Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

Exposure dynamics of Ross River virus in horses - Horses as potential sentinels (a One Health approach).

Ross River virus (RRV), the most medically and economically important arbovirus in Australia, has been the most prevalent arbovirus infections in humans for many years. Infected humans and horses often suffer similar clinical symptoms. We conducted a prospective longitudinal study over a 3.5-year period to investigate the exposure dynamics of RRV in three foal cohorts (n = 32) born in a subtropical region of South East Queensland, Australia, between 2020 and 2022. RRV-specific seroconversion was detected in 56% (n = 18) of foals with a median time to seroconversion, after waning of maternal antibodies, of 429 days (95% CI: 294-582). The median age at seroconversion was 69 weeks (95% CI: 53-57). Seroconversion events were only detected between December and March (Southern Hemisphere summer) over the entire study period. Cox proportion hazards regression analyses revealed that seroconversions were significantly (p < 0.05) associated with air temperature in the month of seroconversion. Time-lags in meteorological variables were not significantly (p > 0.05) associated with seroconversion, except for relative humidity (p = 0.036 at 2-month time-lag). This is in contrast to research results of RRV infection in humans, which peaked between March and May (Autumn) and with a 0-3 month time-lag for various meteorological risk factors. Therefore, horses may be suitable sentinels for monitoring active arbovirus circulation and could be used for early arbovirus outbreak detection in human populations.

Mucosal immunization with dual influenza/COVID-19 single-replication virus vector protects hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic has highlighted the need for mucosal vaccines as breakthrough infections, short-lived immune responses and emergence of new variants have challenged the efficacy provided by the first generation of vaccines against SARS-CoV-2 viruses. M2SR SARS-CoV-2, an M2-deleted single-replication influenza virus vector modified to encode the SARS-CoV-2 receptor binding domain, was evaluated following intranasal delivery in a hamster challenge model for protection against Wuhan SARS-CoV-2. An adjuvanted inactivated SARS-CoV-2 whole virus vaccine administered intramuscularly was also evaluated. The intranasal M2SR SARS-CoV-2 was more effective than the intramuscular adjuvanted inactivated whole virus vaccine in providing protection against SARS-CoV-2 challenge. M2SR SARS-CoV-2 elicited neutralizing serum antibodies against Wuhan and Omicron SARS-CoV-2 viruses in addition to cross-reactive mucosal antibodies. Furthermore, M2SR SARS-CoV-2 generated serum HAI and mucosal antibody responses against influenza similar to an H3N2 M2SR influenza vaccine. The intranasal dual influenza/COVID M2SR SARS-CoV-2 vaccine has the potential to provide protection against both influenza and COVID.

Experimental SARS-CoV-2 Infection of Elk and Mule Deer.

To assess the susceptibility of elk (Cervus canadensis) and mule deer (Odocoileus hemionus) to SARS-CoV-2, we performed experimental infections in both species. Elk did not shed infectious virus but mounted low-level serologic responses. Mule deer shed and transmitted virus and mounted pronounced serologic responses and thus could play a role in SARS-CoV-2 epidemiology.

Kinetics of severe dengue virus infection and development of gut pathology in mice.

IMPORTANCE: Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.

Advax-CpG55.2-adjuvanted monovalent or trivalent SARS-CoV-2 recombinant spike protein vaccine protects hamsters against heterologous infection with Beta or Delta variants.

The ongoing evolution of SARS-CoV-2 variants emphasizes the need for vaccines providing broad cross-protective immunity. This study was undertaken to assess the ability of Advax-CpG55.2 adjuvanted monovalent recombinant spike protein (Wuhan, Beta, Gamma) vaccines or a trivalent formulation to protect hamsters againstBeta or Delta virus infection. The ability of vaccines to block virus transmission to naïve co-housed animals was also assessed. In naïve hosts, the Beta variant induced higher virus loads than the Delta variant, and conversely the Delta variant caused more severe disease and was more likely to be associated with virus transmission. The trivalent vaccine formulation provided the best protection against both Beta and Delta infection and also completely prevented virus transmission. The next best performing vaccine was the original monovalent Wuhan-based vaccine. Notably, hamsters that received the monovalent Gamma spike vaccine had the highest viral loads and clinical disease of all the vaccine groups, a potential signal of antibody dependent-enhancement (ADE). These hamsters were also the most likely to transmit Delta virus to naïve recipients. In murine studies, the Gamma spike vaccine induced the highest total spike protein to RBD IgG ratio and the lowest levels of neutralizing antibody, a context that could predispose to ADE. Overall, the study results confirmed that the current SpikoGen® vaccine based on Wuhan spike protein was still able to protect against clinical disease caused by either the Beta or Delta virus variants but suggested additional protection may be obtained by combining it with extra variant spike proteins to make a multivalent formulation. This study highlights the complexity of optimizing vaccine protection against multiple SARS-CoV-2 variants and stresses the need to continue to pursue new and improved COVID-19 vaccines able to provide robust, long-lasting, and broadly cross-protective immunity against constantly evolving SARS-CoV-2 variants.

