Emergence and spread of Japanese encephalitis virus genotype IV in a novel ecosystem: Australia, 2021-2022

Emergence and spread of Japanese encephalitis virus genotype IV in a novel ecosystem: Australia, 2021-2022 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Emergence and spread of Japanese encephalitis virus genotype IV in a novel ecosystem: Australia, 2021-2022 John-Sebastian Eden, Matthew Neave, Craig Smith, Jana Batovska, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7046873/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract An unprecedented outbreak of the mosquito-borne Japanese encephalitis virus (JEV) occurred in south-eastern Australia in 2022. The outbreak was caused by a novel lineage of JEV genotype IV, which first emerged in Northern Australia in 2021, and resulted in 45 human cases, including 7 deaths, and over 80 infected piggeries during 2021 and 2022. We analyzed 166 whole genomes of JEV from field collected mosquitoes (n=9), humans (n=2), and farmed (n=136) and feral pigs (n=19). The majority of outbreak sequences clustered into two genetically distinct lineages (clades A and B), separated by three formative single nucleotide polymorphisms, which were circulating between February 2021 and August 2022. Both lineages were detected in mosquito and pig samples, while only clade A was detected in the human samples sequenced. We conclude that clades A and B were likely to have been circulating prior to the outbreak. A lack of spatial-temporal phylogenetic structure suggests a rapid dispersal of the outbreak lineages in largely JEV naïve vertebrate populations and competent mosquito vector populations. Ongoing surveillance and genomic characterization of new detections is required to monitor the spread of JEV, the emergence of alternative JEV genotypes or lineages, as well as changes in the viral ecology. Biological sciences/Microbiology/Virology/Viral evolution Biological sciences/Microbiology/Virology/Viral epidemiology Japanese encephalitis genotype IV flavivirus Australia phylodynamics Figures Figure 1 Figure 2 Main Japanese encephalitis virus (JEV) is a single-stranded RNA virus belonging to the species Orthoflavivirus japonicum , family Flaviviridae . Most human infections with JEV are asymptomatic, with the ratio of clinically apparent to inapparent infections ranging from 1:1000 to 1:25 (Vaughn and Hoke, 1992). Despite this, the virus has been estimated to cause approximately 68,000 clinical cases of encephalitis annually worldwide, predominantly in Asia, and in some years this number may exceed 100,000 (Vaughn and Hoke, 1992; Quan et al. , 2020). The estimated mortality rate of human cases of Japanese encephalitis (JE) is 20 – 30%, while an estimated 30 – 50% of survivors have long-term neurological sequelae (van den Hurk, Ritchie and Mackenzie, 2009; Khandaker et al. , 2016; Quan et al ., 2020). These high morbidity and mortality rates make JEV one of the leading causes of encephalitis in Asia (Campbell et al. , 2011). JEV also causes significant disease in domestic animals. In horses, JEV infection can cause potentially fatal encephalitis, while infection in pigs can cause significant reproductive losses, including abortion, congenital defects, birth of weak piglets with neurological signs, or aspermia in boars (Williams et al. , 2019). The natural transmission cycle of JEV involves wild birds, mosquitoes, and domestic and wild pigs (van den Hurk, Ritchie and Mackenzie, 2009). Wild birds, particularly water birds of the family Ardeidae, are considered the main JEV reservoir host in endemic scenarios (Soman et al. , 1977; van den Hurk, Ritchie and Mackenzie, 2009). JEV is transmitted by mosquito vectors, primarily of the genus Culex , to a variety of mammalian hosts. Pigs are considered amplifying hosts for JEV and, when present in sufficient numbers and density, may drive infection dynamics and influence human infection risk (Mulvey et al. , 2021). Other mammals, such as humans, horses and other livestock, do not develop sufficient levels of viremia to infect feeding mosquitoes and are considered dead-end hosts (van den Hurk et al. , 2019). A range of other animals, other than ardeid birds and pigs, have been identified as potential vertebrate hosts that may require further investigation (Levesque et al. , 2024; Moore et al. , 2025). Five phylogenetically distinct genotypes of JEV (I to V) are recognized (Chen, Tesh and Rico-Hesse, 1990; Chen, Rico-Hesse and Tesh, 1992; Satchidanandam and Uchil, 2001; Solomon et al. , 2003). A characteristic of these genotypes has been their changing patterns of geographic distribution and circulation over time (Gao et al. , 2019). In Australia, JE first emerged in 1995 when 3 cases were diagnosed among residents of the Torres Strait Islands in the country’s north (Hanna et al. , 1996). A single additional case occurred in the Torres Strait in 1998, as well as the first case on mainland Australia, in the western Cape York area of Queensland (Hanna et al. , 1999). Accompanying these cases were detections in mosquitoes and pigs (Hanna et al. , 1996, 1999). The 1995 and 1998 viruses belonged to genotype II and were thought to have originated from Papua New Guinea where genetically identical isolates of JEV were reported (Johansen et al. , 2000). Subsequently, isolates obtained in the Torres Strait Islands (2000, 2004) and the Northern Peninsula Area (NPA) of Cape York (2004) belonged to a second genotype, genotype I (Pyke et al. , 2001; van den Hurk et al. , 2006). After 2011, many surveillance activities were discontinued, obscuring the JEV situation in Australia during this period (van den Hurk et al. , 2019). However, opportunistic serological surveillance of livestock in the Torres Strait Islands and NPA provided suggestive evidence for seasonal activity of JEV between 2005 and 2021 (Sikazwe et al. , 2022). More recently, a fatal human case of JE occurred on the Tiwi Islands in February 2021, caused by a strain of the rare genotype IV (Sikazwe et al. , 2022; Waller et al. , 2022). This genotype had previously been isolated from mosquitoes and pigs in Indonesia (Chen, Rico-Hesse and Tesh, 1992; Schuh et al. , 2013; Kuwata et al. , 2020; Faizah et al. , 2021) and caused the death of an Australian tourist who had acquired infection in Bali, in 2019 (Pyke et al. , 2020). Outside Indonesia, the only evidence of JEV genotype IV circulation was a sequence derived from an unconfirmed human case in Vietnam, in 1979 (Mackenzie et al. , 2022). In February 2022, one year after the Tiwi Island case, JEV genotype IV was identified as the cause of stillbirths, piglet deformities and neurological disease in piggeries in the states of New South Wales (NSW), Queensland (QLD) and Victoria (VIC) (Australian Government Department of Health, 2022; Iglesias, 2022). Concurrently, cases of human neurological disease were reported in eastern and south-eastern Australia, which were subsequently diagnosed as JE (Furuya-Kanamori et al. , 2022). Metagenomic sequencing of post-mortem brain samples collected from a patient with meningoencephalitis also led to the detection of JEV genotype IV (Maamary et al. , 2023). These human and swine cases signaled the unprecedented spread and rapid circulation of JEV throughout south-eastern Australia. In this study, we report the genomic sequencing and analysis of JEV genomes from humans, pigs (domestic and feral) and mosquitoes, collected during and prior to the Australian JEV outbreak of 2022. We describe the phylogenetic structure of the Australian genotype IV lineage, the relatedness of viral genomes from vertebrates and mosquitoes and assess the possible origins of the genotype IV incursion into the Australian mainland. Results and discussion Widespread detection of Japanese encephalitis virus in Australia From February 2021 to December 2022, 45 human cases of JE, including seven deaths, and over 80 infected commercial piggeries were reported in Australia (Iglesias 2022; Australian Government Department of Agriculture, Fisheries and Forestry, 2023; Australian Government Department of Health and Aged Care, 2023). We determined 166 JEV genotype IV genomes from mosquitoes and vertebrate hosts across five Australian states and territories (Fig 1) as part of a national One Health outbreak response involving multiple jurisdictions. These data represent the largest genomic epidemiology study of a JEV outbreak, considerably increases the number of publicly available JEV whole genome sequences from 365 (at the time of writing) to 531 and allows an understanding of the molecular epidemiology of an outbreak unparalleled for JEV within a novel ecosystem in Australia or elsewhere. Peaks in the onset of human symptomatic cases in February 2022 preceded the peak of newly infected piggeries (Fig 1, panels B, C). Retrospective testing of stored human clinical samples with undiagnosed neurological impairment identified a case of JE acquired in the Northern Territory (NT) from a Victorian resident in May 2021 (NT Health, 2023). The active