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.

Medically important arboviruses such as dengue, Zika, and chikungunya viruses are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that transmission can be influenced by mosquito viromes. Herein RNA-Seq was used to characterize RNA metaviromes of wild-caught Ae. aegypti from Bangkok (Thailand) and from Cairns (Australia). The two mosquito populations showed a high degree of similarity in their viromes. BLAST searches of assembled contigs suggest up to 27 insect-specific viruses may infect Ae. aegypti, with up to 23 of these currently uncharacterized and up to 16 infecting mosquitoes from both Cairns and Bangkok. Three characterized viruses dominated, Phasi Charoen-like virus, Humaita-Tubiacanga virus and Cell fusing agent virus, and comparisons with other available RNA-Seq datasets suggested infection levels with these viruses may vary in laboratory-reared mosquitoes. As expected, mosquitoes from Bangkok showed higher mitochondrial diversity and carried alleles associated with knock-down resistance to pyrethroids. Blood meal reads primarily mapped to human genes, with a small number also showing homology with rat/mouse and dog genes. These results highlight the wide spectrum of data that can be obtained from such RNA-Seq analyses, and suggests differing viromes may need to be considered in arbovirus vector competence studies.

Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses such as Zika virus and dengue virus has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identify variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono-and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.IMPORTANCE Some flaviviruses are known to cause disease in vertebrates and are typically transmitted by blood-feeding arthropods such as ticks and mosquitoes. While an ever-increasing number of insect-specific flaviviruses have been described, we have a narrow understanding of flavivirus incidence and evolution. To expand this understanding, we discovered a number of novel flaviviruses that infect a range of crustaceans and cephalopod hosts. Phylogenetic analyses of these novel marine flaviviruses suggest that crustacean flaviviruses share a close ancestor to all terrestrial vector-borne flaviviruses, and squid flaviviruses are the most divergent of all known flaviviruses to date. Additionally, our results indicate horizontal transmission of a marine flavivirus between crabs and sharks. Taken together, these data suggest that flaviviruses move horizontally between invertebrates and vertebrates in ocean ecosystems. This study demonstrates that flavivirus invertebrate-vertebrate host associations have arisen in flaviviruses at least twice and may potentially provide insights into the emergence or origin of terrestrial vector-borne flaviviruses. FIG 2 Translation of a complete polyprotein depends on highly st...

Insect specific viruses (ISVs) of the yellow fever mosquito have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of, however, remains poorly understood. In this study, we characterised Aedes anphevirus (AeAV), a negative-sense RNA virus from the order AeAV identified from cell lines were infectious to both and cells, but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available RNA-Seq data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American and Asia-Pacific lineages. The endosymbiotic bacterium restricts positive-sense RNA viruses in Re-analysis of a small RNA library of cells co-infected with AeAV and produces an abundant RNAi response consistent with persistent virus replication. We found enhances replication of AeAV when compared to a tetracycline cleared cell line, and AeAV modestly reduces DENV replication The results from our study improve understanding of the diversity and evolution of the virome of and adds to previous evidence that shows does not restrict a range of negative strand RNA viruses. The mosquito transmits a number of arthropod-borne viruses (arboviruses) such as dengue virus and Zika virus. Mosquitoes also harbour insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a handful of insect-specific viruses described from in the literature. Here, we characterise a novel negative strand virus, Aedes anphevirus (AeAV). Meta-analysis of samples showed that it is present in mosquitoes worldwide and is vertically transmitted. transinfected mosquitoes are currently being used in biocontrol as they effectively block transmission of several positive sense RNA viruses in mosquitoes. Our results demonstrate that enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in as well as providing insight into the complexity of the virus restriction phenotype.

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Influenza viruses (family Orthomyxoviridae) infect a variety of vertebrates, including birds, humans, and other mammals. Recent metatranscriptomic studies have uncovered divergent influenza viruses in amphibians, fish and jawless vertebrates, suggesting that these viruses may be widely distributed. We sought to identify additional vertebrate influenza-like viruses through the analysis of publicly available RNA sequencing data. Accordingly, by data mining, we identified the complete coding segments of five divergent vertebrate influenza-like viruses. Three fell as sister lineages to influenza B virus: salamander influenza-like virus in Mexican walking fish (Ambystoma mexicanum) and plateau tiger salamander (Ambystoma velasci), Siamese algae-eater influenza-like virus in Siamese algae-eater fish (Gyrinocheilus aymonieri) and chum salmon influenza-like virus in chum salmon (Oncorhynchus keta). Similarly, we identified two influenza-like viruses of amphibians that fell as sister lineages to influenza D virus: cane toad influenza-like virus and the ornate chorus frog influenza-like virus, in the cane toad (Rhinella marina) and ornate chorus frog (Microhyla fissipes), respectively. Despite their divergent phylogenetic positions, these viruses retained segment conservation and splicing consistent with transcriptional regulation in influenza B and influenza D viruses, and were detected in respiratory tissues. These data suggest that influenza viruses have been associated with vertebrates for their entire evolutionary history.

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.