Pre-clinical lung squamous cell carcinoma mouse models to identify novel biomarkers and therapeutic interventions.

Primary lung carcinoma or lung cancer (LC) is classified into small-cell or non-small-cell (NSCLC) lung carcinoma. Lung squamous cell carcinoma (LSCC) is the second most common subtype of NSCLC responsible for 30% of all LCs, and its survival remains low with only 24% of patients living for five years or longer post-diagnosis primarily due to the advanced stage of tumors at the time of diagnosis. The pathogenesis of LSCC is still poorly understood and has hampered the development of effective diagnostics and therapies. This review highlights the known risk factors, genetic and epigenetic alterations, miRNA biomarkers linked to the development and diagnosis of LSCC and the lack of therapeutic strategies to target specifically LSCC. We will also discuss existing animal models of LSCC including carcinogen induced, transgenic and xenograft mouse models, and their advantages and limitations along with the chemopreventive studies and molecular studies conducted using them. The importance of developing new and improved mouse models will also be discussed that will provide further insights into the initiation and progression of LSCC, and enable the identification of new biomarkers and therapeutic targets.

A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions.

West Nile virus (WNV) causes skin lesions in farmed crocodiles leading to the depreciation of the value of their hides and significant economic losses. However, there is no commercially available vaccine designed for use in crocodilians against WNV. We tested chimeric virus vaccines composed of the non-structural genes of the insect-specific flavivirus Binjari virus (BinJV) and genes encoding the structural proteins of WNV. The BinJV/WNV chimera, is antigenically similar to wild-type WNV but replication-defective in vertebrates. Intramuscular injection of two doses of BinJV/WNV in hatchling saltwater crocodiles (Crocodylus porosus) elicited a robust neutralising antibody response and conferred protection against viremia and skin lesions after challenge with WNV. In contrast, mock-vaccinated crocodiles became viraemic and 22.2% exhibited WNV-induced lesions. This suggests that the BinJV/WNV chimera is a safe and efficacious vaccine for preventing WNV-induced skin lesions in farmed crocodilians.

In vivo inhibition of nuclear ACE2 translocation protects against SARS-CoV-2 replication and lung damage through epigenetic imprinting.

In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

Beneficial effects of brief early life angiotensin-converting enzyme inhibition wane with time in sheep with solitary functioning kidney.

A child with a congenital solitary functioning kidney (SFK) may develop kidney disease from early in life due to hyperfiltration injury. Previously, we showed in a sheep model of SFK that brief angiotensin-converting enzyme inhibition (ACEi) early in life is reno-protective and increases renal functional reserve (RFR) at 8 months of age. Here we investigated the long-term effects of brief early ACEi in SFK sheep out to 20 months of age. At 100 days gestation (term = 150 days) SFK was induced by fetal unilateral nephrectomy, or sham surgery was performed (controls). SFK lambs received enalapril (SFK+ACEi; 0.5 mg/kg, once daily, orally) or vehicle (SFK) from 4 to 8 weeks of age. At 8, 14 and 20 months of age urinary albumin excretion was measured. At 20 months of age, we examined basal kidney function and RFR via infusion of combined amino acid and dopamine (AA+D). SFK+ACEi resulted in lower albuminuria (∼40%) at 8 months, but not at 14 or 20 months of age compared with vehicle-SFK. At 20 months, basal GFR (∼13%) was lower in SFK+ACEi compared with SFK, but renal blood flow (RBF), renal vascular resistance (RVR) and filtration fraction were similar to SFK. During AA+D, the increase in GFR was similar in SFK+ACEi and SFK animals, but the increase in RBF was greater (∼46%) in SFK+ACEi than SFK animals. Brief ACEi in SFK delayed kidney disease in the short-term but these effects were not sustained long-term.

Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

The swan genome and transcriptome, it is not all black and white.

BACKGROUND: The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related northern hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious diseases, notably infectious diseases from which Australia has been largely shielded. Unlike mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to highly pathogenic avian influenza. Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. RESULTS: Here, we generate the first chromosome-length black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We use these genomes and transcriptomes to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to highly pathogenic avian influenza. We also implicate genetic differences in SLC45A2 gene in the iconic plumage of the black swan. CONCLUSION: Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat, the black swan would be in a significant peril.

