Novel Aquareovirus isolated from channel catfish (Ictalurus punctatus) used in mussel restoration efforts in Wisconsin.

Channel catfish (Ictalurus punctatus) are a food fish extensively reared in aquaculture facilities throughout the world and are also among the most abundant wild catfish species in North America, making them a popular target of anglers. Furthermore, channel catfish are important members of aquatic ecosystems; for example, they serve as a glochidial host for the endangered winged mapleleaf mussel (Quadrula fragosa), making them critical for conserving this species through hatchery-based restoration efforts. During a routine health inspection, a novel aquareovirus was isolated from channel catfish used in mussel propagation efforts at a fish hatchery in Wisconsin. This virus was isolated on brown bullhead cells (ATCC CCL-59) and identified through metagenomic sequencing as a novel member of the family Spinareoviridae, genus Aquareovirus. The virus genome consists of 11 segments, as is typical of the aquareoviruses, with phylogenetic relationships based on RNA-dependent RNA polymerase and major outer capsid protein amino acid sequences showing it to be most closely related to golden shiner virus (aquareovirus C) and aquareovirus C/American grass carp reovirus (aquareovirus G) respectively. The potential of the new virus, which we name genictpun virus 1 (GNIPV-1), to cause disease in channel catfish or other species remains unknown.

Five Species of Wild Freshwater Sport Fish in Wisconsin, USA, Reveal Highly Diverse Viromes.

Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of freshwater fish in Wisconsin, USA: bluegill (Lepomis macrochirus), brown trout (Salmo trutta), lake sturgeon (Acipenser fulvescens), northern pike (Esox lucius), and walleye (Sander vitreus). We analyzed 103 blood serum samples collected during a state-wide survey from 2016 to 2020 and used a metagenomic approach for virus detection to identify known and previously uncharacterized virus sequences. We then characterized viruses phylogenetically and quantified prevalence, richness, and relative abundance for each virus. Within these viromes, we identified 19 viruses from 11 viral families: Amnoonviridae, Circoviridae, Coronaviridae, Hepadnaviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Matonaviridae, Narnaviridae, Nudnaviridae, and Spinareoviridae, 17 of which were previously undescribed. Among these viruses was the first fish-associated coronavirus from the Gammacoronavirus genus, which was present in 11/15 (73%) of S. vitreus. These results demonstrate that, similar to marine fish, freshwater fish also harbor diverse relatives of viruses important to the health of fish and other animals, although it currently remains unknown what effect, if any, the viruses we identified may have on fish health.

Non-Lethal Detection of Ranavirus in Fish.

Emergent infectious diseases have an increasing impact on both farmed animals and wildlife. The ability to screen for pathogens is critical for understanding host-pathogen dynamics and informing better management. Ranavirus is a pathogen of concern, associated with disease outbreaks worldwide, affecting a broad range of fish, amphibian, and reptile hosts, but research has been limited. The traditional screening of internal tissues, such as the liver, has been regarded as the most effective for detecting and quantifying Ranavirus. However, such methodology imposes several limitations from ethical and conservation standpoints. Non-lethal sampling methods of viral detection were explored by comparing the efficacy of both buccal swabbing and fin clipping. The study was conducted on two Iberian, threatened freshwater fish (Iberochondrostoma lusitanicum and Cobitis paludica), and all samples were screened using qPCR. While for C. paludica both methods were reliable in detecting Ranavirus, on I. lusitanicum, there was a significantly higher detection rate in buccal swabs than in fin tissue. This study, therefore, reports that fin clipping may yield false Ranavirus negatives when in small-bodied freshwater fish. Overall, buccal swabbing is found to be good as an alternative to more invasive procedures, which is of extreme relevance, particularly when dealing with a threatened species.

Non-Lethal Detection of Frog Virus 3-Like (RUK13) and Common Midwife Toad Virus-Like (PDE18) Ranaviruses in Two UK-Native Amphibian Species.

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance.

GOPC facilitates the sorting of syndecan-1 in polarized epithelial cells.

