Residue K28 of Zika Virus NS5 Protein Is Implicated in Virus Replication and Antagonism of STAT2.

The identification of four potential nonstructural 5 (NS5) residues-K28, K45, V335, and S749-that share the same amino acid preference in STAT2-interacting flaviviruses [Dengue virus (DENV) and Zika virus (ZIKV)], but not in STAT2-non-interacting flaviviruses [West Nile virus (WNV) and/or Yellow fever virus (YFV)] from an alignment of multiple flavivirus NS5 sequences, implied a possible association with the efficiency of ZIKV to antagonize the human signal transducer and activator of transcription factor 2 (STAT2). Through site-directed mutagenesis and reverse genetics, mutational impacts of these residues on ZIKV growth in vitro and STAT2 antagonism were assessed using virus growth kinetics assays and STAT2 immunoblotting. The results showed that mutations at the residue K28 significantly reduced the efficiency of ZIKV to antagonize STAT2. Further investigation involving residue K28 demonstrated its additional effects on the phenotypes of ZIKV-NS5 nuclear bodies. These findings demonstrate that K28, identified from sequence alignment, is an important determinant of replication and STAT2 antagonism by ZIKV.

Enhancement of cellular immunity following needle-free vaccination of mice with SARS-CoV-2 spike protein.

The COVID-19 pandemic has highlighted the need for vaccines capable of providing rapid and robust protection. One way to improve vaccine efficacy is delivery via microarray patches, such as the Vaxxas high-density microarray patch (HD-MAP). We have previously demonstrated that delivery of a SARS-CoV-2 protein vaccine candidate, HexaPro, via the HD-MAP induces potent humoral immune responses. Here, we investigate the cellular responses induced by HexaPro HD-MAP vaccination. We found that delivery via the HD-MAP induces a type one biassed cellular response of much greater magnitude as compared to standard intramuscular immunization.

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant.

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N-glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.

SARS-CoV-2 omicron BA.5 and XBB variants have increased neurotropic potential over BA.1 in K18-hACE2 mice and human brain organoids.

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology.

Aging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Noncoding RNA of Zika Virus Affects Interplay between Wnt-Signaling and Pro-Apoptotic Pathways in the Developing Brain Tissue.

Zika virus (ZIKV) has a unique ability among flaviviruses to cross the placental barrier and infect the fetal brain causing severe abnormalities of neurodevelopment known collectively as congenital Zika syndrome. In our recent study, we demonstrated that the viral noncoding RNA (subgenomic flaviviral RNA, sfRNA) of the Zika virus induces apoptosis of neural progenitors and is required for ZIKV pathogenesis in the developing brain. Herein, we expanded on our initial findings and identified biological processes and signaling pathways affected by the production of ZIKV sfRNA in the developing brain tissue. We employed 3D brain organoids generated from induced human pluripotent stem cells (ihPSC) as an ex vivo model of viral infection in the developing brain and utilized wild type (WT) ZIKV (producing sfRNA) and mutant ZIKV (deficient in the production of sfRNA). Global transcriptome profiling by RNA-Seq revealed that the production of sfRNA affects the expression of >1000 genes. We uncovered that in addition to the activation of pro-apoptotic pathways, organoids infected with sfRNA-producing WT, but not sfRNA-deficient mutant ZIKV, which exhibited a strong down-regulation of genes involved in signaling pathways that control neuron differentiation and brain development, indicating the requirement of sfRNA for the suppression of neurodevelopment associated with the ZIKV infection. Using gene set enrichment analysis and gene network reconstruction, we demonstrated that the effect of sfRNA on pathways that control brain development occurs via crosstalk between Wnt-signaling and proapoptotic pathways.

Inactivation of SARS-CoV-2 infectivity in platelet concentrates or plasma following treatment with ultraviolet C light or with methylene blue combined with visible light.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be a major transfusion-transmitted pathogen; however, convalescent plasma is a treatment option used in some regions. The risk of transfusion-transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB-plasma and THERAFLEX UV-Platelets systems. Here we examined the capability of these PI systems to inactivate SARS-CoV-2. STUDY DESIGN AND METHODS: SARS-CoV-2 spiked plasma units were treated using the THERAFLEX MB-Plasma system in the presence of methylene blue (~0.8 µmol/L; visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ). SARS-CoV-2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay. RESULTS: Treatment of spiked plasma with the THERAFLEX MB-Plasma system resulted in an average ≥5.03 log10 reduction in SARS-CoV-2 infectivity at one third (40 J/cm2 ) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV-Platelets system resulted in an average ≥5.18 log10 reduction in SARS-CoV-2 infectivity at the standard UVC dose (0.2 J/cm2 ). CONCLUSIONS: SARS-CoV-2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion-transmitted emerging pathogens.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.

