Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission.

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.

Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors.

We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNVKUN-prME and WNVKUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNVKUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNVKUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR-/- MEFs or RNase L-/- MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses.IMPORTANCE The globally important flavivirus pathogens West Nile virus, Zika virus, dengue viruses, and yellow fever virus can infect mosquito vectors and be transmitted to humans and other vertebrate species in which they cause significant levels of disease and mortality. However, the subgroup of closely related flaviviruses, known as lineage II insect-specific flaviviruses (Lin II ISFs), only infect mosquitoes and cannot replicate in cells of vertebrate origin. Our data are the first to uncover the mechanisms that restrict the growth of Lin II ISFs in vertebrate cells and provides new insights into the evolution of these viruses and the mechanisms associated with host switching that may allow new mosquito-borne viral diseases to emerge. The new reagents generated in this study, including the first Lin II ISF-reactive monoclonal antibodies and Lin II ISF mutants and chimeric viruses, also provide new tools and approaches to enable further research advances in this field.

Arbovirus surveillance using FTATM cards in modified CO2 -baited encephalitis virus surveillance traps in the Northern Territory, Australia.

In 2016, modified CO2 -baited encephalitis virus surveillance (EVS) traps were evaluated for flavivirus surveillance in the Northern Territory, Australia. The traps were fitted with honey-soaked nucleic acid preservation cards (FTATM ) for mosquitoes to expectorate virus while feeding on the cards. Cards were tested for the presence of selected arboviruses, with two cards testing positive for Kunjin virus and Alfuy, while sentinel chickens tested in parallel also showed Kunjin virus activity at the same time. The results from the cards and vector mosquito feeding rates indicate that CO2 -baited EVS traps coupled with honey-baited FTATM cards are an effective tool for broad-scale arbovirus surveillance.

Arboviral diseases and malaria in Australia, 2014­15: Annual report of the National Arbovirus and Malaria Advisory Committee

This report describes the epidemiology of mosquito-borne diseases of public health importance in Australia during the 2014­15 season (1 July 2014 to 30 June 2015) and includes data from human notifications, sentinel chicken, vector and virus surveillance programs. The National Notifiable Diseases Surveillance System received notifications for 12,849 cases of disease transmitted by mosquitoes during the 2014­15 season. The Australasian alphaviruses Barmah Forest virus and Ross River virus accounted for 83% (n=10,723) of notifications. However, over-diagnosis and possible false positive diagnostic test results for these two infections mean that the true burden of infection is likely overestimated, and as a consequence, revised case definitions were implemented from 1 January 2016. There were 151 notifications of imported chikungunya virus infection. There were 74 notifications of dengue virus infection acquired in Australia and 1,592 cases acquired overseas, with an additional 34 cases for which the place of acquisition was unknown. Imported cases of dengue were most frequently acquired in Indonesia (66%). There were 7 notifications of Zika virus infection. No cases of locally-acquired malaria were notified during the 2014­15 season, though there were 259 notifications of overseas-acquired malaria and one notification for which no information on the place of acquisition was supplied. Imported cases of malaria were most frequently acquired in southern and eastern Africa (23%) and Pacific Island countries (20%). In 2014­15, arbovirus and mosquito surveillance programs were conducted in most of the states and territories. Surveillance for exotic mosquitoes at international ports of entry continues to be a vital part of preventing the establishment of vectors of mosquito-borne diseases such as dengue to new areas of Australia. In 2014-15, there was a sharp increase in the number of exotic mosquitoes detected at the Australian border, with 36 separate exotic mosquito detections made, representing a 280% increase from the 2013-14 period where there were 13 exotic mosquito detections.

The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo.

Arthropod-borne flaviviruses such as yellow fever (YFV), Zika and dengue viruses continue to cause significant human disease globally. These viruses are transmitted by mosquitoes when a female imbibes an infected blood-meal from a viremic vertebrate host and expectorates the virus into a subsequent host. Bamaga virus (BgV) is a flavivirus recently discovered in Culex sitiens subgroup mosquitoes collected from Cape York Peninsula, Australia. This virus phylogenetically clusters with the YFV group, but is potentially restricted in most vertebrates. However, high levels of replication in an opossum cell line (OK) indicate a potential association with marsupials. To ascertain whether BgV could be horizontally transmitted by mosquitoes, the vector competence of two members of the Cx. sitiens subgroup, Cx. annulirostris and Cx. sitiens, for BgV was investigated. Eleven to thirteen days after imbibing an infectious blood-meal, infection rates were 11.3% and 18.8% for Cx. annulirostris and Cx. sitiens, respectively. Cx. annulirostris transmitted the virus at low levels (5.6% had BgV-positive saliva overall); Cx. sitiens did not transmit the virus. When mosquitoes were injected intrathoracially with BgV, the infection and transmission rates were 100% and 82%, respectively, for both species. These results provided evidence for the first time that BgV can be transmitted horizontally by Cx. annulirostris, the primary vector of pathogenic zoonotic flaviviruses in Australia. We also assessed whether BgV could interfere with replication in vitro, and infection and transmission in vivo of super-infecting pathogenic Culex-associated flaviviruses. BgV significantly reduced growth of Murray Valley encephalitis and West Nile (WNV) viruses in vitro. While prior infection with BgV by injection did not inhibit WNV super-infection of Cx. annulirostris, significantly fewer BgV-infected mosquitoes could transmit WNV than mock-injected mosquitoes. Overall, these data contribute to our understanding of flavivirus ecology, modes of transmission by Australian mosquitoes and mechanisms for super-infection interference.

