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Characterization of Fitzroy River virus and serologic evidence of human and animal infection.

In the state of Western Australia, Australia, active surveillance is conducted for mosquitoborne viruses of major human health significance: alphaviruses Ross River virus (RRV) and Barmah Forest virus (BFV) and flaviviruses Murray Valley encephalitis virus (MVEV) and West Nile virus (subtype Kunjin virus; KUNV). These flaviviruses are endemic and epidemic to the northern and central areas of Australia, where surveillance involves year-round testing for seroconversions in sentinel chickens (1) and virus isolation from mosquito pools collected annually (2,3). More frequent mosquito collection is prevented by the logistical difficulties of accessing remote areas. Commonly isolated arboviruses include the flaviviruses MVEV (and subtype Alfuy virus), KUNV, Kokobera virus (KOKV), and Edge Hill virus (EHV) and the alphaviruses RRV, BFV, and Sindbis virus (4,5). This system occasionally detects viruses that cannot be identified as known viruses, such as Stretch Lagoon virus, an orbivirus isolated in 2002 (6). We describe the detection and characterization of a novel flavivirus named Fitzroy River virus (FRV), isolated from mosquitoes collected in northern Western Australia, and seroepidemiologic evidence of human or animal infection.

Methods

Adult Mosquito Collections

Adult mosquitoes were collected during March and April 2010-2015, at the end of the summer wet season across the Kimberley region of Australia (2) (Figure 1). Mosquitoes were collected in encephalitis vector surveillance traps (7) baited with carbon dioxide and were separated by species and pooled (8-10); blood-fed mosquitoes were excluded from analysis.

Virus Isolation and Identification

Virus isolation from all mosquito pools was performed as previously described (10). In brief, mosquito pools were homogenized and serially passaged from C6/36 (Aedes albopictus mosquito) cells onto Vero (African green monkey kidney) and PSEK (porcine squamous equine kidney) cells. PSEK cells were later replaced by BSR (baby hamster kidney) cells. Viruses were detected and identified by use of microscopy and monoclonal antibody (mAb) binding patterns in ELISA. For flavivirus-reactive samples, a flavivirus group-reactive 1-step reverse transcription PCR assay (Invitrogen, Carlsbad, CA, USA) (11) was used to amplify a 0.6-kb fragment of the nonstructural protein 5 (NS5) and 3' untranslated region (3' UTR) for PCR and sequence confirmation.

Whole-Genome Sequencing

RNA from viral stock (the prototype isolate K73884) was extracted with TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and sequenced on a HiSeq ultra high-throughput sequencing platform (Illumina, San Diego, CA, USA). Trimmed reads were assessed for quality by using PRINSEQ version 0.20.2 (12) before host (metazoan and mosquito) genome subtraction (Bowtie 2) (13) and assembly (MIRA version 4.0) (14). Resulting contiguous sequences and unique singletons were subjected to homology search by using MegaBLAST and blastx (https://blast. ncbi.nlm.nih.gov/Blast.cgi) against the GenBank database. Sequences that were similar to viruses from the yellow fever virus (YFV) group, a monophyletic branch that previously included 3 viruses (Wesselsbron virus [WESSV], Sepik virus [SEPV], and YFV) (15) were manually edited and reassembled by using Geneious version 7.1.5 (16). The complete genome was resequenced by using overlapping PCR and confirmed by bidirectional Sanger sequencing. The sequences for the 5' and 3' UTRs were acquired by using the SMARTer RACE cDNA amplification kit (Takara Bio USA, Mountain View, CA, USA).

Phylogenetic and Recombination Analyses

Nucleotide sequences for the complete polyproteins representing 44 mosquitoborne and tickborne flaviviruses, as well as those that are insect specific or have no known vector, were retrieved from GenBank. Alignments with FRV were performed by using MUSCLE in Geneious version 7.1.5, and a maximum-likelihood phylogenetic tree was constructed by using the general time reversible plus gamma distribution site model of nucleotide substitution with 500 bootstrap replicates (MEGA version 7.0.16) (17). Tamana bat virus was used as the outgroup. For alignments of cleavage recognition sequences from members of the YFV group, previously established sites were identified and aligned (18,19). To assess whether FRV was a recombinant virus, we analyzed polyprotein sequence alignments (as described above) by using default parameters for RDP, GENECONV, BootScan, MaxChi, Chimaera, SisScan, 3SEQ, and Phylpro methods available in the RDP4 program suite (20).

Virus Growth Kinetics in Vitro

Virus replication was assessed in mosquito (C6/36), mammalian (Vero and BSR), and avian (DF-1) cells (21) by using a multiplicity of infection of 0.1 in 2% fetal bovine serum in M199 (C6/36 cells) or DMEM (Vero, BSR, and DF-1 cells). After 1 hour of incubation at 28[degrees]C (C6/36 cells) or 37[degrees]C (Vero, BSR, and DF-1 cells), the inoculum was removed and monolayers were washed before addition of 1 mL of media. Plates were incubated; monolayers examined for cytopathic effect (CPE); and samples removed in triplicate at 0, 1, 2, 3, 4, and 7 days postinoculation (dpi) and stored at -80[degrees]C. The 50% tissue culture infectious dose in each sample was determined by serial dilutions and titration in BSR cells in 96-well tissue culture plates, and titers were calculated (22).

