell monolayer was fixed with 20% (v/v) acetone, 0.2% (w/v) BSA in PBS (phosphate buffered saline). For subsequent ELISA analysis, the plates were blocked with 150 µL per well ELISA blocking buffer (0.05 M Tris/HCl (pH 8.0), 1 mM EDTA, 0.15 M NaCl, 0.05% (v/v) Tween 20, 0.2% w/v casein) for 1 h at room temperature before probing with 50 µL/well mAbs 3G1 and 2G4 (anti-dsRNA) diluted in blocking buffer. Following a 1 h incubation at 37˝C, the plates were washed four times with PBS containing 0.05% tween-20 (PBST). HRP-conjugated goat anti-mouse Ig (DAKO) was diluted 1/2000 in blocking buffer (50 µL/well), was added and incubated at 37˝C for 1 h, prior to washing six times with PBST. Finally, 100 µL/well substrate solution [1 mM 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 3 mM H 2 O 2 in a buffer prepared by mixing 0.1 M citric acid with 0.2 M Na 2 HPO 4 to give a pH of 4.2] was added per well and plates were incubated in the dark at room temperature for 1 h. Absorbance was measured at 405 nm.
Samples that tested positive by the MAVRIC ELISA were further analysed. For detection of PLV sequence, RNA was extracted from 150 µL of culture supernatant using a Macherey-Nagel Nucleospin Viral RNA isolation kit as per the manufacturer's instructions (Macherey-Nagel, Bethlehem, PA, USA). Reverse-transcription PCR (RT-PCR) was performed using the primer pair of CORV_like_F (5 1 -TTATCGGCAGACGGGATTCG) and CORV_like_R (5 1 -CGCTTTCGTTAGCACCATCG) and an Invitrogen Superscript III One-step RT-PCR system with Platinum Taq

Virus Culture
Virus stocks were generated by infecting C6/36 monolayers with virus at a multiplicity of infection (MOI) of 0.1. After incubation at 28˝C for 2 h, inoculum was removed and replaced with fresh growth media containing 2% FBS. Supernatant was harvested at 5 dpi and centrifuged at 3000 rpm, 4˝C for 10 min. Clarified supernatant was passed through a 0.45 µM filter, supplemented with additional FBS to increase total concentration to 10% and stored at´80˝C. Virus stock titers were calculated based on the 50% tissue culture infectious dose [20].

Electron Microscopy
Virions were harvested and concentrated via polyethylene glycol precipitation. Following overnight incubation, the resulting pellet was resuspended in TNE buffer (120 mM NaCl, 30 mM H 3 BO 3 , 1% Triton X-100, 0.1% SDS, 5 mM EDTA, pH: 9.0). Virus-infected supernatant was then underlayed with a 20% sucrose cushion before ultracentrifugation at 28,000 rpm for 2 h at 4˝C. The purified virions were harvested and buffer exchanged into PBS prior to loading onto hydrophilic copper grids and negatively stained with a 1% uranyl acetate solution. Grids were viewed using a F30 transmission electron microscope.

