Arthropods as a source of new RNA viruses.

The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses.

Revisiting the clinal concept of evolution and dispersal for the tick-borne flaviviruses by using phylogenetic and biogeographic analyses.

Tick-borne flaviviruses (TBF) are widely dispersed across Africa, Europe, Asia, Oceania, and North America, and some present a significant threat to human health. Seminal studies on tick-borne encephalitis viruses (TBEV), based on partial envelope gene sequences, predicted a westward clinal pattern of evolution and dispersal across northern Eurasia, terminating in the British Isles. We tested this hypothesis using all available full-length open reading frame (ORF) TBF sequences. Phylogenetic analysis was consistent with current reports. However, linear and nonlinear regression analysis of genetic versus geographic distance combined with BEAST analysis identified two separate clines, suggesting that TBEV spread both east and west from a central point. In addition, BEAST analysis suggested that TBF emerged and dispersed more than 16,000 years ago, significantly earlier than previously predicted. Thus, climatic and ecological changes may have played a greater role in TBF dispersal than humans.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Assessment of gastrointestinal health in racing Alaskan sled dogs using capsule endoscopy and inflammatory cytokines.

OBJECTIVES: Exercise-induced gastrointestinal syndrome occurs in dogs and people and might compromise athlete performance by increasing intestinal permeability and causing gastrointestinal erosions. Racing sled dogs often receive acid suppressant prophylaxis which decreases the incidence of gastric erosions induced by exercise. The objectives were to quantify intestinal injury by measuring serum pro-inflammatory cytokine concentrations before and after exercise and to evaluate gastrointestinal mucosa using video capsule endoscopy after exercise. MATERIALS AND METHODS: Prospective study of 12 racing Alaskan sled dogs receiving approximately 1 mg/kg omeprazole once daily from the day before the race until race completion. Blood was drawn before and 8 to 10 hours after an endurance race for the quantification of cytokines. Gastrointestinal tract mucosa was assessed with video capsule endoscopy immediately post-race. RESULTS: Eight of nine dogs (89%; 95% confidence interval 52 to 100%) had gastric erosions; all dogs (100%, 95% confidence interval 63 to 100%) had small intestinal erosions. Most of the dogs (seven of nine) had straw or foreign material present. Cytokine levels were not different from before to after the race. CLINICAL SIGNIFICANCE: Video capsule endoscopy identified gastrointestinal tract mucosal erosions after exercise in all dogs receiving once-daily omeprazole treatment, though other causes for the lesions besides exercise are possible.

First case of rabbit haemorrhagic disease in Canada: contaminated flying insect, vs. long-term infection hypothesis.

Following the announcement of the first case of rabbit haemorrhagic disease (RHD) in a pet rabbit, housed indoors in Canada for more than 1 year, I submitted an evidence-based explanation to ProMed explaining how RHD might have caused the death of 'one' of the three pet rabbits. I suggested with supporting evidence, that it may have been persistently infected with rabbit haemorrhagic disease virus (RHDV) which may have reactivated to cause the fatal disease. However, in this issue, Peacock et al. have proposed an alternative 'hypothesis' for the appearance of RHD in the pet rabbit. They hypothesise that a non-identified insect or fomite might have become contaminated by a Chinese strain of RHDV somewhere in the US. This insect/fomite then flew or was windborne, from the US to Canada where it entered the house containing three pet rabbits and infected one of them. RHD is non-endemic and is rarely reported in the US, where it has only been observed in domestic European rabbits, held in rabbitries. My proposal was based on the details provided by ProMed, the veterinary report from Canada, where RHDV has never previously been identified and the epidemiological, ecological and evolutionary history of RHDV which includes serological and phylogenetic evidence that ancestral RHDV lineages circulated before 1984. The flying insect hypothesis of Peacock et al. is based on circumstantial evidence and, I believe, has a lower probability of being correct than my evidence-based long-term infection proposal.

The virology, epidemiology, and clinical impact of West Nile virus: a decade of advancements in research since its introduction into the Western Hemisphere.

