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Introduction: Widespread transmission of Japanese encephalitis virus (JEV) genotype four (GIV) occurred across mainland Australia in 2022. This resulted in forty-five human cases, including seven deaths, and the identification of JEV infection in over 80 commercial piggeries. Materials and Methods: We collected mosquitoes which were trapped using CO2-baited light traps deployed near piggeries reporting disease or in regions linked to human cases in the Wide Bay region in the state of Queensland. Mosquitoes from four traps yielded JEV RNA by real-time RT-PCR. Pools containing RNA positive mosquitoes were inoculated onto mosquito cell monolayers. Discussion: A single isolate of JEV was obtained from a pool of mixed mosquito species. Near whole genome sequencing and phylogenetic analysis of the JEV isolate demonstrated its high genomic relatedness with JEV GIV pig sequences sampled from Queensland and the state of New South Wales in 2022. Conclusion: We report the first isolation of JEV GIV from mosquitoes collected in Australia. With only a few JEV GIV isolates available globally, the isolate we report will be essential for future research of JEV host interactions, evolution and disease markers, and development of effective therapies, vaccines, diagnostic assays, and mosquito control strategies. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a zoonotic ailment from the Japanese encephalitis virus (JEV). JEV belongs to the flavivirus genus and is categorized into a solitary serotype consisting of five genetically diverse genotypes (I, II, III, IV, and V). The JEV genotype III (GIII) was the prevailing strain responsible for multiple outbreaks in countries endemic to JEV until 1990. In recent years, significant improvements have occurred in the epidemiology of JE, encompassing the geographical expansion of the epidemic zone and the displacement of prevailing genotypes. The dominant genotype of the JEV has undergone a progressive shift from GIII to GI due to variations in its adaptability within avian populations. From 2021 to 2022, Australia encountered an epidemic of viral encephalitis resulting from infection with the GIV JEV pathogen. The current human viral encephalitis caused by GIV JEV is the initial outbreak since its initial discovery in Indonesia during the late 1970s. Furthermore, following a time frame of 50 years, the detection and isolation of GV JEV have been reported in Culex mosquitoes across China and South Korea. Evidence suggests that the prevalence of GIV and GV JEV epidemic regions may be on the rise, posing a significant threat to public safety and the sustainable growth of animal husbandry. The global approach to preventing and managing JE predominantly revolves around utilizing the GIII strain vaccine for vaccination purposes. Nevertheless, research has demonstrated that the antibodies generated by the GIII strain vaccine exhibit limited capacity to neutralize the GI and GV strains. Consequently, these antibodies cannot protect against JEV challenge caused by animal GI and GV strains. The limited cross-protective and neutralizing effects observed between various genotypes may be attributed to the low homology of the E protein with other genotypes. In addition, due to the GIV JEV outbreak in Australia, further experiments are needed to evaluate the protective efficiency of the current GIII based JE vaccine against GIV JEV. The alteration of the prevailing genotype of JEV and the subsequent enlargement of the geographical extent of the epidemic have presented novel obstacles in JE prevention and control. This paper examines the emerging features of the JE epidemic in recent years and the associated problems concerning prevention and control. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) causes acute viral encephalitis in humans and reproductive disorders in pigs. JEV emerged during the 1870s in Japan, and since that time, JEV has been transmitted exclusively throughout Asia, according to known reporting and sequencing records. A recent JEV outbreak occurred in Australia, affecting commercial piggeries across different temperate southern Australian states, and causing confirmed infections in humans. A total of 47 human cases and 7 deaths were reported. The recent evolving situation of JEV needs to be reported due to its continuous circulation in endemic regions and spread to non-endemics areas. Here, we reconstructed the phylogeny and population dynamics of JEV using recent JEV isolates for the future perception of disease spread. Phylogenetic analysis shows the most recent common ancestor occurred about 2993 years ago (YA) (95% Highest posterior density (HPD), 2433 to 3569). Our results of the Bayesian skyline plot (BSP) demonstrates that JEV demography lacks fluctuations for the last two decades, but it shows that JEV genetic diversity has increased during the last ten years. This indicates the potential JEV replication in the reservoir host, which is helping it to maintain its genetic diversity and to continue its dispersal into non-endemic areas. The continuous spread in Asia and recent detection from Australia further support these findings. Therefore, an enhanced surveillance system is needed along with precautionary measures such as regular vaccination and mosquito control to avoid future JEV outbreaks. [ABSTRACT FROM AUTHOR]