Genome Sequencing and Phylogenetic Analysis
Viral RNA was prepared for sequencing by infecting a monolayer of C6/36 cells at a MOI of 0.1. After incubating at 28˝C for 2 h, inoculum was removed and replaced with fresh growth media containing 2% FBS. Virus was harvested 6 dpi by centrifuging supernatant at 3000 rpm, 4˝C for 10 min before passing it through a 0.45 µM filter. Virus-infected supernatant was then mixed in a 1:4 ratio of 40% polyethylene glycol 8000 (PEG 8000) and incubated on a mixing wheel with slow rotation overnight at 4˝C. The concentrated virions were then pelleted by centrifuging at 10,000ˆg, 4˝C for 1 h before resuspending the resulting pellet in sterile PBS. RNA was then extracted using a Macherey-Nagel Nucleospin Viral RNA isolation kit as per the manufacturer's instructions, with the omission of carrier RNA from the lysis buffer. Next generation sequencing was performed by the AGRF (Brisbane, Australia) using the Illumina HiSeq2000 platform. Reads were assembled using Geneious R8 (8.0.5) software and the CORV MRMI isolate genome as a reference (Accession numbers: KC853042-KC853051). PLV open reading frames (ORFs) were identified using the NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and the ORF sequences deposited in Genbank under accession numbers KU724110-KU724119.
Phylogenetic analysis was performed on highly conserved genes encoding VP1 (Pol), VP2 (T2) and VP7 (T13). Nucleotide sequences of the ORFs were aligned with Geneious R8 (v 8.0.5) using the MUSCLE algorithm on 8 iterations, a distance measure of kmer4_6 for the first iteration, and a distance measure of pctid_kimura for subsequent iterations [21]. Phylogenetic trees based on the alignments were constructed in MEGA-7.0.14 [22] using maximum likelihood, a General Time Reversible substitution model, a gamma distribution (5 discrete gamma categories) and invariant rates among sites (determined to be optimal using jModelTest-2.1.6 software [23,24] minimizing deltaAIC). Bootstrap analysis was performed with 1000 replicates, and the trees were rooted using the divergent orbivirus St. Croix River virus (SCRV) as an outgroup.

Serological Cross-Reactivity Studies
IFA analysis was performed using two rabbit antisera generated against CORV (serum B97 raised to an unknown CORV strain and R167 raised to CORV strain CSIRO109) was performed to assess serological cross-reactivity to PLV. Monolayers of C6/36 cells were grown on glass coverslips before inoculating with PLV, CORV or mock-infected at an MOI of 1. Coverslips were cultured for 4 dpi, before fixing in a solution containing 4% formaldehyde and 0.1% triton X-100 and probed using CORV antiserum, or negative rabbit serum (diluted 1/100), using methods previously detailed [25]. RT-PCR was used to confirm that cross-contamination of CORV in PLV samples had not occurred.

Microneutralisation Assay
Microneutralisation assays were performed to assess if antisera raised against CORV (refer to section 2.7) are able to neutralize PLV. Assays were performed as previously detailed [26] utilizing C6/36 cells, titrating the rabbit serum from a 1/10 dilution and using 450-1000 infectious particles, as determined by back-titration. The neutralization titer was taken as the highest dilution that inhibited all CPE.

Vertebrate Cell Infection Assays
IFA was performed with anti-dsRNA mAb, 3G1, to assess the permissiveness of vertebrate cell lines to infection with PLV. Monolayers of C6/36, BHK and Vero cells were grown on glass coverslips before inoculating with PLV, CORV or mock-infected at an MOI of 1 and were cultured for 3 and 9 dpi. Coverslips were fixed in a PBS solution containing 4% formaldehyde and 0.1% Triton-X 100 before being probed with 3G1 as described [12].

Detection, Isolation and Culture of the Prototype PLV Isolate
As part of routine surveillance for arboviruses, mosquitoes were collected in the northern regions of Western Australia in 2010. A subset of Culex annulirostris mosquito pools that were negative for flaviviruses and alphaviruses and did not cause cytopathic effect (CPE) in vertebrate cells (n = 138) were examined for the presence of insect-specific viruses. The pools were screened using a novel ELISA-based system developed in our laboratory for the detection of viral replication in cell culture (named MAVRIC, [12]). Of the 7 pools that were positive using this system, three (numbers K71435, K71497 and K71551; Table 1) displayed significant CPE five days post infection of C6/36 cell monolayers, causing cell monolayer disturbance, vacuolation and loss of uniformity in size and shape of cells ( Figure 1B). An initial round of Ion Torrent sequencing of one isolate (K71551) identified the virus as an orbivirus (Reoviridae family). The short sequence obtained (approximately 850 bp) was of the gene encoding the viral helicase and NS4 proteins and shared a 77% nucleotide and 68% amino acid identity to the prototype isolate of Corriparta virus, strain MRM1 (CORV-MRM1). The new virus was tentatively named Parry's Lagoon virus (PLV), after the region in which the prototype was first identified.

