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We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response. National Institute of Allergy and Infectious Diseases of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [K22AI125474, R01AI153433, R01AI121452, R24AI120942] Published version This work was supported by grants from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers K22AI125474 and R01AI153433 to AJA, and R01AI121452 and R24AI120942 to SCW. We thank Steven Widen, Thomas Wood and Jill Thompson for assistance with sequencing.

Cetacean morbillivirus (CeMV) is a global threat to cetaceans. We report a novel morbillivirus from a Fraser’s dolphin (Lagenodelphis hosei) that stranded in Maui, Hawaii in 2018 that is dissimilar to the beaked whale morbillivirus previously identified from Hawaii and to other CeMV strains. Histopathological findings included intranuclear inclusions in bile duct epithelium, lymphoid depletion, rare syncytial cells and non-suppurative meningitis. Cerebellum and lung tissue homogenates were inoculated onto Vero.DogSLAMtag cells for virus isolation and cytopathic effects were observed, resulting in the formation of multinucleated giant cells (i.e., syncytia). Transmission electron microscopy of infected cell cultures also revealed syncytial cells with intracytoplasmic and intranuclear inclusions of viral nucleocapsids, consistent with the ultrastructure of a morbillivirus. Samples of the cerebellum, lung, liver, spleen and lymph nodes were positive for morbillivirus using a reverse transcription-polymerase chain reaction. The resulting 559 bp L gene sequence had the highest nucleotide identity (77.3%) to porpoise morbillivirus from Northern Ireland and the Netherlands. The resulting 248 bp P gene had the highest nucleotide identity to porpoise morbillivirus in Northern Ireland and the Netherlands and to a stranded Guiana dolphin (Sotalia guianensis) in Brazil (66.9%). As Fraser’s dolphins are a pelagic species that infrequently strand, a novel strain of CeMV may be circulating in the central Pacific that could have additional population impacts through transmission to other small island-associated cetacean species.

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.

An adult dog was presented for chronic cough and a recent development of ulcerated, erythematous nares with nasal discharge. Cytology of enlarged peripheral lymph nodes revealed many intracellular and extracellular organisms. These round or rarely oval organisms measured approximately 5-9 µm in diameter and frequently contained several globular structures, ranging from deeply basophilic to magenta. A thin, clear halo was present. Smaller 1-2 µm, magenta forms were also observed. Fungal culture yielded small, wet, raised, irregularly shaped, white to pale tan colonies. Microbiologic staining of cultured material revealed features suggestive of algae. Histopathology of the lymph nodes revealed marked granulomatous inflammation with intralesional algal organisms suggestive of Prototheca. Electron microscopic findings were also consistent with protothecosis. Polymerase chain reaction, followed by direct DNA sequencing, identified the organism as Prototheca wickerhamii. A brief literature review discussing protothecosis in veterinary medicine is included.

Alcoholic chronic pancreatitis (ACP) is a fibroinflammatory disease of the pancreas. However, metabolic basis of ACP is not clearly understood. In this study, we evaluated differential pancreatic injury in hepatic alcohol dehydrogenase-deficient (ADH(−)) deer mice fed chronic ethanol (EtOH), chronic plus binge EtOH, and chronic plus binge EtOH and fatty acid ethyl esters (FAEEs, nonoxidative metabolites of EtOH) to understand the metabolic basis of ACP. Hepatic ADH(−) and ADH normal (ADH(+)) deer mice were fed Lieber-DeCarli liquid diet containing 3% (wt/vol) EtOH for 3 mo. One week before the euthanization, chronic EtOH-fed mice were further administered with an oral gavage of binge EtOH with/without FAEEs. Blood alcohol concentration (BAC), pancreatic injury, and inflammatory markers were measured. Pancreatic morphology, ultrastructural changes, and endoplasmic reticulum (ER)/oxidative stress were examined using H&E staining, electron microscopy, immunostaining, and/or Western blot, respectively. Overall, BAC was substantially increased in chronic EtOH-fed groups of ADH(−) versus ADH(+) deer mice. A significant change in pancreatic acinar cell morphology, with mild to moderate fibrosis and ultrastructural changes evident by dilatations and disruption of ER cisternae, ER/oxidative stress along with increased levels of inflammatory markers were observed in the pancreas of chronic EtOH-fed groups of ADH(−) versus ADH(+) deer mice. Furthermore, chronic plus binge EtOH and FAEEs exposure elevated BAC, enhanced ER/oxidative stress, and exacerbated chronic EtOH-induced pancreatic injury in ADH(−) deer mice suggesting a role of increased body burden of EtOH and its metabolism under reduced hepatic ADH in initiation and progression of ACP. NEW & NOTEWORTHY We established a chronic EtOH feeding model of hepatic alcohol dehydrogenase-deficient (ADH(−)) deer mice, which mimics several fibroinflammatory features of human alcoholic chronic pancreatitis (ACP). The fibroinflammatory and morphological features exacerbated by chronic plus binge EtOH and FAEEs exposure provide a strong case for metabolic basis of ACP. Most importantly, several pathological and molecular targets identified in this study provide a much broader understanding of the mechanism and avenues to develop therapeutics for ACP.