circulation of JEV in mosquitoes as early as January 2022 was detected through retrospective testing of stored mosquito pool samples. Proactive surveillance of mosquito populations showed continuing JEV circulation through to April 2022 (Pyke et al., 2024). Culex annulirostris (considered the primary vector of JEV in Australia) was present in all trap collections, although other implicated vectors, including Cx. quinquefasciatus and Cx. gelidus were also collected (van den Hurk et al. , 2022; Supp Table 1). Of the 166 JEV genome sequenced, 93% (154 of 166) were derived from pigs including a single culture isolate (Fig 1D), reflecting the high number of samples received from affected piggeries (n=136) and feral pig surveillance (n=19). Prior to the current outbreak, there were limited JEV surveillance activities in the areas where the virus was subsequently detected. Similarly, there was little pre-existing JEV-specific immunity in humans in the same areas, as JEV vaccines were not routinely used and there had been no recognized JEV activity. Following the outbreak, prospective molecular and serological testing of samples collected from persons in NSW with suspected encephalitis suggested an incidence of 0.15/100,000 over the first three months of the outbreak (Howard-Jones et al. , 2022). Phylodynamics of JEV genotype IV emergence in Australia 2021-22 To examine the dispersal patterns and reveal the possible origins of the outbreak, we performed a phylodynamic analysis of JEV genomes (n=166) collected during the outbreak period and the season prior, spanning February 2021 to August 2022, as well as the previously published genome from the human case on the Tiwi Islands from February 2021 (Fig 2). Sequences were collected from five Australian states and territories, with the majority sampled along the NSW and VIC state border regions in the southeast of the country where the highest concentrations of human and animal cases were identified (Fig 2A). Several genomes were also obtained from northern regions of the NT and QLD that may represent the initial location of introduction of GIV JEV, based on previous detections in these regions (Hanna et al. , 1996, 1999; Sikazwe et al. , 2022). Initially, the genomes were aligned with all available GenBank reference genomes (n=365, as of 31 October 2022) and analyzed using a maximum-likelihood approach (Figure 2B). This showed that the viruses from the Australian outbreak were all genotype IV and formed a single monophyletic group, here termed the genotype IV Australian 2021-22 lineage, which were similar but distinct from genotype IV sequences previously detected in Indonesia and Vietnam. The closest relative on NCBI GenBank was the Indonesian sequence JEV/Swine/Bali-93/2017 (Accession number LC461961) sharing ~96.7% nucleotide identity. Using this sequence as an outgroup, genotype IV Australian 2021-22 lineage-specific phylogenies were then estimated (Figs 2C-D, Supp Fig 1 & 2) to examine genetic, host and temporal structure. The genetic diversity within the outbreak lineage was very low (<1% distance at the nucleotide level), with most sequences falling into two well-supported clades (A & B) that were defined by three synonymous substitutions - positions 539, 5537 and 7241 (relative to JEV/Swine/Bali-93/2017) (Fig 2C). These sites correspond to the pre-membrane (prM), non-structural protein 3 (NS3) and the NS4B protein genes, respectively. An intermediate lineage between clades A and B was also retrospectively identified in the North Queensland region from a case of spontaneous abortion in a piggery in April 2021. A group of putative ancestral sequences were also identified with a distinct mutational signature at the same three sites that included the genome from the earliest known Australian JEV genotype IV case from the Tiwi Islands in February 2021 (Sikazwe et al. , 2022; Waller et al. , 2022) as well as a genome obtained from a feral pig in the NPA (Far North Queensland) in May 2022. A small cluster of three sequences from commercial piggeries located in the Murray River region near the state border of VIC and NSW constituted an intermediate clade between these ancestral lineages and clades A and B. Notably, the phylogeny lacked temporal structure (Supp Figs 3 & 4). Furthermore, there was no apparent clustering of sequences by sampling location, except at some fine spatial scales (specific regions or outbreaks at individual properties). A lack of host-specific phylogenetic clustering was also observed with pig and mosquito detections throughout both major clades (Fig 2C, Supp Figs 1 & 2). JEV sequences derived from the 2022 human cases belonged to clade A, although only a limited number of genomes (n=2) were able to be recovered from patients as most human cases were laboratory confirmed by serology. Together with the star-like phylogeny (Fig 2D), these results suggest that the JEV outbreak in 2022 in southeastern Australia was driven by the concurrent emergence and rapid dispersal of at least two virus lineages in largely JEV naïve populations of vertebrate hosts and competent mosquito vectors. To address the possible role of domestic pig movements in the spread of JEV during the 2022 outbreak, we analyzed genomes derived from 23 infected commercial piggeries in QLD, NSW, VIC and SA, for which information on location and farm operations was available (Supp Table 2). A total of 37 JEV genomes were sequenced and included in the phylogenetic analysis (Supp Fig 5). Genomes belonging to both clades A and B were identified from piggeries located in all states except SA; genomes from only three SA piggeries were available, all clade A. Notably, JEV belonging to both clades were detected within individual production units from QLD, NSW, and VIC, including one NSW farm that was closed to pig movements on or off the premises during the time of study. Another closed farm located in the same region of NSW contained genomes belonging to clade A only, whilst in VIC, closed farms, all located within the same region, contained either clade A or B genomes. One farm in QLD (property 3) that received fortnightly introductions of pigs from another farm located approximately 50 kilometers south (property 2) contained clade A, whereas clade B genome was detected in the donor farm. Thus, this analysis indicates that both clades were co-circulating freely across southeastern Australian piggeries and that there is no evidence to indicate that pig movements between farms contributed significantly to the epidemiology of this outbreak. Consistent with this conclusion was the concurrent detection of clade A and B genomes in mosquitoes sampled in all states with infected piggeries (Supp Table 1). However, we cannot exclude local oronasal transmission of JEV between pigs within individual farming units via direct contact (Ricklin et al. , 2016). In this regard, for some properties from which two or more sequences were obtained, closely related clusters were observed in our phylogenetic analysis (Supp Figs 1 & 2.), suggesting localized circulation of virus, either between pigs and mosquitoes, or, possibly, directly between pigs. Whilst our study does not provide conclusive evidence on the timing of the origins of the 2022 outbreak, the identification of two basal sequences in Northern Australia (Tiwi Islands, February 2021 and Far North Queensland, May 2022) supports the emergence of JEV in Northern Australia, followed by spread to the southeast of the country. Whether these two basal lineages represent two different incursions of JEV into mainland Australia or result from a prolonged period of circulation in Northern Australia, is unclear. An intermediate lineage between clades A and B was also retrospectively identified in the North Queensland region in April 2021, adding further support to the hypothesis that related viruses were already circulating in the lead up to the outbreak in southeastern Australia over the summer of 2021-22. These analyses were unable to shed light on the international origins of the Australian genotype IV lineage beyond the conclusions previously made in relation to the 2021 Tiwi Island index case (Sikazwe et al. , 2022) as our analysis was limited by the lack of earlier JEV genotype IV sequence data from the Indo-Pacific region. Nevertheless, based on the close phylogenetic relationship of the Australian lineage with the most recently isolated Indonesian viruses, the Indonesian archipelago is the probable geographic source. Introduction from Papua New Guinea, from which JEV genotype II is believed to have emerged in Northern Australia in the 1990s (Johansen et al. , 2000), also cannot be discounted as analysis of environmental conditions and waterbird population dynamics suggests large-scale movement of reservoir host species occurred from New Guinea to Northern Australia in 2020 and south through the Murray-Darling Basin in 2021-22 (Purnell, 2022). This southern dispersal may have been assisted by the onset of La Niña dominated weather patterns and concomitant above average