Daniel James Rawle

The cellular and molecular determinants of human neuroinvasive West Nile virus (WNV) disease are currently poorly defined. Previous studies of WNV neural infection relied on rodent models and immortalised cell lines that diverge from human brain tissue. Herein, we established a reproducible human cerebral organoid model of WNV infection and profiled it by virus-inclusive single-cell RNA sequencing (scRNA-Seq) to obtain an understanding of WNV-host interactions in the context of human neural tissue. We found that WNV showed preferential tropism to progenitor cells, immature neurons, and astrocytes, while the mature neurons were largely resistant to infection. Astrocytes were identified as the principal source of interferon (IFN) and the main hub of IFN-signalling, whereas mature neurons expressed little viral RNA-sensing machinery or STAT1 and responded weakly to infection and to IFN. The differential gene expression analysis between WNV-permissive and nonpermissive cells demonstrated that virus-susceptible cells had high baseline expression of the serine synthesis and one-carbon metabolism genes PHGDH , SHMT2 , and MTHFD2 , which were not induced by infection. RNA-interference and pharmacological inhibition of phosphoglycerate dehydrogenase (PHGDH) restricted WNV replication, confirming the requirement of this gene for WNV infection. In addition, the FDA-approved drug disulfiram, known to inhibit PHGDH, also reduced viral replication in neural progenitors and cerebral organoids. Overall, these results provide the first comprehensive single-cell-level characterisation of WNV neural infection in human neural tissue. They identify de novo serine synthesis as a metabolic dependency of WNV in human neural cells that can be targeted as a host-directed antiviral therapy.

Getah virus (GETV) is a mosquito-borne Old World alphavirus that has recently increased its geographic range, particularly in China. GETV causes rheumatic disease in horses, but in recent years its primary impact has been lethal infections in newborn piglets. Herein, we characterize a lethal Ifnar1−/− mouse model of GETV which recapitulates many of the features seen in piglets. These include lethality, high viremia, diarrhoea, and splenomegaly, with histopathological findings aligning with those seen in piglets and indicating severe, acute, multi-organ inflammatory immunopathology. RNA-Seq of spleens from GETV-infected Ifnar1−/− mice and subsequent bioinformatic analyses illustrated a cytokine storm response that included robust type I interferon signatures and a pattern of cytokine/chemokine signatures indicative of viral sepsis/septic shock. Ifnar1−/−mice thus offer a framework and a mouse model for understanding GETV disease in piglets. This model also suggests that viral sepsis/septic shock may be the cause of lethality in GETV-infected piglets.

Getah virus (GETV) is a mosquito borne Old World alphavirus that has recently increased its geographic range, particularly in China. GETV causes rheumatic disease in horses, but in recent years its primary impact has been lethal infections in newborn piglets. Herein we characterize a lethal Ifnar1-/- mouse model of GETV, which recapitulated many of the features seen in piglets. These include lethality, high viremia, diarrhoea, and splenomegaly, with histopathological findings aligning with those seen in piglets and indicating severe, acute, multi-organ inflammatory immunopathology. RNA-Seq of spleens from GETV infected Ifnar1-/- mice and subsequent bioinformatic analyses illustrated a cytokine storm response that included robust type I interferon signatures and a pattern of cytokine/chemokine signatures indicative of viral sepsis/septic shock. Ifnar1-/- mice thus offer a framework for understanding, and a mouse model for, GETV disease in piglets. The model also indicates that lethal GETV infection in piglets ay involve viral sepsis/septic shock.

SUMMARY: Ross River virus (RRV) causes the most mosquito-borne disease notifications in Australia and a considerable burden of non-fatal, yet frequently prolonged rheumatic illness across the Australia-Pacific region, reflected in thousands of notifications each year and notable economic and quality-of-life losses. Research on RRV spans multiple disciplines, encompassing viral genomics, immunopathology, transmission ecology, epidemiology, entomology, and environmental science. This review synthesizes two decades of cross-disciplinary investigations to present an integrated perspective on the biological, clinical, ecological, and environmental dimensions of RRV infection. By consolidating findings from diverse fields, the review enhances understanding of the complex factors influencing RRV transmission and disease outcomes throughout its endemic range.

SUMMARY: Effective control of viral encephalitis requires immune responses that clear infection without causing damaging neuroinflammation, yet the mechanisms governing resolution and recovery remain unclear. Using a Japanese encephalitis virus mouse model spanning asymptomatic, symptomatic, and lethal trajectories, together with single-cell spatial transcriptomics and RNA-seq, we identify apolipoprotein E (ApoE) as a driver of neuroinflammation resolution. Apoe was upregulated in microglia and infiltrating myeloid cells of symptomatic survivors, where Apoe-Trem2-Tyrobp signalling promoted a phagocytic, anti-inflammatory program. In contrast, immune cells in lethal disease failed to induce Apoe and remained pro-inflammatory. ApoE-deficient mice were unable to recover following encephalitis onset, demonstrating that ApoE signalling is essential for resolution and recovery. Analysis of cerebrospinal fluid from acute encephalitis patients linked APOE isoforms to neuroinflammation resolution, with APOEε2 carriers exhibiting reduced neutrophils and shorter hospitalisation. These findings identify ApoE as a critical driver of neuroinflammation resolution and a promising therapeutic target for viral encephalitis.

