Antiviral Wolbachia strains associate with Aedes aegypti endoplasmic reticulum membranes and induce lipid droplet formation to restrict dengue virus replication.

Wolbachia are a genus of insect endosymbiotic bacteria which includes strains wMel and wAlbB that are being utilized as a biocontrol tool to reduce the incidence of Aedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinning the antiviral activity of these Wolbachia strains are not well defined. Here, we generated a panel of Ae. aegypti-derived cell lines infected with antiviral strains wMel and wAlbB or the non-antiviral Wolbachia strain wPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while wPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in wMel- and wAlbB-infected cell lines and mosquito tissues compared to cells infected with wPip or Wolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by wMel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of dengue virus (DENV) replication complexes. Defining Wolbachia's antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale.IMPORTANCEAedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia, reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid droplets. Inhibiting Wolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachia's antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.

Polyphenol rich sugarcane extract (PRSE) has potential antiviral activity against influenza A virus in vitro.

Influenza A virus (IAV) is one of the major global public health concerns but the emerging resistance of IAV to currently available antivirals requires the need to identify potential alternatives. Polyphenol rich sugarcane extract (PRSE) is an extract prepared from the sugarcane plant Saccharum Officinarum. Herein we aimed to determine if PRSE had antiviral activity against IAV. We showed that treatment of IAV-infected cells with PRSE results in a dose-dependent inhibition of virus infection at concentrations that were non-cytotoxic. PRSE treatment limited the early stages of infection, reducing viral genome replication, mRNA transcription and viral protein expression. PRSE did not affect the ability of IAV to bind sialic acid or change the morphology of viral particles. Additionally, PRSE treatment attenuated the replication of multiple IAV strains of the H3N2 and H1N1 subtype. In conclusion, we show that PRSE displays antiviral activity against a broad range of IAV strains, in vitro.

Murine norovirus infection of macrophages induces intrinsic apoptosis as the major form of programmed cell death.

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.

Murine Norovirus: Additional Protocols for Basic and Antiviral Studies.

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents.

Adenovirus vector produced Zika virus-like particles induce a long-lived neutralising antibody response in mice.

Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates.

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Immature Brain Cortical Neurons Have Low Transcriptional Competence to Activate Antiviral Defences and Control RNA Virus Infections.

Virus infections of the central nervous system (CNS) cause important diseases of humans and animals. As in other tissues, innate antiviral responses mediated by type I interferons (IFNs) are crucially important in controlling CNS virus infections. The maturity of neuronal populations is an established critical factor determining the outcome of CNS virus infection. Using primary cultures of mouse cortical neurons, we investigated the relationships between neuronal maturation, type I IFN responses, and the outcome of Semliki Forest virus infection. The virus replicated better, infected more cells, and produced higher titres of infectious viruses in immature neurons. Complete transcriptome analysis demonstrated that resting immature neurons have low transcriptional competence to mount antiviral responses. They had no detectable transcription of the genes Ddx58 and Ifih1, which encode key RNA virus cytoplasmic sensors RIG-I and MDA5, and very low expression of genes encoding key regulators of associated signalling pathways. Upon infection, immature neurons failed to mount an antiviral response as evidenced by their failure to produce chemokines, IFNs, and other cytokines. Treatment of immature neurons with exogenous IFNß prior to infection resulted in antiviral responses and lower levels of virus replication and infectious virus production. In contrast, resting mature neurons generated a robust antiviral response. This was augmented by pretreatment with IFNß. Infection of mature neurons derived from IFNAR-/- mice did not make an antiviral response and replicated virus to high levels.

Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways.

BACKGROUND: The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015-2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses. METHODS: In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. RESULTS: We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. CONCLUSIONS: These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.

Correction: SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

[This corrects the article DOI: 10.1371/journal.ppat.1009800.].

Dual-Gendered Leadership: Gender-Inclusive Scientific-Political Public Health Communication Supporting Government COVID-19 Responses in Atlantic Canada.

This research aims to identify the influence of woman leadership on improving the traditional man-dominated scientific-political communication towards positive COVID-19-driven public health interventions. Across Canada, dual-gendered leadership (women chief medical officers and men prime minister/premiers) at both federal and provincial levels illustrated a positive approach to "flatten the curve" during the first and second waves of COVID-19. With the four provinces of New Brunswick, Newfoundland and Labrador, Nova Scotia, and Prince Edward Island, Atlantic Canada formed the "Atlantic Bubble", which has become a great example domestically and internationally of successfully mitigating the pandemic while maintaining societal operation. Three provinces have benefitted from this complementary dual-gendered leadership. This case study utilized a scoping media coverage review approach, quantitatively examining how gender-inclusive scientific-political cooperation supported effective provincial responses in Atlantic Canada during the first two waves of COVID-19. This case study discovers that (1) at the provincial government level, woman leadership of mitigation, advocating, and coordination encouraged provincial authorities to adapt science-based interventions and deliver consistent and supportive public health information to the general public; and (2) at the community level, this dual-gendered leadership advanced community cohesion toward managing the community-based spread of COVID-19. Future studies may apply a longitudinal, retrospective approach with Canada-wide or cross-national comparison to further evaluate the strengths and weaknesses of dual-gendered leadership.

SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

Liquid Chalk Is an Antiseptic against SARS-CoV-2 and Influenza A Respiratory Viruses.

The COVID-19 pandemic has impacted and enforced significant restrictions within our societies, including the attendance of public and professional athletes in gyms. Liquid chalk is a commonly used accessory in gyms and is comprised of magnesium carbonate and alcohol that quickly evaporates on the hands to leave a layer of dry chalk. We investigated whether liquid chalk is an antiseptic against highly pathogenic human viruses, including SARS-CoV-2, influenza virus, and noroviruses. Chalk was applied before or after virus, inoculum and recovery of infectious virus was determined to mimic the use in the gym. We observed that addition of chalk before or after virus contact led to a significant reduction in recovery of infectious SARS-CoV-2 and influenza virus but had little impact on norovirus. These observations suggest that the use and application of liquid chalk can be an effective and suitable antiseptic for major sporting events, such as the Olympic Games. IMPORTANCE To restrict the potential transmission and infectivity of SARS-CoV-2, the use of liquid chalk has been a requirement in an active gym setting. However, its effectiveness has not been scientifically proven. Here, we show that the application of liquid chalk before or after virus inoculum significantly impacts recovery of infectious SARS-CoV-2 and influenza viruses but not noroviruses. Thus, our study has shown that the implementation and application of liquid chalk in communal social gym settings is effective in reducing the infectivity of respiratory viruses, and this supports the use of liquid chalk in major sporting events to restrict the impact of COVID-19 on our communities.

A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous "organelles" that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Nuclear localisation of West Nile virus NS5 protein modulates host gene expression.

The involvement of the nucleus during flavivirus infection has been observed in only a small number of cases and can be limited to primarily two viral proteins; the structural protein C and the RNA polymerase NS5. Previously we observed that by blocking nuclear transport, WNV strain Kunjin (WNVKUN) replication is severely affected and through mutation of the identified NLS in WNVKUN NS5 protein. In this study, we interrogated the potential nuclear functions of WNVKUN NS5 has on the host transcriptome, by means of RNA sequencing (RNAseq). In a direct comparison between wild type and mutant NS5, it can also be determined that the nuclear translocation of NS5 results in a significant down-regulation of host genes involved in the innate immune response. When compared to published RNAseq data from WNV infection, many of these genes were overlapping indicting the role of NS5 induced transcription during infection.

Lipid droplets and lipid mediators in viral infection and immunity.

Lipid droplets (LDs) contribute to key pathways important for the physiology and pathophysiology of cells. In a homeostatic view, LDs regulate the storage of neutral lipids, protein sequestration, removal of toxic lipids and cellular communication; however, recent advancements in the field show these organelles as essential for various cellular stress response mechanisms, including inflammation and immunity, with LDs acting as hubs that integrate metabolic and inflammatory processes. The accumulation of LDs has become a hallmark of infection, and is often thought to be virally driven; however, recent evidence is pointing to a role for the upregulation of LDs in the production of a successful immune response to viral infection. The fatty acids housed in LDs are also gaining interest due to the role that these lipid species play during viral infection, and their link to the synthesis of bioactive lipid mediators that have been found to have a very complex role in viral infection. This review explores the role of LDs and their subsequent lipid mediators during viral infections and poses a paradigm shift in thinking in the field, whereby LDs may play pivotal roles in protecting the host against viral infection.