rainfall and flooding that facilitated the enhancement of environmental conditions for both reservoir hosts and vectors ahead of the 2021-2022 summer (Walsh et al ., 2023). Notwithstanding the long-range movement of birds, key vector species are also known to disperse many kilometers and potentially facilitated by weather conditions (van den Hurk et al ., 2022), further assisting the geographic spread of JEV. Limitations and future directions A limitation of the present study was the sampling bias of JEV predominantly sourced from pigs, either from lymphoid tissue of adult pigs and aborted fetuses, or neonates that acquired infection in utero . The added complexity of samples derived from fetal or neonatal tissue types is the date of infection in utero can only be estimated based on the gestation period of the infected sow, which do not typically show clinical signs of infection. Additionally, spatial under-sampling, especially in areas that lack farmed pigs but may have large feral pig populations, such as in Northern Australia (Gentle, Wilson and Cuskelly, 2022; Mackenzie et al. , 2022), or a large waterbird population, made it difficult to infer viral origins and estimated timing of introduction, although our analysis is suggestive of at least one introduction into Northern Australia during or prior to the 2020/21 wet season. A lack of JEV whole genome sequences from wild birds or other wildlife also limits inferences on large-scale temporospatial patterns and epidemiology. This highlights the importance of developing improved surveillance systems to understand the viral ecology in wildlife, especially in the context of JEV emerging within a new ecosystem. Multisectoral collaboration, a key underlying principle of One Health (OHHLEP, 2022), was clearly a major feature in the response to the outbreak, as had previously been described for JEV (Impoinvil, Baylis and Solomon , 2013). Ongoing surveillance and genomic characterization of new JEV detections is required to monitor the spread of JEV, the emergence of alternative JEV genotypes or the presence of additional genotype IV genetic lineages, as well as changes in viral ecology. Collectively, this will inform suitable surveillance strategies and diagnostics to support an ongoing One Health approach to the monitoring, control and prevention of JE in Australia. Methods Specimens and Sample selection Clinical specimens for laboratory diagnostic testing of humans were received by public health laboratories (NSW Health Pathology-Institute of Clinical Pathology and Medical Research, Victorian Infectious Diseases Reference Laboratory) and for farmed pigs by the relevant state or national veterinary laboratories (AgriBio Centre, Australian Centre for Disease Preparedness [ACDP], Berrimah Veterinary Laboratory [BVL], Biosecurity Sciences Laboratory, Elizabeth Macarthur Agriculture Institute, Gribbles Veterinary Pathology South Australia). Feral pig samples collected at necropsy were collected as part of Northern Australia Quarantine Strategy surveillance activities in response to the outbreak and sent to ACDP or BVL for diagnostic testing. Mosquitoes were collected in carbon dioxide-baited light traps as part of arbovirus surveillance conducted by the Health Departments of NSW, QLD, VIC and SA (Knope et al. , 2019). Mosquitoes were morphologically identified and placed in pools of mixed species of different sizes prior before being submitted for JEV detection. Samples in which JEV RNA were detected by RT-qPCR using either universal JEV primers or modified genotype IV primers (Shao et al. , 2018) or RT-PCR followed by next generation sequencing (below) were identified for genomic analysis through a muti-sectoral national collaboration. JEV RNA detected in cerebrospinal fluid or brain tissue collected pre- and post-mortem in humans were included for whole genome sequencing (Howard-Jones et al. , 2022; Maamary et al. , 2023). Three types of pig derived samples included: i) farmed pig samples represented by JEV RT-qPCR positive material received from piglets (either aborted or showing neurological signs at birth); ii) feral pig samples consisted of JEV RT-qPCR positive material sourced from the tonsil tissue collected at necropsy; and iii) JEV RT-qPCR positive semen collected from farmed boars. Mosquito samples consisted of homogenized JEV RT-qPCR positive pools. Japanese encephalitis virus genomic sequencing Australian Centre for Disease Preparedness The initial JEV genome from an infected piggery in QLD (JEV/sw-22-00722-11/Qld/2022; GenBank Acc. ON624132) was obtained using a direct metagenomic approach, as previously described (Sikazwe et al. , 2022). First, total RNA was extracted from tonsillar samples using a MagMax 96 Viral RNA Kit (ThermoFisher Scientific). A sequence library was prepared using the TruSeq RNA Library Prep Kit v2 and sequenced with a P2 300 cycle cartridge on a NextSeq2000 instrument (Illumina). Raw reads were cleaned using Trimmomatic v.0.39 (Bolger, Lohse and Usadel, 2014) and mapped to a recent genotype IV genome (GenBank Acc. OM867669) using Bowtie v.2.4.4 (Langmead and Salzberg, 2012). Mapping was manually examined with Geneious Prime v.2020.2.4 and a complete 10,949 bp JEV genome generated. Subsequently, probe hybridization and primer tiling approaches were developed to allow for a more cost-effective whole genome characterization. The probe hybridization procedure was developed using the MyBaits Target Capture Kit (Arbor Biosciences). Oligonucleotide probes for enrichment were designed using the JEV/sw-22-00722-11/Qld/2022 genome, a recent genotype IV genome (GenBank Acc. OM867669), plus a representative selection of JEV genomes from GenBank. The final design consisted of 11,435 probes of 70 bp each with 4x depth over the JEV genomes (Supp material). Sequencing libraries were then prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) and enriched for JEV fragments using the probes as per the manufacturer’s recommendations (Arbor Biosciences). The libraries were reamplified and sequenced using 300 cycle cartridges on either a MiSeq or NextSeq2000 instrument (Illumina). The raw reads were assembled into JEV genomes as described above. Biosecurity Sciences Laboratory Queensland A multiplex RT-PCR for the sequencing of JEV genomes directly from clinical samples was developed using a published method (Quick et al., 2017). Using reference genomes from the 2022 outbreak, JEV/sw-22-00722-11/Qld/2022 and JEV/sw-22-00698-27/Qld/2022, and other genotype IV genomes (LC461961, MT253731, LC579814, KU705228 and AY184212), primers were designed using the online tool Primal Scheme (https://primalscheme.com/), adopting a default amplicon size of 400 bp to produce 34 primer pairs (Supp Table 3). Amplicons from these PCRs were pooled and sequenced on a MinION (Oxford Nanopore Technologies) using a Flongle flow cell and the Rapid Barcoding Kit (RBK004). Westmead Institute for Medical Research and AgriBio Centre for AgriBioscience The JEV genome of the first clinical case in NSW was obtained using a previously described rapid RNA metagenomic approach (Annand et al., 2022; Maamary et al., 2023; Tulloch et al., 2023). Primer tiling approaches were designed using Primal Scheme (https://primalscheme.com/) to sequence JEV genomes using reference genomes (JEV/sw-22-00722-11/Qld/2022; GenBank Acc. ON624132). Adopting a default amplicon size as 500bp to produce 27 primer pairs for the primer set of JEV-SW-500, and as 800bp to produce 16 primer pairs for the primer set of JEV-G4-800 (Supp Table 4). Amplicons from these PCRs were pooled and sequenced either on an iSeq 100 instrument using the Nextera XT DNA Library Preparation Kit and 300 cycle cartridges on iSeq 100 instrument (Illumina) or on a MinION (Oxford Nanopore Technologies) using a flow cell (R9.4.1) and the Rapid Barcoding Kit (RBK110.96). The raw reads were assembled into JEV genomes. Victorian Infectious Diseases Reference Laboratory An almost complete JEV genome was obtained from a Victorian clinical sample using an RNA-directed (DNAse1 treated nucleic acid), sequence-independent, single-primer amplification (SISPA) method as described (Kafetzopoulou et al ., 2018). Sequencing libraries from derived amplicons were created using the Illumina DNA Prep Library kit before sequencing on an Illumina NextSeq550 2 x 150bp mid output cartridge (Illumina). Phylogenetic and phylodynamic analysis The genomes that were sequenced using an Illumina platform were assembled with a Snakemake v.5 pipeline (github.com/neavemj/JEV_assembly_pipe). Briefly, the pipeline trims raw reads with Trimmomatic v.0.39 (Bolger, Lohse and Usadel, 2014), aligns cleaned reads to a reference JEV genome with Bowtie v.2.4 (Langmead and Salzberg, 2012), then masks the tiling primers and generates a consensus genome with iVar v.1.3 (Grubaugh et al. , 2018). Genomic regions with less than 10x coverage were assigned an ‘N’ and genomes with more than 90% Ns were excluded from the analysis. For phylogenetic analysis, the genomes were combined with all