SUMMARY RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3′-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. RBFOX2-mediated APA modulates mRNA levels and/or isoform expression of a collection of genes, including contractile and mitochondrial genes. Depletion of RBFOX2 adversely affects mitochondrial health in myoblasts, correlating with disrupted APA of mitochondrial gene Slc25a4. Mechanistically, RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site, enforcing the use of the proximal polyadenylation site. In sum, our results unveil a role for RBFOX2 in fine-tuning expression of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases., Graphical Abstract, In brief Cao et al. show that depletion of RBFOX2 in rat myoblasts modulates alternative polyadenylation patterns of mitochondrial and contractile genes. RBFOX2 loss adversely affects mitochondrial health.

Cetacean morbillivirus (CeMV) is a global threat to cetaceans. We report a novel morbillivirus from a Fraser's dolphin (Lagenodelphis hosei) that stranded in Maui, Hawaii in 2018 that is dissimilar to the beaked whale morbillivirus previously identified from Hawaii and to other CeMV strains. Histopathological findings included intranuclear inclusions in bile duct epithelium, lymphoid depletion, rare syncytial cells and non-suppurative meningitis. Cerebellum and lung tissue homogenates were inoculated onto Vero.DogSLAMtag cells for virus isolation and cytopathic effects were observed, resulting in the formation of multinucleated giant cells (i.e., syncytia). Transmission electron microscopy of infected cell cultures also revealed syncytial cells with intracytoplasmic and intranuclear inclusions of viral nucleocapsids, consistent with the ultrastructure of a morbillivirus. Samples of the cerebellum, lung, liver, spleen and lymph nodes were positive for morbillivirus using a reverse transcription-polymerase chain reaction. The resulting 559 bp L gene sequence had the highest nucleotide identity (77.3%) to porpoise morbillivirus from Northern Ireland and the Netherlands. The resulting 248 bp P gene had the highest nucleotide identity to porpoise morbillivirus in Northern Ireland and the Netherlands and to a stranded Guiana dolphin (Sotalia guianensis) in Brazil (66.9%). As Fraser's dolphins are a pelagic species that infrequently strand, a novel strain of CeMV may be circulating in the central Pacific that could have additional population impacts through transmission to other small island-associated cetacean species.

As part of an ongoing investigation of harbor seal (Phoca vitulina) mortalities within Puget Sound, Washington State, United States, between October 2007 and July 2008, 25 seal cases were submitted for histopathology and ancillary diagnostic testing, including additional attempted virus isolation. In vitro granular and refractile cytopathic effects (CPE) were consistently observed in Vero.DogSLAMtag cells inoculated with tissue homogenates from three seals. Transmission electron microscopy of infected Vero.DogSLAMtag cells revealed cytoplasmic clusters of icosahedral viral particles morphologically consistent with members of the family Reoviridae. The complete genome of a novel species within the genus Orthoreovirus, tentatively named phocid orthoreovirus 1 (PhRV1), was determined by next-generation sequencing and confirmed by rt-PCR in isolates from the three harbor seals. This is the first report of an orthoreovirus infection associated with dead stranded harbor seals. Aside from the CPE and ultrastructural findings, no consistent signalment, gross pathology, histopathology, or ancillary diagnostic findings were identified with PhRV1 infection. Further research is needed to determine the prevalence, tissue tropism, transmission, pathogenicity, zoonotic potential, and host range of orthoreoviruses in pinnipeds. This study demonstrates the value of thorough necropsy investigations and a multidisciplinary team approach to advance our understanding of marine mammal health.