Vertebrate Cell Infection Assays
IFA was performed with anti-dsRNA mAb, 3G1, to assess the permissiveness of vertebrate cell lines to infection with PLV. Monolayers of C6/36, BHK and Vero cells were grown on glass coverslips before inoculating with PLV, CORV or mock-infected at an MOI of 1 and were cultured for 3 and 9 dpi. Coverslips were fixed in a PBS solution containing 4% formaldehyde and 0.1% Triton-X 100 before being probed with 3G1 as described [12].

Detection, Isolation and Culture of the Prototype PLV Isolate
As part of routine surveillance for arboviruses, mosquitoes were collected in the northern regions of Western Australia in 2010. A subset of Culex annulirostris mosquito pools that were negative for flaviviruses and alphaviruses and did not cause cytopathic effect (CPE) in vertebrate cells (n = 138) were examined for the presence of insect-specific viruses. The pools were screened using a novel ELISA-based system developed in our laboratory for the detection of viral replication in cell culture (named MAVRIC, [12]). Of the 7 pools that were positive using this system, three (numbers K71435, K71497 and K71551; Table 1) displayed significant CPE five days post infection of C6/36 cell monolayers, causing cell monolayer disturbance, vacuolation and loss of uniformity in size and shape of cells ( Figure 1B). An initial round of Ion Torrent sequencing of one isolate (K71551) identified the virus as an orbivirus (Reoviridae family). The short sequence obtained (approximately 850 bp) was of the gene encoding the viral helicase and NS4 proteins and shared a 77% nucleotide and 68% amino acid identity to the prototype isolate of Corriparta virus, strain MRM1 (CORV-MRM1). The new virus was tentatively named Parry's Lagoon virus (PLV), after the region in which the prototype was first identified.

PLV Virions Display Typical Reovirus Morphology
When purified PLV virions were examined by transmission electron microscopy, small, icosahedral non-enveloped particles were observed ( Figure 1D,E). This morphology is consistent to that described for other members of the Reoviridae [9,27,28]. However, with an average diameter of 79 nm, the virions are substantially larger than the 60 nm described for CORV [29], but within the size range generally accepted for orbiviruses (60-80 nm) [1] and may be attributed to the different fixation and staining methods used.

Detection of Other PLV Isolates from Mosquitoes Collected in Western Australia
RT-PCR screening of the other MAVRIC-positive pools from the Parry's Creek 2010 cohort using PLV-specific primers, yielded a total of six PLV isolates ( Table 1). Testing of an additional 15 mosquito pools collected from other locations within close proximity to Parry's Creek, including Kununurra (approximately 80 km south-east) and Billiluna (approximately 500 km south) in 2011 that caused extensive CPE upon inoculation onto C6/36 cells and were positive by the MAVRIC ELISA, returned a further eight PLV isolates. In addition to Culex annulirostris, PLV isolates were also obtained from Culex pullus, Mansonia uniformis and Aedes normanensis mosquito species. Sequencing of a 399-nucleotide region of the helicase segment (seg-9, VP6) of each isolate gave a ě97.5% nucleotide identity (Table 1) to the prototype, suggesting that they are likely to be strains of the same virus species.
It was noted that while pool K75749 was positive for PLV, it also induced CPE at passage 3 upon inoculation onto PS-EK and Vero cells. As no other PLV-positive pools induced CPE in vertebrate cells and sequencing of the 399 bp of segment 9 confirmed the presence of PLV, it is likely that an additional virus is present in that pool. RT-PCR using specific primers to two other reoviruses commonly detected in mosquito pools (Stretch Lagoon virus and Liao ning virus) failed to amplify a product. While this pool was negative for flaviviruses and alphaviruses during the initial isolation using immunological methods, these data were confirmed using flavivirus and alphavirus generic primer sets in RT-PCR.