available JEV complete genomes on NCBI’s GenBank (365 at time of analysis) and aligned using MAFFT v.7.490 (Katoh, 2002) with default parameters. A phylogenetic tree was constructed from the alignment using IQ-TREE v.2.2.0 (Minh et al. , 2020), with the TN+F+G4 model chosen as the best fit and 1,000 bootstrap replicates. For the GIV outbreak phylogenies, the sequences (n=166) were aligned with the Indonesian strain JEV/Swine/Bali-93/2017 (Accession number LC461961) using MAFFT before phylogenetic analysis using both maximum likelihood (ML) and bayesian approaches, with PhyML v3.3.20180214 and MrBayes v3.2.6, respectively. Due to the limited diversity, the HKY+G substitution model was used for both methods. Branch support values for the ML tree were estimated using the Shimodaira–Hasegawa approximate likelihood-based test. The Bayesian phylogeny was estimated using a chain length of 10M, sampled every 10,000 steps. Commercial farmed pig movement metadata Verbal interviews were conducted with representatives from two large commercial pig producers to understand the movement of pigs (sows, gilts, piglets, growers and boars) and semen, within their farming systems and business units. Both companies have commercial pig farming operations across southeastern Australia and collectively produce approximately one third of the fresh pork production in Australia. Thirty-seven genomes from 23 infected piggeries were included in the analysis. Eight of the 37 genomes were derived from infected properties that were closed to the introduction of live animals or semen and considered closed farming systems. Phylogenetic analysis was performed as above, and the placement of the 37 genomes were visually examined within the context of the location of the piggeries and farming practices. Declarations ETHICS This study was conducted for health protection and or diagnostic purposes under the provisions of the relevant legislation in each jurisdiction. Japanese encephalitis is a nationally notifiable disease of humans and animals in Australia (https://www.health.gov.au/topics/communicable-diseases/nationally-notifiable-diseases/list; https://www.agriculture.gov.au/biosecurity-trade/pests-diseases-weeds/animal/state-notifiable). As such, collection and diagnostic testing of clinical specimens was exempt from human or animal research ethics approval. Data Availability Sequence alignments are available via GitHub at https://github.com/jsede/JEV_2022/. The JEV genome sequences generated in this study have been deposited in the NCBI GenBank with accession numbers: PV666440-PV666603. Accessions and metadata are listed in Supplementary Table 5. Code availability The code for assembling and analysing JEV sequences are publicly available via GitHub: https://github.com/jsede/JEV_2022/ Ethics Declaration Competing interests The authors declare no competing financial interests. Acknowledgements Clinicians, veterinarians, animal health technicians and mosquito surveillance field teams, in association with relevant testing laboratories across all Australian states and territories, for providing the JEV positive material; Dr Rachel Iglesias (Office of the Chief Veterinary Officer, Australian Government Department of Agriculture, Fisheries and Forestry) for assistance with compiling and coordinating access to infected piggery data. The NSW samples were provided by the NSW Arbovirus Surveillance Program which is funded by the NSW Department of Health. References Annand, E.J., et al. 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(2023) ‘RAPID prep : A simple, fast protocol for RNA metagenomic sequencing of clinical samples’, Viruses, 15(4), 1006. https://doi.org/10.3390/v15041006. van den Hurk, A.F. et al. (2006) ‘Short report: the first isolation of Japanese encephalitis virus from mosquitoes collected from mainland Australia’, The American Journal of Tropical Medicine and Hygiene , 75(1), pp. 21–25. van den Hurk, A.F. et al. (2019) ‘Japanese Encephalitis Virus in Australia: From Known Known to Known Unknown’, Tropical Medicine and Infectious Disease , 4(1), p. 38. Available at: https://doi.org/10.3390/tropicalmed4010038. van den Hurk, A.F. et al. (2022) ‘The Emergence of Japanese Encephalitis Virus in Australia in 2022: Existing Knowledge of Mosquito Vectors’, Viruses , 14(6), p. 1208. Available at: https://doi.org/10.3390/v14061208. van den Hurk, A.F., Ritchie, S.A. and Mackenzie, J.S. (2009) ‘Ecology and Geographical Expansion of Japanese Encephalitis Virus’, Annual Review of Entomology , 54(1), pp. 17–35. Available at: https://doi.org/10.1146/annurev.ento.54.110807.090510.Waller, C. et al. (2022) ‘Japanese Encephalitis in Australia — A Sentinel Case’, New England Journal of Medicine , 387(7), pp. 661–662. Available at: https://doi.org/10.1056/NEJMc2207004. Vaughn D.W., C.H. Hoke. (1992) ‘The epidemiology of Japanese encephalitis: prospects for prevention’, Epidemiologic Review s , 14(1), pp.197-221. Available at: https://doi.org/10.1093/oxfordjournals.epirev.a036087. Walsh, M.G., Webb, C. and Brookes, V. (2023). An evaluation of the landscape structure and La Niña climatic anomalies associated with Japanese encephalitis virus outbreaks reported in Australian piggeries in 2022. One Health , 16 , p.100566. Available at: https://doi.org/10.1016/j.onehlt.2023.100566 Williams, D.T., MacKenzie, J.S. and Bingham, J. (2019). ‘Flaviviruses’, In Diseases of Swine (eds J.J. Zimmerman, L.A. Karriker, A. Ramirez, K.J. Schwartz, G.W. Stevenson and J. Zhang). Available at: https://doi.org/10.1002/9781119350927.ch33 Additional Declarations There is NO Competing Interest. Supplementary Files JEVAustraliaSupplementaryInformation040725.docx Supplementary Material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7046873","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":488816749,"identity":"f793f69a-6992-4ea1-80f0-a5c3dffaeeef","order_by":0,"name":"John-Sebastian Eden","email":"","orcid":"https://orcid.org/0000-0003-1374-3551","institution":"University of Sydney","correspondingAuthor":false,"prefix":"","firstName":"John-Sebastian","middleName":"","lastName":"Eden","suffix":""},{"id":488816750,"identity":"353479a4-4c89-41a8-8b07-dc71b416aa3e","order_by":1,"name":"Matthew Neave","email":"","orcid":"","institution":"CSIRO, Australian Centre for 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12:40:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7046873/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7046873/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87703453,"identity":"5be6ea47-6b19-4808-a5d0-8e2ca4bca652","added_by":"auto","created_at":"2025-07-28 07:42:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJapanese encephalitis (JE) cases in Australia (2021-2022) and JEV genome sequences from vertebrates and mosquitoes\u003c/strong\u003e. (A) JEV whole genome sequences generated by each jurisdiction from this study and others (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022). (B) Human JE epidemic curve by month of symptom onset and jurisdiction, with date notification received used where date of symptom onset was not available (Australian Government Department of Health and Aged Care, 2023). (C) JEV infected commercial piggeries by jurisdiction and month of report, with exception of the April 2021 case in Queensland, which was retrospectively identified in 2022 (data provided by the Chief Veterinary Offices of New South Wales, Queensland, South Australia and Victoria). (D) Sampling timeline of vertebrates and mosquitoes that yielded whole genome sequences. Coloring is based on government regions as shown in the legend; VIC = Victoria, NSW = New South Wales, QLD = Queensland, NT = Northern Territory, SA = South Australia.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7046873/v1/a82455070ff2e7aee05ed7d0.png"},{"id":87703452,"identity":"e720ce28-dd7a-4f56-9f86-f17dd60ecf28","added_by":"auto","created_at":"2025-07-28 07:42:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":297784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetics of the JEV outbreak in Australia. \u003c/strong\u003e(A) Geographic origins of\u003cstrong\u003e \u003c/strong\u003ethe JEV genomes analyzed. The size of each source location symbol (circle) is proportional to the number of sequences from that location. Symbols are colored based on government regions as shown in the legend; VIC = Victoria, NSW = New South Wales, QLD = Queensland, NT = Northern Territory, SA = South Australia. (B) Maximum likelihood (ML) tree estimated using an alignment of all available GenBank reference genomes, showing the different JEV genotypes. ML trees of the genotype IV Australian lineage 2021-22 rooted (C) using the Indonesian strain JEV/Swine/Bali-93/2017 (Accession number LC461961) and unrooted (D), with clades A and B indicated. Terminal branch symbols in (C) are colored according to the source region with the shape corresponding to the host as depicted in the legend. Branch support values (\u0026gt; 0.7) using the Shimodaira–Hasegawa approximate likelihood-based test are shown at nodes with black diamonds. Scale bars are proportional to the number of substitutions per site.