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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 impactN-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 additionalN-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 binding. Glycan site-occupancy analyses revealed that the mutation resulted inN-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.

The emergence of novel disease-causing viruses in mammals is part of the long evolutionary history of viruses. Tracing these evolutionary histories contextualises virus spill over events and may help to elucidate how and why they occur. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the orderArticulavirales, which includes the influenza viruses. From this, we identified the first instance ofArticulaviralesin the Cnidaria (including corals), constituting a novel and divergent family that we tentatively named theCnidenomoviridae. This may be the basal group within theArticulavirales. We also extended the known evolutionary history of the influenza virus lineage by identifying a highly divergent, sturgeon-associated influenza virus. This suggests that fish were among the first hosts of influenza viruses. Finally, we substantially expanded the known diversity of quaranjaviruses and proposed that this genus be reclassified as a family (theQuaranjaviridae). We find evidence that vertebrate infectingQuaranjaviridaemay have initially evolved in crustaceans before spilling into terrestrial Chelicerata (i.e., ticks). Together, our findings indicate that theArticulaviraleshas evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of this order was not shaped by strict virus-host codivergence, but rather by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

The endosymbiotic bacterium Wolbachia pipientis confers RNA virus refractoriness in Drosophila and Aedes mosquitoes. Questions remain about the Wolbachia-virus restriction phenotype and how extensive this phenomenon may be within other arthropods. Here, we generated two Spodoptera frugiperda cell lines stably transinfected with two strains of Wolbachia, wAlbB and wMelPop-CLA. Despite the high density of Wolbachia in stably infected Sf9 cells, RT-PCR indicated the presence of the negative-sense RNA virus Spodoptera frugiperda rhabdovirus (SfRV) in Wolbachia-infected and uninfected cell lines. No differences in the replication of SfRV between Sf9 and Wolbachia-infected cells was found. RNA-Seq analysis of the parental Sf9 cells supported SfRV's presence in these cells with abundant 20 nt virus-derived small RNAs indicating active replication of SfRV in these cells. Overall, this study supports a growing body of evidence that Wolbachia does not restrict negative-sense RNA viruses and generates an in vitro model to examine Lepidoptera-Wolbachia virus interactions.

Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cellsin vivostrongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence; for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected individuals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2.In vivotreatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.

A frequently repeated premise is that viruses evolve to become less pathogenic. This appears also to be true for SARS-CoV-2, although the increased level of immunity in human populations makes it difficult to distinguish between reduced intrinsic pathogenicity and increasing protective immunity. The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described and appears to be due to reduced utilization of TMPRRS2. That this reduced pathogenicity remains true for omicron BA.5 was recently reported. In sharp contrast, we show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infection showing increased neurovirulence, encephalitis and mortality, similar to that seen for an original ancestral isolate. BA.5 also infected human cortical brain organoids to a greater extent than a BA.1 and original ancestral isolate. Neurons were the target of infection, with increasing evidence of neuron infection in COVID-19 patients. These results argue that while omicron virus may be associated with reduced respiratory symptoms, BA.5 shows increased neurovirulence compared to earlier omicron sub-variants.

The flaviviruses (family Flaviviridae) are a group of positive-strand RNA viruses, many of which pose serious risks to human health on a global scale. Here, we calibrate the timeline of flavivirus evolution using flavivirus-derived DNA sequences identified in animal genomes. We demonstrate that the family is at least 100 million years old and show that this timing can be integrated with dates inferred from co-phylogenetic analysis and paleontological records to produce a cohesive overview of flavivirus evolution in which the main subgroups originate early in animal evolution and broadly co-diverge with animal phyla. In addition, we show that the arthropod-borne ‘classical’ flaviviruses first evolved from tick-specific viruses, and only later adapted to become insect-borne. Our findings demonstrate that the biological properties of flaviviruses have been acquired over many millions of years of evolution, implying that broad-scale comparative analysis can reveal fundamental insights into flavivirus biology. We implement a novel approach to computational genomic studies of viruses that can support these efforts by enabling more efficient utilization of evolution-related domain knowledge in virus research. Significance Understanding how pathogenic viruses evolved can provide vital insights into their biology. In this study we use genomic data to show that flaviviruses – a group of viruses that includes important pathogens such as Dengue virus and hepatitis C virus - arose through an extended history of evolutionary interaction with host and vector species. Our findings show that comparative studies can productively utilise genomic data to reveal insights into flavivirus biology, which will help to facilitate the development of more effective antiviral treatments and strategies.

Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa., Perdomo et al. fed mosquitos (Aedes aegypti) a diet spiked with human blood miRNA, hsa-miR-21-5p and used proteomics analysis to show differential protein expression in female Ae. aegypti mosquitoes after feeding. They showed that hsa-miR-21-5p positively regulated vitellogenin, which plays a role in egg production, thus could have effects on Ae. aegypti progeny production.

The Aedes aegypti mosquito is the primary vector of several medically important arboviruses. The endosymbiotic bacterium, Wolbachia pipientis , has emerged as a means of blocking transmission of arboviruses such as dengue and Zika viruses. One Wolbachia strain that has shown potential in field trials is wAlbB, a naturally occurring Wolbachia strain of the Asian tiger mosquito Aedes albopictus. When transinfected into Ae. aegypti, wAlbB exhibits strong virus inhibition. In addition to modulating arboviruses, Wolbachia also modulates some insect-specific viruses. Here, we explored the effect of Wolbachia on the virome of the Ae. albopictus cell line Aa23 naturally infected with wAlbB and also a stably transinfected recipient Ae. aegypti cell line (Aag2.wAlbB). RNA sequencing and bioinformatic analysis on both cell lines revealed an 11 kb genome of a single-stranded positive-sense RNA negev-like virus related to the recently proposed negevirus taxon. We denoted this novel virus as Aedes albopictus negev-like virus (AalNLV). Tetracycline clearance of Wolbachia from Aa23 cells did not significantly affect AalNLV levels, while in Aag2.wAlbB cells, a significant increase in virus genome RNA copies was observed. We further investigated the inhibitory effect of wAlbB on AalNLV and another positive-sense RNA virus, cell fusing agent virus, which is present in Aag2 cells and known to be suppressed by Wolbachia. wAlbB suppressed both viruses, with the effect on AalNLV being more striking. The findings from this study further supplement our understanding of the complex interaction between Wolbachia , host and virome.

Flavivirids (family

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.

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 utilising a model of human monocyte-derived microglia. We identified that SARS-CoV-2 isolates can bind and enter microglia, triggering inflammasome activation in the absence of viral replication. Mechanistically, microglial NLRP3 could be both primed and activated with SARS-CoV-2 spike glycoprotein in a NF-κB and ACE2-dependent manner. Notably, virus- and spike protein-mediated inflammasome activation in microglia was significantly enhanced in the presence of α-synuclein fibrils, which was entirely ablated by NLRP3-inhibition. These results support a possible mechanism of microglia activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in certain COVID-19 infected individuals, and a potential therapeutic avenue for intervention.SIGNIFICANCE STATEMENTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) principally affects the lungs, however there is evidence that the virus can also reach the brain and lead to chronic neurological symptoms. In this study, we examined the interaction SARS-CoV-2 with brain immune cells, by using an ex-vivo model of human monocyte-derived microglia. We identified robust activation of the innate immune sensor complex, NLRP3 inflammasome, in cells exposed to SARS-CoV-2. This was dependent on spike protein-ACE2 receptor interaction and was potentiated in the presence of α-synuclein. We therefore identify a possible mechanism for SARS-CoV-2 and increased vulnerability to developing neurological dysfunction. These findings support a potential therapeutic avenue for treatment of SARS-CoV-2 driven neurological manifestations, through use of NLRP3 inflammasome or ACE2 inhibitors.

