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.

N-linked glycosylation of flavivirus envelope proteins is widely viewed as being required for optimal folding, processing and/or transit of envelope proteins, and the assembling virons, through the endoplasmic reticulum (ER) and the Golgi. Zika virus (ZIKV) has a single N-linked envelope glycan located adjacent to the fusion loop. Herein we show that independent serial passage of ZIKVNatal in Rag1 -/- mice for 223 or 386 days generated two unique envelope glycan-deficient mutants, ZIKV-V153D and ZIKV-N154D, respectively. Surprisingly, these mutants grew to titres ∼1 to 2.6 logs higher than the glycosylated parental ZIKVNatal in Vero E6 cells and human brain organoids. RNA-Seq of infected organoids suggested that this increased replication fitness was associated with upregulation of the unfolded protein response (UPR). Cell death, cellular viral RNA, and viral protein levels were not significantly affected, arguing that these glycan mutants enjoyed faster ER/Golgi folding, processing, assembly, transit, and virion egress, assisted by an upregulated UPR. Thus, ZIKV envelope N-linked glycosylation is not essential for promoting envelope folding, assembly, and transit through the ER/Golgi, since aspartic acid (D) substitutions in the glycosylation motif can achieve this with significantly greater efficiency. Instead, the evolution of glycan mutants in Rag1 -/- mice indicates that such envelope glycosylation can have a fitness cost in an environment devoid of virus-specific antibody responses. The V153D and N154D mutations, generated by natural selection in Rag1 -/- mice, have to date not been employed in orthoflavivirus envelope glycosylation studies. Instead, genetic engineering has been used to generate mutant viruses that, for instance, contain a N154A substitution. The latter may impart confounding unfavourable properties, such as envelope protein insolubility, that have a detrimental impact on virus replication. The V153D and N154D substitutions may avoid imparting unfavourable properties by preserving the surface negative charge provided by the glycan moiety in the parental ZIKVNatal envelope protein. In Ifnar1 -/- mice ZIKV-V153D and -N154D showed faster viremia onsets, but reduced viremic periods, than the parental ZIKVNatal, consistent with an established contention that such glycans have evolved to delay neutralizing antibody activity.

Certain insect-specific viruses (ISVs), specifically the mosquito alphaviruses, Eilat and Yada Yada viruses, and orthoflaviviruses, Binjari, Aripo, YN15-283-02 and Chaoyang viruses, have emerged as potential platforms for generation of whole virus vaccines for human and veterinary applications. These ISVs are remarkably tolerant of the substitution of their structural polyproteins with those of alphaviruses and orthoflaviviruses that are pathogenic in humans and/or animals. The resulting ISV-based chimeric vaccines have been evaluated in mouse models and have demonstrated safety and efficacy in non-human primates, crocodiles and pigs. Targets include chikungunya, Venezuelan and eastern equine encephalitis, dengue, Zika, yellow fever, Japanese encephalitis and West Nile viruses. ISV-based chimeric vaccines provide authentically folded tertiary and quaternary whole virion particle structures to the immune system, a key feature for induction of protective antibody responses. These vaccines are manufactured in C6/36 or C7-10 mosquito cell lines, where they grow to high titers, but they do not replicate in vertebrate vaccine recipients. This review discusses the progress of these emerging technologies and addresses challenges related to adjuvanting, safety, and manufacturing.

Murray Valley encephalitis virus (MVEV) is a zoonotic flavivirus endemic to Australia and Papua New Guinea. A recent outbreak of MVEV has prompted renewed concerns regarding the potential for MVEV to generate disease outbreaks. Currently, nine full length sequences of MVEV are publicly available, divided into four genotypes (G1-G4). Herein, we sequenced MVEV isolates from the Ralph Doherty Virus Collection, a virus bank with Australian field isolates dating between the 1950s-1980s, and determined their phylogenetic relationship with existing isolates to provide insights into virus evolution and genetic diversity. Additionally, we characterised isolates from different genotypes both in vitro using human neuronal cells, and in vivo using C57BL/6J mice, to provide additional insight into MVEV pathogenicity and establish models of MVEV disease that recapitulate MVEV human disease. We found 15 new full length sequences of MVEV, which primarily clustered into the dominant genotype, G1. Additionally, we show MVEV can be lethal and neuroinvasive in C57BL/6J mice, recapitulating histological lesions identified in human infection. Overall, our study contributes significant genomic sequences to the current MVEV database and establishes mouse models of disease and infection which can be used for mechanistic studies and evaluation of new interventions. ### Competing Interest Statement The authors have declared no competing interest. National Health and Medical Research Council, https://ror.org/011kf5r70, APP1173880

