Influence of an extreme event-the COVID-19 pandemic-On establishment of and data collection by a citizen science project.

The recent rising incidence of extreme natural events may significantly influence the implementation of citizen science projects, including the success of outreach strategies and the quality and scope of data collection. The MassMammals Watch and subsidiary MassBears citizen science projects, initiated during the height of the pandemic, recruit volunteers to submit sightings of black bears and other mammals. In this study, we evaluated the methods we employed for engaging and retaining community volunteers during a period of intense social restrictions, and we assessed whether such conditions were associated with spatial biases in our collected data. Newspaper features were more likely to recruit volunteers who engaged with the project multiple times, but social media and internet presence were important for reaching a larger audience. Bear sighting submissions peaked in number and were more likely to be in forested areas during 2020, the height of the pandemic, compared to later years, a pattern which we suggest stems from an increased desire to participate in outdoor activities in light of social distancing measures during that year. Such shifts in patterns of data collection are likely to continue, particularly in response to increasing extreme weather events associated with climate change. Here, we both make recommendations on optimal outreach strategies for others initiating citizen science programs and illustrate the importance of assessing potential biases in data collection imposed by extreme circumstances.

Results of the 2020 Genomic Proficiency Test for the network of European Union Reference Laboratory for Antimicrobial Resistance assessing whole-genome-sequencing capacities.

The global surveillance and outbreak investigation of antimicrobial resistance (AMR) is amidst a paradigm shift from traditional biology to bioinformatics. This is due to developments in whole-genome-sequencing (WGS) technologies, bioinformatics tools, and reduced costs. The increased use of WGS is accompanied by challenges such as standardization, quality control (QC), and data sharing. Thus, there is global need for inter-laboratory WGS proficiency test (PT) schemes to evaluate laboratories' capacity to produce reliable genomic data. Here, we present the results of the first iteration of the Genomic PT (GPT) organized by the Global Capacity Building Group at the Technical University of Denmark in 2020. Participating laboratories sequenced two isolates and corresponding DNA of Salmonella enterica, Escherichia coli and Campylobacter coli, using WGS methodologies routinely employed at their laboratories. The participants' ability to obtain consistently good-quality WGS data was assessed based on several QC WGS metrics. A total of 21 laboratories from 21 European countries submitted WGS and meta-data. Most delivered high-quality sequence data with only two laboratories identified as overall underperforming. The QC metrics, N50 and number of contigs, were identified as good indicators for high-sequencing quality. We propose QC thresholds for N50 greater than 20 000 and 25 000 for Campylobacter coli and Escherichia coli, respectively, and number of contigs >200 bp greater than 225, 265 and 100 for Salmonella enterica, Escherichia coli and Campylobacter coli, respectively. The GPT2020 results confirm the importance of systematic QC procedures, ensuring the submission of reliable WGS data for surveillance and outbreak investigation to meet the requirements of the paradigm shift in methodology.

Daily life coping-Helping stress-afflicted people manage everyday activities.

BACKGROUND: Symptoms of stress are widespread in the working population and associated with long-term sickness absence and poor work functioning. Occupational therapy (OT)-based interventions are effective in improving return-to-work (RTW)-rates in stress-afflicted long-term sickness benefit beneficiaries (SBBs). No Danish standard on OT-RTW for stress-afflicted SSBs exists. AIMS: To describe and evaluate the feasibility of a seven-week OT-RTW intervention for stress-afflicted SBBs. MATERIALS AND METHODS: Daily Life Coping (DLC) utilizes peer-learning and salutogenetic perspectives to address occupational imbalances experienced by long-term SBBs. Stress-afflicted SBBs (n = 54) in the municipality of Hilleroed were enrolled. Self-efficacy and well-being were measured before and after intervention using the general self-efficacy (GSE) scale and WHO-5 Well-Being Index (WHO-5). RTW-rates were measured at one-year follow-up. Participant feedback was collected using standardized evaluation forms. RESULTS: Following the intervention, WHO-5 improved by 13.7 ± 16.5 points and GSE improved by 4.3 ± 4.8 points. Participant feedback was positive. At one-year follow-up, employment status data were available for 51 SSBs of these 58.8% achieved employment. High post-test GSE and WHO-5 scores were associated with employment at follow-up. CONCLUSION: DLC led to significant improvements in self-efficacy and well-being. Preliminary results indicate benefits on RTW-rates. SIGNIFICANCE: This study provides evidence of the feasibility of DLC in a municipal setting.

