Metatranscriptomic Sequencing of Medically Important Mosquitoes Reveals Extensive Diversity of RNA Viruses and Other Microbial Communities in Western Australia.

Mosquitoes harbor a wide diversity of microorganisms, including viruses that are human pathogens, or that are insect specific. We used metatranscriptomics, an unbiased high-throughput molecular approach, to describe the composition of viral and other microbial communities in six medically important mosquito species from across Western Australia: Aedes vigilax, Culex annulirostris, Cx. australicus, Cx. globocoxitus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. We identified 42 viral species, including 13 novel viruses, from 19 families. Culex mosquitoes exhibited a significantly higher diversity of viruses than Aedes mosquitoes, and no virus was shared between the two genera. Comparison of mosquito populations revealed a heterogenous distribution of viruses between geographical regions and between closely related species, suggesting that geography and host species may play a role in shaping virome composition. We also detected bacterial and parasitic microorganisms, among which Wolbachia bacteria were detected in three members of the Cx. pipiens complex, Cx. australicus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. In summary, our unbiased metatranscriptomics approach provides important insights into viral and other microbial diversity in Western Australian mosquitoes that vector medically important viruses.

Specific IgA, But Not IgG, in Human Milk From COVID-19-infected Mothers Neutralizes SARS-CoV-2.

This study highlights the importance of human milk in providing anti-severe acute respiratory syndrome coronavirus 2 immunity to newborns. The highest protective activity of human milk against COVID-19 was found in colostrum from infected mothers. Neutralizing activity was associated with high levels of specific IgA. Depletion of IgA, but not IgG, from milk samples completely abolished the ability of human milk to neutralize severe acute respiratory syndrome coronavirus 2.

The APPRISE Virtual Biobank for Infectious Diseases.

The Australian Partnership for Preparedness Research on InfectiouS disease Emergencies (APPRISE) has developed a virtual biobank to support infectious disease research in Australia. The virtual biobank (https://apprise.biogrid.org.au) integrates access to existing distributed infectious disease biospecimen collections comprising multiple specimen types, including plasma, serum, and peripheral blood mononuclear cells. Through the development of a common data model, multiple collections can be searched simultaneously via a secure web portal. The portal enhances the visibility and searchability of existing collections within their current governance and custodianship arrangements. The portal is easily scalable for integration of additional collections.

Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae.

This review provides a summary of the recently ratified changes to genus and species nomenclature within the virus family Flaviviridae along with reasons for these changes. First, it was considered that the vernacular terms "flaviviral", "flavivirus", and "flaviviruses" could under certain circumstances be ambiguous due to the same word stem "flavi" in the taxon names Flaviviridae and Flavivirus; these terms could either have referred to all viruses classified in the family Flaviviridae or only to viruses classified in the included genus Flavivirus. To remove this ambiguity, the genus name Flavivirus was changed to Orthoflavivirus by the International Committee on Taxonomy of Viruses (ICTV). Second, all species names in the family were changed to adhere to a newly ICTV-mandated binomial format (e.g., Orthoflavivirus zikaense, Hepacivirus hominis) similar to nomenclature conventions used for species elsewhere in biology. It is important to note, however, that virus names remain unchanged. Here we outline the revised taxonomy of the family Flaviviridae as approved by the ICTV in April 2023.

Defining the pediatric response to SARS-CoV-2 variants.

The global population has been severely affected by the coronavirus disease 2019 (COVID-19) pandemic, however, with older age identified as a risk factor, children have been underprioritized. This article discusses the factors contributing to the less severe response observed in children following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including, differing viral entry receptor expression and immune responses. It also discusses how emerging and future variants could present a higher risk to children, including those with underlying comorbidities, in developing severe disease. Furthermore, this perspective discusses the differential inflammatory markers between critical and non-critical cases, as well as discussing the types of variants that may be more pathogenic to children. Importantly, this article highlights where more research is urgently required, in order to protect the most vulnerable of our children.

Genomic Analysis of Sindbis Virus Reveals Uncharacterized Diversity within the Australasian Region, and Support for Revised SINV Taxonomy.

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.

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

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.

Hyperendemic Dengue and Possible Zika Circulation in the Westernmost Region of the Indonesian Archipelago.

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.

Co-Circulation of Chikungunya and Multiple DENV Serotypes and Genotypes, Western Indonesia 2015-2016.

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.

Genome Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea Indicates Long-Term, Local Evolution.

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: A Minireview.

Dengue, caused by infection of any of four dengue virus serotypes (DENV-1 to DENV-4), is a mosquito-borne disease of major public health concern associated with significant morbidity, mortality, and economic cost, particularly in developing countries. Dengue incidence has increased 30-fold in the last 50 years and over 50% of the world's population, in more than 100 countries, live in areas at risk of DENV infection. We reviews DENV biology, epidemiology, transmission dynamics including circulating serotypes and genotypes, the immune response, the pathogenesis of the disease as well as updated diagnostic methods, treatments, vector control and vaccine developments.