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7046873/v1/3fc68f228970b7b7a9536ec6.png"},{"id":88122833,"identity":"ccc995eb-ca6c-42d1-83fb-ebdf42cf39bc","added_by":"auto","created_at":"2025-08-01 16:14:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1060486,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7046873/v1/df8f4b07-8c4a-4e3e-9058-e55556d38620.pdf"},{"id":87703454,"identity":"150b9379-9bb4-406d-8791-925701acb512","added_by":"auto","created_at":"2025-07-28 07:42:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1055480,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"JEVAustraliaSupplementaryInformation040725.docx","url":"https://assets-eu.researchsquare.com/files/rs-7046873/v1/9de810c2d3fc01f8005d7d6a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Emergence and spread of Japanese encephalitis virus genotype IV in a novel ecosystem: Australia, 2021-2022","fulltext":[{"header":"Main","content":"\u003cp\u003eJapanese encephalitis virus (JEV) is a single-stranded RNA virus belonging to the species \u003cem\u003eOrthoflavivirus japonicum\u003c/em\u003e, family \u003cem\u003eFlaviviridae\u003c/em\u003e. Most human infections with JEV are asymptomatic, with the ratio of clinically apparent to inapparent infections ranging from 1:1000 to 1:25 (Vaughn and Hoke, 1992). Despite this, the virus has been estimated to cause approximately 68,000 clinical cases of encephalitis annually worldwide, predominantly in Asia, and in some years this number may exceed 100,000 (Vaughn and Hoke, 1992; Quan \u003cem\u003eet al.\u003c/em\u003e, 2020). The estimated mortality rate of human cases of Japanese encephalitis (JE) is 20 \u0026ndash; 30%, while an estimated 30 \u0026ndash; 50% of survivors have long-term neurological sequelae (van den Hurk, Ritchie and Mackenzie, 2009; Khandaker \u003cem\u003eet al.\u003c/em\u003e, 2016; Quan \u003cem\u003eet al\u003c/em\u003e., 2020). These high morbidity and mortality rates make JEV one of the leading causes of encephalitis in Asia (Campbell \u003cem\u003eet al.\u003c/em\u003e, 2011). JEV also causes significant disease in domestic animals. In horses, JEV infection can cause potentially fatal encephalitis, while infection in pigs can cause significant reproductive losses, including abortion, congenital defects, birth of weak piglets with neurological signs, or aspermia in boars (Williams \u003cem\u003eet al.\u003c/em\u003e, 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe natural transmission cycle of JEV involves wild birds, mosquitoes, and domestic and wild pigs (van den Hurk, Ritchie and Mackenzie, 2009). Wild birds, particularly water birds of the family Ardeidae, are considered the main JEV reservoir host in endemic scenarios (Soman \u003cem\u003eet al.\u003c/em\u003e, 1977; van den Hurk, Ritchie and Mackenzie, 2009). JEV is transmitted by mosquito vectors, primarily of the genus \u003cem\u003eCulex\u003c/em\u003e, to a variety of mammalian hosts. Pigs are considered amplifying hosts for JEV and, when present in sufficient numbers and density, may drive infection dynamics and influence human infection risk (Mulvey \u003cem\u003eet al.\u003c/em\u003e, 2021). Other mammals, such as humans, horses and other livestock, do not develop sufficient levels of viremia to infect feeding mosquitoes and are considered dead-end hosts (van den Hurk \u003cem\u003eet al.\u003c/em\u003e, 2019). A range of other animals, other than ardeid birds and pigs, have been identified as potential vertebrate hosts that may require further investigation (Levesque \u003cem\u003eet al.\u003c/em\u003e, 2024; Moore \u003cem\u003eet al.\u003c/em\u003e, 2025). Five phylogenetically distinct genotypes of JEV (I to V) are recognized (Chen, Tesh and Rico-Hesse, 1990; Chen, Rico-Hesse and Tesh, 1992; Satchidanandam and Uchil, 2001; Solomon \u003cem\u003eet al.\u003c/em\u003e, 2003). A characteristic of these genotypes has been their changing patterns of geographic distribution and circulation over time (Gao \u003cem\u003eet al.\u003c/em\u003e, 2019).\u003c/p\u003e\n\u003cp\u003eIn Australia, JE first emerged in 1995 when 3 cases were diagnosed among residents of the Torres Strait Islands in the country\u0026rsquo;s north (Hanna \u003cem\u003eet al.\u003c/em\u003e, 1996). A single additional case occurred in the Torres Strait in 1998, as well as the first case on mainland Australia, in the western Cape York area of Queensland (Hanna \u003cem\u003eet al.\u003c/em\u003e, 1999). Accompanying these cases were detections in mosquitoes and pigs (Hanna \u003cem\u003eet al.\u003c/em\u003e, 1996, 1999). The 1995 and 1998 viruses belonged to genotype II and were thought to have originated from Papua New Guinea where genetically identical isolates of JEV were reported (Johansen \u003cem\u003eet al.\u003c/em\u003e, 2000). Subsequently, isolates obtained in the Torres Strait Islands (2000, 2004) and the Northern Peninsula Area (NPA) of Cape York (2004) belonged to a second genotype, genotype I (Pyke \u003cem\u003eet al.\u003c/em\u003e, 2001; van den Hurk \u003cem\u003eet al.\u003c/em\u003e, 2006). After 2011, many surveillance activities were discontinued, obscuring the JEV situation in Australia during this period (van den Hurk \u003cem\u003eet al.\u003c/em\u003e, 2019). However, opportunistic serological surveillance of livestock in the Torres Strait Islands and NPA provided suggestive evidence for seasonal activity of JEV between 2005 and 2021 (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022). More recently, a fatal human case of JE occurred on the Tiwi Islands in February 2021, caused by a strain of the rare genotype IV (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022; Waller \u003cem\u003eet al.\u003c/em\u003e, 2022). This genotype had previously been isolated from mosquitoes and pigs in Indonesia (Chen, Rico-Hesse and Tesh, 1992; Schuh \u003cem\u003eet al.\u003c/em\u003e, 2013; Kuwata \u003cem\u003eet al.\u003c/em\u003e, 2020; Faizah \u003cem\u003eet al.\u003c/em\u003e, 2021) and caused the death of an Australian tourist who had acquired infection in Bali, in 2019 (Pyke \u003cem\u003eet al.\u003c/em\u003e, 2020). Outside Indonesia, the only evidence of JEV genotype IV circulation was a sequence derived from an unconfirmed human case in Vietnam, in 1979 (Mackenzie \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e\n\u003cp\u003eIn February 2022, one year after the Tiwi Island case, JEV genotype IV was identified as the cause of stillbirths, piglet deformities and neurological disease in piggeries in the states of New South Wales (NSW), Queensland (QLD) and Victoria (VIC)\u0026nbsp;(Australian Government Department of Health, 2022; Iglesias, 2022). Concurrently, cases of human neurological disease were reported in eastern and south-eastern Australia, which were subsequently diagnosed as JE (Furuya-Kanamori \u003cem\u003eet al.\u003c/em\u003e, 2022). Metagenomic sequencing of post-mortem brain samples collected from a patient with meningoencephalitis also led to the detection of JEV genotype IV (Maamary \u003cem\u003eet al.\u003c/em\u003e, 2023). These human and swine cases signaled the unprecedented spread and rapid circulation of JEV throughout south-eastern Australia. In this study, we report the genomic sequencing and analysis of JEV genomes from humans, pigs (domestic and feral) and mosquitoes, collected during and prior to the Australian JEV outbreak of 2022. We describe the phylogenetic structure of the Australian genotype IV lineage, the relatedness of viral genomes from vertebrates and mosquitoes and assess the possible origins of the genotype IV incursion into the Australian mainland.\u003c/p\u003e"},{"header":"Results and discussion ","content":"\u003cp\u003e\u003cem\u003eWidespread detection of Japanese encephalitis virus in Australia\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFrom February 2021 to December 2022, 45 human cases of JE, including seven deaths, and over 80 infected commercial piggeries were reported in Australia (Iglesias 2022; Australian Government Department of Agriculture, Fisheries and Forestry, 2023; Australian Government Department of Health and Aged Care, 2023). We determined 166 JEV genotype IV genomes from mosquitoes and vertebrate hosts across five Australian states and territories (Fig 1) as part of a national One Health outbreak response involving multiple jurisdictions. These data represent the largest genomic epidemiology study of a JEV outbreak, considerably increases the number of publicly available JEV whole genome sequences from 365 (at the time of writing) to 531 and allows an understanding of the molecular epidemiology of an outbreak unparalleled for JEV within a novel ecosystem in Australia or elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePeaks in the onset of human symptomatic cases in February 2022 preceded the peak of newly infected piggeries (Fig 1, panels B, C). Retrospective testing of stored human clinical samples with undiagnosed neurological impairment identified a case of JE acquired in the Northern Territory (NT) from a Victorian resident in May 2021 (NT Health, 2023). The active circulation of JEV in mosquitoes as early as January 2022 was detected through retrospective testing of stored mosquito pool samples. Proactive surveillance of mosquito populations showed continuing JEV circulation through to April 2022 (Pyke \u003cem\u003eet al.,\u003c/em\u003e 2024). \u003cem\u003eCulex annulirostris\u003c/em\u003e (considered the primary vector of JEV in Australia) was present in all trap collections, although other implicated vectors, including \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e and \u003cem\u003eCx. gelidus\u003c/em\u003e were also collected (van den Hurk \u003cem\u003eet al.