Mosquito and Virus Surveillance as a Predictor of Human Ross River Virus Infection in South-West Western Australia: How Useful Is It?

Mosquito and virus surveillance systems are widely used in Western Australia (WA) to support public health efforts to reduce mosquito-borne disease. However, these programs are costly to maintain on a long-term basis. Therefore, we aimed to assess the validity of mosquito numbers and Ross River virus (RRV) isolates from surveillance trap sites as predictors of human RRV cases in south-west WA between 2003 and 2014. Using negative binomial regression modeling, mosquito surveillance was found to be a useful tool for predicting human RRV cases. In eight of the nine traps, when adjusted for season, there was an increased risk of RRV cases associated with elevated mosquito numbers detected 1 month before the onset of human cases for at least one quartile compared with the reference group. The most predictive urban trap sites were located near saltmarsh mosquito habitat, bushland that could sustain macropods and densely populated residential suburbs. This convergence of environments could allow enzootic transmission of RRV to spillover and infect the human population. Close proximity of urban trap sites to each other suggested these sites could be reduced. Ross River virus isolates were infrequent at some trap sites, so ceasing RRV isolation from mosquitoes at these sites or where isolates were not predictive of human cases could be considered. In future, trap sites could be reduced for routine surveillance, allowing other environments to be monitored to broaden the understanding of RRV ecology in the region. A more cost-effective and efficient surveillance program may result from these modifications.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction.

Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

Characterization of Fitzroy River Virus and Serologic Evidence of Human and Animal Infection.

In northern Western Australia in 2011 and 2012, surveillance detected a novel arbovirus in mosquitoes. Genetic and phenotypic analyses confirmed that the new flavivirus, named Fitzroy River virus, is related to Sepik virus and Wesselsbron virus, in the yellow fever virus group. Most (81%) isolates came from Aedes normanensis mosquitoes, providing circumstantial evidence of the probable vector. In cell culture, Fitzroy River virus replicated in mosquito (C6/36), mammalian (Vero, PSEK, and BSR), and avian (DF-1) cells. It also infected intraperitoneally inoculated weanling mice and caused mild clinical disease in 3 intracranially inoculated mice. Specific neutralizing antibodies were detected in sentinel horses (12.6%), cattle (6.6%), and chickens (0.5%) in the Northern Territory of Australia and in a subset of humans (0.8%) from northern Western Australia.

Discovery and Characterisation of Castlerea Virus, a New Species of Negevirus Isolated in Australia.

With advances in sequencing technologies, there has been an increase in the discovery of viruses that do not group with any currently described virus families. The newly described taxon Negevirus encompasses a group of viruses displaying an insect-specific phenotype which have been isolated from multiple host species on numerous continents. Using a broad-spectrum virus screening assay based on the detection of double-stranded RNA and next-generation sequencing, we have detected a novel species of negevirus, from Anopheles, Culex, and Aedes mosquitoes collected in 4 geographically separate regions of Australia. Bioinformatic analysis of the virus, tentatively named Castlerea virus, revealed that it is genetically distinct from previously described negeviruses but clusters in the newly proposed Nelorpivirus clade within this taxon. Analysis of virions confirmed the presence of 2 proteins of 24 and 40 kDa which support previous bioinformatic predictions of negevirus structural proteins.

Analysis of Arbovirus Isolates from Australia Identifies Novel Bunyaviruses Including a Mapputta Group Virus from Western Australia That Links Gan Gan and Maprik Viruses.

The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia (K10441, SW27571, K13190, and K42904) and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.

Virulence and Evolution of West Nile Virus, Australia, 1960-2012.