Effects of El Niño Southern Oscillation and Dipole Mode Index on Chikungunya Infection in Indonesia.

The aim of this study was to assess the possible association of El Niño Southern Oscillation (ENSO) and Dipole Mode Index (DMI) on chikungunya incidence overtime, including the significant reduction in cases that was observed in 2017 in Indonesia. Monthly nation-wide chikungunya case reports were obtained from the Indonesian National Disease Surveillance database, and incidence rates (IR) and case fatality rate (CFR) were calculated. Monthly data of Niño3.4 (indicator used to represent the ENSO) and DMI between 2011 and 2017 were also collected. Correlations between monthly IR and CFR and Niño3.4 and DMI were assessed using Spearman's rank correlation. We found that chikungunya case reports declined from 1972 cases in 2016 to 126 cases in 2017, a 92.6% reduction; the IR reduced from 0.67 to 0.05 cases per 100,000 population. No deaths associated with chikungunya have been recorded since its re-emergence in Indonesia in 2001. There was no significant correlation between monthly Niño3.4 and chikungunya incidence with r = -0.142 (95%CI: -0.320-0.046), p = 0.198. However, there was a significant negative correlation between monthly DMI and chikungunya incidence, r = -0.404 (95%CI: -0.229--0.554) with p < 0.001. In conclusion, our initial data suggests that the climate variable, DMI but not Niño3.4, is likely associated with changes in chikungunya incidence. Therefore, further analysis with a higher resolution of data, using the cross-wavelet coherence approach, may provide more robust evidence.

The Diversity and Distribution of Viruses Associated with Culex annulirostris Mosquitoes from the Kimberley Region of Western Australia.

Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.

Phylogenetic and Timescale Analysis of Barmah Forest Virus as Inferred from Genome Sequence Analysis.

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.

Discovery of Jogalong virus, a novel hepacivirus identified in a Culex annulirostris (Skuse) mosquito from the Kimberley region of Western Australia.

The discovery of hepaciviruses in non-human hosts has accelerated following the advancement of high-throughput sequencing technology. Hepaciviruses have now been described in reptiles, fish, birds, and an extensive array of mammals. Using metagenomic sequencing on pooled samples of field-collected Culex annulirostris mosquitoes, we discovered a divergent hepacivirus-like sequence, named Jogalong virus, from the Kimberley region in northern Western Australia. Using PCR, we screened the same 300 individual mosquitoes and found just a single positive sample (1/300, 0.33%). Phylogenetic analysis of the hepacivirus NS5B protein places Jogalong virus within the genus Hepacivirus but on a distinct and deeply rooted monophyletic branch shared with duck hepacivirus, suggesting a notably different evolutionary history. Vertebrate barcoding PCR targeting two mitochondrial genes, cytochrome c oxidase subunit I and cytochrome b, indicated that the Jogalong virus-positive mosquito had recently fed on the tawny frogmouth (Podargus strigoides), although it is currently unknown whether this bird species contributes to the natural ecology of this virus.

Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977-2014.

Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977-2014) and during the substantial Pacific Islands RRV epidemic (1979-1980). Our final data set comprised isolates sampled over 59 years (1959-2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis.IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia and causes a significant burden of suffering to infected individuals as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history have so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust whole-genome analysis has not yet been conducted. This study generated 94 novel near-whole-genome sequences to investigate the evolutionary history of RRV to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance, and potential future vaccine design.

Nasal Cytokine Profiles of Patients Hospitalised with Respiratory Wheeze Associated with Rhinovirus C.

BACKGROUND: Rhinovirus C is an important pathogen of asthmatic and non-asthmatic children hospitalised with episodic wheeze. Previous studies on other respiratory viruses have shown that several host cytokines correlate with duration of hospitalisation, but this has yet to be investigated in children with RV-C infection. We determined the nasal cytokine profiles of these children and investigated their relationship with RV-C load and clinical outcome. Flocked nasal swabs were collected from children aged 24-72 months presenting to the Emergency Department at Princess Margaret Hospital with a clinical diagnosis of acute wheeze and an acute upper respiratory tract viral infection. RV-C load was determined by quantitative RT-PCR and cytokine profiles were characterised by a commercial human cytokine 34-plex panel. RV-C was the most commonly detected virus in pre-school-aged children hospitalised with an episodic wheeze. RV-C load did not significantly differ between asthmatic and non-asthmatic patients. Both groups showed a Th2-based cytokine profile. However, Th17 response cytokines IL-17 and IL-1ß were only elevated in RV-C-infected children with pre-existing asthma. Neither RV-C load nor any specific cytokines were associated illness severity in this study. Medically attended RV-C-induced wheeze is characterised by a Th2 inflammatory pattern, independent of viral load. Any therapeutic interventions should be aimed at modulating the host response following infection.