Chikungunya virus (CHIKV) is a positive sense RNA Alphavirus that continues to pose major public health threats throughout the world. CHIKV is primarily transmitted via the Aedes genus mosquito; however, has also exhibited transmission routes via blood transfusion and vertical transmission (mother to child). With only one approved vaccine thus far and no approved medicines or specific therapeutics, early detection is crucial in mitigating potential CHIKV outbreaks. Here, we designed and evaluated a sensitive and specific CHIKV diagnostic using reverse transcription-recombinase aided amplification (RT-RAA) coupled lateral flow strip detection (LFD) targeting a highly conserved region of the CHIKV E1 gene. Our results demonstrate that using our simple sample preparation reagent (TNA-Cifer-E), we can inactivate live CHIKV in two minutes at room temperature, whilst also sustaining viable viral RNA. Our specificity analysis demonstrates the Iso-CHIKV-Dx does not detect any closely related Alphaviruses nor any of the common co-circulating Flaviviruses. Proof-of-concept evaluation using urine spiked with CHIKV exhibited that in CHIKV infected urine samples, our Iso-CHIKV-Dx can detect as low as 570 copies/µL of CHIKV RNA in 30 minutes under isothermal conditions. Contrary to conventional RT-qPCR, our Iso-CHIKV-Dx does not require expensive machinery, advanced instrumentation or extensively trained personnel. Further performance comparisons also show that our Iso-CHIKV-Dx is four times faster than conventional RNA isolation and RT-qPCR. As such, pre-clinical, proof-of-concept evaluation demonstrates that our Iso-CHIKV-Dx has the potential to act as a robust, point of care CHIKV diagnostic that could prove to be highly beneficial in place of, or in the absence of conventional diagnostic approaches such as RT-qPCR.

Zika virus (ZIKV) caused unprecedented outbreaks in South America and the Caribbean in 2015-2016, leading primarily to a series of abnormalities in neonates termed congenital Zika syndrome. The threat of ZIKV reemergence has seen the development of multiple ZIKV vaccines that are at the preclinical stage or in early-stage clinical trials. Herein, we describe a pathway to the development of ZIKV vaccines generated using a baculovirus-insect cell expression system, which is widely applied for the manufacture of biologics for human use. Virus-like particle (VLP) vaccines comprising CprME and subviral particle (SVP) vaccines comprising prME were evaluated for their ability to mediate protection against ZIKV challenge in Ifnar1-/- mice. Initial attempts resulted in VLP and SVP vaccines that failed to present quaternary epitopes and did not provide effective protection. To improve the SVP vaccine, two modifications were introduced: firstly, an alanine to cysteine substitution (A264C) in the E domain II region to promote the formation of stabilized E homodimers and, secondly, the use of Spodoptera frugiperda Sf9 insect cells that had been adapted to grow and produce vaccine at a neutral pH of 7. E homodimers largely retain their pre-fusion conformation at pH 7, which is a requirement for the induction of effective neutralizing antibody responses. The stabilized SVP-A26C vaccine induced high levels of neutralizing antibodies and protected male Ifnar1-/- mice against viremia and testicular damage. Our study reiterates the need to present the immune system with E dimers arranged in authentic quaternary conformations and provides a scalable production method for this novel ZIKV vaccine.IMPORTANCEWe describe the generation of a subviral particle (SVP) vaccine comprising prME proteins of ZIKV, with an envelope protein substitution, A264C, that stabilizes E dimer formation. The SVP vaccine was produced in a novel Sf9 insect cell line adapted to grow in suspension at pH 7. The study highlights the importance of challenge experiments to ascertain whether the responses induced by an experimental vaccine actually mediate protection against virus infection and disease. The study also reiterates the contention that effective flavivirus vaccines need to present the immunogen in an authentic tertiary and quaternary structure with a pre-fusion conformation.