West Nile virus (WNV) is now endemic in the USA. After the widespread surge of virus activity across the USA, research has flourished, and our knowledge base has significantly expanded over the past 10 years since WNV was first recognized in New York City. This article provides a review of the virology of WNV, history, epidemiology, clinical features, pathology of infection, the innate and adaptive immune response, host risk factors for developing severe disease, clinical sequelae following severe disease, chronic infection, and the future of prevention.

Mathematically aggregating experts' predictions of possible futures.

Structured protocols offer a transparent and systematic way to elicit and combine/aggregate, probabilistic predictions from multiple experts. These judgements can be aggregated behaviourally or mathematically to derive a final group prediction. Mathematical rules (e.g., weighted linear combinations of judgments) provide an objective approach to aggregation. The quality of this aggregation can be defined in terms of accuracy, calibration and informativeness. These measures can be used to compare different aggregation approaches and help decide on which aggregation produces the "best" final prediction. When experts' performance can be scored on similar questions ahead of time, these scores can be translated into performance-based weights, and a performance-based weighted aggregation can then be used. When this is not possible though, several other aggregation methods, informed by measurable proxies for good performance, can be formulated and compared. Here, we develop a suite of aggregation methods, informed by previous experience and the available literature. We differentially weight our experts' estimates by measures of reasoning, engagement, openness to changing their mind, informativeness, prior knowledge, and extremity, asymmetry or granularity of estimates. Next, we investigate the relative performance of these aggregation methods using three datasets. The main goal of this research is to explore how measures of knowledge and behaviour of individuals can be leveraged to produce a better performing combined group judgment. Although the accuracy, calibration, and informativeness of the majority of methods are very similar, a couple of the aggregation methods consistently distinguish themselves as among the best or worst. Moreover, the majority of methods outperform the usual benchmarks provided by the simple average or the median of estimates.

Pathogenic flaviviruses.

Haemorrhagic disease, encephalitis, biphasic fever, flaccid paralysis, and jaundice are typical manifestations of diseases in human beings after infections by mosquito-borne or tick-borne flaviviruses such as yellow fever, dengue, West Nile, St Louis encephalitis, Japanese encephalitis, tick-borne encephalitis, Kyasanur Forest disease, and Omsk haemorrhagic fever. Although the characteristics of these viruses are well defined, they are still unpredictable with increases in disease severity, unusual clinical manifestations, unexpected methods of transmission, long-term persistence, and the discovery of new species. This Seminar will compare the epidemiological and clinical features of the medically important flaviviruses, consider the effect of human activity on their evolution and dispersal, and draw attention to new findings and some of the unanswered questions, unresolved issues, and controversies that remain.

Neurogenesis in the neocortex of adult primates.

In primates, prefrontal, inferior temporal, and posterior parietal cortex are important for cognitive function. It is shown that in adult macaques, new neurons are added to these three neocortical association areas, but not to a primary sensory area (striate cortex). The new neurons appeared to originate in the subventricular zone and to migrate through the white matter to the neocortex, where they extended axons. These new neurons, which are continually added in adulthood, may play a role in the functions of association neocortex.

A low symptomatic form of neurodegeneration in younger carriers of the FMR1 premutation, manifesting typical radiological changes.

Fragile X-associated tremor/ataxia (FXTAS) is a late onset disorder caused by a premutation in the FMR1 gene, in which neurological symptoms are associated with white matter (wm) changes, especially within the middle cerebellar peduncles (MCP sign), seen on magnetic resonance images (MRIs). We report a discrepancy between obvious radiological presentations and minimal clinical involvement in two younger male premutation carriers. These carriers, aged 52 and 39 years, showed distinct MCP sign, but reported no neurological symptoms. If this discrepancy represents the initial stage of FXTAS, our findings suggest the possibility of early diagnosis from MRI scans.

Use of a non-ICU specialty ward for immediate post-operative management of head and neck free flaps; a randomized controlled trial.

OBJECTIVES: Compare length of stay, flap failure rate, medical and surgical complications and cost when patients undergoing head and neck free flap reconstruction are monitored in an intensive care unit (ICU) versus a specialty ward unit postoperatively. MATERIALS AND METHODS: A prospective, non-inferiority, randomized controlled trial was conducted from 7/22/2016 to 9/12/2018 at a single institution. Patients were randomized to the ICU or specialty ward unit. Flap check protocols were identical between the groups. Perioperative and postoperative outcome variables were assessed and compared. RESULTS: 131 patients were enrolled in the study and 118 ultimately underwent head and neck free flap reconstruction. 57 were randomized to the ICU and 61 to the specialty ward unit. There were no significant differences between the ICU and specialty ward unit groups with regard to demographic variables including age, gender, co-morbidities, tobacco or alcohol use, prior chemotherapy or radiation therapy treatment. There were no significant differences in perioperative variables including need for transfusion, tracheostomy, ischemia time, blood loss, fluid administration or post-operative antibiotic use. There was no significant difference in the primary outcome variable, length of stay. There were no significant differences in the number of the medical or surgical complications, flap failure rate, or hospital costs. CONCLUSION: In this prospective, randomized controlled trial, head and neck free-flap patients cared for on a specialty ward in the immediate post-operative period had equivalent outcomes to those cared for in the ICU.

Does antiviral therapy have a role in the control of Japanese encephalitis?

Approximately 2 billion people live in countries where Japanese encephalitis (JE) presents a significant risk to humans and animals, particularly in China and India, with at least 700 million potentially susceptible children. The combined effects of climate change, altered bird migratory patterns, increasing movement of humans, animals and goods, increasing deforestation and development of irrigation projects will inevitably lead to further geographic dispersal of the virus and an enhanced threat. Although most human infections are mild or asymptomatic, some 50% of patients who develop encephalitis suffer permanent neurologic defects, and 25% die. Vaccines have reduced the incidence of JE in some countries. No specific antiviral therapy is currently available. Interferon alpha-2a was tested in a double-blind placebo-controlled trial on children with Japanese encephalitis, but with negative results. There is thus a real need for antivirals that can reduce the toll of death and neurological sequelae resulting from infection with JE virus. Here we briefly review the epidemiological problems presented by this virus, the present state of drug development and the contributory role that antiviral therapy might play in developing future control strategies for JE.

The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Preliminary evaluation of exotic tick species and exotic pathogens imported on migratory birds into the British Isles.

Field studies were carried out to determine whether ticks are being imported into the British Isles on migratory birds. During spring and autumn migration 2004, ticks were collected from ringed birds at 11 bird observatories and 3 inland Riparia riparia colonies. A total of 38 ticks of 4 species (Ixodes ricinus, I. frontalis, I. lividus, I. arboricola) were collected from 12 species of bird. Ticks were tested for viruses in the Flavivirus and Nairovirus genera, with no positives found. This data demonstrates that ticks are being imported into the British Isles on migratory birds with future work recommended to determine the quantity of ticks imported and to detect low prevalence pathogens.

Learning enhances adult neurogenesis in the hippocampal formation.

Thousands of hippocampal neurons are born in adulthood, suggesting that new cells could be important for hippocampal function. To determine whether hippocampus-dependent learning affects adult-generated neurons, we examined the fate of new cells labeled with the thymidine analog bromodeoxyuridine following specific behavioral tasks. Here we report that the number of adult-generated neurons doubles in the rat dentate gyrus in response to training on associative learning tasks that require the hippocampus. In contrast, training on associative learning tasks that do not require the hippocampus did not alter the number of new cells. These findings indicate that adult-generated hippocampal neurons are specifically affected by, and potentially involved in, associative memory formation.

The effect of ketamine/xylazine and carbon dioxide on plasma luteinizing hormone releasing hormone and testosterone concentrations in the male Norway rat.

The effect of a commonly used anaesthetic, ketamine/xylazine and/or carbon dioxide (CO(2)) on plasma luteinizing hormone releasing hormone (LHRH) and testosterone concentrations was determined in male Sprague-Dawley rats. These values were compared with values obtained from pre-anaesthetic control samples. Ketamine/xylazine treatment did not significantly affect testosterone concentrations. In contrast, LHRH started to decrease one hour after ketamine/xylazine administration and continued to significantly decrease after 24 h. In addition, in the CO(2) euthanasia-only group, LHRH concentrations were also significantly decreased. These results suggest that ketamine/xylazine anaesthesia followed by CO(2) euthanasia 24 h later is exerting a significant effect on LHRH concentrations 24 h after anaesthetizing, while only having a slight effect on testosterone, and that CO(2) is exerting an immediate significant effect on LHRH. In conclusion, LHRH analysis should be avoided after ketamine/xylazine anaesthesia and CO(2) euthanasia.

Exploratory re-encoding of yellow fever virus genome: new insights for the design of live-attenuated viruses.

Virus attenuation by genome re-encoding is a pioneering approach for generating effective live-attenuated vaccine candidates. Its core principle is to introduce a large number of synonymous substitutions into the viral genome to produce stable attenuation of the targeted virus. Introduction of large numbers of mutations has also been shown to maintain stability of the attenuated phenotype by lowering the risk of reversion and recombination of re-encoded genomes. Identifying mutations with low fitness cost is pivotal as this increases the number that can be introduced and generates more stable and attenuated viruses. Here, we sought to identify mutations with low deleterious impact on the in vivo replication and virulence of yellow fever virus (YFV). Following comparative bioinformatic analyses of flaviviral genomes, we categorised synonymous transition mutations according to their impact on CpG/UpA composition and secondary RNA structures. We then designed seventeen re-encoded viruses with 100-400 synonymous mutations in the NS2A-to-NS4B coding region of YFV Asibi and Ap7M (hamster-adapted) genomes. Each virus contained a panel of synonymous mutations designed according to the above categorisation criteria. The replication and fitness characteristics of parent and re-encoded viruses were compared in vitro using cell culture competition experiments. In vivo laboratory hamster models were also used to compare relative virulence and immunogenicity characteristics. Most of the re-encoded strains showed no decrease in replicative fitness in vitro. However, they showed reduced virulence and, in some instances, decreased replicative fitness in vivo. Importantly, the most attenuated of the re-encoded strains induced robust, protective immunity in hamsters following challenge with Ap7M, a virulent virus. Overall, the introduction of transitions with no or a marginal increase in the number of CpG/UpA dinucleotides had the mildest impact on YFV replication and virulence in vivo. Thus, this strategy can be incorporated in procedures for the finely tuned creation of substantially re-encoded viral genomes.

The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

Pseudocowpox virus: the etiological agent of contagious ecthyma (Auzdyk) in camels (Camelus dromedarius) in the Arabian peninsula.

We have determined the nucleotide sequence of DNA extracted from pustules, saliva, and blood of camels presenting with contagious ecthyma, in Bahrain and also from a sample (SACamel) of infected tissue from a camel that had presented with contagious ecthyma in 1998 in Saudi Arabia (1). Sequence homologies and phylogenetic analysis showed that this extracted DNA was more closely related to Pseudocowpox virus (PCPV) than Orf virus (ORFV), which infects sheep, goats, and other animal species. The phylogeny also demonstrated that PCPV in Arabian camels was phylogenetically distinct from, and circulates independently of, ruminant-associated PCPV from Europe.

Tremor/ataxia syndrome and fragile X premutation: diagnostic caveats.

The fragile X-associated tremor/ataxia syndrome (FXTAS) is a newly discovered late-onset neurodegenerative disorder caused by a premutation in the FMR1 X-linked gene. We present examples of a discrepancy between obvious brain changes observed on MRI, and minimal clinical neurological manifestations in three older carriers of this premutation. This discrepancy occurred in three of nine carriers ascertained in an unbiased manner. If the systematic follow-up studies of adult carriers confirm this trend, this will have an impact on early diagnosis of neurological involvement and possible prevention. If MRI changes precede clinical manifestation of FXTAS this may explain the low detection rate of fragile X carriers among patients with neurological syndromes associated with tremor/ataxia.