The trans-Golgi network must coordinate sorting and secretion of proteins and lipids to intracellular organelles and the plasma membrane. During polarization of epithelial cells, changes in the lipidome and the expression and distribution of proteins contribute to the formation of apical and basolateral plasma membrane domains. Previous studies using HeLa cells show that the syndecan-1 transmembrane domain confers sorting within sphingomyelin-rich vesicles in a sphingomyelin secretion pathway. In polarized Madin-Darby canine kidney cells, we reveal differences in the sorting of syndecan-1, whereupon the correct trafficking of the protein is not dependent on its transmembrane domain and changes in sphingomyelin content of cells during polarization. Instead, we reveal that correct basolateral targeting of syndecan-1 requires a full-length PDZ motif in syndecan-1 and the PDZ domain golgin protein GOPC. Moreover, we reveal changes in Golgi morphology elicited by GOPC overexpression. These results suggest that the role of GOPC in sorting syndecan-1 is indirect and likely due to GOPC effects on Golgi organization.

Cargo sorting at the trans-Golgi network at a glance.

The Golgi functions principally in the biogenesis and trafficking of glycoproteins and lipids. It is compartmentalized into multiple flattened adherent membrane sacs termed cisternae, which each contain a distinct repertoire of resident proteins, principally enzymes that modify newly synthesized proteins and lipids sequentially as they traffic through the stack of Golgi cisternae. Upon reaching the final compartments of the Golgi, the trans cisterna and trans-Golgi network (TGN), processed glycoproteins and lipids are packaged into coated and non-coated transport carriers derived from the trans Golgi and TGN. The cargoes of clathrin-coated vesicles are chiefly residents of endo-lysosomal organelles, while uncoated carriers ferry cargo to the cell surface. There are outstanding questions regarding the mechanisms of protein and lipid sorting within the Golgi for export to different organelles. Nonetheless, conceptual advances have begun to define the key molecular features of cargo clients and the mechanisms underlying their sorting into distinct export pathways, which we have collated in this Cell Science at a Glance article and the accompanying poster.

A Springboard for physicians returning to practice.

BACKGROUND: Many physicians take time out of training and have decreased confidence and poor performance ratings on their return. Courses employing multiple educational methods have been shown to be effective in easing learners into new clinical roles during transition periods but, to date, there is limited evidence for courses to support trainees returning to practice (RTP). METHODS: A 2-day course, named Springboard, was developed, specifically to address the needs of trainee physicians RTP. It employed a blended, multi-modal approach to learning, including lectures, workshops, case-based sessions, interactive panel discussions, small group teaching, peer-led practical advice sessions and simulation training. Springboard was delivered eight times between 2014 and 2019 with a total of 540 doctors attending. We analysed participant pre-and post-course questionnaire feedback. RESULTS: Reasons for doctors taking time out of training included parental leave, research, fellowships in education and leadership, health-related absence and career breaks. Time out of training ranged between 3 months and 6 years. A significant pre/post-course increase in candidates' self-reported leadership skills and confidence in being prepared to return to practice was demonstrated alongside an appreciation of a multi-modal, 'boot camp' course delivered by expert faculty and a networking experience. DISCUSSION: Dedicated training courses tailored to the needs of physicians RTP provide an opportunity for improving confidence relating to many areas of clinical and non-clinical practice as well as providing an environment for networking and sharing experiences. Further work would be valuable to establish the potential of providing this on a larger scale.

A qPCR-MPN method for rapid quantification of Escherichia coli in bivalve molluscan shellfish.

Faecal contamination of bivalve molluscan shellfish (BMS) can lead to infections from enteric pathogens if consumed. Across Europe, the faecal indicator bacteria Escherichia coli, is used to determine contamination of BMS harvesting areas. The reference most probable number (MPN) method for E. coli in BMS takes around 48 h from sample receipt to result. In this study, an alternative method was developed in which the final, E. coli confirmation step in the MPN method (usually carried out on chromogenic TBX agar) was replaced by presence/absence real-time PCR (qPCR). This qPCR-MPN method was directly compared with the reference TBX-MPN method using 194 BMS samples consisting of mussels (Mytilus spp.), Pacific oysters (Crassostrea gigas) and common cockles (Cerastoderma edule). The qPCR-MPN method correlated positively with the TBX-MPN method (Kendall's tau coefficient = 0.812). However, the strength of this correlation varied between BMS species, with mussels having the poorest correlation (0.677) followed by Pacific oysters (0.795) and common cockles (0.890). There were some samples for which the difference between the two methods was higher than might be expected by statistical probability alone. Variations in the way in which the two confirmation methods work may account for much of this variation. This method may serve as an ad hoc, rapid assessment method that is complementary to the official reference method and could be easily implemented in many official control laboratories.