Genome Sequence, Organisation and Phylogenetics
Next generation sequencing was performed on PLV RNA using the Illumina HiSeq 2000 platform and assembled using the CORV-MRM1 genome as a reference sequence. Partial sequence was obtained from 10 genome segments, consistent with the number of segments of an orbivirus genome (Accession numbers KU724110-KU724119). Complete sequence of each ORF was elucidated and further analysis of these ORFs revealed that 8 of the 10 segments (seg-1 to seg-8) consisted of a single ORF while the remaining two segments (seg-9 and seg-10) consisted of two overlapping ORFs, encoding VP6/NS4 and NS3/NS3a respectively, consistent with other orbiviruses. Pairwise alignments between the ORFs of each segment of the genomes of CORV and PLV demonstrated nucleotide identities ranging between 74.7% (VP3; OC1) and 81.2% (NS3/NS3a) and amino acid identities of 72.6% (VP6/NS4) and 95.3% (VP5; OC2) ( Table 2).

Genome Sequence, Organisation and Phylogenetics
Next generation sequencing was performed on PLV RNA using the Illumina HiSeq 2000 platform and assembled using the CORV-MRM1 genome as a reference sequence. Partial sequence was obtained from 10 genome segments, consistent with the number of segments of an orbivirus genome (Accession numbers KU724110-KU724119). Complete sequence of each ORF was elucidated and further analysis of these ORFs revealed that 8 of the 10 segments (seg-1 to seg-8) consisted of a single ORF while the remaining two segments (seg-9 and seg-10) consisted of two overlapping ORFs, encoding VP6/NS4 and NS3/NS3a respectively, consistent with other orbiviruses. Pairwise alignments between the ORFs of each segment of the genomes of CORV and PLV demonstrated nucleotide identities ranging between 74.7% (VP3; OC1) and 81.2% (NS3/NS3a) and amino acid identities of 72.6% (VP6/NS4) and 95.3% (VP5; OC2) ( Table 2).   Phylogenetic trees were constructed using ORFs available for VP1, T2 and T13 proteins. In each case PLV clustered with CORV within the mosquito-associated orbivirus clade (Figure 2), with strong bootstrap support (>95). A separate phylogenetic tree prepared for the partial sequence of T13 was also supportive of inclusion of PLV within the mosquito-associated clade ( Figure S1). PLV and CORV clustered together indicating a closer genetic relationship between these viruses, which was consistent with their higher amino acid identity (Table 3). An amino acid identity for T2 (subcore) of <91% has previously been proposed as a possible value for demarcation of species within the Orbivirus genus [30]. Based on this parameter, a 94.3% and 92% amino acid identity with CORV, respectively, would suggest that both PLV and CMPV are strains of the virus species CORV, as has been previously suggested [11].  Phylogenetic trees were constructed using ORFs available for VP1, T2 and T13 proteins. In each case PLV clustered with CORV within the mosquito-associated orbivirus clade (Figure 2), with strong bootstrap support (>95). A separate phylogenetic tree prepared for the partial sequence of T13 was also supportive of inclusion of PLV within the mosquito-associated clade ( Figure S1). PLV and CORV clustered together indicating a closer genetic relationship between these viruses, which was consistent with their higher amino acid identity (Table 3). An amino acid identity for T2 (subcore) of <91% has previously been proposed as a possible value for demarcation of species within the Orbivirus genus [30]. Based on this parameter, a 94.3% and 92% amino acid identity with CORV, respectively, would suggest that both PLV and CMPV are strains of the virus species CORV, as has been previously suggested [11].

