Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia

Abstract

Background A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). Methods Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. Results Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7–96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998–2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. Conclusion This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.

Growth, Pathogenesis, and Serological Characteristics of the Japanese Encephalitis Virus Genotype IV Recent Strain 19CxBa-83-Cv

“…A phylogenetic tree of JEV was constructed using the nucleotide sequence of the E gene of the GI–GV JEV strains ( Figure 1 ). Strain 19CxBa-83-Cv was clustered in the GIV and showed higher homology to the Bali2019 strain isolated from an Australian JE patient infected in Bali in 2019, compared to the strains JEV/Aus/NT_Tiwi Islands/2021 (Hu_Tiwi 2021; OM867669) and sw-22-00722-11 (ON624132) identified in Australia in 2021 and 2022, respectively [ 30 , 31 , 38 , 40 ].…”

“…Genetic analysis of the JEV isolates from mosquitoes and pigs indicated that the isolates in 1998 also belonged to GII, whereas the isolate from Badu in the Torres Strait Islands in 2004 belonged to GI [ 36 , 37 ]. A JE patient who was thought to be infected with GIV JEV in the Tiwi islands in northern Australia was confirmed as infected in Australia in 2021 following a 20-year absence of JE [ 38 , 39 , 40 ]. In 2022, JEV was widespread in Australia.…”

“…The outbreak of JEV was first identified in swine and subsequently in humans, mosquitoes, and sentinel chickens [ 42 ]. Genetic analysis of the virus associated with the epidemics in 2021 and 2022 indicated that the virus was GIV JEV [ 38 , 40 , 43 ].…”

Japanese encephalitis virus: an emerging and re-emerging virus in Australia

“…A fatal case of JE occurred in March 2021 in an inhabitant from the Tiwi Islands. 43,44 Sequence studies confirmed that it was JEV belonging to genotype IV, the first time this genotype was acquired outside Indonesia. It was also the second known fatal human infection due to GIV, the first having been reported in an Australian tourist to Bali.…”

RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples

“…We present a simple yet robust workflow for the mNGS of RNA from clinical respiratory samples. Our RAPIDprep assay has been designed specifically as a rapid response tool, and has proven to be effective in novel disease investigations, including identifying the first cases of emergent Japanese encephalitis virus during the 2021-22 outbreak in south-eastern Australia [42,43], and characterising the first cases of COVID-19 in NSW [33]. The assay has also been used to investigate non-human diseases, and was critical to the genome recovery of a novel Hendra virus variant detected initially from a fatal equine infection by pan-paramyxovirus RT-PCR [1].…”