Determination of Virulence in Mice

All procedures using animals were approved by The University of Queensland Animal Ethics Committee. Groups of 10 mice (18-19-day weanling CD1 mice, equal numbers of each sex) were challenged by intraperitoneal injection of 50 [micro]L or intracranial injection of 20 [micro]L of either 100 or 1,000 50% tissue culture dose infectious units (IU) of FRV. Groups of 3 mice were mock challenged intraperitoneally or intracranially. Veterinarians monitored the mice twice daily for 19 days and then daily through 21 dpi (23,24). At 21 dpi, all mice were deeply anesthetized, bled by cardiac puncture, and killed by cervical dislocation. No animals required euthanasia during the experiment. FRV-specific antibodies were detected by fixed-cell ELISA (25), and the brains of mice with mild clinical signs were fixed in 10% neutral-buffered formaldehyde and processed for histopathologic and immunohistochemical examination (23,24).

Serologic Surveys

Human serologic studies were performed with approval from The University of Western Australia Human Ethics Committee. We used human serum samples that were submitted to PathWest Laboratory Medicine WA (Nedlands, Western Australia, Australia) for arbovirus serologic testing and that were positive by flavivirus hemagglutination inhibition assay. We also used serum samples that were submitted for alphavirus testing only (RRV and BFV) and that were serologically negative. Deidentified information about patient age, sex, ZIP code, and test results were provided. Samples from residents of regions in northern Western Australia where FRV had been detected in mosquitoes or where Aedes normanensis mosquitoes are abundant were targeted for this survey. Samples were tested in a flavivirus epitope blocking ELISA that used mAb 3H6 (26). Serum containing flavivirus antibodies in ELISA were subsequently tested by serum cross-neutralization assay for antibodies to FRV, MVEV, KUNV, Alfuy virus, KOKV, Stratford virus, and EHV as described previously (27). As controls, we used polyclonal rabbit or mouse serum previously raised to these viruses.

Animal serologic studies were approved by The Charles Darwin University Animal Ethics Committee. Animal serum (sentinel cattle, horses, chickens, and wallabies) from the Northern Territory of Australia was tested for antibodies to FRV by neutralization tests (28) without prior testing by flavivirus epitope blocking ELISA. Cross-neutralizations with SEPV were conducted on a subset of positive samples to confirm antibody specificity.

Results

Viruses and mAb Binding Patterns

We saw little or no visual evidence of infection of C6/36 cell monolayers during the isolation of FRV, and it grew slowly in Vero cells. Cytopathic evidence of infection was most marked in PSEK and BSR cells. Isolates were initially typed by their mAb binding profile against a panel of flavivirus- and alphavirus-reactive mAbs in fixed-cell ELISA. All isolates of FRV reacted with the flavivirus-reactive mAb 4G2 but failed to react with other flavivirus- and alphavirus-reactive mAbs (data not shown). Preliminary analyses of the nucleotide sequence of the NS5-3' UTR showed 75%-80% identity to SEPV and WESSV, the viruses most closely related to YFV (15,19,29,30). Identity between all FRV isolates in the NS5-3' UTR was 98.9%100%. The mAb binding profile differed from EHV and SEPV, and positive reactions to FRV were detected with mAbs 4G2 and 4G4 only (Table 1).

Whole-Genome Sequences and Phylogeny

Unbiased high-throughput sequencing results provided >99% of the FRV genome with only partial UTRs not obtained. The completed full-genome length of FRV was 10,807 nt with a single 10,218-nt open reading frame flanked by a 117-nt 5' UTR and a 472-nt 3' UTR (Table 2; online Technical Appendix Figure, panel A, https://www.nc.cdc. gov/EID/article/23/8/16-1440-Techapp1.pdf) and has been deposited in GenBank under accession no. KM361634. At the amino acid level, FRV was more similar to SEPV than to WESSV in most regions, with the exception of the short 2k peptide (70% vs. 91% aa homology), NS4a (87% vs. 92% aa homology), and NS5 (88% vs. 89% aa homology). Because there was a sharp change in amino acid homology between FRV and SEPV across the 2k peptide (70%) and NS4b (94%), we assessed aligned polyprotein sequences for recombination breakpoints by using 8 algorithms in the RDP4 suite, but we found no evidence suggesting that recombination had occurred (data not shown).