\u003c/em\u003e, 2022; Supp Table 1). Of the 166 JEV genome sequenced, 93% (154 of 166) were derived from pigs including a single culture isolate (Fig 1D), reflecting the high number of samples received from affected piggeries (n=136) and feral pig surveillance (n=19). Prior to the current outbreak, there were limited JEV surveillance activities in the areas where the virus was subsequently detected. Similarly, there was little pre-existing JEV-specific immunity in humans in the same areas, as JEV vaccines were not routinely used and there had been no recognized JEV activity. Following the outbreak, prospective molecular and serological testing of samples collected from persons in NSW with suspected encephalitis suggested an incidence of 0.15/100,000 over the first three months of the outbreak (Howard-Jones \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhylodynamics of JEV genotype IV emergence in Australia 2021-22\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the dispersal patterns and reveal the possible origins of the outbreak, we performed a phylodynamic analysis of JEV genomes (n=166) collected during the outbreak period and the season prior, spanning February 2021 to August 2022, as well as the previously published genome from the human case on the Tiwi Islands from February 2021 (Fig 2). Sequences were collected from five Australian states and territories, with the majority sampled along the NSW and VIC state border regions in the southeast of the country where the highest concentrations of human and animal cases were identified (Fig 2A). Several genomes were also obtained from northern regions of the NT and QLD that may represent the initial location of introduction of GIV JEV, based on previous detections in these regions (Hanna \u003cem\u003eet al.\u003c/em\u003e, 1996, 1999; Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022). Initially, the genomes were aligned with all available GenBank reference genomes (n=365, as of 31 October 2022) and analyzed using a maximum-likelihood approach (Figure 2B). This showed that the viruses from the Australian outbreak were all genotype IV and formed a single monophyletic group, here termed the genotype IV Australian 2021-22 lineage, which were similar but distinct from genotype IV sequences previously detected in Indonesia and Vietnam. The closest relative on NCBI GenBank was the Indonesian sequence JEV/Swine/Bali-93/2017 (Accession number LC461961) sharing ~96.7% nucleotide identity. Using this sequence as an outgroup, genotype IV Australian 2021-22 lineage-specific phylogenies were then estimated (Figs 2C-D, Supp Fig 1 \u0026amp; 2) to examine genetic, host and temporal structure. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe genetic diversity within the outbreak lineage was very low (\u0026lt;1% distance at the nucleotide level), with most sequences falling into two well-supported clades (A \u0026amp; B) that were defined by three synonymous substitutions - positions 539, 5537 and 7241 (relative to JEV/Swine/Bali-93/2017) (Fig 2C). These sites correspond to the pre-membrane (prM), non-structural protein 3 (NS3) and the NS4B protein genes, respectively. An intermediate lineage between clades A and B was also retrospectively identified in the North Queensland region from a case of spontaneous abortion in a piggery in April 2021. A group of putative ancestral sequences were also identified with a distinct mutational signature at the same three sites that included the genome from the earliest known Australian JEV genotype IV case from the Tiwi Islands in February 2021 (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022; Waller \u003cem\u003eet al.\u003c/em\u003e, 2022) as well as a genome obtained from a feral pig in the NPA (Far North Queensland) in May 2022. A small cluster of three sequences from commercial piggeries located in the Murray River region near the state border of VIC and NSW constituted an intermediate clade between these ancestral lineages and clades A and B. Notably, the phylogeny lacked temporal structure (Supp Figs 3 \u0026amp; 4). Furthermore, there was no apparent clustering of sequences by sampling location, except at some fine spatial scales (specific regions or outbreaks at individual properties). A lack of host-specific phylogenetic clustering was also observed with pig and mosquito detections throughout both major clades (Fig 2C, Supp Figs 1 \u0026amp; 2). JEV sequences derived from the 2022 human cases belonged to clade A, although only a limited number of genomes (n=2) were able to be recovered from patients as most human cases were laboratory confirmed by serology. Together with the star-like phylogeny (Fig 2D), these results suggest that the JEV outbreak in 2022 in southeastern Australia was driven by the concurrent emergence and rapid dispersal of at least two virus lineages in largely JEV na\u0026iuml;ve populations of vertebrate hosts and competent mosquito vectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address the possible role of domestic pig movements in the spread of JEV during the 2022 outbreak, we analyzed genomes derived from 23 infected commercial piggeries in QLD, NSW, VIC and SA, for which information on location and farm operations was available (Supp Table 2). A total of 37 JEV genomes were sequenced and included in the phylogenetic analysis (Supp Fig 5). Genomes belonging to both clades A and B were identified from piggeries located in all states except SA; genomes from only three SA piggeries were available, all clade A. Notably, JEV belonging to both clades were detected within individual production units from QLD, NSW, and VIC, including one NSW farm that was closed to pig movements on or off the premises during the time of study. Another closed farm located in the same region of NSW contained genomes belonging to clade A only, whilst in VIC, closed farms, all located within the same region, contained either clade A or B genomes. One farm in QLD (property 3) that received fortnightly introductions of pigs from another farm located approximately 50 kilometers south (property 2) contained clade A, whereas clade B genome was detected in the donor farm. Thus, this analysis indicates that both clades were co-circulating freely across southeastern Australian piggeries and that there is no evidence to indicate that pig movements between farms contributed significantly to the epidemiology of this outbreak. Consistent with this conclusion was the concurrent detection of clade A and B genomes in mosquitoes sampled in all states with infected piggeries (Supp Table 1). However, we cannot exclude local oronasal transmission of JEV between pigs within individual farming units via direct contact (Ricklin \u003cem\u003eet al.\u003c/em\u003e, 2016). In this regard, for some properties from which two or more sequences were obtained, closely related clusters were observed in our phylogenetic analysis (Supp Figs 1 \u0026amp; 2.), suggesting localized circulation of virus, either between pigs and mosquitoes, or, possibly, directly between pigs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhilst our study does not provide conclusive evidence on the timing of the origins of the 2022 outbreak, the identification of two basal sequences in Northern Australia (Tiwi Islands, February 2021 and Far North Queensland, May 2022) supports the emergence of JEV in Northern Australia, followed by spread to the southeast of the country. Whether these two basal lineages represent two different incursions of JEV into mainland Australia or result from a prolonged period of circulation in Northern Australia, is unclear. An intermediate lineage between clades A and B was also retrospectively identified in the North Queensland region in April 2021, adding further support to the hypothesis that related viruses were already circulating in the lead up to the outbreak in southeastern Australia over the summer of 2021-22. These analyses were unable to shed light on the international origins of the Australian genotype IV lineage beyond the conclusions previously made in relation to the 2021 Tiwi Island index case (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022) as our analysis was limited by the lack of earlier JEV genotype IV sequence data from the Indo-Pacific region. Nevertheless, based on the close phylogenetic relationship of the Australian lineage with the most recently isolated Indonesian viruses, the Indonesian archipelago is the probable geographic source. Introduction from Papua New Guinea, from which JEV genotype II is believed to have emerged in Northern Australia in the 1990s (Johansen \u003cem\u003eet al.