Distinct contributions of the innate immune receptors TLR2 and RP105 to formation and architecture of structured lung granulomas in mice infected with Mycobacterium tuberculosis.

Granulomas are key histopathological features of Mycobacterium tuberculosis (Mtb) infection, with complex roles in pathogen control and dissemination. Thus, understanding drivers and regulators of granuloma formation is important for improving tuberculosis diagnosis, treatment, and prevention. Yet, molecular mechanisms underpinning granuloma formation and dynamics remain poorly understood. Here we used low-dose Mtb infection of C57BL/6 mice, which elicits structured lung granulomas composed of central macrophage clusters encased by a lymphocyte mantle, alongside the disorganized lymphocyte and macrophage clusters commonly observed in Mtb-infected mice. Using gene-deficient mice, we observed that Toll-like receptor (TLR) 2 and the TLR-related Radioprotective 105 kDa protein (RP105) contributed to the extent and spatial positioning of pathology in infected lung tissues, consistent with functional cooperation between TLR2 and RP105 in the innate immune recognition of Mtb. In mice infected with the highly virulent Mtb clinical isolate HN878, TLR2, but not RP105, positively regulated the extent of central macrophage regions within structured granulomas. Moreover, RP105, but not TLR2, promoted the formation of structured lung granulomas, suggesting that the functions of RP105 as an innate immune sensor for Mtb reach beyond its roles as TLR2 co-receptor. TLR2 and RP105 contributions to lung pathology are governed by Mtb biology, as neither receptor affected the frequency or architecture of structured granulomas in mice infected with the reference strain Mtb H37Rv. Thus, by revealing distinctive as well as cooperative functions of TLR2 and RP105 in lung pathology, our data identify TLRs as molecular determinants of TB granuloma formation and architecture, and expand understanding of how interactions between innate immune receptors and Mtb shape TB disease manifestation.

GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

RATIONALE: Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. OBJECTIVES: To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. METHODS: Preclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. RESULTS: Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte/macrophage infiltration to the lung during influenza A virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of Gpr183 or treatment with a GPR183 antagonist reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single-cell RNA-sequencing data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. CONCLUSION: This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

Immunogenicity and Efficacy of Monovalent and Bivalent Formulations of a Virus-Like Particle Vaccine against SARS-CoV-2.

Virus-like particles (VLPs) offer great potential as a safe and effective vaccine platform against SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 VLPs can be generated by expression of the four viral structural proteins in a mammalian expression system. Immunization of mice with a monovalent VLP vaccine elicited a potent humoral response, showing neutralizing activity against multiple variants of SARS-CoV-2. Subsequent immunogenicity and efficacy studies were performed in the Golden Syrian hamster model, which closely resembles the pathology and progression of COVID-19 in humans. Hamsters immunized with a bivalent VLP vaccine were significantly protected from infection with the Beta or Delta variant of SARS-CoV-2. Vaccinated hamsters showed reduced viral load, shedding, replication, and pathology in the respiratory tract. Immunized hamsters also showed variable levels of cross-neutralizing activity against the Omicron variant. Overall, the VLP vaccine elicited robust protective efficacy against SARS-CoV-2. These promising results warrant further study of multivalent VLP vaccines in Phase I clinical trials in humans.

BCGΔBCG1419c increased memory CD8+ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis.

Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L-), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.

Epidemiological Study of Multiple Zoonotic Mosquito-Borne Alphaviruses in Horses in Queensland, Australia (2018-2020).

The increased frequency of extreme weather events due to climate change has complicated the epidemiological pattern of mosquito-borne diseases, as the host and vector dynamics shift to adapt. However, little is known about the seroprevalence of common mosquito-borne virus infections in horses in Australia. In this study, serological surveys for multiple alphaviruses were performed on samples taken from 622 horses across two horse populations (racehorses and horses residing on The University of Queensland (UQ) campus) in Queensland using the gold standard virus neutralization test. As is the case in humans across Australia, Ross River virus (RRV) is the most common arbovirus infection in horses, followed by Barmah Forest virus, with an overall apparent seroprevalence of 48.6% (302/622) and 4.3% (26/607), respectively. Horses aged over 6 years old (OR 1.86, p = 0.01) and residing at UQ (OR 5.8, p < 0.001) were significantly associated with seroconversion to RRV. A significant medium correlation (r = 0.626, p < 0.001) between RRV and Getah virus (GETV) neutralizing antibody titers was identified. Collectively, these results advance the current epidemiological knowledge of arbovirus exposure in a susceptible host in Australia. The potential use of horses as sentinels for arbovirus monitoring should be considered. Furthermore, since GETV is currently exotic to Australia, antibodies cross-reactivity between RRV and GETV should be further investigated for cross-protection, which may also help to inform vaccine developments.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome.

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.