FRV was highly similar to SEPV across the structural viral proteins, including the membrane (96%) and envelope (96%) proteins. Over the full genome, FRV displayed the highest nucleotide identity to SEPV (79%), WESSV (77%), and YFV (62%). These lower nucleotide identities are in contrast to the polyprotein amino acid homologies (SEPV 91%, WESSV 89%, YFV 61%), indicating that a large proportion of nucleotide differences between FRV and SEPV or WESSV were synonymous. The level of nucleotide identity was higher in the 5' and 3' UTRs (94% and 86%, respectively) than in structural proteins (up to 81%) for FRV and SEPV, reflecting the functional importance of the UTRs for virus replication. Closer analysis of several conserved features of flavivirus UTRs is shown in the online Technical Appendix Figure. Although comparisons of the cyclization sequences (online Technical Appendix Figure, panel C) indicate a high level of conservation among all members of the YFV group, alignments of the upstream AUG region (online Technical Appendix Figure, panel D) highlight a clear separation of FRV, SEPV, and WESSV from YFV. When we assessed the string of tandem repeats in the 3' UTR, some differences between FRV, SEPV, and WESSV emerged. We identified 3 highly conserved repeats in the 3' UTR of FRV (RFR1, RFR2, and RFR3; online Technical Appendix Figure, panel B), which are most similar to the previously described sequences identified in YFV (RYF1, RYF2, and RYF3). Strikingly, WESSV (69.7%-90.6%) and FRV (72.7%-90.6%) retained homologous sequences to RYF1, RYF2, and RYF3, and SEPV retained only a vestigial repeat sequence (RSEP3) that is highly divergent from all other members of the YFV group, including FRV.

FRV shares a common ancestor with SEPV (with 100% bootstrap support) (Figure 2) and is located in the distinct YFV group according to International Committee on Taxonomy of Viruses classification (15). Analysis of the 12 cleavage sites located within the polyprotein open reading frame, following the scheme described by Kuno and Chang (19), supported the phylogenetic structure of the YFV group; YFV displayed marked divergence from FRV, SEPV, and WESSV in several cleavage sites, including Ci/PrM, NS1/NS2a, NS2a/NS2b, and NS2b/NS3 (online Technical Appendix Figure, panel E).

Viruses Isolated from Mosquitoes

Mosquitoes yielding isolates of FRV were collected at Fitzroy Crossing in the West Kimberley region in 2011 (Table 3); FRV was isolated from 2 pools of Ae. normanensis and 1 pool of Anopheles amictus mosquitoes. In 2012, a similar level of sampling in the same geographic area (data not shown) showed a shift of activity away from Fitzroy Crossing to a broader area in the eastern and southern Kimberley region, encompassing Billiluna, Kununurra, and Wyndham (Table 3). Sixteen isolates were obtained, most (81.2%) from Ae. normanensis mosquitoes (Table 3) and all from female mosquitoes. Additional virus was isolated from An. amictus, Culex annulirostris, and a pool of damaged and unidentifiable Aedes spp. mosquitoes. The minimum infection rate was greatest at Billiluna (2.5 FRV-infected mosquitoes/1,000 mosquitoes; Table 4). Other arboviruses detected during these seasons included MVEV, KUNV, KOKV, RRV, and Sindbis virus (Table 3).

In Vitro Virus Replication

FRV replicated in all 4 cell lines tested. At all time points, the FRV titer grew higher in BSR than in other cell lines, with the exception of Vero cells on day 7; the difference was usually significant (Table 5). Mild CPE was not apparent until day 4 in BSR cells and day 7 in Vero and DF-1 cells; no CPE was evident in C6/36 cells.

Virus Virulence in Mice

Two female mice in the 1,000 IU intracerebrally inoculated group and 1 female mouse in the 100 IU intracerebrally inoculated group had hind limb weakness, intermittent photophobia, and/or mild retrobulbar swelling between 5 and 12-13 dpi; however, only 1 female mouse in each intracerebrally inoculated group received a score of 1 on 1-2 days (days 5 and 9). By 13-14 dpi, all mice appeared to be clinically healthy. The only abnormality in intraperitoneally inoculated mice was mild photophobia in 1 mouse in the 1,000 IU group at 6 dpi. Mock-challenged animals showed no clinical abnormalities throughout the study period. All intracerebrally inoculated mice seroconverted; antibody titers were [greater than or equal to] 160. In the intraperitoneally inoculated group, 6 mice in the 100 IU group seroconverted and 8 mice in the 1,000 IU group seroconverted (titers 40 to [greater than or equal to] 160), indicating successful FRV replication.

For the 3 mice in which subtle clinical signs developed, we processed the heads for histopathology. We found histopathologic signs of meningoencephalitis in all 3 (Figure 3, panels A-D). The lesions were most notable in the 1,000 IU intracerebrally inoculated group, corroborating the mild clinical signs observed. The least severe lesions were seen in the mouse from the 100 IU intracerebrally inoculated group; however, the lesions were unilateral in the hemisphere not inoculated. In the other 2 mice, the trend was toward greater severity in the inoculated hemisphere; however, in the other hemisphere and distant from the inoculation site, we found leukocyte infiltration, gliosis, and neuronal degeneration. No viral antigen was detected in the affected brains by immunohistochemistry, suggesting that FRV was cleared at the time of euthanasia (21 dpi).