PLV Does Not Replicate in Vertebrate Cells
No CPE was observed in PS-EK cells at the third passage during initial isolations of PLV (Table 1). This contrasted with earlier studies of CORV that indicated it grows readily in porcine cells and causes CPE [29]. To investigate further, other cells of vertebrate origin were assessed for permissiveness to PLV (isolate K71551) and CORV (MRM1) by infecting monolayers of each cell line at an MOI of 1 and fixing after 3 days. IFA analysis was performed using anti-dsRNA mAbs to detect viral genomic and replicative dsRNA in the cytoplasm. We have previously reported the detection of the replicative dsRNA of other reoviruses such as Bluetongue virus and Liao ning virus using this protocol [12]. While PLV replication was clearly detected by the anti-dsRNA mAbs in C6/36 cells, there was no staining of BHK, DF-1 or Vero cells ( Figure 3A) and no CPE observed. In contrast, CORV replicated in both C6/36, BHK and DF-1 cells as indicated by clear anti-dsRNA mAb-binding in IFA and by marked CPE (in the C6/36 and BHK cells) ( Figure 3A). Consistent with previous reports, the replication of CORV was not detected in Vero cells until 9 dpi ( Figure 3B). Cultures of Vero cells inoculated with PLV in parallel were negative by IFA under the same culturing conditions. Together, these data suggest that PLV displays a tropism that is substantially different to CORV and is likely to be restricted to insect cells.

PLV Does Not Replicate in Vertebrate Cells
No CPE was observed in PS-EK cells at the third passage during initial isolations of PLV (Table 1). This contrasted with earlier studies of CORV that indicated it grows readily in porcine cells and causes CPE [29]. To investigate further, other cells of vertebrate origin were assessed for permissiveness to PLV (isolate K71551) and CORV (MRM1) by infecting monolayers of each cell line at an MOI of 1 and fixing after 3 days. IFA analysis was performed using anti-dsRNA mAbs to detect viral genomic and replicative dsRNA in the cytoplasm. We have previously reported the detection of the replicative dsRNA of other reoviruses such as Bluetongue virus and Liao ning virus using this protocol [12]. While PLV replication was clearly detected by the anti-dsRNA mAbs in C6/36 cells, there was no staining of BHK, DF-1 or Vero cells ( Figure 3A) and no CPE observed. In contrast, CORV replicated in both C6/36, BHK and DF-1 cells as indicated by clear anti-dsRNA mAb-binding in IFA and by marked CPE (in the C6/36 and BHK cells) ( Figure 3A). Consistent with previous reports, the replication of CORV was not detected in Vero cells until 9 dpi ( Figure 3B). Cultures of Vero cells inoculated with PLV in parallel were negative by IFA under the same culturing conditions. Together, these data suggest that PLV displays a tropism that is substantially different to CORV and is likely to be restricted to insect cells.   . IFA analysis was performed with anti-dsRNA mAb 3G1 to detect replicating virus via dsRNA products [12]. Cell nuclei were stained with Hoechst 33342. Images were taken at ×40 magnification. . Serological Cross-Reactively Studies. C6/36 cells were inoculated with CORV or PLV at an MOI of 1, or mock-infected and fixed with 4% formaldehyde containing 0.1% triton X-100 after 4 days. IFA was performed by probing with rabbit sera raised against CORV CSIRO109l or naïve rabbit serum (negative) to assess cross-reactivity with PLV. Images were taken using ×40 magnification. . IFA analysis was performed with anti-dsRNA mAb 3G1 to detect replicating virus via dsRNA products [12]. Cell nuclei were stained with Hoechst 33342. Images were taken atˆ40 magnification.  . IFA analysis was performed with anti-dsRNA mAb 3G1 to detect replicating virus via dsRNA products [12]. Cell nuclei were stained with Hoechst 33342. Images were taken at ×40 magnification. Figure 4. Serological Cross-Reactively Studies. C6/36 cells were inoculated with CORV or PLV at an MOI of 1, or mock-infected and fixed with 4% formaldehyde containing 0.1% triton X-100 after 4 days. IFA was performed by probing with rabbit sera raised against CORV CSIRO109l or naïve rabbit serum (negative) to assess cross-reactivity with PLV. Images were taken using ×40 magnification. IFA was performed by probing with rabbit sera raised against CORV CSIRO109l or naïve rabbit serum (negative) to assess cross-reactivity with PLV. Images were taken usingˆ40 magnification.