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen–host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

Rickettsial species have independently lost several genes owing to reductive evolution while retaining those predominantly implicated in virulence, survival, and biosynthetic pathways. In this study, we have identified a previously uncharacterized Rickettsia conorii gene RC0497 as an N-acetylmuramoyl-L-alanine amidase constitutively expressed during infection of cultured human microvascular endothelial cells at the levels of both mRNA transcript and encoded protein. A homology-based search of rickettsial genomes reveals that RC0497 homologs, containing amidase_2 family and peptidoglycan binding domains, are highly conserved among the spotted fever group (SFG) rickettsiae. The recombinant RC0497 protein exhibits α-helix secondary structure, undergoes a conformational change in the presence of zinc, and exists as a dimer at higher concentrations. We have further ascertained the enzymatic activity of RC0497 via demonstration of its ability to hydrolyze Escherichia coli peptidoglycan. Confocal microscopy on E. coli expressing RC0497 and transmission immunoelectron microscopy of R. conorii revealed its localization predominantly to the cell wall, septal regions of replicating bacteria, and the membrane of vesicles pinching off the cell wall. In summary, we have identified and functionally characterized RC0497 as a peptidoglycan hydrolase unique to spotted fever rickettsiae, which may potentially serve as a novel moonlighting protein capable of performing multiple functions during host-pathogen interactions.

Stem cell transplantation to the liver is a promising therapeutic strategy for a variety of disorders. Hepatocyte transplantation has short-term efficacy but can be problematic due to portal hypertension, inflammation, and sinusoidal thrombosis. We have previously transplanted small mouse endoderm progenitor (EP) cells to successfully reverse a murine model of hemophilia B, and labeling these cells with iron nanoparticles renders them responsive to magnetic fields, which can be used to enhance engraftment. The mechanisms mediating progenitor cell migration from the sinusoidal space to the hepatocyte compartment are unknown. Here we find human EP and hepatic progenitor (HP) cells can be produced from human embryonic stem cells with high efficiency, and they also readily uptake iron nanoparticles. This provides a simple manner through which one can readily identify transplanted cells in vivo using electron microscopy, shortly after delivery. High resolution imaging shows progenitor cell morphologies consistent with epithelial-to-mesenchymal transition (EMT) mediating invasion into the hepatic parenchyma. This occurs in as little as 3 h, which is considerably faster than observed when hepatocytes are transplanted. We confirmed activated EMT in transplanted cells in vitro, as well as in vivo 24 h after transplantation. We conclude that EMT naturally occurs concurrent with EP and HP cell engraftment, which may mediate the rate, safety, and efficacy of early cell engraftment in the undamaged quiescent liver.

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

How microbe–microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host–microbe interactions and provides evidence that microbial interactions influence mosquito behavior.

SUMMARYRBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression/function in muscle cells, contributing to disease pathology. Here, we employed both 3’-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts. We found that RBFOX2-mediated APA modulates both mRNA levels and isoform expression of a collection of genes including contractile and mitochondrial genes. We identified the key muscle-specific contractile gene, Tropomyosin 1 and essential mitochondrial gene, Slc25a4 as APA targets of RBFOX2. Unexpectedly, depletion of RBFOX2 adversely affected mitochondrial health in myoblasts that is in part mediated by disrupted APA of mitochondrial gene Slc25a4. Mechanistically, we found that RBFOX2 regulation of Slc25a4 APA is mediated through consensus RBFOX2 binding motifs near the distal polyadenylation site enforcing the use of the proximal polyadenylation site. In sum, our results unveiled a new role for RBFOX2 in fine tuning expression levels of mitochondrial and contractile genes via APA in myoblasts relevant to heart diseases.