The N-terminal region (VP4) of the foot-and-mouth disease capsid precursor (P1-2A) is not required during its synthesis to allow subsequent processing by the 3C protease.

The capsid precursor (P1-2A) of foot-and-mouth disease virus is processed by the 3C protease (3Cpro) to VP0, VP3 and VP1 plus 2A. During capsid assembly, the VP0 is cleaved to VP4 plus VP2. Single amino acid changes in a conserved motif (YCPRP) near the C-terminus of VP1 can block processing of the capsid precursor by the 3Cpro, although the cleavage sites are located hundreds of amino acids distant from this motif, presumably due to misfolding. In contrast, we show here that the absence of the VP4 sequence during the synthesis of the capsid precursor does not affect its subsequent processing. Cleavage of this truncated precursor by 3Cpro at the VP3/VP1 and VP2/VP3 junctions occurred efficiently. Thus, in contrast to the presence of the YCPRP motif in VP1, there are no critical motifs near the N-terminus of the precursor, within VP4, required for correct cleavage by 3Cpro.

Diploidy within a Haploid Genus of Entomopathogenic Fungi.

Fungi in the genus Metarhizium are soil-borne plant-root endophytes and rhizosphere colonizers, but also potent insect pathogens with highly variable host ranges. These ascomycete fungi are predominantly asexually reproducing and ancestrally haploid, but two independent origins of persistent diploidy within the Coleoptera-infecting Metarhizium majus species complex are known and has been attributed to incomplete chromosomal segregation following meiosis during the sexual cycle. There is also evidence for infrequent sexual cycles in the locust-specific pathogenic fungus Metarhizium acridum (Hypocreales: Clavicipitaceae), which is an important entomopathogenic biocontrol agent used for the control of grasshoppers in agricultural systems as an alternative to chemical control. Here, we show that the genome of the M. acridum isolate ARSEF 324, which is formulated and commercially utilized is functionally diploid. We used single-molecule real-time sequencing technology to complete a high-quality assembly of ARSEF 324. K-mer frequencies, intragenomic collinearity between contigs and single nucleotide variant read depths across the genome revealed the first incidence of diploidy described within the species M. acridum. The haploid assembly of 44.7 Mb consisted of 20.8% repetitive elements, which is the highest proportion described of any Metarhizium species. The long-read diploid genome assembly sheds light on past research on this strain, such as unusual high UVB tolerance. The data presented here could fuel future investigation into the fitness landscape of fungi with infrequent sexual reproduction and aberrant ploidy levels, not least in the context of biocontrol agents.

Foot-and-mouth disease virus: Prospects for using knowledge of virus biology to improve control of this continuing global threat.

Understanding of the biology of foot-and-mouth disease virus (FMDV) has grown considerably since the nucleotide sequence of the viral RNA was determined. The ability to manipulate the intact genome and also to express specific parts of the genome individually has enabled detailed analyses of viral components, both RNA and protein. Such studies have identified the requirements for specific functional elements for virus replication and pathogenicity. Furthermore, information about the functions of individual virus proteins has enabled the rational design of cDNA cassettes to express non-infectious empty capsid particles that can induce protective immunity in the natural host animals and thus represent new vaccine candidates. Similarly, attempts to block specific virus activities using antiviral agents have also been performed. However, currently, only the well-established, chemically inactivated FMDV vaccines are commercially available and suitable for use to combat this important disease of livestock animals. These vaccines, despite certain shortcomings, have been used very successfully (e.g. in Europe) to control the disease but it still remains endemic in much of Africa, southern Asia and the Middle East. Hence there remains a significant risk of reintroduction of the disease into highly susceptible animal populations with enormous economic consequences.