In 2022, Australia saw an unprecedented outbreak of Japanese encephalitis virus genotype IV (JEV GIV). The outbreak involved 42 human cases with 7 fatalities, as well as affecting >80 pig farms in New South Wales and Queensland. Herein, we designed, constructed, and tested two JEV GIV mRNA vaccines encoding prME, which provided protection against a lethal JEV GIV challenge in an Ifnar-/- mouse model. The vaccines were not codon optimized and included either the Native (full-length) or a Shorter signal peptide, with the latter missing the N-terminal n-region. Two vaccinations with 5 µg of the Shorter vaccine provided neutralizing antibody responses that were significantly lower but overlapped with those seen after vaccination with Imojev, a live attenuated vaccine approved for use in humans. Both mRNA vaccines provided approximately a five to six log reduction in viremia, ≥80% protection against overt disease and weight loss, and mortality. The paper illustrates in-country mRNA vaccine generation in response to a local outbreak, with JEV mRNA vaccines potentially emerging to be easier to manufacture, cheaper, and more suitable for immunocompromised individuals.

We describe RNA-Seq analyses conducted on nasopharyngeal swabs collected from 37 patients admitted to an Australian intensive care unit from October 2022 to August 2023. During this time, the dominant omicron sublineage infections broadly progressed from BA.5 to BA.2-like, to XBB-like, then XBC, consistent with global trends. Viral load and patient metadata correlations indicated this cohort was broadly representative of severe COVID-19 patients. Human gene expression analyses were complicated by the large range (>5 log) and variability in viral reads. Nevertheless, the comparison of XBC and BA.5 samples that had comparable viral read counts, revealed differentially expressed genes and a cellular deconvolution signature that indicated increased targeting of ciliated epithelial cells by XBC. To obtain more evidence for increased targeting of ciliated epithelial cells by the later omicron sublineage viruses, a series of mouse strains were infected with a BA.5 or a XBB isolate. Increased infection of the nasal turbinates and ciliated epithelial cells by XBB was demonstrated by viral titrations and immunohistochemistry, respectively. Compared with previous lineages, the omicron lineage showed increased targeting of ciliated epithelia in the upper respiratory tract, with the data presented herein suggesting this trend continued for the omicron sublineages.

COVID-19 diagnostic testing generally involves collection of patient samples via nasopharyngeal swabs. Herein we describe viral and human gene expression data available from such samples using RNA-Seq and bioinformatics. Swabs were collected from 38 patients admitted to an Australian intensive care unit (ICU) from October 2022 to August 2023. During this time the omicron sublineages changed from BA.5, BA.2-like, XBB-like to XBC. RNA-Seq viral read data correlating well with RTPCR-based quantitation and sublineage identification. High viral loads were associated with patients >64 years of age. Treatment with corticosteroids or baricitinib did not significantly influence viral loads. Viral loads did not correlate with length of stay in hospital or ICU, or with vaccination status. Analyses of human gene expression data were complicated by the very large range (>5 log) and variability in viral reads. Nevertheless, comparison of XBC and BA.5 samples that had comparable viral read counts, revealed differentially expressed genes and a cellular deconvolution signature that indicated increased infection of ciliated epithelial cells by XBC. A key feature that differentiated the omicron lineage from previous lineages was the increased targeting of ciliated epithelia in the upper respiratory track. The data presented herein argue that this evolutionary trend continued in the omicron sublineages.

Herein, we explore the potential influence of Schistosoma mansoni Sambon, 1907 soluble egg antigen (SmSEA) on the immunopathology of COVID-19 in K18-hACE2 mice infected with an Omicron BA.5 isolate of SARS-CoV-2. SmSEA treatment was delivered in a single dose by intraperitoneal injection, shortly after intrapulmonary inoculation of SARS-CoV-2. RNA-seq identified 36 differentially expressed genes in the spleens of virus-infected mice treated with SmSEA vs. PBS on day 5 post infection. Ingenuity Pathway Analysis of these genes suggested marginal modulation of cytokine responses, with upregulation of the IL-10 and IL-4 signatures and downregulation of the IFNγ signature. However, cytokine responses and histopathology in the lungs were largely unaffected. Future work will require purification of active helminth compounds and dosing and scheduling optimisation.

The Australasian Virology Society (AVS) holds premier biennial virology meetings that foster multidisciplinary research and collaboration and promote equity and inclusion of early-career researchers. The 12th AVS meeting (AVS12), convened by M. Tate, J. Fraser, and G. Moseley, was held from 2 to 5 December 2024 on Dja Dja Wurrung country at the RACV Goldfields Resort in Creswick, Victoria, Australia. In this report, we give a brief overview of the history of AVS and outline the current and developing priorities for the society. We provide a summary of the insightful panel discussions held to address career development and Indigenous virology, highlight the presentations given by international plenary speakers Joe Grove and Chantal Abergel, and celebrate the recipients of the numerous awards.