Antibodies in Humans and Animals

A total of 366 serum samples from humans from northern Western Australia, submitted to PathWest Laboratory Medicine WA for alphavirus or flavivirus serologic testing from March through May in 2014 and 2015, were tested for antibodies to FRV (online Technical Appendix Table). Overall, the prevalence of antibodies to flaviviruses in the ELISA was 33.6%, of which initial screening showed an FRV neutralization titer [greater than or equal to] 10 in 9 samples. For 3 of these samples, cross-neutralization titers showed FRV antibody titers [greater than or equal to] 40 and at least a 4-fold difference between antibody titer to FRV and other flaviviruses from Australia (Table 6), yielding an FRV positivity rate of 0.8% (3/336) of all samples tested and 2.4% (3/123) of samples with evidence of a flavivirus infection by ELISA. All 3 FRV antibody-positive samples were from the West Kimberley (Broome) region.

Serum from 227 sentinel cattle, 87 horses, and 178 sentinel chickens from the Northern Territory sampled from 2009/10 through 2014/15 were tested for antibodies to FRV. Neutralizing antibodies to FRV were detected in horses (12.6%) and cattle (6.6%). FRV and SEPV cross-neutralization tests on a subsample of FRV anti-body-positive serum samples indicated that the FRV infections were not the result of serologic cross-infection with closely related SEPV (data not shown), which occurs in neighboring Papua New Guinea. One sentinel chicken had a low FRV antibody titer. Most FRV infections in domestic animals (n = 24; 77%) were from 2012/13. We also tested serum from 25 wallabies, 1 wallaroo, and 1 bandicoot from the Northern Territory, collected from 2006/07 through 2013/14. Low levels of FRV antibodies were found in 1 wallaby from the Darwin region in January 2007. We found no association between infection and clinical disease in any cattle, horses, chickens, or marsupials tested.

Discussion

The new flavivirus from northern Australia, for which we proposed the name Fitzroy River virus, was first isolated from Ae. normanensis mosquitoes collected near the Fitzroy River. Phylogenetic analysis of isolate K73884 demonstrates that FRV belongs to the YFV group (15). Cleavage recognition sequence analysis groups FRV together with WESSV and SEPV, but distinct from YFV, reflecting the pattern of amino acid homology of members of the YFV group across the polyprotein. When homology of individual viral proteins is assessed, FRV is most closely related to SEPV in all regions excluding some nonstructural components that have a required role in replication (36,37), notably NS4a and 2k, where homology to WESSV was higher. We found no evidence of recombination breakpoints occurring along the FRV genome, and the low (70%) amino acid homology observed in the 2k peptide is most likely the result of a collection of nonsynonymous mutations. However, this recombination analysis was limited to 44 reference flavivirus sequences; a larger collection that includes more YFV group isolates from the Southeast Asia region may reveal further insights into the evolutionary history of FRV.

The phylogenetic placement of FRV in the YFV group is further supported by analysis of features in the flavivirus UTRs (cyclization sequence, upstream AUG region, and tandem repeats), sequences that are functionally necessary for enhancing replication through formation of secondary RNA structures (38). Although we observed near complete consensus in the cyclization sequence at each terminus between these 4 viruses, FRV had higher levels of identity to SEPV and WESSV than YFV across the respective upstream AUG regions. Conversely, the tandem repeats found in the 3' UTR showed consistently high nucleotide identities (>80% across all 3 sites) with YFV rather than WESSV and SEPV. Together, these data indicate that FRV possesses a unique collection of sequence signatures that distinguish it from other members of the YFV group.

The origin of FRV is unknown. Although increased surveillance in neighboring countries is needed, arbovirus and mosquito monitoring has been pursued in northern Western Australia since the early 1970s (39,40) with only minor changes in strategy. FRV was not detected by virus culture from earlier mosquito collections, a finding consistent with recent introduction into Western Australia and possibly elsewhere in Australia, thus highlighting the value of ongoing surveillance activities. We cannot exclude the possibility that FRV was circulating in mosquitoes of species (or other insect vectors) not commonly collected in traps routinely used for surveillance of adult mosquitoes in Western Australia and that genetic changes enabled the virus to adapt to a new host species, as has been seen with chikungunya virus (41).

Phylogenetic analysis indicated that FRV is most closely related to SEPV in the YFV group, which is currently found only in Papua New Guinea, and WESSV, which occurs in Africa and Thailand. Recent experience with introduction of likely or suspected arbovirus and arbovirus vectors into northern Australia suggests that FRV was probably introduced from Southeast Asia. Included are introductions by mosquitoes such as Aedes aegypti (L.), Aedes vexans, and Culex gelidus (42,43) and introductions of viruses including Japanese encephalitis virus from Papua New Guinea (44), bluetongue viruses from Southeast Asia (45,46), and epizootic hemorrhagic disease virus 1 from Indonesia (47).

Most (81%) FRV has been isolated from Ae. normanensis mosquitoes, providing circumstantial evidence that this species may be the dominant vector. Mosquito collections at each locality were conducted [approximately equal to] 2-3 weeks after a period of high rainfall following a relatively dry period. These conditions favor an abundance of Ae. normanensis mosquitoes because these mosquitoes rapidly hatch from desiccation-resistant eggs (48). The detection of antibodies to FRV in sentinel animals from the Northern Territory is consistent with the range and feeding behavior of Ae. normanensis mosquitoes and indicates a wide distribution of FRV in northern Australia.