Corriparta Antiserum Cross-Reacts with and Neutralizes Parry's Lagoon Virus
In order to determine if PLV is antigenically related to CORV, serological cross-reactivity studies and microneutralization assays were performed. Cross-reaction of CORV antiserum with PLV-infected cells in IFA confirmed that PLV shares similar antigenic structure to CORV, although the staining for PLV was not as intense as that for CORV (Figure 4). Similarly, two rabbit antisera produced to different strains of CORV also neutralized PLV-infection of C6/36 cell monolayers, but to different extents (Table 4). A four-fold difference between neutralizing titers of CORV and PLV by antiserum B97 was observed (80 vs. 20 respectively) indicating superior neutralization of CORV by this antiserum. However, when the second antiserum produced to the CORV CSIRO109 strain was assessed, this antiserum neutralized PLV at dilutions of at least 4-fold greater than CORV (PLV >1280 vs. CORV 320) and may suggest subtle antigenic differences even between strains previously assigned to the CORV species. Since no genome sequencing of CORV CSIRO109 is available to elucidate its genetic relatedness to PLV, it must be highlighted that CORV CSIRO109 was isolated using BHK [6] cells and thus does not display restricted host range. As expected, a negative control rabbit antiserum did not neutralize either virus.

Discussion
The Orbivirus genus is the largest of the Reoviridae family. These viruses are able to infect a wide range of arthropod and vertebrate species. During an investigation to determine the prevalence and biodiversity of insect-specific viruses in mosquitoes of northern Western Australia, a novel orbivirus was identified in pools of Culex annulirostris, Culex pullus, Mansonia uniformis and Aedes normanensis mosquitoes collected in 2010 and 2011 that had previously been screened for vertebrate-infecting viruses. PLV was genetically closely related to Corriparta virus (CORV), a virus that has also been isolated from mosquitoes in this region [8] and from mosquitoes and birds in other regions of northern Australia [31]. Despite high amino acid sequence identities over the polymerase, capsid and core proteins (86% (Outer capsid protein 1) to 95% (Outer capsid protein 2)) between PLV and CORV, PLV did not replicate in Vero, BHK or DF-1 cells. This contrasts with the efficient replication of CORV in each of these cell lines.
An amino acid identity for T2 of <91% has previously been proposed as a possible value for demarcation of species within the Orbivirus genus [30]. If this criterion alone is taken into consideration, a 94.3% amino acid identity for the T2 inner core protein between PLV and CORV-MRM1 would suggest that PLV should be included in the CORV species. Traditionally, orbiviruses were classified based on complement-fixation tests, group-specific ELISAs or agar-gel-immuno-diffusion tests [1,32]. Using these serology-based methods, viruses designated to the Corriparta serogroup include other viruses similarly isolated from Culex mosquito species, including CORV-MRM1 (Australia), Acado virus (Ethiopia) and Jacareacanga virus (Brazil). Isolates have also been identified in Aedeomyia catastica mosquitoes captured from the Ord River region of northern Western Australia (approximately 70 km from the Parry's Creek region) between 1972 and 1976 [8]. CMPV was similarly isolated from Culex mosquitoes (Culex tarsalis) in North America, but has been proposed to become a member of the CORV species based on genetic sequence analysis [11]. Indeed, our own serological assessment of PLV with CORV antiserum confirms that PLV is antigenically related to CORV, but with neutralization values of 4 fold or greater between the two viruses. The current lack of full-genome sequencing data for other CORV serotypes apart from the prototype strain (and now for PLV) precludes accurate identification and classification of new serotypes as part of the CORV species or serogroup. Furthermore, with the ability of segmented viruses to reassort, taxonomic demarcation based purely on one segment may be insufficient. Indeed, studies have demonstrated extensive reassortment within the CORV serocomplex members [33]. To ensure more accurate taxonomic classification of PLV, future studies will also focus on the elucidation of the conserved terminal nucleotides of each genome segment, as these termini are often conserved within a single species [1].