Ranaviruses are emerging pathogens associated with worldwide epizootics in farmed and wild ectothermic vertebrates. In this study, we determined the full genomes of eight ranaviruses isolated from marbled sleeper goby ( Oxyeleotris marmorata), goldfish ( Carassius auratus), guppy ( Poecilia reticulata), tiger frog ( Hoplobatrachus tigerinus), Asian grass frog ( Fejervarya limnocharis), and East Asian bullfrog ( H. rugulosus) cultured or imported into Thailand. These ranaviral isolates induced the same cytopathic effects (i.e., progression of coalescing round plaques) in epithelioma papulosum cyprini (EPC) cell cultures. Transmission electron microscopy of infected EPC cells revealed cytoplasmic viral particles with ultrastructural features typical for ranaviruses. Pairwise genetic comparisons of the complete major capsid protein coding sequences from the Thai ranaviruses displayed the highest identity (99.8%–100%) to a ranavirus (tiger frog virus; TFV) isolated from diseased tiger frogs cultured in China, a slightly lower identity (99.3%–99.4%) to a ranavirus (Wamena virus; WV) isolated from diseased green tree pythons ( Morelia viridis) illegally exported from Papua New Guinea, and a lower identity to 35 other ranaviruses (93.7%–98.6%). Phylogenomic analyses supported the eight Thai ranaviruses, Chinese TFV, and WV as a subclade within a larger frog virus 3 clade. Our findings confirm the spread of TFV among cultured fish and amphibians in Asia and likely in reptiles in Oceania. Biosecurity measures are needed to ensure TFV does not continue to spread throughout Southeast Asia and to other parts of the world via international trade.

The seal picornavirus 1, species Aquamavirus A, is currently the only recognized member of the genus Aquamavirus within the family Picornaviridae. The bear picornavirus 1 was recently proposed as the second species in the genus under the name aquamavirus B. Herein, we determined the complete genomes of two novel pinniped picornaviruses, the harbor seal picornavirus (HsPV) and the ribbon seal picornavirus (RsPV). The HsPV and the RsPV were isolated in Vero.DogSLAMtag cells from samples collected from stranded harbor (Phoca vitulina) and ribbon (Histriophoca fasciata) seals. RsPV-infected Vero.DogSLAMtag cells displaying extensive cytopathic effects were processed for transmission electron microscopy and revealed non-enveloped viral particles aggregated into paracrystalline arrays in the cytoplasm. A next-generation sequencing approach was used to recover the complete genomes of the HsPV and the RsPV (6,709 bp and 6,683 bp, respectively). Phylogenetic and genetic analyses supported the HsPV and the RsPV as members of the Aquamavirus genus. Based on these results, RsPV represents a novel strain of Aquamavirus A, while the HsPV is a novel strain of the proposed species aquamavirus B. These discoveries provide information on the evolutionary relationships and ultrastructure of aquamaviruses and expands the known host range of those viruses. Our results underscore the importance of the application of classical virology and pathology techniques coupled with high-throughput sequencing technologies for the discovery and characterization of pathogens in wild marine mammals.

Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolayer lysis in the initial inoculation and upon repassage. Transmission electron microscopy of infected SSN-1 cells revealed small round particles (mean diameter=20.0–21.7 nm) within the cytoplasm, consistent with the ultrastructure of a picornavirus. Full-genome sequencing of the purified virus revealed a novel picornavirus most closely related to the bluegill picornavirus and other members of the genus Limnipivirus. Additionally, pairwise protein alignments between the clownfish picornavirus (CFPV) and other known members of the genus Limnipivirus yielded results in accordance with the current International Committee on Taxonomy of Viruses criteria for members of the same genus. Thus, CFPV represents a proposed new limnipivirus species. Future experimental challenge studies are needed to determine the role of CFPV in disease.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behaviour; Serratia exposure to Ae. aegypti disrupted their feeding behaviour, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behaviour.

This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of Aedes sollicitans mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus Entomobirnavirus, family Birnaviridae. PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.

The commercial production of lumpfish

Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry's Lagoon viruses (Reoviridae), Bontang virus (Mesoniviridae), and Culex theileri flavivirus (Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease.