Rationale and Objectives: Iron availability and metabolism are important in the pathogenesis of bacterial infections. More recently, links have been reported between iron and the severity of viral infections. In this study, we characterize a crucial relationship between iron metabolism and IAV infection and disease. Methods: Iron-related gene expression was assessed in human airway epithelial cells (AEC) infected with IAV. AECs were cultured with ferric iron, iron-loaded transferrin, or iron chelator, deferoxamine (DFO), prior to infection with IAV. Mice were placed on a high iron diet for 8 weeks prior to infection with IAV or treated with anti-transferrin receptor-1 (TFR1) antibody during IAV infection. The effects of iron modulation and depletion of TFR1-mediated responses on IAV infection were assessed. Measurements and main results: Iron-related gene expression and metabolism are altered systemically and in lung tissues and AECs during IAV infections. Increasing iron availability increases viral titer in AECs, while DFO protects against iron-induced increased susceptibility to infection. Increasing systemic iron loading, which increases iron levels in the lung, increases viral titer, proinflammatory responses, airway inflammation, and worsens IAV-induced disease in terms of lung function and weight loss in vivo. Inhibition of TFR1 protects against IAV-induced disease in vivo. Conclusion: IAV infections remain a major threat to human health and global economies. Strategies that boost protective, or reduce pathogenic, host responses may provide broadly effective, long-term therapeutic options. We have identified a key role for iron metabolism in modifying host responses to IAV that can be harnessed to protect against disease. ### Competing Interest Statement The authors have declared no competing interest.

Chikungunya virus (CHIKV) is a mosquito-transmitted, arthritogenic alphavirus that causes sporadic outbreaks of often debilitating rheumatic disease. The recently approved CHIKV vaccine, IXCHIQ, is based on a live-attenuated CHIKV strain (VLA1553), with viraemic vaccine recipients theoretically able to transmit VLA1553 to mosquitoes with ensuing onward transmission. We thus evaluated VLA1553 transmission from artificial blood meals to Aedes albopictus mosquitoes, and onward transmission to mice. Female A. albopictus mosquitoes were fed on defibrinated sheep blood containing wild-type CHIKV (viral titre: 7.50 log10CCID50/mL) or VLA1553 (viral titres: 7.85, 5.72, 4.58, and 3.79 log10CCID50/mL). Viral titres in mosquito bodies and saliva were determined using CCID50 assays 7–8 days after the blood meal. After providing CHIKV or VLA1553 (viral titres 7–8 log10CCID50/mL) in blood meals to mosquitoes, infected mosquitoes were fed on highly susceptible Irf3/7−/− mice (n = 3 per group). Data were re-analysed using the same reverse transcription quantitative polymerase chain reaction (RT-qPCR) as for an earlier VLA1553 phase 1 clinical trial, to allow correlations between blood meal titres and viraemia in vaccine recipients. Mosquito body viral titres were significantly higher (P < 0.0001) for CHIKV versus VLA1553-fed mosquitoes at blood meal viral titres of 7–8 log10CCID50/mL. Mosquito body VLA1553 titres decreased with reducing blood meal titres, but there was no dose-dependent effect on saliva viral titres. No dissemination to salivary glands was seen at blood meal titres ≤ 3.875 log10CCID50/mL. CHIKV-fed mosquitoes were able to transmit virus, and induce viraemia in, 3/3 Irf3/7−/− mice via mosquito bites. In contrast, 0/3 Irf3/7−/− mice became infected after bites from VLA1553-fed mosquitoes. RT-qPCR comparisons with phase 1 clinical data for VLA1553-vaccinated individuals indicated that VLA1553 viraemia was at or below the aforementioned threshold for transmission. The evidence presented herein argue that the low viraemia in VLA1553-vaccinated individuals would mitigate against transmission. In addition, replication of VLA1553 in mosquito bodies was also significantly attenuated. Overall, mosquito-borne transmission of VLA1553 from vaccinated individuals to others appears improbable.

Antibody-dependent enhancement (ADE) of infection is a concern for flavivirus vaccine development. Poorly neutralising cross-reactive antibodies that target viral envelope can result in ADE. Vaccine strategies that harness T cell immunity should be explored. Nonstructural protein 1 (NS1) is a promising vaccine antigen that abrogates ADE risk. We developed a ZIKV NS1 DNA vaccine that is protective and immunogenic in mice. We evaluated NS1 vaccine in the pregnancy model of ZIKV infection showing protection of fetuses from IUGR, microcephaly, and brain damage. Vaccination of male IFNAR-/- mice prevented ZIKV-induced testicular damage and viral persistence. Protection was mediated by T cells. Vaccination of rhesus macaques with PharmaJet Tropis device showed that the vaccine is highly immunogenic and protective against ZIKV infection. Next, we developed NS1 mRNA vaccine and evaluated its protective efficacy. Inclusion of additional antigens can increase the breadth of T cell responses providing enhanced protection. To select additional antigens, we comprehensively evaluated in in vivo effector, early and late memory T cell responses after ZIKV infection. Complementing clinical data, we show that ZIKV NS3 and NS4 are the dominant T cell targets post-infection. Next we evaluated efficacy and immunogenicity of multivalent ZIKV NS1, NS3/4 mRNA vaccine. Our results have important implications for the development of multivalent ZIKV vaccines, that abrogate the risk of flavivirus ADE The Hospital Research Foundation Group (Australia), Medical research Future Fund (Australia) and National Foundation for Medical Research and Innovation (Australia) Vaccines and Immunotherapy (VAC)