RationaleSevere 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.ObjectivesTo 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.MethodsPreclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.ResultsOxidised 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 thatCH25H,CYP7B1andGPR183are significantly upregulated in macrophages during COVID-19.ConclusionThis study demonstrates that oxysterols drive inflammation in the lungviaGPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

The yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus are the two most significant vectors of arthropod-borne viruses including dengue, yellow fever, Zika and Chikungunya viruses. Considerable progress has been made towards determining mosquito host factors controlling competence for transmission of arboviruses. Recent studies have demonstrated that insect-specific viruses (ISVs) can modulate refractoriness of Ae. aegypti and Ae. albopictus to arbovirus infection in vitro and in vivo. While research is ongoing to implicate ISVs as potential pro or antiviral host factors, we have limited understanding of the composition and diversity of viruses associated with these mosquitoes.One promising strategy for the control of arboviruses in these mosquitoes is the stable transinfection of the endosymbiotic bacterium Wolbachia pipientis into Ae. aegypti and Ae. albopictus mosquitoes. Wolbachia has been demonstrated to restrict a range of RNA viruses in Drosophila melanogaster and both arboviruses and ISVs in Ae. aegypti and Ae. albopictus, suggesting complex multi-factorial contributions of the virome and bacterial composition to mosquito vector competence.To unpack these complex interactions, we first set out to uncover previously unknown virome diversity in the mosquitoes using a metagenomics survey approach through the analysis of ~3000 published RNA-Seq libraries from wild-caught, laboratory colonies and cell line samples. We demonstrate that common laboratory colonies and cell lines harbour numerous novel mononegaviruses, orthomyxoviruses, and negeviruses. Additionally, we analysed ~100 individual mosquito transcriptomes and demonstrate that inter-host variation of ISVs exists between mosquitoes as well as heterogeneity between different laboratory strains. The results of which providing a resource for the further assessment of the ecology, evolution and interaction of ISVs with their mosquito hosts and arboviruses they transmit.One virus uncovered from these analyses was Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales. AeAV was present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. We experimentally demonstrated the host range of AeAV as being infectious to Aedes cells, but not to three mammalian cell lines. Reanalysis of a small RNA library of Ae. aegypti cells coinfected with AeAV and Wolbachia indicated an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces dengue virus replication in vitro.In addition to the interactions between AeAV, Ae. aegypti and Wolbachia, we also explored the effect of Wolbachia infection on Aedes albopictus densovirus (AalDNV-1), a single-stranded DNA virus. We demonstrated that Wolbachia enhances AalDNV-1 in Aedes cell lines in a density-dependent manner. Further, we found that in Wolbachia-infected Aag2 cells greater absolute abundance of virus-derived short interfering RNAs was produced compared to uninfected cells. Additionally, we found the production of virus-derived PIWI-like RNAs (vpiRNA) in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA U1 bias signature, whereas the typical “ping-pong” signature (U1 - A10) was evident in vpiRNAs from the cytoplasmic fractions. These findings are the first report of the density-dependent enhancement of a DNA virus by Wolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time. The results from both of these studies add to previous evidence indicating Wolbachia does not restrict a range of negative-strand RNA viruses or DNA viruses.Finally, using the pipeline described for the data-mining of ISVs associated with Aedes mosquitoes, we explored the ability to uncover further host associations of divergent flaviviruses. Most flaviviruses are disease-causing pathogens of vertebrates maintained between mosquitoes or ticks and vertebrate hosts. However poor sampling of flaviviruses outside the vector-borne flaviviruses has presented a narrow understanding of flavivirus diversity and evolution. In the final study presented here, 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. Also, we identified variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNAi response. Taken together the evidence suggests Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. These data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expanding our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.

There is interest in understanding the mechanisms that underlie reports that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain PCR positive many weeks after initial infection. The recent paper by Zhang et al. (1) suggests a potential explanation of this phenomenon by claiming that SARS-CoV-2 RNA can integrate into the genome of infected human cells. The authors also reanalyze RNA sequencing (RNA-seq) data and report that SARS-CoV-2−host chimeric reads are present in cells and patient tissues. Given the potential implications of this research on the long-term impacts of COVID-19, we feel that it’s necessary to scrutinize the evidence presented. To determine whether SARS-CoV-2 RNA might be retrotranscribed … [↵][1]1To whom correspondence may be addressed. Email: r.parry{at}uq.edu.au or lachlan.coin{at}unimelb.edu.au. [1]: #xref-corresp-1-1

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.

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., Here the authors describe a simple reverse genetics method that relies on overlapping cDNA fragments for generation of positive-strand viruses including SARS-CoV-2 and characterize them in vitro and in vivo.

Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress the coconut rhinoceros beetle (Oryctes rhinoceros) in Southeast Asia and the Pacific Islands. A new wave of O. rhinoceros incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. In this study, chronically infected beetles were collected from Philippines, Fiji, Papua New Guinea (PNG), and the Solomon Islands (SI). RNA sequencing (RNA-seq) was performed to investigate the global viral gene expression profiles and for comparative genomic analysis of structural variations. Maximum likelihood phylogenic analysis indicated that OrNV strains from the SI and Philippines are closely related, while OrNV strains from PNG and Fiji formed a distinct adjacent clade. We detected several polymorphic sites with a frequency higher than 35% in 892 positions of the viral genome. Nonsynonymous mutations were detected in several hypothetical proteins and 15 nudivirus core genes, such as gp034, lef-8, lef-4, and vp91. We found limited evidence of variation in viral gene expression among geographic populations. Only a few genes, such as gp01, gp022, and gp107, were differentially expressed among different strains. Additionally, small RNA sequencing from the SI population suggested that OrNV is targeted by the host RNA interference (RNAi) response with abundant 21-nucleotide small RNAs. Some of these genomic changes are specific to the geographic population and could be related to particular phenotypic characteristics of the strain, such as viral pathogenicity or transmissibility, and this requires further investigation. IMPORTANCE Oryctes rhinoceros nudivirus has been an effective biocontrol agent against the coconut rhinoceros beetle in Southeast Asia and the Pacific Islands for decades. The recent outbreak of these beetles in many South Pacific islands has had a significant impact on livelihoods in the region. It has been suggested that the resurgence and spread of the pest are related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. We examined viral genomic and transcriptional variations in chronically infected beetles from different geographical populations. A high number of polymorphic sites among several geographical strains of OrNV were identified, but potentially only a few of these variations in the genome are involved in functional changes and can potentially alter the typical function. These findings provide valuable resources for future studies to improve our understanding of the OrNV genetic variations in different geographic regions and their potential link to virus pathogenicity.

Due to the scope and impact of the COVID-19 pandemic there exists a strong desire to understand where the SARS-CoV-2 virus came from and how it jumped species boundaries to humans. Molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. However, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. Here, we dispute the conclusions of Xia (2020. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol. doi:10.1093/molbev/masa095) that dogs are a likely intermediate host of a SARS-CoV-2 ancestor. We highlight major flaws in Xia’s inference process and his analysis of CpG deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. Bats and pangolins currently have the greatest support as ancestral hosts of SARS-CoV-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited.

Oryctes rhinoceros nudivirus (OrNV) is a large circular double-stranded DNA virus which has been used as a biological control agent to suppress Coconut Rhinoceros Beetle (Oryctes rhinoceros) in Southeast Asia and the Pacific Islands. Recently a new wave of O. rhinoceros incursions in Oceania in previously non-infested areas is thought to be related to the presence of low virulence isolates of OrNV or virus tolerant haplotypes of beetles. In this study, chronically infected O. rhinoceros adults were field collected from the Philippines, Fiji, Papua New Guinea and the Solomon Islands. We extracted total RNA from these samples to investigate the global viral gene expression profiles and comparative genomic analysis of structural variations between the four different populations. Maximum likelihood phylogenic analysis indicated that OrNV strains from the Solomon Islands and the Philippines are closely related to while OrNV strains from PNG and Fiji formed a distinct adjacent clade. We detected several polymorphic sites with a frequency higher than 35% in 892 positions of the viral genome. The highest number of structural variants, including single nucleotide variants (SNV), insertion, deletion and non-synonymous mutations, were found in strains from Fiji and PNG when compared to complete recently sequenced Solomon Islands OrNV reference genome. Non-synonymous mutations were detected in several hypothetical proteins, and 15 nudivirus core genes such as OrNV_gp034 (DNA Helicase), lef-8, lef-4 and vp91. For examination of the global gene expression profile of OrNV in chronically infected populations, we found limited evidence of variation between geographic populations. Only a few genes such as OrNV_gp01 (DNA polymerase B), OrNV_gp022 and OrNV_gp107 (Pif-3) were differentially expressed among different strains. Additionally, small RNA sequencing from the Solomon Islands population suggests that OrNV is targeted by the host RNA interference (RNAi) response with abundant 21nt small RNAs. Additionally, we identified a highly abundant putative 22 nt miRNA from the 3’ of a pre-miRNA-like hairpin originating from OrNV-gp-098. These findings provide valuable resources for future studies to improve our understanding of the OrNV genetic variation. Some of these structural changes are specific to the geographic population and could be related to particular phenotypic characteristics of the strain, such as viral pathogenicity or transmissibility, and this requires further investigation.

