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

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

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

Dengue is a mosquito-borne disease of public health concern affecting tropical and subtropical countries, including Indonesia. Although studies on dengue epidemiology have been undertaken in Indonesia, data are lacking in many areas of the country. The aim of this study was to determine dengue virus (DENV) and chikungunya virus (CHIKV) molecular epidemiology in western regions of the Indonesian archipelago. A one-year prospective study was conducted in Aceh and Jambi in 2015 and 2016, respectively, where patients with dengue-like illness were enrolled. Of 205 patients recruited, 29 and 27 were confirmed with dengue in Aceh and Jambi, respectively, and three from Jambi were confirmed with chikungunya. DENV-1 was the predominant serotype identified in Aceh while DENV-2 was predominant in Jambi. All DENV-1 and DENV-2 from both regions were classified as Genotype I and Cosmopolitan genotype, respectively, and all DENV-3 viruses from Jambi were Genotype I. Some viruses, in particular DENV-1, displayed a distinct lineage distribution, where two DENV-1 lineages from Aceh were more closely related to viruses from China instead of Jambi highlighting the role of travel and flight patterns on DENV transmission in the region. DENV-2 from both Aceh and Jambi and DENV-3 from Jambi were all closely related to Indonesian local strains. All three CHIKV belonged to Asian genotype and clustered closely with Indonesian CHIKV strains including those previously circulating in Jambi in 2015, confirming continuous and sustainable transmission of CHIKV in the region. The study results emphasize the importance of continuous epidemiological surveillance of arboviruses in Indonesia and simultaneous testing for CHIKV among dengue-suspected patients.

The transmission of dengue and other medically important mosquito-borne viruses in the westernmost region of Indonesia is not well described. We assessed dengue and Zika virus seroprevalence in Aceh province, the westernmost area of the Indonesian archipelago. Serum samples collected from 199 randomly sampled healthy residents of Aceh Jaya in 2017 were analyzed for neutralizing antibodies by plaque reduction neutralization test (PRNT). Almost all study participants (198/199; 99.5%) presented with multitypic profiles of neutralizing antibodies to two or more DENV serotypes, indicating transmission of multiple DENV in the region prior to 2017. All residents were exposed to one or more DENV serotypes by the age of 30 years. The highest geometric mean titers were measured for DENV-4, followed by DENV-1, DENV-2 and DENV-3. Among a subset of 116 sera, 27 neutralized ZIKV with a high stringency (20 with PRNT₉₀ > 10 and 7 with PRNT₉₀ > 40). This study showed that DENV is hyperendemic in the westernmost region of the Indonesian archipelago and suggested that ZIKV may have circulated prior to 2017.