An outbreak of viral encephalitis caused by a Japanese encephalitis virus (JEV) genotype IV infection occurred in Australia between 2021 and 2022. A total of 47 cases and seven deaths were reported as of November 2022. This is the first outbreak of human viral encephalitis caused by JEV GIV since it was first isolated in Indonesia in the late 1970s. Here, a comprehensive phylogenetic analysis based on the whole genome sequences of JEVs revealed it emerged 1037 years ago (95% HPD: 463 to 2100 years). The evolutionary order of JEV genotypes is as follows: GV, GIII, GII, GI, and GIV. The JEV GIV emerged 122 years ago (95% HPD: 57–233) and is the youngest viral lineage. The mean substitution rate of the JEV GIV lineage was 1.145 × 10−3 (95% HPD values, 9.55 × 10−4, 1.35 × 10−3), belonging to rapidly evolving viruses. A series of amino acid mutations with the changes of physico-chemical properties located in the functional important domains within the core and E proteins distinguished emerging GIV isolates from old ones. These results demonstrate the JEV GIV is the youngest JEV genotype at a rapid evolution stage and has good host/vector adaptability for introduction to non-endemic areas. Thus, surveillance of JEVs is highly recommended. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is an arboviral, encephalitogenic, zoonotic flavivirus characterized by its complex epidemiology whose transmission cycle involves reservoir and amplifying hosts, competent vector species and optimal environmental conditions. Although typically endemic in Asia and parts of the Pacific Islands, unprecedented outbreaks in both humans and domestic pigs in southeastern Australia emphasize the virus' expanding geographical range. To estimate areas at highest risk of JEV transmission in Australia, ecological niche models of vectors and waterbirds, a sample of piggery coordinates and feral pig population density models were combined using mathematical and geospatial mapping techniques. These results highlight that both coastal and inland regions across the continent are estimated to have varying risks of enzootic and/or epidemic JEV transmission. We recommend increased surveillance of waterbirds, feral pigs and mosquito populations in areas where domestic pigs and human populations are present. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is transmitted by Culex species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. Cx. annulirostris has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian Cx. annulirostris with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian Cx. annulirostris is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.

A widespread outbreak of Japanese encephalitis virus (JEV) was detected in mainland Australia in 2022 in a previous non-endemic area. Given JEV is known to be transfusion-transmissible, a rapid blood-safety risk assessment was performed using a simple deterministic model to estimate the risk to blood safety over a 3-month outbreak period during which 234,212 donors attended. The cumulative estimated incidence in donors was 82 infections with an estimated 4.26 viraemic components issued, 1.58 resulting in transfusion-transmission and an estimated risk of encephalitis of 1 in 4.3 million per component transfused over the risk period. Australia has initiated a robust public health response, including vector control, animal control and movement, and surveillance. Unlike West Nile virus, there is an effective vaccine that is being rolled-out to those at higher risk. Risk evaluation considered options such as restricting those potentially at risk to plasma for fractionation, which incorporates additional pathogen reduction, introducing a screening test, physicochemical pathogen reduction, quarantine, post donation illness policy changes and a new donor deferral. However, except for introducing a new deferral to potentially cover rare flavivirus risks, no option resulted in a clear risk reduction benefit but all posed threats to blood sufficiency or cost. Therefore, the blood safety risk was concluded to be tolerable without specific mitigations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• JEV serological assays show good clinical performance in low endemicity areas. • Convalescent testing is important in case finding. • Cross reactivity between flavivirus confirmatory assays is common. • Assay validation should include clinical and epidemiological findings. The unexpected recent emergence of Japanese encephalitis virus (JEV) genotype IV in multiple southern states of Australia necessitated an evaluation of JEV serological tests suitable for diagnosing acute infection and for seroprevalence studies. This study examined the analytical and clinical performance of two high-throughput JEV assays, Euroimmun immunofluorescence assay (IFA) and Euroimmun enzyme-linked immunosorbent assay (ELISA), across four cohorts; (1) surveillance of piggery workers in outbreak areas, (2) surveillance of residents in outbreak areas, (3) acute JEV infection and (4) post-JEV vaccination. ELISA and IFA IgM demonstrated minimal cross-reactivity (0–1.8%) with other endemic flaviviruses, with high sensitivity (100%) for acute JEV infection in this low endemicity setting. Differences in IgG serodynamics between the two assays suggest convalescent and paired testing with IgM are critical in diagnosing acute infection. High assay concordance was observed between ELISA and IFA when used in serosurveillance (97.4% agreement, Cohen' κ 0.74 [95% CI 0.614–0.860]) and vaccination cohorts (91.1% agreement, Cohen's κ 0.806 [95% CI 0.672–0.941]). In conclusion, this study highlights the clinical & epidemiological applications and limitations of these two commercial JEV assays. [ABSTRACT FROM AUTHOR]