The horn fly, Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between $1 billion and $2.5 billion (U.S. dollars) annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, TX, and Alberta, Canada, but it has also been identified in wild-caught samples from Canada, the United States, Mexico, and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularized 1.3-Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1,249 coding genes, 34 tRNAs, 3 rRNAs, and 5 prophage regions. Comparative genomics and whole-genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggested that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicated that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggested two phylogenetically distinct CI loci and acquisition of another cifB homolog from phylogenetically distant supergroup A Wolbachia strains, suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies. IMPORTANCE Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction, such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, TX, horn fly (wIrr). Annotation of wIrr suggests its unique features, including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study provides the foundation for future studies of Wolbachia-induced biological effects for control of horn flies.

Dengue infection along with its related disease conditions poses a significant threat to human health. The pathogen responsible for this infection is dengue virus (DENV) which is primarily transmitted to humans through the bites of Aedes aegypti mosquitoes. Unavailability of a potent vaccine has recently sparked renewed research endeavours aimed at vector control. To date, Wolbachia as an endosymbiotic bacterium has shown promise as a novel biocontrol agent to restrict DENV replication in the vector, although the underlying antiviral mechanism remains elusive. Recent studies have demonstrated the potential role of Vago as a novel secretory protein involved in cross-talk between the innate immune pathways in Culex quinquefasciatus mosquitoes to restrict West Nile virus replication. In this study, we have identified two homologs of the Vago protein in Ae. aegypti and looked into their modulation in the case of Wolbachia wMelPop strain infection. Furthermore, we have investigated the role of AeVago1, that is highly induced by Wolbachia, in the context of Wolbachia-mosquito-DENV interactions. Knockdown studies of the AeVago1 gene in Wolbachia-infected cells led to significant increases in DENV replication, with no effect on Wolbachia density. Our results suggest that the Wolbachia-induced AeVago1 in Ae. aegypti may function as a host factor to suppress DENV replication in the mosquito.

The

The horn fly,Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between USD 1-2.5 billion annually in North America and Brazil. The endosymbiotic bacteriumWolbachia pipientisis a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, USA and Alberta, Canada, but has also been identified in wild-caught samples from Canada, USA, Mexico and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the KerrvilleH. i. irritansgenome project allowed for a complete and circularised 1.3 MbWolbachiagenome (wHae). Annotation ofwHae yielded 1249 coding genes, 34 tRNAs, three rRNAs, and five prophage regions. Comparative genomics and whole genome Bayesian evolutionary analysis ofwHae compared to publishedWolbachiagenomes suggests thatwHae is most closely related to and diverged fromWolbachiasupergroup A strains known to infectDrosophilaspp. Whole-genome synteny analyses betweenwHae and closely related genomes indicates thatwHae has undergone convoluted genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes ofwHae suggests two phylogenetically distinct CI loci and acquisition of anotherCifBhomolog from phylogenetically distant supergroup AWolbachiastrains suggesting horizontal acquisition of these loci. ThewHae genome provides a resource for future examination of the impactWolbachiamay have in both biocontrol and potential insecticide resistance of horn flies.ImportanceHorn flies,Haematobia irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control.Wolbachia pipientisis an endosymbiont bacterium known to have a range of effects on host reproduction such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts on vector transmission. These characteristics ofWolbachiahave been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of theWolbachiastrain of the Kerrickville, USA horn fly (wHae). Annotation ofwHae suggests its unique features including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study will provide the foundation for futureWolbachia-induced biological effect studies for control of horn flies.

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.