Identification of plasticity and interactions of a highly conserved motif within a picornavirus capsid precursor required for virus infectivity.

The picornavirus family includes poliovirus (PV) (genus: enterovirus), human rhinoviruses (enterovirus) and foot-and-mouth disease virus (FMDV) (aphthovirus). These are responsible for important human and animal health concerns worldwide including poliomyelitis, the common cold and foot-and-mouth disease (FMD) respectively. In picornavirus particles, the positive-sense RNA genome (ca. 7-9 kb) is packaged within a protein shell (capsid) usually consisting of three surface exposed proteins, VP1, VP2 and VP3 plus the internal VP4, which are generated following cleavage of the capsid precursor by a virus-encoded protease. We have previously identified a motif near the C-terminus of FMDV VP1 that is required for capsid precursor processing. This motif is highly conserved among other picornaviruses, and is also likely to be important for their capsid precursor processing. We have now determined the plasticity of residues within this motif for virus infectivity and found an important interaction between FMDV residue VP1 R188 within this conserved motif and residue W129 in VP2 that is adjacent in the virus capsid. The FMDV (VP1 R188A) mutant virus has only been rescued with the secondary substitution VP2 W129R. This additional change compensates for the defect resulting from the VP1 R188A substitution and restored both capsid precursor processing and virus viability.

Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites.

Many picornaviruses cause important diseases in humans and other animals including poliovirus, rhinoviruses (causing the common cold) and foot-and-mouth disease virus (FMDV). These small, non-enveloped viruses comprise a positive-stranded RNA genome (ca. 7-9 kb) enclosed within a protein shell composed of 60 copies of three or four different capsid proteins. For the aphthoviruses (e.g. FMDV) and cardioviruses, the capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to generate VP0, VP3 and VP1 plus 2A. For enteroviruses, e.g. poliovirus, the capsid precursor is P1 alone, which is cleaved by the 3CD protease to generate just VP0, VP3 and VP1. The sequences required for correct processing of the FMDV capsid protein precursor in mammalian cells were analyzed. Truncation of the P1-2A precursor from its C-terminus showed that loss of the 2A peptide (18 residues long) and 27 residues from the C-terminus of VP1 (211 residues long) resulted in a precursor that cannot be processed by 3Cpro although it still contained two unmodified internal cleavage sites (VP0/VP3 and VP3/VP1 junctions). Furthermore, introduction of small deletions within P1-2A identified residues 185-190 within VP1 as being required for 3Cpro-mediated processing and for optimal accumulation of the precursor. Within this C-terminal region of VP1, five of these residues (YCPRP), are very highly conserved in all FMDVs and are also conserved amongst other picornaviruses. Mutant FMDV P1-2A precursors with single amino acid substitutions within this motif were highly resistant to cleavage at internal junctions. Such substitutions also abrogated virus infectivity. These results can explain earlier observations that loss of the C-terminus (including the conserved motif) from the poliovirus capsid precursor conferred resistance to processing. Thus, this motif seems essential for maintaining the correct structure of picornavirus capsid precursors prior to processing and subsequent capsid assembly; it may represent a site that interacts with cellular chaperones.

Cleavages at the three junctions within the foot-and-mouth disease virus capsid precursor (P1-2A) by the 3C protease are mutually independent.

The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3Cpro and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3Cpro while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3Cpro independently.

Spatial genetic structure in American black bears (Ursus americanus): female philopatry is variable and related to population history.