Our finding of serologic evidence of human infection by FRV, despite low prevalence and apparent confinement to the West Kimberley region, is noteworthy. We detected FRV more extensively across northern Western Australia, so further human infections are likely. Because these samples had been sent for routine diagnostic arbovirus testing, it is presumed that most persons had a clinical illness of concern; however, we did not have access to detailed clinical information. Also, because the samples were single rather than paired acute- and convalescent-phase samples, we could not determine whether the FRV antibodies are the result of acute or previous infections. The antibody titers to FRV in humans were low, and although the cross-neutralizations included all known Australian flaviviruses that replicate in the cell lines we used, these persons may have been infected with an unrecognized flavivirus.

The close relationship of FRV with WESSV and SEPV may indicate potential for FRV to affect domestic animals such as cattle, goats, and sheep. Cattle stations are a dominant agricultural feature of northern Australia. Given that most FRV was isolated from Ae. normanensis mosquitoes, that mosquitoes of this species readily feed on cattle and horses, and that the FRV antibody prevalence in sentinel cattle and horses in the Northern Territory was high, we believe that the enzootic transmission cycle for FRV probably involves Ae. normanensis mosquitoes and domestic animals such as cattle and horses. Infection with FRV was not associated with clinical disease in animals but could potentially be disguised by other arbovirus infections, such as bovine ephemeral fever (49).

The finding of mild clinical signs in FRV-infected weanling mice, more often in those that were intracerebrally infected, indicates that severe clinical disease may be unlikely unless the health of the animal host is compromised. Further research is required to determine if FRV causes clinical disease in humans or domestic animals. The outcomes of this study demonstrate the value of surveillance for mosquitoborne viruses in the detection, characterization, and impact assessment of novel and known arboviruses.

Acknowledgments

We thank Michael Lindsay, Peter Neville, and Susan Harrington for assistance with mosquito collections. The assistance of Sarah Power, Margaret Wallace, and Michael Burley with mosquito identification and virus detection is gratefully acknowledged. Technical staff at PathWest provided assistance with PCR and sequencing, and Stephen Davis kindly performed the neutralization assays on animal serum from the Northern Territory. Willy Suen, Caitlin O'Brien, and Agathe Colmant assisted with animal experiments. We also thank John Mackenzie for helpful discussion and the Medical Entomology Branch of the WA Department of Health for funding.

Additional financial support was provided by the Department of Health through the Funding Initiative for Mosquito Management in Western Australia. S.H.W. and W.I.L. were supported by the National Institutes of Health (grant no. AI109761, Center for Research and Diagnostics and Discovery, Center for Excellence in Translational Research). D.T.W. was funded by the Australian Government Department of Agriculture and Water Resources.

Dr. Johansen is a senior medical scientist at PathWest Laboratory Medicine WA and adjunct senior research fellow at The University of Western Australia; her research interests include mosquito and arbovirus surveillance and ecology of arboviruses and vectors. Mr. Williams is a staff associate at the Center for Infection and Immunity, Columbia University, New York, USA; his research interests include rodent and arthropodborne viruses and disease transmission.

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Address for correspondence: Cheryl A. Johansen, PathWest Laboratory Medicine Western Australia-Microbiology, PP Block, QEII Medical Centre, Nedlands, WA 6909, Australia; email: cheryl.johansen@uwa.edu.au

Cheryl A. Johansen, [1] Simon H. Williams, [1] Lorna F. Melville, Jay Nicholson, Roy A. Hall, Helle Bielefeldt-Ohmann, Natalie A. Prow, Glenys R. Chidlow, Shani Wong, Rohini Sinha, David T. Williams, W. Ian Lipkin, David W. Smith

Author affiliations: The University of Western Australia, Nedlands, Western Australia, Australia (C.A. Johansen, J. Nicholson, S. Wong, D.W. Smith); PathWest Laboratory Medicine Western Australia, Nedlands (C.A. Johansen, G.R. Chidlow, D.W. Smith); Columbia University, New York, New York, USA (S.H. Williams, R. Sinha, W.I. Lipkin); The Northern Territory Government, Darwin, Northern Territory, Australia (L.F. Melville); The University of Queensland, St. Lucia, Queensland, Australia (R.A. Hall, H. Bielefeldt-Ohmann, N.A. Prow); The University of Queensland, Gatton, Queensland, Australia (H. Bielefeldt-Ohmann); CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia (D.T. Williams)

DOI: https://doi.org/ 10.3201/eid2308.161440

[1] These authors contributed equally to this article.

Caption: Figure 1. Locations where Fitzroy River virus-positive mosquitoes were collected (black dots), Western Australia, Australia, 2011 and 2012. Perth (asterisk), the capital city and most densely populated area of Western Australia, is shown to indicate its distance from the Kimberley region.

Caption: Figure 2. Phylogenetic tree of the genus Flavivirus, based on full polyprotein nucleotide sequences. Asterisk * indicates Fitzroy River virus. Scale bar indicates nucleotide substitutions per site. YFV, yellow fever virus.

