Diverse RNA viruses of parasitic nematodes can elicit antibody responses in vertebrate hosts.

Parasitic nematodes have an intimate, chronic and lifelong exposure to vertebrate tissues. Here we mined 41 published parasitic nematode transcriptomes from vertebrate hosts and identified 91 RNA viruses across 13 virus orders from 24 families in ~70% (28 out of 41) of parasitic nematode species, which include only 5 previously reported viruses. We observe widespread distribution of virus-nematode associations across multiple continents, suggesting an ancestral acquisition event and host-virus co-evolution. Characterization of viruses of Brugia malayi (BMRV1) and Onchocerca volvulus (OVRV1) shows that these viruses are abundant in reproductive tissues of adult parasites. Importantly, the presence of BMRV1 RNA in B. malayi parasites mounts an RNA interference response against BMRV1 suggesting active viral replication. Finally, BMRV1 and OVRV1 were found to elicit antibody responses in serum samples from infected jirds and infected or exposed humans, indicating direct exposure to the immune system.

Detection of Dengue Virus 1 and Mammalian Orthoreovirus 3, with Novel Reassortments, in a South African Family Returning from Thailand, 2017.

In July 2017, a family of three members, a 46-year-old male, a 45-year-old female and their 8-year-old daughter, returned to South Africa from Thailand. They presented symptoms consistent with mosquito-borne diseases, including fever, headache, severe body aches and nausea. Mosquito bites in all family members suggested recent exposure to arthropod-borne viruses. Dengue virus 1 (Genus Orthoflavivirus) was isolated (isolate no. SA397) from the serum of the 45-year-old female via intracerebral injection in neonatal mice and subsequent passage in VeroE6 cells. Phylogenetic analysis of this strain indicated close genetic identity with cosmopolitan genotype 1 DENV1 strains from Southeast Asia, assigned to major lineage K, minor lineage 1 (DENV1I_K.1), such as GZ8H (99.92%) collected in November 2018 from China, and DV1I-TM19-74 isolate (99.72%) identified in Bangkok, Thailand, in 2019. Serum samples from the 46-year-old male yielded a virus isolate that could not be confirmed as DENV1, prompting unbiased metagenomic sequencing for virus identification and characterization. Illumina sequencing identified multiple segments of a mammalian orthoreovirus (MRV), designated as Human/SA395/SA/2017. Genomic and phylogenetic analyses classified Human/SA395/SA/2017 as MRV-3 and assigned a tentative genotype, MRV-3d, based on the S1 segment. Genomic analyses suggested that Human/SA395/SA/2017 may have originated from reassortments of segments among swine, bat, and human MRVs. The closest identity of the viral attachment protein σ1 (S1) was related to a human isolate identified from Tahiti, French Polynesia, in 1960. This indicates ongoing circulation and co-circulation of Southeast Asian and Polynesian strains, but detailed knowledge is hampered by the limited availability of genomic surveillance. This case represents the rare concurrent detection of two distinct viruses with different transmission routes in the same family with similar clinical presentations. It highlights the complexity of diagnosing diseases with similar sequelae in travelers returning from tropical areas.

Multiple novel caliciviruses identified from stoats (Mustela erminea) in the United Kingdom.

The Caliciviridae family, comprising positive-sense RNA viruses, is characterised by its non-enveloped, small virions, broad host range, and notable tendency for host switching. These viruses are primarily associated with gastroenteric disease, though they can lead to haemorrhagic or respiratory infections. Our study employed a metagenomics analysis of faecal samples from stoats (Mustela erminea), identifying two novel calicivirus species, named stoat vesivirus and stoat valovirus. Stoat vesivirus was identified in three samples (ST008, ST006, ST004), exhibiting a genome wide nucleotide identity of approximately 92 %. The complete coding sequences of these samples were 8471 (ST004) and 8322 (ST006) nucleotides in length, respectively. Each comprised three open reading frames (ORF), closely resembling the Vesivirus mink calicivirus (China/2/2016), with 70-72 % similarity in ORF1, 61-62 % in ORF2 and 71 % in ORF3. Phylogenetic analysis robustly supported stoat vesivirus as belonging within the Vesivirus genus. The second calivicirus (stoat valovirus), detected solely in sample ST008, was 6527 nucleotides in length and with complete coding sequences present. It shared highest similarity with St-Valérien swine virus and marmot norovirus HT16, showing 39.5 and 38.8 % protein identity with ORF1 and 43.3 and 42.9 % for VP1. Stoat valovirus is borderline for meeting the ICTV criteria for a new genus, demonstrating 60 % divergence in ORF1 compared to the other valovirus', however it clusters basally within the Valovirus genus, supporting leaving it included in this genus.

A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

The RNA interference response to alphanodavirus replication in Phlebotomus papatasi sand fly cells.

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi-derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo-siRNA) pathway, with minimal engagement from the PIWI-interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus-derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo-siRNA pathway.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

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.

Evidence for an ancient aquatic origin of the RNA viral order Articulavirales.