SARS-CoV-2 triggers complement activation through interactions with heparan sulfate.

Objectives: To determine whether SARS-CoV-2 can trigger complement activation, the pathways that are involved and the functional significance of the resultant effect. Methods: SARS-CoV-2 was inoculated into a human lepirudin-anticoagulated whole blood model, which contains a full repertoire of complement factors and leukocytes that express complement receptors. Complement activation was determined by measuring C5a production with an ELISA, and pretreatment with specific inhibitors was used to identify the pathways involved. The functional significance of this was then assessed by measuring markers of C5a signalling including leukocyte C5aR1 internalisation and CD11b upregulation with flow cytometry. Results: SARS-CoV-2 inoculation in this whole blood model caused progressive C5a production over 24 h, which was significantly reduced by inhibitors for factor B, C3, C5 and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface interactions with heparan sulfate. Functional analysis of this phenomenon revealed that C5aR1 signalling and CD11b upregulation in granulocytes and monocytes was delayed and only occurred after 24 h. Conclusion: SARS-CoV-2 is a noncanonical alternative pathway activator that progressively triggers complement activation through interactions with heparan sulfate.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Reporter Flaviviruses as Tools to Demonstrate Homologous and Heterologous Superinfection Exclusion.

Binjari virus (BinJV) is a lineage II or dual-host affiliated insect-specific flavivirus previously demonstrated as replication-deficient in vertebrate cells. Previous studies have shown that BinJV is tolerant to exchanging its structural proteins (prM-E) with pathogenic flaviviruses, making it a safe backbone for flavivirus vaccines. Here, we report generation by circular polymerase extension reaction of BinJV expressing zsGreen or mCherry fluorescent protein. Recovered BinJV reporter viruses grew to high titres (107-8 FFU/mL) in Aedes albopictus C6/36 cells assayed using immunoplaque assays (iPA). We also demonstrate that BinJV reporters could be semi-quantified live in vitro using a fluorescence microplate reader with an observed linear correlation between quantified fluorescence of BinJV reporter virus-infected C6/36 cells and iPA-quantitated virus titres. The utility of the BinJV reporter viruses was then examined in homologous and heterologous superinfection exclusion assays. We demonstrate that primary infection of C6/36 cells with BinJVzsGreen completely inhibits a secondary infection with homologous BinJVmCherry or heterologous ZIKVmCherry using fluorescence microscopy and virus quantitation by iPA. Finally, BinJVzsGreen infections were examined in vivo by microinjection of Aedes aegypti with BinJVzsGreen. At seven days post-infection, a strong fluorescence in the vicinity of salivary glands was detected in frozen sections. This is the first report on the construction of reporter viruses for lineage II insect-specific flaviviruses and establishes a tractable system for exploring flavivirus superinfection exclusion in vitro and in vivo.

Skin-patch delivered subunit vaccine induces broadly neutralising antibodies against SARS-CoV-2 variants of concern.

The ongoing SARS-CoV-2 pandemic continues to pose an enormous health challenge globally. The ongoing emergence of variants of concern has resulted in decreased vaccine efficacy necessitating booster immunizations. This was particularly highlighted by the recent emergence of the Omicron variant, which contains over 30 mutations in the spike protein and quickly became the dominant viral strain in global circulation. We previously demonstrated that delivery of a SARS-CoV-2 subunit vaccine via a high-density microarray patch (HD-MAP) induced potent immunity resulting in robust protection from SARS-CoV-2 challenge in mice. Here we show that serum from HD-MAP immunized animals maintained potent neutralisation against all variants tested, including Delta and Omicron. These findings highlight the advantages of HD-MAP vaccine delivery in inducing high levels of neutralising antibodies and demonstrates its potential at providing protection from emerging viral variants.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

The distinguishing NS5-M114V mutation in American Zika virus isolates has negligible impacts on virus replication and transmission potential.

During 2015-2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/ß receptor knockout (Ifnar-/-) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo, albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates but had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential.

Dermal Delivery of a SARS-CoV-2 Subunit Vaccine Induces Immunogenicity against Variants of Concern.

The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.

Peptide-Based Vaccine against SARS-CoV-2: Peptide Antigen Discovery and Screening of Adjuvant Systems.

The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1-B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution.

Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3'UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3'UTRs of all classical ISFs, except Anopheles spp-asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3'UTRs of dual host-associated and Anopheles-associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp-associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding.