Diversity, evolution, and emergence of fish viruses.

The production of aquatic animals has more than doubled over the last 50 years and is anticipated to continually increase. While fish are recognized as a valuable and sustainable source of nutrition, particularly in the context of human population growth and climate change, the rapid expansion of aquaculture coincides with the emergence of highly pathogenic viruses that often spread globally through aquacultural practices. Here, we provide an overview of the fish virome and its relevance for disease emergence, with a focus on the insights gained through metagenomic sequencing, noting potential areas for future study. In particular, we describe the diversity and evolution of fish viruses, for which the majority have no known disease associations, and demonstrate how viruses emerge in fish populations, most notably at an expanding domestic-wild interface. We also show how wild fish are a powerful and tractable model system to study virus ecology and evolution more broadly and can be used to identify the major factors that shape vertebrate viromes. Central to this is a process of virus-host co-divergence that proceeds over many millions of years, combined with ongoing cross-species virus transmission.

Host adaptive radiation is associated with rapid virus diversification and cross-species transmission in African cichlid fishes.

Adaptive radiations are generated through a complex interplay of biotic and abiotic factors. Although adaptive radiations have been widely studied in the context of animal and plant evolution, little is known about how they impact the evolution of the viruses that infect these hosts, which in turn may provide insights into the drivers of cross-species transmission and hence disease emergence. We examined how the rapid adaptive radiation of the cichlid fishes of African Lake Tanganyika over the last 10 million years has shaped the diversity and evolution of the viruses they carry. Through metatranscriptomic analysis of 2,242 RNA sequencing libraries, we identified 121 vertebrate-associated viruses among various tissue types that fell into 13 RNA and 4 DNA virus groups. Host-switching was commonplace, particularly within the Astroviridae, Metahepadnavirus, Nackednavirus, Picornaviridae, and Hepacivirus groups, occurring more frequently than in other fish communities. A time-calibrated phylogeny revealed that hepacivirus diversification was not constant throughout the cichlid radiation but accelerated 2-3 million years ago, coinciding with a period of rapid cichlid diversification and niche packing in Lake Tanganyika, thereby providing more closely related hosts for viral infection. These data depict a dynamic virus ecosystem within the cichlids of Lake Tanganyika, characterized by rapid virus diversification and frequent host jumping, and likely reflecting their close phylogenetic relationships that lower the barriers to cross-species virus transmission.

Dark Chocolate Intake Positively Modulates Gut Permeability in Elite Football Athletes: A Randomized Controlled Study.

Gut barrier disruption can lead to enhanced intestinal permeability, which allows endotoxins, pathogens, and other proinflammatory substances to move through the intestinal barrier into circulation. Intense exercise over a prolonged period increases intestinal permeability, which can be further worsened by the increased production of reactive oxygen species (ROS) and pro-inflammatory cytokines. The aim of this study was to assess the degree of intestinal permeability in elite football players and to exploit the effect of cocoa polyphenols on intestinal permeability induced by intensive physical exercise. Biomarkers of intestinal permeability, such as circulating levels of zonulin, a modulator of tight junctions, occludin, a tight junction protein, and LPS translocation, were evaluated in 24 elite football players and 23 amateur athletes. Moreover, 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (n = 12) or a control group (n = 12) for 30 days in a randomized controlled trial. Biochemical analyses were performed at baseline and after 30 days of chocolate intake. Compared to amateur athletes, elite football players showed increased intestinal permeability as indicated by higher levels of zonulin, occludin, and LPS. After 30 days of dark chocolate intake, decreased intestinal permeability was found in elite athletes consuming dark chocolate. In the control group, no changes were observed. In vitro, polyphenol extracts significantly improved intestinal damage in the human intestinal mucosa cell line Caco-2. These results indicate that chronic supplementation with dark chocolate as a rich source of polyphenols positively modulates exercise-induced intestinal damage in elite football athletes.

Limited cross-species virus transmission in a spatially restricted coral reef fish community.

The Great Barrier Reef (GBR)-the largest coral reef ecosystem in the world-supports over 1,200 fish species with some of the highest population densities and diversities observed in vertebrates, offering a high potential for virus transmission among species. As such, the GBR represents an exceptional natural ecosystem to determine the impact of host community diversity on virus evolution and emergence. In recent decades, the GBR has also experienced significant threats of extinction, making it one of the most vulnerable ecosystems on the planet. Despite the global importance of the GBR, our understanding of virus diversity and connectivity in tropical reef fishes remains poor. Here, we employed metatranscriptomic sequencing to reveal the viromes of sixty-one reef fish species. This identified transcripts representing 132 putative viral sequences, 38 of which exhibited strong phylogenetic relationships with known vertebrate-associated viral genera, including a novel Santee-Cooper ranavirus (Iridoviridae). We found little evidence for virus transmission between fish species living within a very restricted geographical space-a 100-m2 coral reef ecosystem-suggesting that there might be important host barriers to successful cross-species transmission despite regular exposure. We also identified differences in virome composition among reef fish families, such that cryptobenthic reef fishes-characterized by small body sizes and short life spans-exhibited greater virome richness compared to large reef fishes. This study suggests that there are important barriers to cross-species virus transmission and that successful emergence in a reef fish community likely requires active host adaptation, even among closely related host species.