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2.

SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 5 2020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

Isolation and characterization of potentially pathogenic Vibrio species in a temperate, higher latitude hotspot.

The recent emergence of Vibrio infections at high latitudes represents a clear human health risk attributable to climate change. Here, we investigate the population dynamics of three Vibrio species: Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae within a British coastal estuarine site, with contrasting salinity and temperature regimes during an intense heatwave event. Water samples were collected weekly through the summer of 2018 and 2019 and filtered using membrane filtration and subsequently grown on selective media. Suspected vibrios were confirmed using a conventional species-specific PCR assay and further analysed for potential pathogenic markers. Results showed that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were present at high concentrations throughout both years, with their populations at substantially greater abundances corresponding to conditions of higher water temperatures during the heatwave of 2018 and at lower salinity sites, which is comparable to the results of previous studies. A subset of strains isolated during the extreme heatwave event in 2018 (46 Vibrio parahaemolyticus, 11 Vibrio cholerae and 4 Vibrio vulnificus) were genomically sequenced. Analysis of these 63 sequenced strains revealed a broad phenotypic and genomic diversity of strains circulating in the environment. An analysis of pathogenicity attributes identified a broad array of virulence genes across all three species, including a variety of genes associated with human disease. This study highlights the importance of the need for an increased Vibrio spp. surveillance system in temperate regions and the potential impact warming events such as heatwaves may have on the abundance of potentially pathogenic bacteria in the environment.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Phosphorus Characterization and Contribution from Eroding Streambank Soils of Vermont's Lake Champlain Basin.

Streambank erosion is an important contributor to sediment and nutrient export. This study determined total P (TP), soil-test P (Modified Morgan's, MM-P), and the degree of P saturation (DPS) in eroding riparian soils along four Lake Champlain Basin stream corridors. We investigated the relationship between these data and soil texture and with a series of GIS-derived landscape metrics. We also quantified the potential P load from eroding streambanks using remote sensing. Soil samples were taken from 76 erosion features to a depth of 90 cm on four streams in Chittenden County, Vermont. Mean concentrations of TP and MM-P were similar among the watersheds and through depth. Neither TP, MM-P, nor DPS were well related to texture. Metrics from available spatial databases for parent material, soil series, and landscape position were somewhat useful in predicting TP and MM-P. Eroding streambank soil from 2004 through 2007 in the four streams was estimated to contain from 0.5 to 3.9 Mg of TP and 1.4 to 10.9 kg MM-P. The mean DPS in each watershed was <18% and, along with low MM-P concentrations, suggests that eroded streambanks may act as sinks rather than sources of P. The portion of total nonpoint P export potentially contributed by streambank erosion ranged from 6% in the stream with the lowest erosion rate to 30% in the stream with the highest erosion rate. Based on TP values, the P contribution of these streambanks could be considerable, and more information is needed on their actual contribution to bioavailable P in receiving waters.

Host-pathogen reorganisation during host cell entry by Chlamydia trachomatis.

Chlamydia trachomatis is obligate intracellular bacterial pathogen that remains a significant public health burden worldwide. A critical early event during infection is chlamydial entry into non-phagocytic host epithelial cells. Like other Gram-negative bacteria, C. trachomatis uses a type III secretion system (T3SS) to deliver virulence effector proteins into host cells. These effectors trigger bacterial uptake and promote bacterial survival and replication within the host cell. In this review, we highlight recent cryo-electron tomography that has provided striking insights into the initial interactions between Chlamydia and its host. We describe the polarised structure of extracellular C. trachomatis elementary bodies (EBs), and the supramolecular organisation of T3SS complexes on the EB surface, in addition to the changes in host and pathogen architecture that accompany bacterial internalisation and EB encapsulation into early intracellular vacuoles. Finally, we consider the implications for further understanding the mechanism of C. trachomatis entry and how this might relate to those of other bacteria and viruses.