Primary isolations of CORV were historically performed through inoculation of mosquito homogenate in suckling mouse brain [31]. However, with the advent of cell-culture based systems, more recent CORV isolates were identified through the inoculation of mosquito homogenate onto BHK or BSR cell monolayers and subsequent visualization of CPE [6,34]. In this context, the inability of PLV to replicate in vertebrate cells precluded its discovery using standard surveillance methods and highlights the utility of virus detection through the use of monoclonal antibodies to dsRNA [12].
While Australian CORV isolates have been derived from mosquitoes of both Culex and Aedeomyia genera, considering the proposed insect-specific tropism of PLV, it was interesting that PLV was also isolated from multiple mosquito genera. Given that vertical transmission has either been proposed or demonstrated for other insect-specific viruses [35][36][37], the presence of a virus in multiple mosquito genera is not consistent with mosquito-specific tropism. However, the isolation of other species of insect-specific viruses from mosquitoes of various genera has also been reported for viruses of other virus families, such as the Bunyaviridae [38,39]. Further studies assessing the growth of PLV in a broader range of vertebrate cell lines and mice will also assist in determining whether cryptic vertebrate hosts could be involved in the maintenance and transmission of PLV between mosquito species. Indeed, other reoviruses such as Yunnan orbivirus, Eyach virus, Cimodo virus, Fako virus and Aedes psuedoscutellaris reovirus similarly did not replicate in vertebrate cells in vitro during initial investigations, including Vero and BHKs [27,28,[40][41][42]. However, both Yunnan orbivirus and Eyach virus were capable of infecting mice (10 week old intraperitoneal inoculation and suckling mice intracranial inoculation respectively) [27,41]. Members of the Reoviridae family also participate in a transmission cycle between insect hosts such as planthoppers or leafhoppers and the plants on which these insects feed on [43]. Hence, the ability of PLV to replicate in plants should also be considered, especially since mosquitoes feed on the nectar of plants as a sugar source. It is likewise plausible that the virus may be transmitted in the aquatic environment during the larval feeding stage. In any case, further investigations should be carried out to elucidate the mechanisms for host range restriction and persistence of PLV in nature.
The striking difference in cell tropism between PLV and CORV, despite the close genetic relatedness was unexpected. However, dramatic shifts in host cell tropism between closely related mosquito-borne viruses has been observed previously. A notable example is the lack of growth of Rabensburg virus (RaBV-a tentative lineage of the West Nile species complex) in vertebrate cells at 37˝C [44,45]. While other West Nile virus strains replicate efficiently in a range of vertebrate cell lines, RaBV failed to replicate significantly in vertebrate cells when incubated at 37˝C with growth only apparent when incubated at 34˝C or below [45]. Suggestions that RaBV may have adapted to a transmission cycle without vertebrate hosts or used hosts with lower body temperatures, such as reptiles or amphibians are similarly applicable hypotheses for PLV. Future studies investigating the inability of PLV to infect vertebrate cells should consider temperature sensitivity as a possible restriction factor in its host tropism.

Conclusions
In summary, we have identified and characterised a new virus (PLV), belonging to the Reoviridae family. Analysis of highly conserved segments as well as serological cross-reactivity and neutralization analyses indicate that PLV belongs to the Corriparta serocomplex. However, in a fundamental difference to CORV, PLV failed to replicate in the vertebrate cell lines tested here, which may warrant its classification as a new species within the serocomplex. While these findings do suggest that PLV represents the first isolation and characterisation of an insect-specific member of the Orbivirus genus discovered in Australia, future in vivo and in vitro studies are warranted to confirm a mosquito-only life cycle. The discovery and characterisation of additional mosquito-associated orbiviruses will provide valuable insights into genetic divergence, extending and enhancing our understanding of the mosquito virome.