Arthritogenic alphaviruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-borne viruses that can cause debilitating polyarthritis/polyarthralgia in humans. Although two CHIKV vaccines have been licensed, there are no licensed vaccines for RRV. Herein we generate a host-restricted, insect-specific alphavirus, Yada Yada virus (YYV), chimeric vaccine for CHIKV (YYV-CHIKVMauritius) and for RRV (YYV-RRVTT). YYV-CHIKVMauritius and YYV-RRVTT was able to replicate in C6/36 mosquito cells to similar titres as wild-type CHIKV and RRV. YYV-CHIKVMauritius was also neutralised by CHIKV monoclonal antibodies to the same titres as wild-type CHIKV, indicating its potential as a diagnostic antigen to detect neutralising CHIKV antibodies in human or animal sera. YYV-CHIKVMauritius further demonstrated protection against CHIKV infection and disease in a wild-type mouse model. Two doses of YYV-CHIKVMauritius showed anti-CHIKV ELISA and neutralising antibody responses, with protection against foot swelling, viraemia and viral feet tissue titres. Protection against CHIKV histopathology including myositis, tendonitis, arthritis, subcutaneous oedema and haemorrhage was also observed. YYV-RRVTT also demonstrated protection against RRV infection and disease in a wild-type mouse model, with two vaccine doses inducing anti-RRV ELISA and neutralising antibody responses. Protection against foot swelling, viraemia and viral feet tissue titres and RRV histopathology including myositis, tendonitis, arthritis and subcutaneous oedema was also observed. Cross-protection was also evaluated between YYV-CHIKVMauritius and RRV. Although cross-reactive total IgG were observed for YYV-CHIKVMauritius vaccinated mice, this offered no cross-neutralising antibodies and no protection against RRV infection and disease. Overall, our findings show that YYV-CHIKVMauritius and YYV-RRVTT are safe and efficacious vaccines against CHIKV and RRV, respectively, but do not offer cross-protection. ### Competing Interest Statement The authors have declared no competing interest. Advance Queensland Industry Research Fellowship, AQIRF067-2020-CV University of Queensland Research Training Program Stipend QIMR Berghofer

Currently, no approved antiviral drugs target dengue virus (DENV) infection, leaving treatment reliant on supportive care. DENV vaccine efficacy varies depending on the vaccine type, the circulating serotype, and vaccine coverage. We investigated defective interfering particles (DIPs) and lipid nanoparticles (LNPs) to deliver DI290, an anti-DENV DI RNA. Both DIPs and DI290-loaded LNPs (LNP-290) effectively suppressed DENV infection in human primary monocyte-derived macrophages (MDMs), THP-1 macrophages, and fibroblasts-natural DENV targets. Inhibiting interferon (IFN) signaling with a Janus kinase 1/2 inhibitor or an IFN-c/13 receptor 1 (IFNAR1)binding antibody blocked DIP and LNP-290 antiviral activity. LNP-290 demonstrated a greater than log10 inhibition of DENV viral loads in IFNAR-deficient (Ifnar-/-) and IFN regulatory factor (IRF) 3 and 7 double knockout (Irf3/7-/-) mice. Pathway analysis of RNA sequencing data from LNP-treated C57BL/6J mice, Ifnar-/- mice, and human MDMs treated with LNPs or DENV DIPs indicated DI290 treatment enhanced IFN responses, suggesting IFN-A and IFN-y provided antiviral activity when IFN-c/13 responses were diminished. While viral interference by DI290 is possible, results did not support RNA replication competition as an inhibition mechanism. These findings suggest that DI290 may be a promising DENV therapeutic by activating the innate immune system.

