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ABSTRACT Precise mosquito identification is integral to effective arbovirus surveillance. Nonetheless, the conventional morphological approach to identifying mosquito species is laborious, demands expertise and presents challenges when specimens are damaged. DNA barcoding offers a promising alternative, surmounting challenges inherent in morphological identification. To integrate DNA barcoding into arbovirus surveillance effectively, a robust dataset of mosquito barcode sequences is required. This study established a comprehensive repository of Cytochrome Oxidase I (COI) barcodes, encompassing 177 samples representing 45 mosquito species from southern and northern Western Australia (WA), including 16 species which have not been previously barcoded. The average intraspecific and interspecific genetic distances were 1% and 6.8%, respectively. Anopheles annulipes sensu lato had the highest intraspecific distance at 9.1%, signifying a genetically diverse species. While validating the potential of COI barcodes to accurately differentiate mosquito species, we identified that some species pairs have low COI divergence. This includes Aedes clelandi and Ae. hesperonotius, Tripteroides atripes and Tp. punctolaeralis and Ae. turneri and Ae. stricklandi. In addition, we observed ambiguity in identification of the members of Culex sitiens subgroup (Cx. annulirostris, Cx. palpalis and Cx. sitiens) and three members of Cx. pipiens complex (Cx. australicus, Cx. globocoxitus, Cx. quinquefasciatus). In summary, despite presenting challenges in the identification of some mosquito species, the COI barcode accurately identified most of the species and generated a valuable resource that will support the WA arbovirus surveillance program and enhance public health intervention strategies for mosquito‐borne disease control.

Abstract The global demand from multi-sectoral partners for operational tools for One Health implementation and capacity building is increasing, yet a validated global inventory of One Health tools did not exist. Here, we map and analyze available One Health tools and assess their suitability to support One Health implementation, including the One Health Joint Plan of Action 2022–2026 (OH JPA). Our objectives were to identify (i) publicly available One Health tools to support capacity building and OH JPA implementation; (ii) optimal outcomes for countries/regions using available One Health tools; (iii) linkages to OH JPA Action Tracks and pathways in the One Health Theory of Change (TOC); and (iv) gaps and priorities for the development of additional One Health tools. One Health High Level Expert Panel (OHHLEP) members compiled information on One Health tools that were publicly available and released up to June 30, 2023, via online sources and partner networks including the Quadripartite organizations. Inclusion criteria addressed One Health relevance, use at the national, subnational, or regional level in ≥5 locations, and publicly available information. Tools were assessed for applicability by OH JPA action track, TOC pathway, scope, and intended outcomes, as well as the extent to which tools addressed gender equality, social inclusion, and environmental dimensions of One Health. Of 132 candidate tools, 50 (38%) met the inclusion criteria. These tools addressed all six OH JPA Action Tracks, but relatively fewer tools addressed Action Tracks 4 (Food Safety), 5 (Antimicrobial Resistance), and 6 (Environmental Integration). Tools were available to support all three TOC outcome pathways, and many addressed more than one Action Track and TOC outcome pathway. Most available One Health tools addressed assessment and to a lesser extent implementation, with fewer tools available for action planning, prioritization, and monitoring. Gaps and opportunities for improving One Health tools were identified, including the integration of the environment dimension, gender equality, and social inclusion. Ultimately, our findings will contribute to further the advancement of One Health globally, including via OH JPA implementation, while spurring adjustments to existing One Health tools and the development of new ones to address key gaps. One Health impact statement The One Health approach is gaining momentum globally, and this study represents the first integrated mapping and analysis of globally available One Health tools. Our findings aim to improve the quality, applicability, and availability of tools to support One Health implementation at the subnational, national, regional, and global levels, including through the Quadripartite’s One Health Joint Plan of Action. By using the OHHLEP definition of One Health to assess available One Health tools and map them onto the One Health Joint Plan of Action, we identify the need for a systematic approach and enhanced integration across dimensions of One Health to lead to sustainable One Health systems.

The health of humans, domestic and wild animals, plants, and the environment are inter-dependent. Global anthropogenic change is a key driver of disease emergence and spread and leads to biodiversity loss and ecosystem function degradation, which are themselves drivers of disease emergence. Pathogen spill-over events and subsequent disease outbreaks, including pandemics, in humans, animals and plants may arise when factors driving disease emergence and spread converge. One Health is an integrated approach that aims to sustainably balance and optimize human, animal and ecosystem health. Conventional disease surveillance has been siloed by sectors, with separate systems addressing the health of humans, domestic animals, cultivated plants, wildlife and the environment. One Health surveillance should include integrated surveillance for known and unknown pathogens, but combined with this more traditional disease-based surveillance, it also must include surveillance of drivers of disease emergence to improve prevention and mitigation of spill-over events. Here, we outline such an approach, including the characteristics and components required to overcome barriers and to optimize an integrated One Health surveillance system.