Caption: Figure 3. Photomicrographs of Fitzroy River virus (FRV)-induced meningoencephalitis in weanling mice inoculated with 1,000 infectious units of FRV. Panels show multifocal mild to severe perivascular and neuropil infiltration of lymphocytes and monocytes (blue arrows in A-C); meningitis in a sulcus (black arrow in A); glial cell activation with notable astrocytosis, neuron degeneration, and neuronophagia (arrowhead in B); occasional hemorrhage (blue arrow in D); mild periventricular spongiosis (blue arrows in C); and meningitis (black arrow in C). Hematoxylin and eosin staining. Original magnifications: A) x40, B) x400, C) x100, D) x400.
Table 1. Monoclonal antibody binding pattern of FRV isolates from
Western Australia *

Virus                 Monoclonal antibody ([dagger])

               4G2   4G4   6F7   7C6   8G2   6A9   3D11

FRV ([double    +     +     -    --    --    --     --
  dagger])
SEPV            +     +     +    --    --    --     --
YFV             +     +    --    --    --    --     --
EHV             +    --     +     +     +     +     +

Virus             Monoclonal antibody
                     ([dagger])

               3B11   3G1   5D3   7C3

FRV ([double    --    --    --    --
  dagger])
SEPV            --    --    --    --
YFV             --    --    --    --
EHV             +      +     +     +

* EHV, Edge Hill virus; FRV, Fitzroy River virus; SEPV, Sepik virus;
YFV, yellow fever virus; +, positive (optical density of [greater
than or equal to] 0.2 and at least 2 times the mean of negative
control wells); -, negative.

([dagger]) Original descriptions of monoclonal antibody from (31)
(4G2), (32) (4G4), (33) (6F7), and (34) (7C6, 8G2, 6A9,
3D11, 3B11, 3G1, 5D3, and 7C3).

([double dagger]) Monoclonal antibody binding patterns of all FRV
isolates were identical to those of the prototype isolate K73884.

Table 2. Comparison of genomic region lengths and similarities
between members of the YFV group and FRV *

Genomic                        Virus
region
                    FRV                SEPV

                nt      aa         nt            aa

5'UTR          117      NA      116 (94)         NA
Capsid         348      116     348 (77)      116 (84)
Premembrane    267      89      267 (78)      89 (94)
Membrane       225      75      225 (80)      75 (96)
Envelope      1,470     490    1,470 (81)     490 (96)
NS1           1,059     353    1,059 (79)     353 (93)
NS2a           678      226     678 (78)      226 (88)
NS2b           390      130     390 (78)      130 (88)
NS3           1,869     623    1,869 (79)     623 (93)
NS4a           378      126     378 (75))     126 (87)
2k              69      23       69 (71)      23 (70)
NS4b           744      248     744 (79)      248 (94)
NS5           2,721     906    2,721 (78)     906 (88)
3'UTR          472      NA      459 (86)         NA
Polyprotein   10,218   3,405   10,218 (79)   3,405 (91)
Full genome   10,807    NA     10,793 (79)       NA

Genomic                               Virus
region
                       WESSV                       YFV

                  nt            aa           nt           aa

5'UTR          118 (92)         NA        118 (52)        NA
Capsid         348 (74)      116 (78)     363 (54)     121 (39)
Premembrane    267 (73)      89 (87)      267 (59)      89 (59)
Membrane       225 (74)      75 (87)      225 (58)      75 (47)
Envelope      1,470 (79)     490 (93)    1,479 (63)    493 (54)
NS1           1,059 (76)     353 (87)    1,056 (63)    352 (64)
NS2a           678 (77)      226 (85)     672 (54)     224 (40)
NS2b           390 (76)      130 (88)     390 (56)     130 (50)
NS3           1,869 (77)     623 (92)    1,869 (67)    623 (71)
NS4a           378 (75)      126 (92)     378 (61)     126 (57)
2k              69 (74)      23 (91)       69 (61)      23 (57)
NS4b           744 (77)      248 (91)     750 (66)     250 (64)
NS5           2,721 (76)     906 (89)    2,718 (66)    905 (68)
3'UTR          478 (84)         NA        508 (65)        NA
Polyprotein   10,218 (77)   3,405 (89)   10,236 (63)   3,411 (61
Full genome   10,814 (77)       NA       10,862 (62)      NA

* Values are sequence length (% identity with FRV). FRV, Fitzroy
River virus (GenBank accession no. KM631634); NS, nonstructural;
SEPV, Sepik virus (GenBank accession no. NC008719); UTR, untranslated
region; WESSV, Wesselsbron virus (accession no. JN226796); YFV,
yellow fever virus (X03700); NA, not applicable.

Table 3. Mosquito species collected and arboviruses isolated from the
Kimberley region of Western Australia, Australia, 2011 and 2012 *

Year, location, mosquito            No. (%)         No.      No. pools
species                            collected     processed   processed

2011
  Fitzroy Crossing
    Ae. (Ochlerotatus)           4,657 (38.4)      2,497        110
      normanensis
    An. (Cellia) amictus           750 (6.2)        504         29
    An. (Cellia) annulipes       2,879 (23.7)      1,898        84
      s.l.
    Cx. (Culex) annulirostris    3,202 (26.4)      1,773        79

    Other                          635 (5.2)        482         100
    Subtotal                     12,123 (100)      7,154        402

2012
  Billiluna
    Ae. (Macleaya) tremulus        252 (2.0)        135         14
    Ae. (Ochlerotatus)           1,679 (13.4)      1,244        58
      normanensis
    An. (Cellia) amictus           650 (5.2)        508         42
    An. (Cellia) annulipes       3,456 (27.5)      1,555        74
      s.l.
    An. (Cellia) novaguinensis     247 (2.0)        152         18
    Cx. (Culex) annulirostris    5,608 (44.6)      3,424        148
    Damaged Anopheles spp.         131 (1.0)        83          14
    Damaged Culex spp.             218 (1.7)        111         15
    Other                          326 (2.6)        199         84
    Subtotal                     12,567 (100)      7,411        467

  Kununurra
    Ae. (Finlaya) notoscriptus     455 (1.3)        381         31
    Ae. (Neomellanoconion)        3,457 (9.9)      1540         80
      lineatopennis
    Ae. (Ochlerotatus)           12,632 (36.2)     4917         219
      normanensis
    An. (Anopheles) bancroftii    2,428 (7.0)       723         52
    An. (Cellia) annulipes        1,758 (5.0)      1025         67
      s.l.
    An. (Cellia) meraukensis      2,717 (7.8)       863         60
    Cq. (Coquillettidia)           931 (2.7)        796         55
      xanthogaster
    Cx. (Culex) annulirostris    7,600 (21.8)      4195         193
    Ve. (Verrallina) reesi         468 (1.3)        287         33
    Damaged Culex spp.             350 (1.0)        253         30
    Other                         2,111 (6.0)      1528         369
    Subtotal                     34,907 (100)      16508       1189

  Wyndham
    Ae. (Ochlerotatus)            1,661 (5.4)       551         30
      normanensis
    An. (Anopheles) bancroftii     532 (1.7)        122         14
    An. (Cellia) amictus          1,589 (5.1)       380         26
    An. (Cellia) annulipes         982 (3.2)        262         20
      s.l.
    An. (Cellia) meraukensis      1,677 (5.4)       450         27
    Cx. (Culex) annulirostris    21,388 (69.1)     5,357        224
    Cx. (Culex) crinicauda         330 (1.1)        62          14
    Damaged Culex spp.             907 (2.9)        247         17
    Other                         1,881 (6.1)       898         206
    Subtotal                     30,947 (100)      8329         578

Total                               90,544        39,402       2,636

Year, location, mosquito               No. virus isolates
species

2011
  Fitzroy Crossing
    Ae. (Ochlerotatus)                  2 FRV, 3 non A/F
      normanensis
    An. (Cellia) amictus                1 FRV, 4 non A/F
    An. (Cellia) annulipes                  6 non A/F
      s.l.
    Cx. (Culex) annulirostris            2 MVEV, 1 KUNV,
                                           1 KUNV+SINV,
    Other                        6 non A/F 1 non A/F ([dagger])
    Subtotal

2012
  Billiluna
    Ae. (Macleaya) tremulus
    Ae. (Ochlerotatus)                        3 FRV
      normanensis
    An. (Cellia) amictus
    An. (Cellia) annulipes
      s.l.
    An. (Cellia) novaguinensis
    Cx. (Culex) annulirostris                2 MVEV
    Damaged Anopheles spp.
    Damaged Culex spp.
    Other
    Subtotal

  Kununurra
    Ae. (Finlaya) notoscriptus
    Ae. (Neomellanoconion)
      lineatopennis
    Ae. (Ochlerotatus)                    7 FRV, 2 RRV
      normanensis
    An. (Anopheles) bancroftii
    An. (Cellia) annulipes
      s.l.
    An. (Cellia) meraukensis
    Cq. (Coquillettidia)
      xanthogaster
    Cx. (Culex) annulirostris                 1 RRV
    Ve. (Verrallina) reesi
    Damaged Culex spp.                        1 RRV
    Other                            4 RRV ([double dagger])
    Subtotal

  Wyndham
    Ae. (Ochlerotatus)                    1 FRV, 1 RRV
      normanensis
    An. (Anopheles) bancroftii
    An. (Cellia) amictus
    An. (Cellia) annulipes
      s.l.
    An. (Cellia) meraukensis
    Cx. (Culex) annulirostris         1 FRV, 2 KOKV, 4 RRV
    Cx. (Culex) crinicauda
    Damaged Culex spp.                        1 RRV
    Other                           1 FRV, 1 RRV ([section])
    Subtotal

Total

* Only mosquito collection locations that yielded isolates of FRV are
shown; species collected at abundance of <1.0% are grouped as
"other"; named species are female mosquitoes only. Results from male
mosquitoes are included in "other." Ae., Aedes; An., Anopheles; Cq.,
Coquillettidia; Cx., Culex; FRV, Fitzroy River virus; KOKV, Kokobera
virus; KUNV, West Nile (Kunjin) virus; non A/F, not an alphavirus or
flavivirus and identity is yet to be determined; MVEV, Murray Valley
encephalitis virus; RRV, Ross River virus; SINV, Sindbis virus; Ve.,
Verrallina.