Worldwide, West Nile virus (WNV) causes encephalitis in humans, horses, and birds. The Kunjin strain of WNV (WNVKUN) is endemic to northern Australia, but infections are usually asymptomatic. In 2011, an unprecedented outbreak of equine encephalitis occurred in southeastern Australia; most of the ≈900 reported cases were attributed to a newly emerged WNVKUN strain. To investigate the origins of this virus, we performed genetic analysis and in vitro and in vivo studies of 13 WNVKUN isolates collected from different regions of Australia during 1960-2012. Although no disease was recorded for 1984, 2000, or 2012, isolates collected during those years (from Victoria, Queensland, and New South Wales, respectively) exhibited levels of virulence in mice similar to that of the 2011 outbreak strain. Thus, virulent strains of WNVKUN have circulated in Australia for >30 years, and the first extensive outbreak of equine disease in Australia probably resulted from a combination of specific ecologic and epidemiologic conditions.

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.

Arboviral diseases and malaria in Australia, 2012-13: Annual report of the National Arbovirus and Malaria Advisory Committee.

This report describes the epidemiology of mosquito-borne diseases of public health importance in Australia during the 2012-13 season (1 July 2012 to 30 June 2013) and includes data from human notifications, sentinel chicken, vector and virus surveillance programs. The National Notifiable Diseases Surveillance System received notifications for 9,726 cases of disease transmitted by mosquitoes during the 2012-13 season. The Australasian alphaviruses Barmah Forest virus and Ross River virus accounted for 7,776 (80%) of total notifications. However, over-diagnosis and possible false positive diagnostic test results for these 2 infections mean that the true burden of infection is likely overestimated, and as a consequence, the case definitions were revised, effective from 1 January 2016. There were 96 notifications of imported chikungunya virus infection. There were 212 notifications of dengue virus infection acquired in Australia and 1,202 cases acquired overseas, with an additional 16 cases for which the place of acquisition was unknown. Imported cases of dengue were most frequently acquired in Indonesia. No locally-acquired malaria was notified during the 2012-13 season, though there were 415 notifications of overseas-acquired malaria. There were no cases of Murray Valley encephalitis virus infection in 2012-13. In 2012-13, arbovirus and mosquito surveillance programs were conducted in most jurisdictions with a risk of vectorborne disease transmission. Surveillance for exotic mosquitoes at the border continues to be a vital part of preventing the spread of mosquito-borne diseases such as dengue to new areas of Australia, and in 2012-13, there were 7 detections of exotic mosquitoes at the border.

Discovery and characterisation of a new insect-specific bunyavirus from Culex mosquitoes captured in northern Australia.

Insect-specific viruses belonging to significant arboviral families have recently been discovered. These viruses appear to be maintained within the insect population without the requirement for replication in a vertebrate host. Mosquitoes collected from Badu Island in the Torres Strait in 2003 were analysed for insect-specific viruses. A novel bunyavirus was isolated in high prevalence from Culex spp. The new virus, provisionally called Badu virus (BADUV), replicated in mosquito cells of both Culex and Aedes origin, but failed to replicate in vertebrate cells. Genomic sequencing revealed that the virus was distinct from sequenced bunyavirus isolates reported to date, but phylogenetically clustered most closely with recently discovered mosquito-borne, insect-specific bunyaviruses in the newly proposed Goukovirus genus. The detection of a functional furin cleavage motif upstream of the two glycoproteins in the M segment-encoded polyprotein suggests that BADUV may employ a unique strategy to process the virion glycoproteins.

Arboviral diseases and malaria in Australia, 2013-14: Annual report of the National Arbovirus and Malaria Advisory Committee.

This report describes the epidemiology of mosquito-borne diseases of public health importance in Australia during the 2013-14 season (1 July 2013 to 30 June 2014) and includes data from human notifications, sentinel chicken, vector and virus surveillance programs. The National Notifiable Diseases Surveillance System received notifications for 8,898 cases of disease transmitted by mosquitoes during the 2013-14 season. The Australasian alphaviruses Barmah Forest virus and Ross River virus accounted for 6,372 (72%) total notifications. However, over-diagnosis and possible false positive diagnostic test results for these 2 infections mean that the true burden of infection is likely overestimated, and as a consequence, the case definitions have been amended. There were 94 notifications of imported chikungunya virus infection and 13 cases of imported Zika virus infection. There were 212 notifications of dengue virus infection acquired in Australia and 1,795 cases acquired overseas, with an additional 14 cases for which the place of acquisition was unknown. Imported cases of dengue were most frequently acquired in Indonesia (51%). No cases of locally-acquired malaria were notified during the 2013-14 season, though there were 373 notifications of overseas-acquired malaria. In 2013-14, arbovirus and mosquito surveillance programs were conducted in most jurisdictions. Surveillance for exotic mosquitoes at international ports of entry continues to be a vital part of preventing the spread of vectors of mosquito-borne diseases such as dengue to new areas of Australia, with 13 detections of exotic mosquitoes at the ports of entry in 2013-14.

A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia.

To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the diversity and prevalence of ISFs in Australian mosquitoes, samples from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.