Microbiota within mosquitoes influence nutrition, immunity, fecundity, and the capacity to transmit pathogens. Despite their importance, we have a limited understanding of host-microbiota interactions, especially at the cellular level. It is evident bacterial symbionts that are localized within the midgut also infect other organs within the mosquito; however, the route these symbionts take to colonize other tissues is unknown. Here, utilizing the gentamicin protection assay, we showed that the bacterial symbionts Cedecea and Serratia have the capacity to invade and reside intracellularly within mosquito cells. Symbiotic bacteria were found within a vacuole and bacterial replication was observed in mosquito cell by transmission electron microscopy, indicating bacteria were adapted to the intracellular milieu. Using gene silencing, we determined that bacteria exploited host factors, including actin and integrin receptors, to actively invade mosquito cells. As microbiota can affect pathogens within mosquitoes, we examined the influence of intracellular symbionts on Zika virus (ZIKV) infection. Mosquito cells harbouring intracellular bacteria had significantly less ZIKV compared to uninfected cells or cells exposed to non-invasive bacteria. Intracellular bacteria were observed to substantially upregulate the Toll and IMD innate immune pathways, providing a possible mechanism mediating these anti-viral effects. Examining mono-axenically infected mosquitoes using transmission electron and fluorescent microscopy revealed that bacteria occupied an intracellular niche in vivo. Our results provided evidence that bacteria that associate with the midgut of mosquitoes have intracellular lifestyles which likely have implications for mosquito biology and pathogen infection. This study expands our understanding of host-microbiota interactions in mosquitoes, which is important as symbiont microbes are being exploited for vector control strategies.

We report the detection and gene coding sequences of three novel Orbivirus species found in six dead farmed white-tailed deer in the United States. Phylogenetic analyses indicate that the new orbiviruses are genetically closely related to the Guangxi, Mobuck, Peruvian horse sickness, and Yunnan orbiviruses, which are thought to be solely borne by mosquitos. However, four of the six viruses analyzed in this work were found as co-infecting agents along with a known cervid pathogen, epizootic hemorrhagic disease virus-2 (EHDV-2), raising questions as to whether the new viruses are primary pathogens or secondary pathogens that exacerbate EHDV-2 infections. Moreover, EHDV-2 is known to be a Culicoides-borne virus, raising additional questions as to whether Culicoides species can also serve as vectors for the novel orbiviruses, if mosquitoes can vector EHDV-2, or whether the deer were infected through separate bites by the insects. Our findings expand knowledge of the possible viral pathogens of deer in the United States. Moreover, due to the close genetic relatedness of the three new orbiviruses to viruses that are primary pathogens of cattle and horses, our findings also underscore a crucial need for additional research on the potential role of the three new orbiviruses as pathogens of other animals.

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections., Author summary Lymphopenia is a common characteristic of the disease caused by EBOV. We determined that despite the apparent lack of productive infection, EBOV is capable of entering T cells and producing both viral RNAs and proteins. Furthermore, we demonstrate that EBOV causes an abortive infection in T cells due to the presence of a cellular restriction factor. The abortive infection was associated with cell death following ER-stress induced autophagy. Collectively, these findings suggest that abortive infection in T cells is likely to contribute to lymphopenia during Ebola virus disease, which is uniformly linked with the severity of the disease. All EBOV vaccine candidates utilize GP as the sole antigen inducing a protective antibody response and in some clinical trials were shown to induce adverse side effects. The present study suggests that these effects can be associated with GP, which may lead to abortive infection of the vaccine construct in T cells contributing to the inflammatory response to the vaccines.