Sindbis virus (SINV) is a widely dispersed mosquito-borne alphavirus. Reports of Sindbis disease are largely restricted to northern Europe and South Africa. SINV is frequently sampled in Australian mosquito-based arbovirus surveillance programs, but human disease has rarely been reported. Molecular epidemiological studies have characterized six SINV genotypes (G1–G6) based on E2 gene phylogenies, mostly comprising viruses derived from the African–European zoogeographical region and with limited representation of Australasian SINV. In this study, we conducted whole genome sequencing of 66 SINV isolates sampled between 1960 and 2014 from countries of the Australasian region: Australia, Malaysia, and Papua New Guinea. G2 viruses were the most frequently and widely sampled, with three distinct sub-lineages defined. No new G6 SINV were identified, confirming geographic restriction of these viruses to south-western Australia. Comparison with global SINV characterized large-scale nucleotide and amino acid sequence divergence between African–European G1 viruses and viruses that circulate in Australasia (G2 and G3) of up to 26.83% and 14.55%, respectively, divergence that is sufficient for G2/G3 species demarcation. We propose G2 and G3 are collectively a single distinct alphavirus species that we name Argyle virus, supported by the inapparent or mild disease phenotype and the higher evolutionary rate compared with G1. Similarly, we propose G6, with 24.7% and 12.61% nucleotide and amino acid sequence divergence, is a distinct alphavirus species that we name Thomson’s Lake virus.

Japanese encephalitis virus (JEV) first emerged in the Torres Strait of north-eastern Australia in 1995, with three human cases, and widespread infection of pigs on a number of islands. The virus was shown to belong to genotype II. Further cases occurred in 1998, including the first case on mainland Australia on Cape York. A second genotype of JEV, genotype Ia, was reported in mosquitoes and pigs in 2000–04, possibly displacing genotype II. JEV re-emerged in Australia with a fatal human case on the Tiwi Islands, Northern Territory, in 2021, and shown to belong to genotype IV. This case was followed about a year later by a large outbreak of JE; first detected in piggeries in four states, Queensland, New South Wales, Victoria, and South Australia, resulting in reproductive losses affecting 80 piggeries and 42 human cases, with seven fatal cases. The wide geographic spread of cases suggested that the virus had been circulating for a number of months or even years prior to detection, and has led to significant concern that the virus will become endemic to Australia, in a similar ecology to Murray Valley encephalitis virus. Known competent mosquito vectors and ardeid birds, as maintenance hosts, occur in Australia, and it is probable that feral pigs will provide an additional wildlife reservoir of virus. Little is known of the properties of genotype IV, but it is expected to have a similar ecology and pathogenesis to other JEV genotypes.

A fatal case of Japanese encephalitis (JE) occurred in northern Australia in early 2021. Sequence studies showed that the virus belonged to genotype IV (GIV), a genotype previously believed to be restricted to the Indonesian archipelago. This was the first locally acquired case of Japanese encephalitis virus (JEV) GIV to occur outside Indonesia, and the second confirmed fatal human case caused by a GIV virus. A closely related GIV JEV strain subsequently caused a widespread outbreak in eastern Australia in 2022 that was first detected by fetal death and abnormalities in commercial piggeries. Forty-two human cases also occurred with seven fatalities. This has been the first major outbreak of JEV in mainland Australia, and geographically the largest virgin soil outbreak recorded for JEV. This outbreak provides an opportunity to discuss and document the factors involved in the virus’ spread and its ecology in a novel ecological milieu in which other flaviviruses, including members of the JE serological complex, also occur. The probable vertebrate hosts and mosquito vectors are discussed with respect to virus spread and its possible endemicity in Australia, and the need to develop a One Health approach to develop improved surveillance methods to rapidly detect future outbreak activity across a large geographical area containing a sparse human population. Understanding the spread of JEV in a novel ecological environment is relevant to the possible threat that JEV may pose in the future to other receptive geographic areas, such as the west coast of the United States, southern Europe or Africa.