Transcriptome mining extends the host range of the Flaviviridae to non-bilaterians.

The flavivirids (family Flaviviridae) are a group of positive-sense RNA viruses that include well-documented agents of human disease. Despite their importance and ubiquity, the timescale of flavivirid evolution is uncertain. An ancient origin, spanning millions of years, is supported by their presence in both vertebrates and invertebrates and by the identification of a flavivirus-derived endogenous viral element in the peach blossom jellyfish genome (Craspedacusta sowerbii, phylum Cnidaria), implying that the flaviviruses arose early in the evolution of the Metazoa. To date, however, no exogenous flavivirid sequences have been identified in these hosts. To help resolve the antiquity of the Flaviviridae, we mined publicly available transcriptome data across the Metazoa. From this, we expanded the diversity within the family through the identification of 32 novel viral sequences and extended the host range of the pestiviruses to include amphibians, reptiles, and ray-finned fish. Through co-phylogenetic analysis we found cross-species transmission to be the predominate macroevolutionary event across the non-vectored flavivirid genera (median, 68 per cent), including a cross-species transmission event between bats and rodents, although long-term virus-host co-divergence was still a regular occurrence (median, 23 per cent). Notably, we discovered flavivirus-like sequences in basal metazoan species, including the first associated with Cnidaria. This sequence formed a basal lineage to the genus Flavivirus and was closer to arthropod and crustacean flaviviruses than those in the tamanavirus group, which includes a variety of invertebrate and vertebrate viruses. Combined, these data attest to an ancient origin of the flaviviruses, likely close to the emergence of the metazoans 750-800 million years ago.

Genetic Reassortment between Endemic and Introduced Macrobrachium rosenbergii Nodaviruses in the Murray-Darling Basin, Australia.

Macrobrachium rosenbergii nodavirus (MrNV)-the aetiological agent of white tail disease-is a major limiting factor of crustacean aquaculture as it causes up to 100% mortality in M. rosenbergii larvae and juveniles. Despite the importance of MrNV, there have been few studies on the phylogenetic diversity and geographic range of this virus in Australian waterways. Here, we detected MrNV genomes in common carp (Cyprinus carpio) metatranscriptomes sampled at five freshwater sites across the Murray-Darling Basin (MDB), Australia. We identified genetic divergence of the RNA-dependent RNA polymerase gene between MrNV sequences identified in the northern and southern rivers of the MDB. Northern viruses exhibited strong phylogenetic clustering with MrNV from China, whereas the southern viruses were more closely related to MrNV from Australia. However, all five viruses were closely related in the capsid protein, indicative of genetic reassortment of the RNA1 and RNA2 segments between Australian and introduced MrNV. In addition, we identified Macrobrachium australiense in two of the five MrNV-positive libraries, suggesting that these species may be important reservoir hosts in the MDB. Overall, this study reports the first occurrence of MrNV outside of the Queensland region in Australia and provides evidence for genetic reassortment between endemic and introduced MrNV.

Platelet Activation Favours NOX2-Mediated Muscle Damage in Elite Athletes: The Role of Cocoa-Derived Polyphenols.

Mechanisms of exercise-induced muscle injury with etiopathogenesis and its consequences have been described; however, the impact of different intensities of exercise on the mechanisms of muscular injury development is not well understood. The aim of this study was to exploit the relationship between platelet activation, oxidative stress and muscular injuries induced by physical exercise in elite football players compared to amateur athletes. Oxidant/antioxidant status, platelet activation and markers of muscle damage were evaluated in 23 elite football players and 23 amateur athletes. Compared to amateurs, elite football players showed lower antioxidant capacity and higher oxidative stress paralleled by increased platelet activation and muscle damage markers. Simple linear regression analysis showed that sNOX2-dp and H2O2, sCD40L and PDGF-bb were associated with a significant increase in muscle damage biomarkers. In vitro studies also showed that plasma obtained from elite athletes increased oxidative stress and muscle damage in human skeletal muscle myoblasts cell line compared to amateurs' plasma, an effect blunted by the NOX2 inhibitor or by the cell treatment with cocoa-derived polyphenols. These results indicate that platelet activation increased muscular injuries induced by oxidative stress. Moreover, NOX2 inhibition and polyphenol extracts treatment positively modulates redox status and reduce exercise-induced muscular injury.

Cardiovascular effects of COVID-19 lockdown in professional football players.

BACKGROUND: The COVID-19 pandemic with the stay-at-home orders and lockdown has dramatically forced athletes to stop team training and competitions, causing deep changes in habits and lifestyle. Aim of this study was to evaluate in a retrospective single center study the cardiovascular (CV) health and fitness of elite football player after COVID-19 lockdown in Italy and to compare such findings with the 2019 off-season period, in order to identify potential differences in the CV features and outcomes. METHODS: All 29 professional football players of the first male team were enrolled before resuming training and competition after COVID-19 lockdown and underwent several exams including physical examination, resting and stress electrocardiography (ECG), echocardiography, spirometry and blood tests. RESULTS: Median age was 27 years (23; 31), with no athlete being COVID-19 positive at the time of the evaluation. In comparison with the usual off-season 2-month detraining, significant differences were found for left ventricular (LV) mass (189 g [172; 212] vs. 181 g [167; 206], P=0.024) and LV Mass Index for body surface area (94 g/m2 [85; 104] vs. 88 g/m2 [79.5; 101.5], P=0.017), while LV mass/fat free mass remained unchanged (2.8 g/kg [2.6; 2.9] vs. 2.9 g/kg [2.6; 3.2], P=0.222). Respiratory function and metabolic profile were improved, while no significant changes were found in ECG findings, at rest and during exercise. CONCLUSIONS: Prolonged abstinence from training and competitions induced by lockdown elicited significant changes in comparison with off-season in parameters ascribable to detraining, as the changes in LV mass, in respiratory function and in metabolic profile.

Slippery when wet: cross-species transmission of divergent coronaviruses in bony and jawless fish and the evolutionary history of the Coronaviridae.

The Nidovirales comprise a genetically diverse group of positive-sense single-stranded RNA virus families that infect a range of invertebrate and vertebrate hosts. Recent metagenomic studies have identified nido-like virus sequences, particularly those related to the Coronaviridae, in a range of aquatic hosts including fish, amphibians, and reptiles. We sought to identify additional members of the Coronaviridae in both bony and jawless fish through a combination of total RNA sequencing (meta-transcriptomics) and data mining of published RNA sequencing data and from this reveal more of the long-term patterns and processes of coronavirus evolution. Accordingly, we identified a number of divergent viruses that fell within the Letovirinae subfamily of the Coronaviridae, including those in a jawless fish-the pouched lamprey. By mining fish transcriptome data, we identified additional virus transcripts matching these viruses in bony fish from both marine and freshwater environments. These new viruses retained sequence conservation in the RNA-dependant RNA polymerase across the Coronaviridae but formed a distinct and diverse phylogenetic group. Although there are broad-scale topological similarities between the phylogenies of the major groups of coronaviruses and their vertebrate hosts, the evolutionary relationship of viruses within the Letovirinae does not mirror that of their hosts. For example, the coronavirus found in the pouched lamprey fell within the phylogenetic diversity of bony fish letoviruses, indicative of past host switching events. Hence, despite possessing a phylogenetic history that likely spans the entire history of the vertebrates, coronavirus evolution has been characterised by relatively frequent cross-species transmission, particularly in hosts that reside in aquatic habitats.

Erratum: Virome composition in marine fish revealed by meta-transcriptomics.

[This corrects the article DOI: 10.1093/ve/veab005.].

Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia.

Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to ninety per cent of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control agent. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of thirty-six RNA libraries representing ten species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified eighteen vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further twenty-seven that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fish sampled here, suggesting that there is little virus transmission from common carp to native fish species, despite co-existing for over fifty years. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.

Virome composition in marine fish revealed by meta-transcriptomics.

Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterized the viromes of nineteen wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterization. From this, we identified twenty-five new vertebrate-associated viruses and a further twenty-two viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, negative-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae, and Flaviviridae, and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition-viral richness, abundance, and diversity-can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi, and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

Articular-sided rotator cuff tears: which is the best repair? A three-year prospective randomised controlled trial.