Previously, American black bears (Ursus americanus) were thought to follow the pattern of female philopatry and male-biased dispersal. However, recent studies have identified deviations from this pattern. Such flexibility in dispersal patterns can allow individuals greater ability to acclimate to changing environments. We explored dispersal and spatial genetic relatedness patterns across ten black bear populations-including long established (historic), with known reproduction >50 years ago, and newly established (recent) populations, with reproduction recorded <50 years ago-in the Interior Highlands and Southern Appalachian Mountains, United States. We used spatially explicit, individual-based genetic simulations to model gene flow under scenarios with varying levels of population density, genetic diversity, and female philopatry. Using measures of genetic distance and spatial autocorrelation, we compared metrics between sexes, between population types (historic and recent), and among simulated scenarios which varied in density, genetic diversity, and sex-biased philopatry. In empirical populations, females in recent populations exhibited stronger patterns of isolation-by-distance (IBD) than females and males in historic populations. In simulated populations, low-density populations had a stronger indication of IBD than medium- to high-density populations; however, this effect varied in empirical populations. Condition-dependent dispersal strategies may permit species to cope with novel conditions and rapidly expand populations. Pattern-process modeling can provide qualitative and quantitative means to explore variable dispersal patterns, and could be employed in other species, particularly to anticipate range shifts in response to changing climate and habitat conditions.

Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

Expression study of GLUT4 translocation-related genes in a porcine pre-diabetic model.

Obesity is a world-wide exponentially growing health problem that increases the risk of co-morbidities including metabolic syndrome, pre-diabetes, Type 2 Diabetes Mellitus (T2DM), and cancer. These co-morbidities are all complex conditions constituting a big challenge when searching for susceptibility genes. Identification of relevant genes, which could contribute to an earlier identification of individuals prone to develop diabetes, is urgently needed as many long-term complications can be avoided by preventive measures. Pre-diabetes is mainly associated with hyperglycemia; thus studying this phenotype might provide knowledge on relevant genes implicated in molecular mechanisms underlying pre-diabetes, and contributing to the development of T2DM. In the present study, two groups of pigs with high (HGG, N=6) and low (NGG, N=6) fasting plasma glucose level respectively were selected from a large pig population. Transcriptional levels of seven genes involved in the glucose transporter 4 (GLUT4) translocation pathway were studied by quantitative real-time PCR (qPCR) in diabetes relevant tissues (pancreas, adipose tissue, skeletal muscle, liver and kidney). Three of the genes, TBC (Tre-2, BUB2, CDC16) 1 Domain Family Member 4 (TBC1D4), insulin receptor and GLUT4 showed altered expression in some of the tissues. The expression pattern observed is in agreement with what has previously been reported in pre-diabetic humans confirming the pre-diabetic status of our pigs. Moreover, a novel isoform of TBC1D4 was detected by Western blotting using protein extracted from pancreas. The expression level of this novel isoform was further verified by qPCR in all tissues, showing the highest expression in the pancreas.

Influence of drift and admixture on population structure of American black bears (Ursus americanus) in the Central Interior Highlands, USA, 50 years after translocation.

Bottlenecks, founder events, and genetic drift often result in decreased genetic diversity and increased population differentiation. These events may follow abundance declines due to natural or anthropogenic perturbations, where translocations may be an effective conservation strategy to increase population size. American black bears (Ursus americanus) were nearly extirpated from the Central Interior Highlands, USA by 1920. In an effort to restore bears, 254 individuals were translocated from Minnesota, USA, and Manitoba, Canada, into the Ouachita and Ozark Mountains from 1958 to 1968. Using 15 microsatellites and mitochondrial haplotypes, we observed contemporary genetic diversity and differentiation between the source and supplemented populations. We inferred four genetic clusters: Source, Ouachitas, Ozarks, and a cluster in Missouri where no individuals were translocated. Coalescent models using approximate Bayesian computation identified an admixture model as having the highest posterior probability (0.942) over models where the translocation was unsuccessful or acted as a founder event. Nuclear genetic diversity was highest in the source (AR = 9.11) and significantly lower in the translocated populations (AR = 7.07-7.34; P = 0.004). The Missouri cluster had the lowest genetic diversity (AR = 5.48) and served as a natural experiment showing the utility of translocations to increase genetic diversity following demographic bottlenecks. Differentiation was greater between the two admixed populations than either compared to the source, suggesting that genetic drift acted strongly over the eight generations since the translocation. The Ouachitas and Missouri were previously hypothesized to be remnant lineages. We observed a pretranslocation remnant signature in Missouri but not in the Ouachitas.