This research was funded by grant R24AI120942 and contracts N01-AI30027, HHS-N2722010000401, and HHSN2720004/D04 from the U.S. National Institutes of Health, and a CNPq grant 302032/2011-8 from the Brazilian National Council for Research and Development. MAC was supported by a Ph.D. scholarship from the Colombian Department of Science (Colciencias Convocatoria 567). The University of Queensland. School of Biological Sciences. Australia. University of Texas Medical Branch. Department of Pathology. Galveston, TX, USA / University of Texas Medical Branch. Center for Biodefense and Emerging Infectious Diseases. Galveston, TX, USA / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TX, USA / University of Texas Medical Branch. Institute for Human Infection and Immunity. Galveston, TX, USA / University of Texas Medical Branch. World Reference Center for Emerging Viruses and Arboviruses, Galveston, TX, USA. University of Texas Medical Branch. Department of Pathology. Galveston, TX, USA / University of Texas Medical Branch. Institute for Human Infection and Immunity. Galveston, TX, USA / University of Texas Medical Branch. World Reference Center for Emerging Viruses and Arboviruses, Galveston, TX, USA. University of Texas Medical Branch. Department of Pathology. Galveston, TX, USA / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TX, USA / University of Texas Medical Branch. Institute for Human Infection and Immunity. Galveston, TX, USA / University of Texas Medical Branch. World Reference Center for Emerging Viruses and Arboviruses, Galveston, TX, USA. University of Texas Medical Branch. Department of Pathology. Galveston, TX, USA / University of Texas Medical Branch. Institute for Human Infection and Immunity. Galveston, TX, USA / University of Texas Medical Branch. World Reference Center for Emerging Viruses and Arboviruses, Galveston, TX, USA. Harris County Public Health and Environmental Services. Mosquito and Vector Control Division. Houston, TX, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / University of S?o Paulo. Ribeir?o Preto School of Medicine. Virology Research Center. Ribeir?o Preto, SP, Brasil. University of Antioquia and National University of Colombia. Program for Study and Control of Tropical Diseases. Medellin, Colombia. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Harris County Public Health and Environmental Services. Mosquito and Vector Control Division. Houston, TX, USA. Harris County Public Health and Environmental Services. Mosquito and Vector Control Division. Houston, TX, USA. Harris County Public Health and Environmental Services. Mosquito and Vector Control Division. Houston, TX, USA. University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Department of Biochemistry and Molecular Biology. Galveston, TX, USA. University of Texas Medical Branch. Department of Pathology. Galveston, TX, USA / University of Texas Medical Branch. Center for Biodefense and Emerging Infectious Diseases. Galveston, TX, USA / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TX, USA / University of Texas Medical Branch. Institute for Human Infection and Immunity. Galveston, TX, USA / University of Texas Medical Branch. World Reference Center for Emerging Viruses and Arboviruses, Galveston, TX, USA. This report describes and characterizes three novel RNA viruses isolated from dead birds collected during West Nile virus surveillance in Harris County, TX, USA (the Houston metropolitan area). The novel viruses are identified as members of the families Nyamaviridae, Orthomyxoviridae, and Peribunyaviridae and have been designated as San Jacinto virus, Mason Creek virus, and Buffalo Bayou virus, respectively. Their potential public health and/or veterinary importance are still unknown.

ZIKV is a recently emerged mosquito-borne flavivirus that can cause devastating congenital Zika syndrome in pregnant women and Guillain-Barré syndrome in adults. The molecular mechanism of ZIKV virion assembly is largely unknown. Here, we report that ZIKV NS2A plays a central role in recruiting viral RNA, structural protein prM/E, and viral NS2B/NS3 protease to the virion assembly site and orchestrating virion morphogenesis. One mutation that impairs these interactions does not significantly affect viral RNA replication but selectively abolishes virion assembly, demonstrating the specific role of these interactions in virus morphogenesis. We also show that the 3ʹ UTR of ZIKV RNA may serve as a “recruitment signal” through binding to NS2A to enter the virion assembly site. Following a coordinated cleavage of C-prM-E at the virion assembly site, NS2A may present the viral RNA to C protein for nucleocapsid formation followed by envelopment with prM/E proteins. The results have provided new insights into flavivirus virion assembly., The flavivirus virion consists of an envelope outer layer, formed by envelope (E) and membrane (M) proteins on a lipid bilayer, and an internal core, formed by capsid (C) protein and genomic RNA. The molecular mechanism of flavivirus assembly is not well understood. Here, we show that Zika virus (ZIKV) NS2A protein recruits genomic RNA, the structural protein prM/E complex, and the NS2B/NS3 protease complex to the virion assembly site and orchestrates virus morphogenesis. Coimmunoprecipitation analysis showed that ZIKV NS2A binds to prM, E, NS2B, and NS3 (but not C, NS4B, or NS5) in a viral RNA-independent manner, whereas prM/E complex does not interact with NS2B/NS3 complex. Remarkably, a single-amino-acid mutation (E103A) of NS2A impairs its binding to prM/E and NS2B/NS3 and abolishes virus production, demonstrating the indispensable role of NS2A/prM/E and NS2A/NS2B/NS3 interactions in virion assembly. In addition, RNA-protein pulldown analysis identified a stem-loop RNA from the 3ʹ untranslated region (UTR) of the viral genome as an “RNA recruitment signal” for ZIKV assembly. The 3ʹ UTR RNA binds to a cytoplasmic loop of NS2A protein. Mutations of two positively charged residues (R96A and R102A) from the cytoplasmic loop reduce NS2A binding to viral RNA, leading to a complete loss of virion assembly. Collectively, our results support a virion assembly model in which NS2A recruits viral NS2B/NS3 protease and structural C-prM-E polyprotein to the virion assembly site; once the C-prM-E polyprotein has been processed, NS2A presents viral RNA to the structural proteins for virion assembly.