PURPOSE: To compare two groups of patients who underwent two different arthroscopic procedures for repair of articular-sided partial-thickness rotator cuff tears (PTRCTs). MATERIALS: This is a comparative prospective study of two methods for repair of partial cuff tears: Thirty-two patients underwent arthroscopic rotator cuff repair with a transtendon technique (group 1); 28 underwent arthroscopic full-thickness conversion and repair of the lesion (group 2). ROM measures, clinical findings, MRI features (tendon healing and re-tear), Constant-Murley and ASES scores were assessed pre- and postoperatively and compared. Patients were also asked about return to sport and level of activity. RESULTS: At the last appointment, patients of both the groups were significantly improved for clinical findings, ROM measures, imaging features, Constant-Murley and ASES scores than at baseline, without any significant inter-group difference. In group 1, 15 of 20 patients (75 %) who practiced recreational sport activities had returned to sport at the same level as before the onset of symptoms, without any discomfort. In group 2, 12 of 18 patients (67 %) had returned to the same level of sport activity they practiced before symptoms. At the last follow up, MRI showed rotator cuff healing in 31 patients of Group 1 and 27 patients of Group 2 (p = 0.83). CONCLUSIONS: The two procedures are safe, effective, and comparable.

Cross-cultural adaptation and validation with reliability, validity, and responsiveness of the Italian version of the Oxford Hip Score in patients with hip osteoarthritis.

PURPOSE: The purpose of this study was to translate the Oxford Hip Score (OHS) into Italian and to evaluate the psychometric properties by testing the feasibility, internal consistency, reproducibility, construct validity, and responsiveness in patients with hip osteoarthritis (OA). METHODS: The OHS Italian version was developed and evaluated in Italian-speaking patients with hip OA. The psychometric properties assessed were feasibility with floor and ceiling effect; internal consistency with Cronbach's Alpha; reproducibility with intraclass correlation coefficient (ICC); construct validity with evaluating correlation with the SF-36 and visual analogue scale (VAS) for pain; responsiveness to intra-articular hyaluronate injections. The standardized effect size (ES) and standardized response mean (SRM) were also evaluated. RESULTS: A total of 96 patients were recruited (mean age 62.7 ± 5.8 years, range 49-77). No floor or ceiling effect was observed prior to or after treatment with HA injections. Cronbach's alpha value was 0.85 at baseline and 0.89 after treatment. The reproducibility was good with an ICC of 0.89. A strong correlation was found between OHS, VAS, and some SF-36 subscales, indicating good cross-sectional and longitudinal construct validity. The mean OHS improved from 16.4 ± 5.8 to 28.0 ± 9.0 after hyaluronate injections (P < 0.01). The ES and SRM values were 1.98 and 1.12, respectively. CONCLUSIONS: The Italian OHS questionnaire is valid, reliable, and responsive for use in Italian patients with symptomatic hip OA receiving hyaluronate injections.

Effectiveness of a mucolythic agent as a local adjuvant in revision lumbar spine surgery.

Aim of this prospective randomized trial was to analyze the effectiveness of MESNA in chemical dissection of peridural fibrosis in patients who underwent revision lumbar spine surgery. Between January 2003 and October 2006, 30 patients who underwent revision lumbar spine surgery were enrolled in the study. Patients were randomly assigned to one of two groups: a study group (A) and a control group (B). Once peridural fibrosis was exposed, MESNA (Uromixetan MESNA, 50 mg/ml) was intraoperatively applied on the fibrous tissue (Group A) to ease tissue dissection and enter the canal. In patients of Group B, saline solution was used. Surgical time, preoperative and 1 week postoperative hemoglobin (Hb), length of hospitalization (days), and incidence of perioperative complications were evaluated. The blinded surgeon assigned the surgeries to one of four categories as none, minimal, moderate, and severe basing on intraoperative difficulty in dissecting the fibrous tissue and intraoperative bleeding. Statistical analysis used chi-square analysis to evaluate the difference in surgery difficulty and the incidence of intraoperative complications between the two groups. The analysis of surgical time and hemoglobin levels was performed using a one-sample Wilcoxon test and Mann-Whitney U test. Patients in whom MESNA was used intraoperatively (Group A) presented better intraoperative and perioperative parameters with respect to the control group. Average surgical time and decrease in Hb postoperatively were more in the saline group (B) respect to MESNA (A) (P = 0.004 and P = 0.001, respectively), while no difference in average hospital stay was reported between the two groups. Surgeon-blinded intraoperative report on surgical difficulty showed a significant difference between the two groups (P < 0.05). Postoperatively, no complications directly attributable to the use of MESNA were experienced. The incidence of dural tears and intraoperative bleeding from epidural veins were significantly less in Group A with respect to the control group. MESNA contributed significantly to reduce the operative complications, with a diminution of the surgical time and the grade of difficult for the surgeon, confirming its ability as chemical dissector also for epidural fibrosis in revision lumbar spine surgery.