The emergence of previously unknown disease-causing viruses in mammals is in part the result of a long-term evolutionary process. Reconstructing the deep phylogenetic histories of viruses helps identify major evolutionary transitions and contextualizes the emergence of viruses in new hosts. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the RNA virus order Articulavirales, which includes the influenza viruses. We identified instances of Articulavirales in the invertebrate phylum Cnidaria (including corals), constituting a novel and divergent family that we provisionally named the "Cnidenomoviridae." We further extended the evolutionary history of the influenza virus lineage by identifying four divergent, fish-associated influenza-like viruses, thereby supporting the hypothesis that fish were among the first hosts of influenza viruses. In addition, we substantially expanded the phylogenetic diversity of quaranjaviruses and proposed that this genus be reclassified as a family-the "Quaranjaviridae." Within this putative family, we identified a novel arachnid-infecting genus, provisionally named "Cheliceravirus." Notably, we observed a close phylogenetic relationship between the Crustacea- and Chelicerata-infecting "Quaranjaviridae" that is inconsistent with virus-host codivergence. Together, these data suggest that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of the Articulavirales was likely shaped by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

Egg protein profile and dynamics during embryogenesis in Haemaphysalis flava ticks.

Tick eggs contain all essential proteins for embryogenesis, and egg proteins are a potential reservoir of tick-protective antigens. However, the protein profile and dynamics during embryonic development remain unknown. This study aimed to depict the protein profile and dynamics in tick embryogenesis, further providing protein candidates for targeted interventions. Eggs from Haemaphysalis flava ticks were incubated at 28 °C and 85% relative humidity. On days 0 (newly laid eggs without incubation), 7, 14 and 21, eggs were collected, dewaxed and subject to protein extraction. Extracted proteins were digested by filter-aided sample preparation and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). MS data were searched against an in-house H. flava protein database for tick-derived protein identification. Abundances of 40 selected high-confidence proteins were further quantified by LC-parallel reaction monitoring (PRM)/MS analysis throughout egg incubation. A total of 93 high-confidence proteins were identified in eggs on 0-day incubation. Identified proteins belonged to seven functional categories: transporters, enzymes, proteinase inhibitors, immunity-related proteins, cytoskeletal proteins, heat shock proteins and uncharacterized proteins. The enzyme category contained the most types of proteins. Neutrophil elastase inhibitors represented the most abundant proteins in terms of intensity-based absolute-protein-quantification. LC-PRM/MS revealed that the abundances of 20 proteins increased including enolase, calreticulin, actin, GAPDH et cetera, and the abundances of 11 proteins decreased including vitellogenins, neutrophil elastase inhibitor, carboxypeptidase Q, et cetera from 0- to 21-day incubation. This study provides the most comprehensive egg protein profile and dynamics during tick embryogenesis. Further investigations are needed to test the tick-control efficacy by targeting the egg proteins.

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.

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.

Comparative analysis of genome-encoded viral sequences reveals the evolutionary history of flavivirids (family Flaviviridae).

Flavivirids (family Flaviviridae) are a group of positive-strand ribonucleic acid (RNA) viruses that pose serious risks to human and animal health on a global scale. Here, we use flavivirid-derived deoxyribonucleic acid (DNA) sequences, identified in animal genomes, to reconstruct the long-term evolutionary history of family Flaviviridae. We demonstrate that flavivirids are >100 million years old and show that this timing can be combined with dates inferred from co-phyletic analysis to produce a cohesive overview of their evolution, distribution, and diversity wherein the main flavivirid subgroups originate in early animals and broadly co-diverge with major animal phyla. In addition, we reveal evidence that the 'classical flaviviruses' of vertebrates, most of which are transmitted via blood-feeding arthropod vectors, originally evolved in haematophagous arachnids and later acquired the capacity to be transmitted by insects. Our findings imply that the biological properties of flavivirids have been acquired gradually over the course of animal evolution. Thus, broad-scale comparative analysis will likely reveal fundamental insights into their biology. We therefore published our results via an open, extensible, database (Flavivirid-GLUE), which we constructed to facilitate the wider utilisation of genomic data and evolution-related domain knowledge in flavivirid research.

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia.

In Australia, there is a paucity of data about the extent and impact of zoonotic tick-related illnesses. Even less is understood about a multifaceted illness referred to as Debilitating Symptom Complexes Attributed to Ticks (DSCATT). Here, we describe a research plan for investigating the aetiology, pathophysiology, and clinical outcomes of human tick-associated disease in Australia. Our approach focuses on the transmission of potential pathogens and the immunological responses of the patient after a tick bite. The protocol is strengthened by prospective data collection, the recruitment of two external matched control groups, and sophisticated integrative data analysis which, collectively, will allow the robust demonstration of associations between a tick bite and the development of clinical and pathological abnormalities. Various laboratory analyses are performed including metagenomics to investigate the potential transmission of bacteria, protozoa and/or viruses during tick bite. In addition, multi-omics technology is applied to investigate links between host immune responses and potential infectious and non-infectious disease causations. Psychometric profiling is also used to investigate whether psychological attributes influence symptom development. This research will fill important knowledge gaps about tick-borne diseases. Ultimately, we hope the results will promote improved diagnostic outcomes, and inform the safe management and treatment of patients bitten by ticks in Australia.

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.