([dagger]) Isolated from female Ae. lineatopennis mosquito.

([double dagger]) Isolated from female Aedeomyia catasticta (1),
Anopheles amictus (1), and Mansonia uniformis (2) mosquitoes.

([section]) FRV isolated from a pool of damaged female Aedes spp.
mosquitoes, RRV isolated from female An. bancroftii mosquitoes.

Table 4. Minimum infection rates of mosquitoes infected with FRV,
Western Australia, Australia, 2011 and 2012 *

Year, location, mosquito species       No.       Minimum
                                     isolates   infection
                                                   rate
                                                ([dagger])

2011
  Fitzroy Crossing
    Ae. (Ochlerotatus) normanensis      2          0.8
    An. (Cellia) amictus                1          2.0

2012

  Billiluna
    Ae. (Ochlerotatus) normanensis      3          2.5

  Kununurra
    Ae. (Ochlerotatus) normanensis      7          1.4

  Wyndham
    Ae. (Ochlerotatus) normanensis      1          1.8
    Cx. (Culex) annulirostris           1          0.2
    Damaged Aedes spp.                  1          1.1

* Ae., Aedes; An., Anopheles; Cx, Culex; FRV, Fitzroy River virus.

([dagger]) No. FRV/infected mosquitoes/1,000 mosquitoes; calculated
according to (35).

Table 5. Fitzroy River virus replication in 4 cell lines

             Mean Fitzroy River virus titer *

Day           C6/36                     Vero

1             0 (a)                    0 (a)
2     4.68 [+ or -] 0.21 (a)   3.78 [+ or -] 0.05 (b)
3     5.58 [+ or -] 0.05 (a)   4.72 [+ or -] 0.02 (b)
4     6.66 [+ or -] 0.09 (a)   5.02 [+ or -] 0.07 (b)
7     4.41 [+ or -] 0.14 (a)   7.01 [+ or -] 0.01 (b)

             Mean Fitzroy River virus titer *

Day            BSR                      DF-1

1     3.07 [+ or -] 0.06 (b)           0 (a)
2     5.13 [+ or -] 0.06 (c)   3.81 [+ or -] 0.3 (ab)
3     6.67 [+ or -] 0.08 (c)   5.5 [+ or -] 0.08 (a)
4     6.72 [+ or -] 0.05 (a)   6.26 [+ or -] 0.15 (a)
7     6.6 [+ or -] 0.21 (b)    4.25 [+ or -] 0.23 (a)

* Statistical significance of log transformed arithmetic means was
determined with 2-way analysis of variance with correction for
multiple comparisons and using the Tukey method for pairwise multiple
comparisons (GraphPad Prism version 6.0; GraphPad Software Inc, San
Diego, CA, USA). Means [+ or -] SE in the same row followed by the same
superscript letter did not differ significantly (p>0.05). Results at
day 0 were excluded because virus detected at this time point
represented residual inoculum.

Table 6. Serologic test results for 9 serum samples from humans from
northern Western Australia, which contained FRV neutralizing
antibodies at initial testing *

         ELISA, %    FRV initial      Cross-neutralization titers
          block     neutralization

Sample                  titer        FRV   MVEV   KUNV   ALFV

2014-1      79            10         <10   <10    <10    <10
2014-2      87            20         <10    10     10    <10
2014-3      86            40         <10   <10    <10    <10
2014-4      84           160         40    <10     10    <10
2015-1      90            80         <10   <10    160    <10
2015-2      92            20         <10   <10     80    <10
2015-3      98            40         80    <10     10    <10
2015-4      95            80         80     10     20    <10
2015-5      98            10         <10   <10     10    <10

         Cross-neutralization   Infecting
               titers             virus

Sample   KOKV   STRV   EHV

2014-1   <10    <10    <10        UD
2014-2    10     10    40         EHV
2014-3   <10     20    80         EHV
2014-4   <10     10    <10        FRV
2015-1   <10    <10    <10       KUNV
2015-2   <10    <10    <10       KUNV
2015-3   <10    <10    <10        FRV
2015-4    10     10    20         FRV
2015-5   <10    <10    <10        UD

* ALFV, Alfuy virus (K74157); EHV, Edge Hill virus (K74003); FRV,
Fitzroy River virus (K73884); KOKV, Kokobera virus (K69949); KUNV,
West Nile (Kunjin) virus (K81136); MVEV, Murray Valley encephalitis
virus (K68150); STRV, Stratford virus (C338); UD, undetermined.
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Title Annotation:RESEARCH
Author:Johansen, Cheryl A.; Williams, Simon H.; Melville, Lorna F.; Nicholson, Jay; Hall, Roy A.; Bielefeld
Publication:Emerging Infectious Diseases
Geographic Code:8AUST
Date:Aug 1, 2017
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