\u003c/em\u003e, 2000), also cannot be discounted as analysis of environmental conditions and waterbird population dynamics suggests large-scale movement of reservoir host species occurred from New Guinea to Northern Australia in 2020 and south through the Murray-Darling Basin in 2021-22 (Purnell, 2022). This southern dispersal may have been assisted by the onset of La Ni\u0026ntilde;a dominated weather patterns and concomitant above average rainfall and flooding that facilitated the enhancement of environmental conditions for both reservoir hosts and vectors ahead of the 2021-2022 summer (Walsh \u003cem\u003eet al\u003c/em\u003e., 2023). Notwithstanding the long-range movement of birds, key vector species are also known to disperse many kilometers and potentially facilitated by weather conditions (van den Hurk \u003cem\u003eet al\u003c/em\u003e., 2022), further assisting the geographic spread of JEV.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLimitations and future directions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA limitation of the present study was the sampling bias of JEV predominantly sourced from pigs, either from lymphoid tissue of adult pigs and aborted fetuses, or neonates that acquired infection \u003cem\u003ein utero\u003c/em\u003e. The added complexity of samples derived from fetal or neonatal tissue types is the date of infection \u003cem\u003ein utero\u0026nbsp;\u003c/em\u003ecan only be estimated based on the gestation period of the infected sow, which do not typically show clinical signs of infection. Additionally, spatial under-sampling, especially in areas that lack farmed pigs but may have large feral pig populations, such as in Northern Australia (Gentle, Wilson and Cuskelly, 2022; Mackenzie \u003cem\u003eet al.\u003c/em\u003e, 2022), or a large waterbird population, made it difficult to infer viral origins and estimated timing of introduction, although our analysis is suggestive of at least one introduction into Northern Australia during or prior to the 2020/21 wet season. A lack of JEV whole genome sequences from wild birds or other wildlife also limits inferences on large-scale temporospatial patterns and epidemiology. This highlights the importance of developing improved surveillance systems to understand the viral ecology in wildlife, especially in the context of JEV emerging within a new ecosystem.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMultisectoral collaboration, a key underlying principle of One Health (OHHLEP, 2022), was clearly a major feature in the response to the outbreak, as had previously been described for JEV (Impoinvil, Baylis and Solomon\u003cem\u003e,\u0026nbsp;\u003c/em\u003e2013). Ongoing surveillance and genomic characterization of new JEV detections is required to monitor the spread of JEV, the emergence of alternative JEV genotypes or the presence of additional genotype IV genetic lineages, as well as changes in viral ecology. Collectively, this will inform suitable surveillance strategies and diagnostics to support an ongoing One Health approach to the monitoring, control and prevention of JE in Australia.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eSpecimens and Sample selection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eClinical specimens for laboratory diagnostic testing of humans were received by public health laboratories (NSW Health Pathology-Institute of Clinical Pathology and Medical Research, Victorian Infectious Diseases Reference Laboratory) and for farmed pigs by the relevant state or national veterinary laboratories (AgriBio Centre, Australian Centre for Disease Preparedness [ACDP], Berrimah Veterinary Laboratory [BVL], Biosecurity Sciences Laboratory, Elizabeth Macarthur Agriculture Institute, Gribbles Veterinary Pathology South Australia). Feral pig samples collected at necropsy were collected as part of Northern Australia Quarantine Strategy surveillance activities in response to the outbreak and sent to ACDP or BVL for diagnostic testing. Mosquitoes were collected in carbon dioxide-baited light traps as part of arbovirus surveillance conducted by the Health Departments of NSW, QLD, VIC and SA (Knope \u003cem\u003eet al.\u003c/em\u003e, 2019). Mosquitoes were morphologically identified and placed in pools of mixed species of different sizes prior before being submitted for JEV detection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples in which JEV RNA were detected by RT-qPCR using either universal JEV primers or modified genotype IV primers (Shao \u003cem\u003eet al.\u003c/em\u003e, 2018) or RT-PCR followed by next generation sequencing (below) were identified for genomic analysis through a muti-sectoral national collaboration. JEV RNA detected in cerebrospinal fluid or brain tissue collected pre- and post-mortem in humans were included for whole genome sequencing (Howard-Jones \u003cem\u003eet al.\u003c/em\u003e, 2022; Maamary \u003cem\u003eet al.\u003c/em\u003e, 2023). Three types of pig derived samples included: i) farmed pig samples represented by JEV RT-qPCR positive material received from piglets (either aborted or showing neurological signs at birth); ii) feral pig samples consisted of JEV RT-qPCR positive material sourced from the tonsil tissue collected at necropsy; and iii) JEV RT-qPCR positive semen collected from farmed boars. Mosquito samples consisted of homogenized JEV RT-qPCR positive pools.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJapanese encephalitis virus genomic sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAustralian Centre for Disease Preparedness\u003c/p\u003e\n\u003cp\u003eThe initial JEV genome from an infected piggery in QLD (JEV/sw-22-00722-11/Qld/2022; GenBank Acc. ON624132) was obtained using a direct metagenomic approach, as previously described (Sikazwe \u003cem\u003eet al.\u003c/em\u003e, 2022). First, total RNA was extracted from tonsillar samples using a MagMax 96 Viral RNA Kit (ThermoFisher Scientific). A sequence library was prepared using the TruSeq RNA Library Prep Kit v2 and sequenced with a P2 300 cycle cartridge on a NextSeq2000 instrument (Illumina). Raw reads were cleaned using Trimmomatic v.0.39 (Bolger, Lohse and Usadel, 2014) and mapped to a recent genotype IV genome (GenBank Acc. OM867669) using Bowtie v.2.4.4 (Langmead and Salzberg, 2012). Mapping was manually examined with Geneious Prime v.2020.2.4 and a complete 10,949 bp JEV genome generated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, probe hybridization and primer tiling approaches were developed to allow for a more cost-effective whole genome characterization.\u003c/p\u003e\n\u003cp\u003eThe probe hybridization procedure was developed using the MyBaits Target Capture Kit (Arbor Biosciences). Oligonucleotide probes for enrichment were designed using the JEV/sw-22-00722-11/Qld/2022 genome, a recent genotype IV genome (GenBank Acc. OM867669), plus a representative selection of JEV genomes from GenBank. The final design consisted of 11,435 probes of 70 bp each with 4x depth over the JEV genomes (Supp material). Sequencing libraries were then prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) and enriched for JEV fragments using the probes as per the manufacturer\u0026rsquo;s recommendations (Arbor Biosciences). The libraries were reamplified and sequenced using 300 cycle cartridges on either a MiSeq or NextSeq2000 instrument (Illumina). The raw reads were assembled into JEV genomes as described above.\u003c/p\u003e\n\u003cp\u003eBiosecurity Sciences Laboratory Queensland\u003c/p\u003e\n\u003cp\u003eA multiplex RT-PCR for the sequencing of JEV genomes directly from clinical samples was developed using a published method (Quick et al., 2017). Using reference genomes from the 2022 outbreak, JEV/sw-22-00722-11/Qld/2022 and JEV/sw-22-00698-27/Qld/2022, and other genotype IV genomes (LC461961, MT253731, LC579814, KU705228 and AY184212), primers were designed using the online tool Primal Scheme (https://primalscheme.com/), adopting a default amplicon size of 400 bp to produce 34 primer pairs (Supp Table 3). Amplicons from these PCRs were pooled and sequenced on a MinION (Oxford Nanopore Technologies) using a Flongle flow cell and the Rapid Barcoding Kit (RBK004).