In early 2022, the Japanese encephalitis virus (JEV) was identified as the cause of stillborn and mummified piglets in pig farms in southeastern Australia. Human cases and additional pig farms with infected piglets were subsequently identified across a widespread area encompassing four states. To inform surveillance and control programs, we synthesized existing information on Australian vectors of JEV, much of which was generated in response to incursions of JEV into the northern state of Queensland between 1995 and 2005. Members of the Culex sitiens subgroup, particularly Culex annulirostris, should be considered the primary vectors of JEV in Australia, as they yielded >87% of field detections of JEV, were highly efficient laboratory vectors of the virus, readily fed on pigs and birds (the key amplifying hosts of the virus) when they were available, and are widespread and often occur in large populations. Three introduced species, Culex quinquefasciatus, Culex gelidus and Culex tritaeniorhynchus may also serve as vectors, but more information on their geographical distribution, abundance and bionomics in the Australian context is required. Mosquitoes from other genera, such as Aedes and Verrallina, whilst considered relatively poor vectors, could play a regional or supplemental role in transmission, especially facilitating vertical transmission as a virus overwintering mechanism. Additional factors that could impact JEV transmission, including mosquito survival, dispersal and genetics, are also discussed. Possible directions for investigation are provided, especially in the context of the virus emerging in a region with different mosquito fauna and environmental drivers than northern Australia.

Ross River virus (RRV) is the most medically significant mosquito-borne virus of Australia, in terms of human morbidity. RRV cases, characterised by febrile illness and potentially persistent arthralgia, have been reported from all Australian states and territories. RRV was the cause of a large-scale epidemic of multiple Pacific Island countries and territories (PICTs) from 1979 to 1980, involving at least 50,000 cases. Historical evidence of RRV seropositivity beyond Australia, in populations of Papua New Guinea (PNG), Indonesia and the Solomon Islands, has been documented. We describe the genomic characterisation and timescale analysis of the first isolate of RRV to be sampled from PNG to date. Our analysis indicates that RRV has evolved locally within PNG, independent of Australian lineages, over an approximate 40 year period. The mean time to most recent common ancestor (tMRCA) of the unique PNG clade coincides with the initiation of the PICTs epidemic in mid-1979. This may indicate that an ancestral variant of the PNG clade was seeded into the region during the epidemic, a period of high RRV transmission. Further epidemiological and molecular-based surveillance is required in PNG to better understand the molecular epidemiology of RRV in the general Australasian region.

Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.

Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1–3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state’s history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections continue to be a serious emerging disease problem internationally with well over 1000 cases and a major outbreak outside of the Middle East region. While the hypothesis that dromedary camels are the likely major source of MERS-CoV infection in humans is gaining acceptance, conjecture continues over the original natural reservoir host(s) and specifically the role of bats in the emergence of the virus. Dromedary camels were imported to Australia, principally between 1880 and 1907 and have since become a large feral population inhabiting extensive parts of the continent. Here we report that during a focussed surveillance study, no serological evidence was found for the presence of MERS-CoV in the camels in the Australian population. This finding presents various hypotheses about the timing of the emergence and spread of MERS-CoV throughout populations of camels in Africa and Asia, which can be partially resolved by testing sera from camels from the original source region, which we have inferred was mainly northwestern Pakistan. In addition, we identify bat species which overlap (or neighbour) the range of the Australian camel population with a higher likelihood of carrying CoVs of the same lineage as MERS-CoV. Both of these proposed follow-on studies are examples of “proactive surveillance”, a concept that has particular relevance to a One Health approach to emerging zoonotic diseases with a complex epidemiology and aetiology.

In recent years, it has become evident that a generational gap has developed in the community of arbovirus research. This apparent gap is due to the dis-investment of training for the next generation of arbovirologists, which threatens to derail the rich history of virus discovery, field epidemiology, and understanding of the richness of diversity that surrounds us. On the other hand, new technologies have resulted in an explosion of virus discovery that is constantly redefining the virosphere and the evolutionary relationships between viruses. This paradox presents new challenges that may have immediate and disastrous consequences for public health when yet to be discovered arboviruses emerge. In this review we endeavor to bridge this gap by providing a historical context for the work being conducted today and provide continuity between the generations. To this end, we will provide a narrative of the thrill of scientific discovery and excitement and the challenges lying ahead.

Japanese encephalitis virus (JEV) is a major cause of neurological disease in Asia. It is a zoonotic flavivirus transmitted between water birds and/or pigs by Culex mosquitoes; humans are dead-end hosts. In 1995, JEV emerged for the first time in northern Australia causing an unprecedented outbreak in the Torres Strait. In this article, we revisit the history of JEV in Australia and describe investigations of JEV transmission cycles in the Australian context. Public health responses to the incipient outbreak included vaccination and sentinel pig surveillance programs. Virus isolation and vector competence experiments incriminated Culex annulirostris as the likely regional vector. The role this species plays in transmission cycles depends on the availability of domestic pigs as a blood source. Experimental evidence suggests that native animals are relatively poor amplifying hosts of JEV. The persistence and predominantly annual virus activity between 1995 and 2005 suggested that JEV had become endemic in the Torres Strait. However, active surveillance was discontinued at the end of 2005, so the status of JEV in northern Australia is unknown. Novel mosquito-based surveillance systems provide a means to investigate whether JEV still occurs in the Torres Strait or is no longer a risk to Australia.