Human infections with the Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEVNSW/22) obtained from a stillborn piglet brain. Herein we conduct a thorough characterization of JEVNSW/22 in three different mouse strains and in human cortical brain organoids (hBOs), and determined the ability of JEVNSW/22 to be neutralized by sera from humans vaccinated with IMOJEV. JEVNSW/22 was less virulent than JEVFU (genotype 2) and JEVNakayama (genotype 3) in C57BL/6J mice and in interferon regulatory factor 7 deficient (Irf7-/-) mice, with infection of wild-type and knockout murine embryonic fibroblasts indicating JEVNSW/22 is more sensitive to type I interferon responses. Irf7-/- mice provide a new model for JEVNSW/22, showing higher viremia levels compared to C57BL/6J mice, and allowing for lethal neuroinvasive infection. All JEV strains were universally lethal in Ifnar-/- mice by day 3, with histological signs of brain hemorrhage, but no other lesions. There were no indications of brain infection in Ifnar-/- mice, with viral protein detected in blood vessels, but not neurons. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEVNSW/22. IMOJEV vaccination in humans induced antibodies capable of neutralizing JEVNSW/22, although, for all JEV strains, cross-neutralization titers declined with increasing divergence from IMOJEV in the envelope amino acid sequences. Overall, our study establishes JEVNSW/22 mouse and hBO models of infection, allowing for possible lethal neuroinvasive infection in mice that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases. In this Review, the authors discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 mu g vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.

Presently, no approved antiviral drug targets dengue virus (DENV) infection. Treatment mainly relies on supportive measures, while DENV vaccines’ efficacy varies based on factors like vaccine type, circulating DENV serotypes and vaccinated population. This study explores using defective interfering particles (DIPs) and lipid nanoparticles (LNPs) to deliver an anti-DENV defective interfering RNA, known as DI290. Results showed that both DIPs and DI290 loaded LNPs (LNP-290) effectively suppressed DENV infection in human primary monocyte-derived macrophages (MDMs), THP-1 macrophages and human fibroblasts, representing cell types naturally targeted by DENV. Furthermore, LNP-290 demonstrated >log10 inhibition of DENV viral loads in IFNAR-deficient mice, which lack functional type I interferon (IFN) receptors. DI290-mediated inhibition was also effective in IFN regulatory factor 3 and 7 double knockout mice. RNA-Seq data from LNP-treated C57BL/6J mice, IFNAR-deficient mice and human MDMs treated with LNPs or DENV DIPs illustrated DI290 treatment heightened IFN responses, particularly IFNγ, as well as IFNα/β and IFNλ. DI290 thus induces a broad range of IFN responses, with IFNγ and IFNλ providing anti-viral activity when IFNα/β responses are absent. Mice administered LNP-290 also did not manifest acute overt clinical signs. In summary, these experiments suggest DI290’s potential as a therapeutic approach for combating DENV infection.### Competing Interest StatementThe authors have declared no competing interest.

Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARSCoV-2 mouse adapted 1 (SARS-CoV-2MA1), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2MA1 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2MA1 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar-'- mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2MA1 does not replicate in the Ace2-'- mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2MA1 is likely an in vitro phenomenon with no apparent implications for infection in vivo.

IntroductionThe severity of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often dictated by a range of comorbidities. A considerable literature suggests iron deficiency and iron overload may contribute to increased infection, inflammation and disease severity, although direct causal relationships have been difficult to establish.MethodsHere we generate iron deficient and iron loaded C57BL/6 J mice by feeding standard low and high iron diets, with mice on a normal iron diet representing controls. All mice were infected with a primary SARS-CoV-2 omicron XBB isolate and lung inflammatory responses were analyzed by histology, immunohistochemistry and RNA-Seq.ResultsCompared with controls, iron deficient mice showed no significant changes in lung viral loads or histopathology, whereas, iron loaded mice showed slightly, but significantly, reduced lung viral loads and histopathology. Transcriptional changes were modest, but illustrated widespread dysregulation of inflammation signatures for both iron deficient vs. controls, and iron loaded vs. controls. Some of these changes could be associated with detrimental outcomes, whereas others would be viewed as beneficial.DiscussionDiet-associated iron deficiency or overload thus induced modest modulations of inflammatory signatures, but no significant histopathologically detectable disease exacerbations.

IntroductionGlobal microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis, with a growing body of concern surrounding the potential impacts on human health.MethodsUsing a mouse model of mild COVID-19, we describe herein the effects of azide-free 1 μm polystyrene MP beads, co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum. The effect of MPs on the host response to SARS-CoV-2 infection was analysed using histopathology and RNA-Seq at 2 and 6 days post-infection (dpi).ResultsAlthough infection reduced clearance of MPs from the lung, virus titres and viral RNA levels were not significantly affected by MPs, and overt MP-associated clinical or histopathological changes were not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 dpi and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the ‘cytokine release syndrome’ signature observed in some COVID-19 patients.DiscussionThe findings are consistent with the recent finding that MPs can inhibit phagocytosis of apoptotic cells via binding of Tim4. They also add to a growing body of literature suggesting that MPs can dysregulate inflammatory processes in specific disease settings.