The introduction and subsequent rapid spread of Japanese encephalitis virus genotype IV across all Australian mainland states and the Northern Territory since late 2021 has increased the risk of an incursion of this mosquito-transmitted zoonotic virus disease into New Zealand, with serious implications for both animal and human health. The potential modes of entry are through introduction of infected mosquitoes as hitchhikers on ships or aircraft, windborne transfer of mosquitoes, or arrival of infected reservoir bird species. A competent vector mosquito, Culex quinquefasciatus , is endemic in New Zealand and other mosquito species may also become involved. If infection becomes established in New Zealand, the scale of transmission may be considerably less than has occurred in Australia because climatic and epidemiological factors are not so favourable. Early evidence of an incursion could come from detection of clinical disease in horses or pigs, or from human cases. Targeted surveillance to confirm or refute indications of an incursion could be undertaken by antibody detection in a number of species. Dogs have been shown to be a particularly valuable sentinel species due to their cohabitation with people and high seroconversion rate. Other novel methods of surveillance could include reverse transcriptase PCR (RT-PCR) on oronasal secretions of pigs. Should evidence of the disease be detected, prompt action would be required to vaccinate at-risk human populations and clarify the epidemiological situation with respect to mammalian hosts and mosquito vector species, including whether a new mosquito species had arrived in the country. Abbreviations: AHL: Animal Health Laboratory; JE: Japanese encephalitis disease; JEV: Japanese encephalitis virus; RT-PCR: Reverse transcriptase PCR.

In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus prevalent in east and southeast Asia, the Western Pacific, and northern Australia. Since viruses are obligatory intracellular pathogens, the dynamic processes of viral entry, replication, and assembly are dependent on numerous host-pathogen interactions. Efforts to identify JEV-interacting host factors are ongoing because their identification and characterization remain incomplete. Three enzymatic activities of flavivirus non-structural protein 3 (NS3), including serine protease, RNA helicase, and triphosphatase, play major roles in the flaviviruses lifecycle. To identify cellular factors that interact with NS3, we screened a human brain cDNA library using a yeast two-hybrid assay, and identified eight proteins that putatively interact with NS3: COPS5, FBLN5, PPP2CB, CRBN, DNAJB6, UBE2N, ZNF350, and GPR137B. We demonstrated that the DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6) colocalizes and interacts with NS3, and has a negative regulatory function in JEV replication. We also show that loss of DNAJB6 function results in significantly increased viral replication, but does not affect viral binding or internalization. Moreover, the time-course of DNAJB6 disruption during JEV infection varies in a viral load-dependent manner, suggesting that JEV targets this host chaperone protein for viral benefit. Deciphering the modes of NS3-interacting host proteins functions in virion production will shed light on JEV pathogenic mechanisms and may also reveal new avenues for antiviral therapeutics. [ABSTRACT FROM AUTHOR]

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans., (© 2013 Elsevier Inc. All rights reserved.)