Abstract Objective: To assess evidence of recent and past exposure to Murray Valley encephalitis virus (MVEV) and West Nile clade Kunjin virus (KUNV) in residents of the Murray Valley, Victoria, during a period of demonstrated activity of both viruses in early 2011. Methods: A cross‐sectional serosurvey using two convenience samples: stored serum specimens from a diagnostic laboratory in Mildura and blood donors from the Murray Valley region. Specimens were collected between April and July 2011. The main outcome measure was total antibody (IgM and IgG) reactivity against MVEV and KUNV measured using an enzyme immunoassay and defined as inhibiting binding of monoclonal antibodies by >50%, when compared to negative controls. Evidence of recent exposure was measured by the presence of MVEV and KUNV IgM detected by immunofluorescence. Results: Of 1,115 specimens, 24 (2.2%, 95% CI 1.3–3.0%) were positive for MVEV total antibody, and all were negative for MVEV IgM. Of 1,116 specimens, 34 (3.1%, 95% CI 2.0–4.0%) were positive for KUNV total antibody, and 3 (0.27%) were KUNV IgM positive. Total antibody seroprevalence for both viruses was higher in residents born before 1974. Conclusions: Despite widespread MVEV and KUNV activity in early 2011, this study found that seroprevalence of antibodies to both viruses was low (

Edge Hill virus (EHV) is a mosquito-borne flavivirus isolated throughout Australia during mosquito surveillance programs. While not posing an immediate threat to the human population, EHV is a taxonomically interesting flavivirus since it remains the only member of the yellow fever virus (YFV) sub-group to be detected within Australia. Here we present both an antigenic and genetic investigation of collected isolates, and confirm taxonomic classification of the virus within the YFV-group. Isolates were not clustered based on geographical origin or time of isolation, suggesting that minimal genetic evolution of EHV has occurred over geographic distance or time within the EHV cluster. However, two isolates showed significant differences in antigenic reactivity patterns, and had a much larger divergence from the EHV prototype (19% nucleotide and 6% amino acid divergence), indicating a distinct subtype or variant within the EHV subgroup.

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

China holds the key to solving many questions crucial to global control of severe acute respiratory syndrome (SARS). The disease appears to have originated in Guangdong Province, and the causative agent, SARS coronavirus, is likely to have originated from an animal host, perhaps sold in public markets. Epidemiologic findings, integral to defining an animal-human linkage, may then be confirmed by laboratory studies; once animal host(s) are confirmed, interventions may be needed to prevent further animal-to-human transmission. Community seroprevalence studies may help determine the basis for the decline in disease incidence in Guangdong Province after February 2002. China will also be able to contribute key data about how the causative agent is transmitted and how it is evolving, as well as identifying pivotal factors influencing disease outcome. There must be support for systematically addressing these fundamental questions in China and rapidly disseminating results.

The rapid succession of the pandemic of arbovirus diseases, such as dengue, West Nile fever, chikungunya, and Zika fever, has intensified research on these and other arbovirus diseases worldwide. Investigating the unique mode of vector-borne transmission requires a clear understanding of the roles of vertebrates. One major obstacle to this understanding is the ambiguity of the arbovirus definition originally established by the World Health Organization. The paucity of pertinent information on arbovirus transmission at the time contributed to the notion that vertebrates played the role of reservoir in the arbovirus transmission cycle. Because this notion is a salient feature of the arbovirus definition, it is important to reexamine its validity. This review addresses controversial issues concerning vertebrate reservoirs and their role in arbovirus persistence in nature, examines the genesis of the problem from a historical perspective, discusses various unresolved issues from multiple points of view, assesses the present status of the notion in light of current knowledge, and provides options for a solution to resolve the issue.

Until recently, West Nile (WN) and Kunjin (KUN) viruses were classified as distinct types in the Flavivirus genus. However, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. To better define this relationship, we performed sequence analyses on 32 isolates of KUN virus and 28 isolates of WN virus from different geographic areas, including a WN isolate from the recent outbreak in New York. Sequence comparisons showed that the KUN virus isolates from Australia were tightly grouped but that the WN virus isolates exhibited substantial divergence and could be differentiated into four distinct groups. KUN virus isolates from Australia were antigenically homologous and distinct from the WN isolates and a Malaysian KUN virus. Our results suggest that KUN and WN viruses comprise a group of closely related viruses that can be differentiated into subgroups on the basis of genetic and antigenic analyses.

To determine whether relocating domestic pigs, the amplifying host of Japanese encephalitis virus (JEV), decreased the risk for JEV transmission to humans in northern Australia, we collected mosquitoes for virus detection. Detection of JEV in mosquitoes after pig relocation indicates that pig relocation did not eliminate JEV risk.