The Molecular Epidemiology and Evolution of Murray Valley Encephalitis Virus: Recent Emergence of Distinct Sub-lineages of the Dominant Genotype 1.

BACKGROUND: Recent increased activity of the mosquito-borne Murray Valley encephalitis virus (MVEV) in Australia has renewed concerns regarding its potential to spread and cause disease. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the genetic relationships between earlier and more recent circulating strains, patterns of virus movement, as well as the molecular basis of MVEV evolution, complete pre-membrane (prM) and Envelope (Env) genes were sequenced from sixty-six MVEV strains from different regions of the Australasian region, isolated over a sixty year period (1951-2011). Phylogenetic analyses indicated that, of the four recognized genotypes, only G1 and G2 are contemporary. G1 viruses were dominant over the sampling period and found across the known geographic range of MVEV. Two distinct sub-lineages of G1 were observed (1A and 1B). Although G1B strains have been isolated from across mainland Australia, Australian G1A strains have not been detected outside northwest Australia. Similarly, G2 is comprised of only Western Australian isolates from mosquitoes, suggesting G1B and G2 viruses have geographic or ecological restrictions. No evidence of recombination was found and a single amino acid substitution in the Env protein (S332G) was found to be under positive selection, while several others were found to be under directional evolution. Evolutionary analyses indicated that extant genotypes of MVEV began to diverge from a common ancestor approximately 200 years ago. G2 was the first genotype to diverge, followed by G3 and G4, and finally G1, from which subtypes G1A and G1B diverged between 1964 and 1994. CONCLUSIONS/SIGNIFICANCE: The results of this study provides new insights into the genetic diversity and evolution of MVEV. The demonstration of co-circulation of all contemporary genetic lineages of MVEV in northwestern Australia, supports the contention that this region is the enzootic focus for this virus.

Rainfall and sentinel chicken seroconversions predict human cases of Murray Valley encephalitis in the north of Western Australia.

BACKGROUND: Murray Valley encephalitis virus (MVEV) is a flavivirus that occurs in Australia and New Guinea. While clinical cases are uncommon, MVEV can cause severe encephalitis with high mortality. Sentinel chicken surveillance is used at many sites around Australia to provide an early warning system for risk of human infection in areas that have low population density and geographical remoteness. MVEV in Western Australia occurs in areas of low population density and geographical remoteness, resulting in logistical challenges with surveillance systems and few human cases. While epidemiological data has suggested an association between rainfall and MVEV activity in outbreak years, it has not been quantified, and the association between rainfall and sporadic cases is less clear. In this study we analysed 22 years of sentinel chicken and human case data from Western Australia in order to evaluate the effectiveness of sentinel chicken surveillance for MVEV and assess the association between rainfall and MVEV activity. METHODS: Sentinel chicken seroconversion, human case and rainfall data from the Kimberley and Pilbara regions of Western Australia from 1990 to 2011 were analysed using negative binomial regression. Sentinel chicken seroconversion and human cases were used as dependent variables in the model. The model was then tested against sentinel chicken and rainfall data from 2012 and 2013. RESULTS: Sentinel chicken seroconversion preceded all human cases except two in March 1993. Rainfall in the prior three months was significantly associated with both sentinel chicken seroconversion and human cases across the regions of interest. Sentinel chicken seroconversion was also predictive of human cases in the models. The model predicted sentinel chicken seroconversion in the Kimberley but not in the Pilbara, where seroconversions early in 2012 were not predicted. The latter may be due to localised MVEV activity in isolated foci at dams, which do not reflect broader virus activity in the region. CONCLUSIONS: We showed that rainfall and sentinel chickens provide a useful early warning of MVEV risk to humans across endemic and epidemic areas, and that a combination of the two indicators improves the ability to assess MVEV risk and inform risk management measures.

The seroprevalence and factors associated with Ross river virus infection in western grey kangaroos (Macropus fuliginosus) in Western Australia.

A serosurvey was undertaken in 15 locations in the midwest to southwest of Western Australia (WA) to investigate the seroprevalence of Ross River virus (RRV) neutralizing antibodies and factors associated with infection in western grey kangaroos (Macropus fuliginosus). The estimated seroprevalence in 2632 kangaroo samples, using a serum neutralization test, was 43.9% (95% CI 42.0, 45.8). Location was significantly associated with seroprevalence (p<0.001). There was a strong positive correlation between seroprevalence and the average log-transformed neutralizing antibody titer (r=0.98, p<0.001). The seroprevalence among adult kangaroos was significantly higher than in subadult kangaroos (p<0.05). No significant association was observed between seroprevalence and the sex of kangaroos (p>0.05). The results of this study indicate that kangaroos in WA are regularly infected with RRV and may be involved in the maintenance and transmission of RRV.