# Genetic diversity of Murray Valley encephalitis virus 1951–2020 identified via phylogenetic and evolutionary analyses

## TL;DR

This study reveals the genetic diversity and evolution of Murray Valley encephalitis virus in Australia and Papua New Guinea over nearly 70 years.

## Contribution

The study identifies a new sub-lineage of MVEV and shows its geographic spread, enhancing understanding of the virus's enzootic focus and evolution.

## Key findings

- MVEV genetic diversity is higher than previously recognized, with sub-lineage G1B predominant in Western Australia.
- A new sub-lineage, G1C, was identified and circulated in the 1990s before reappearing in 2003.
- MVEV transmission is more widespread and frequent than previously understood, with WA as the likely enzootic focus.

## Abstract

Murray Valley encephalitis virus (MVEV) is a mosquito-borne orthoflavivirus endemic to Australia that can cause fatal neurological disease. The enzootic focus of MVEV is believed to reside in northern Western Australia (WA). We sequenced whole genomes of 70 MVEV sampled over 51 years, 1969–2020, from locations across Australia and Papua New Guinea (PNG) and identified greater MVEV diversity than previously recognized. Genotype 1 (G1) demonstrated greatest intra-genotype diversity and was predominant over the sampling period with sub-lineage G1B circulating in WA and seeding activity across Australia. G1A included viruses sampled across northern WA, as well as the Northern Territory (NT). A newly identified sub-lineage G1C circulated in northern WA in 1993 and was detected again in 2003. G2 viruses were distributed across the Kimberley and Pilbara regions of northern WA, and in the NT. Although no new G3 and G4 viruses, previously identified only in PNG, were detected in the present study, other MVEV originating in PNG clustered with G1A. We confirm MVEV is enzootic in northern WA, with transmission occurring more frequently and across a wider geographical area than previously recognised. Additionally, we identify evidence of regular genotype replacement that has occurred over many decades where the major genotypes G1 and G2 have circulated in northern WA since the late 1960s. We also show that WA MVEV likely seeded an MVE outbreak in Victoria in 1974, further supporting the notion that the enzootic focus of MVEV lies in northern WA. Recent increases in MVEV detections, MVE cases and deaths in WA and across Australia highlight the need for enhanced surveillance and more frequent sampling to understand viral origin and genomic diversity, to identify potential virulence motifs, and to understand the ecological drivers that determine emergence of MVEV in northern WA and movement of MVEV across the country.

We analysed MVEV sequenced from archived virus isolates and mosquito homogenates collected since 1972 by the Western Australia Arbovirus Surveillance Program and by various research projects, with other published whole and partial genome sequences, to understand MVEV genetic diversity, evolution, and geographic distribution over a 69-year period 1951–2020. We amplified MVEV from virus isolates and developed an MVEV tiling amplicon PCR assay to analyse mosquito homogenates, and sequenced whole genomes of 70 viruses to create the largest and most geographically and temporally diverse MVEV dataset published to date. We describe a new sub-lineage G1C that circulated unrecognised in the 1990s, highlighting the value of more regular and strategic virus sampling and genome sequencing to capture the true diversity and evolutionary history of MVEV.