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWestmead Institute for Medical Research and AgriBio Centre for AgriBioscience\u003c/p\u003e\n\u003cp\u003eThe JEV genome of the first clinical case in NSW was obtained using a previously described rapid RNA metagenomic approach (Annand \u003cem\u003eet al.,\u003c/em\u003e 2022; Maamary \u003cem\u003eet al.,\u0026nbsp;\u003c/em\u003e2023; Tulloch \u003cem\u003eet al.,\u0026nbsp;\u003c/em\u003e2023). Primer tiling approaches were designed using Primal Scheme (https://primalscheme.com/) to sequence JEV genomes using reference genomes\u0026nbsp;(JEV/sw-22-00722-11/Qld/2022; GenBank Acc. ON624132). Adopting a default amplicon size as 500bp to produce 27 primer pairs for the primer set of JEV-SW-500, and as 800bp to produce 16 primer pairs for the primer set of JEV-G4-800 (Supp Table 4). Amplicons from these PCRs were pooled and sequenced either on an iSeq 100 instrument using the Nextera XT DNA Library Preparation Kit and 300 cycle cartridges on iSeq 100 instrument (Illumina) or on a MinION (Oxford Nanopore Technologies) using a flow cell (R9.4.1) and the Rapid Barcoding Kit (RBK110.96). The raw reads were assembled into JEV genomes.\u003c/p\u003e\n\u003cp\u003eVictorian Infectious Diseases Reference Laboratory\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn almost complete JEV genome was obtained from a Victorian clinical sample using an RNA-directed (DNAse1 treated nucleic acid), sequence-independent, single-primer amplification (SISPA) method as described (Kafetzopoulou \u003cem\u003eet al\u003c/em\u003e., 2018). \u0026nbsp;Sequencing libraries from derived amplicons were created using the Illumina DNA Prep Library kit before sequencing on an Illumina NextSeq550 2 x 150bp mid output cartridge (Illumina).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic and phylodynamic analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomes that were sequenced using an Illumina platform were assembled with a Snakemake v.5 pipeline (github.com/neavemj/JEV_assembly_pipe). Briefly, the pipeline trims raw reads with Trimmomatic v.0.39 (Bolger, Lohse and Usadel, 2014), aligns cleaned reads to a reference JEV genome with Bowtie v.2.4 (Langmead and Salzberg, 2012), then masks the tiling primers and generates a consensus genome with iVar v.1.3 (Grubaugh \u003cem\u003eet al.\u003c/em\u003e, 2018). Genomic regions with less than 10x coverage were assigned an \u0026lsquo;N\u0026rsquo; and genomes with more than 90% Ns were excluded from the analysis.\u003c/p\u003e\n\u003cp\u003eFor phylogenetic analysis, the genomes were combined with all available JEV complete genomes on NCBI\u0026rsquo;s GenBank (365 at time of analysis) and aligned using MAFFT v.7.490 (Katoh, 2002) with default parameters. A phylogenetic tree was constructed from the alignment using IQ-TREE v.2.2.0 (Minh \u003cem\u003eet al.\u003c/em\u003e, 2020), with the TN+F+G4 model chosen as the best fit and 1,000 bootstrap replicates. For the GIV outbreak phylogenies, the sequences (n=166) were aligned with the Indonesian strain JEV/Swine/Bali-93/2017 (Accession number LC461961) using MAFFT before phylogenetic analysis using both maximum likelihood (ML) and bayesian approaches, with PhyML v3.3.20180214 and MrBayes v3.2.6, respectively. Due to the limited diversity, the HKY+G substitution model was used for both methods. Branch support values for the ML tree were estimated using the Shimodaira\u0026ndash;Hasegawa approximate likelihood-based test. The Bayesian phylogeny was estimated using a chain length of 10M, sampled every 10,000 steps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommercial farmed pig movement metadata\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVerbal interviews were conducted with representatives from two large commercial pig producers to understand the movement of pigs (sows, gilts, piglets, growers and boars) and semen, within their farming systems and business units. Both companies have commercial pig farming operations across southeastern Australia and collectively produce approximately one third of the fresh pork production in Australia. Thirty-seven genomes from 23 infected piggeries were included in the analysis. Eight of the 37 genomes were derived from infected properties that were closed to the introduction of live animals or semen and considered closed farming systems. Phylogenetic analysis was performed as above, and the placement of the 37 genomes were visually examined within the context of the location of the piggeries and farming practices. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eETHICS This study was conducted for health protection and or diagnostic purposes under the provisions of the relevant legislation in each jurisdiction. Japanese encephalitis is a nationally notifiable disease of humans and animals in Australia (https://www.health.gov.au/topics/communicable-diseases/nationally-notifiable-diseases/list; https://www.agriculture.gov.au/biosecurity-trade/pests-diseases-weeds/animal/state-notifiable). As such, collection and diagnostic testing of clinical specimens was exempt from human or animal research ethics approval.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence alignments are available via GitHub at https://github.com/jsede/JEV_2022/. The JEV genome sequences generated in this study have been deposited in the NCBI GenBank with accession numbers: PV666440-PV666603. Accessions and metadata are listed in Supplementary Table 5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe code for assembling and analysing JEV sequences are publicly available via GitHub: https://github.com/jsede/JEV_2022/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinicians, veterinarians, animal health technicians and mosquito surveillance field teams, in association with relevant testing laboratories across all Australian states and territories, for providing the JEV positive material; Dr Rachel Iglesias (Office of the Chief Veterinary Officer, Australian Government Department of Agriculture, Fisheries and Forestry) for assistance with compiling and coordinating access to infected piggery data. The NSW samples were provided by the NSW Arbovirus Surveillance Program which is funded by the NSW Department of Health.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnnand, E.J., \u003cem\u003eet al.\u003c/em\u003e (2022) \u0026lsquo;Novel Hendra virus variant detected by sentinel surveillance of horses in Australia\u0026rsquo; \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e, 28, pp. 693\u0026ndash;704. Available at: https://doi.org/10.3201/eid2803.211245 \u003c/li\u003e\n\u003cli\u003eAustralian Government Department of Agriculture, Fisheries and Forestry (2023) \u003cem\u003eJapanese encephalitis virus\u003c/em\u003e, \u003cem\u003eJapanese encephalitis virus\u003c/em\u003e. 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Ramirez, K.J. Schwartz, G.W. Stevenson and J. Zhang). Available at: https://doi.org/10.1002/9781119350927.ch33\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Japanese encephalitis, genotype IV, flavivirus, Australia, phylodynamics","lastPublishedDoi":"10.21203/rs.3.rs-7046873/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7046873/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn unprecedented outbreak of the mosquito-borne Japanese encephalitis virus (JEV) occurred in south-eastern Australia in 2022. The outbreak was caused by a novel lineage of JEV genotype IV, which first emerged in Northern Australia in 2021, and resulted in 45 human cases, including 7 deaths, and over 80 infected piggeries during 2021 and 2022. We analyzed 166 whole genomes of JEV from field collected mosquitoes (n=9), humans (n=2), and farmed (n=136) and feral pigs (n=19). The majority of outbreak sequences clustered into two genetically distinct lineages (clades A and B), separated by three formative single nucleotide polymorphisms, which were circulating between February 2021 and August 2022. Both lineages were detected in mosquito and pig samples, while only clade A was detected in the human samples sequenced. We conclude that clades A and B were likely to have been circulating prior to the outbreak. A lack of spatial-temporal phylogenetic structure suggests a rapid dispersal of the outbreak lineages in largely JEV naïve vertebrate populations and competent mosquito vector populations. Ongoing surveillance and genomic characterization of new detections is required to monitor the spread of JEV, the emergence of alternative JEV genotypes or lineages, as well as changes in the viral ecology.\u003c/p\u003e","manuscriptTitle":"Emergence and spread of Japanese encephalitis virus genotype IV in a novel ecosystem: Australia, 2021-2022","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 07:42:41","doi":"10.21203/rs.3.rs-7046873/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7a9c3a5c-2f51-4e8d-ba81-d888b3ec1d20","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51890128,"name":"Biological sciences/Microbiology/Virology/Viral evolution"},{"id":51890129,"name":"Biological sciences/Microbiology/Virology/Viral epidemiology"}],"tags":[],"updatedAt":"2025-12-16T04:00:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 07:42:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7046873","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7046873","identity":"rs-7046873","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}