The endosymbiotic bacteriumWolbachia pipientishas been shown to restrict a range of RNA viruses inDrosophila melanogasterand transinfected dengue mosquito,Aedes aegypti. Here, we show thatWolbachiainfection enhances replication of Aedes albopictus densovirus (AalDNV-1), a single stranded DNA virus, inAedescell lines in a density-dependent manner. Analysis of previously produced small RNAs of Aag2 cells showed thatWolbachia-infected cells produced greater proportions of viral derived short interfering RNAs as compared to uninfected cells. Additionally, we found production of viral derived PIWI-like RNAs (vpiRNA) produced in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA signature U1bias whereas the typical “ping-pong” signature (U1- A10) was evident in the cytoplasmic fraction. This is the first report of the density-dependent enhancement of DNA viruses byWolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time.

The endosymbiotic bacterium Wolbachia pipientis has been shown to restrict a range of RNA viruses in Drosophila melanogaster and transinfected dengue mosquito, Aedes aegypti. Here, we show that Wolbachia infection enhances replication of Aedes albopictus densovirus (AalDNV-1), a single stranded DNA virus, in Aedes cell lines in a density-dependent manner. Analysis of previously produced small RNAs of Aag2 cells showed that Wolbachia-infected cells produced greater absolute abundance of virus-derived short interfering RNAs compared to uninfected cells. Additionally, we found production of virus-derived PIWI-like RNAs (vpiRNA) produced in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA signature U1 bias whereas the typical "ping-pong" signature (U1 - A10) was evident in vpiRNAs from the cytoplasmic fractions. This is the first report of the density-dependent enhancement of DNA viruses by Wolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time.

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti 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 A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus 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 transcriptome sequencing (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 A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-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 Wolbachia 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 A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

Insect specific viruses (ISVs) of the yellow fever mosquitoAedes aegyptihave 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 ofAe. aegypti, however, remains poorly understood. In this study, we characterised Aedes anphevirus (AeAV), a negative-sense RNA virus from the orderMononegavirales. AeAV identified fromAedescell lines were infectious to bothAe. aegyptiandAedes albopictuscells, 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 theAe. aegyptimosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American and Asia-Pacific lineages. The endosymbiotic bacteriumWolbachia pipientisrestricts positive-sense RNA viruses inAe. aegypti. Re-analysis of a small RNA library ofAe. aegypticells co-infected with AeAV andWolbachiaproduces an abundant RNAi response consistent with persistent virus replication. We foundWolbachiaenhances replication of AeAV when compared to a tetracycline cleared cell line, and AeAV modestly reduces DENV replicationin vitro. The results from our study improve understanding of the diversity and evolution of the virome ofAe. aegyptiand adds to previous evidence that showsWolbachiadoes not restrict a range of negative strand RNA viruses.

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.

Introduction Smokefree street policies are relatively rare, and little has been published on the methods for establishing an evidence base to inform such policy making. We aimed to (a) pilot methods for such data collection in New Zealand, a country where local governments are actively pursuing outdoor smokefree policies and (a) to provide data on smoking behavior, attitudes toward smokefree policies, and levels of smoke exposure on streets in Wellington. Methods Three methods were piloted: (a) systematic observation of smoking behavior by observers walking a standard route of major streets, the "Golden Mile" (GM) in Wellington (n = 42 observation runs); (b) measurement of fine particulate levels (PM(2.5)) along this route and with purposeful sampling in selected settings; and (c) an attitudinal survey of pedestrians along sections of this route. Results Each of the 3 methods proved to be feasible in this urban setting. A total of 932 smokers were observed during 21 hr of observation, an average of 7 smokers every 10 min of walking. Air monitoring indicated fine particulate exposure. Levels of (mean) PM(2.5) were 1.5 times higher during periods when smoking was observed than when they were not (9.3 vs. 6.3 μg/m(3), p = .002). Dose-response patterns were observed for smoking proximity and for smoker numbers. Surveying pedestrians (n = 220) with a brief questionnaire achieved an 81% response rate and was able to identify variation in support for a smokefree GM by different groups (overall support was 55.9%, 95% CI = 49.3%-62.4%). Reasons for support were also identified, for example, perceived health hazards, at 34.1%, was the main reason. Conclusions These methods can provide information that may contribute to the smokefree streets policymaking process and may also be relevant to informing other smokefree outdoor policies.