Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >= 2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.Peer reviewe

Molecular epidemiology and evolutionary trajectory of emerging echovirus 30, Europe

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >= 2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Anthropogenic land-use signals propagate through stream food webs in a California, USA, watershed

a b s t r a c t Human development of watersheds can change aquatic ecosystems via multiple pathways. For instance, human rural development may add nutrients to ecosystems. We used naturally occurring stable isotopes in stream food webs to investigate how land use affects stream ecosystems across a gradient of land development in the San Lorenzo watershed, California. Road density was used as a proxy for land development. We found that streams in watersheds with higher road densities had elevated concentrations of phosphate and nitrate. Furthermore, algal ␦ 15 N values increased as a function of nitrate concentration, but saturated at approximately 6‰. This saturating pattern was consistent with a two-source mixing model with anthropogenic and watershed sources, fit using Bayesian model fitting. In sites that had >2.6 km roads km −2 , anthropogenic sources of N were estimated to represent >90% of the N pool. This anthropogenic N signal was propagated to stream consumers: rainbow trout (Oncorhynchus mykiss), signal crayfish (Pacifasticus leniusculus), and benthic invertebrate ␦ 15 N were positively correlated with algal ␦ 15 N. Even relatively low density rural human land use may have substantial impacts on nutrient cycling of stream ecosystems. © 2014 Elsevier GmbH. All rights reserved. Introduction Human land-use impacts freshwater ecosystems via multiple pathways, such as through nutrient loading and habitat alteration Stable isotopes are increasingly used to investigate how anthropogenic land-use alters aquatic ecosystems. For example, nitrogen stable isotope ratios (␦ 15 N) can identify potential sources of nitrogen as well as inform rates of nutrient transformations The ecological effects of human land use can be illuminated through the study of gradients that span urban to rural developments Materials and methods Study system We examined 12 sites within the San Lorenzo River watershed (Santa Cruz County, CA, USA) that spanned a gradient of human land-use intensity. Sites were located on the numerous relatively small (first and second order) streams within the watershed and were part of a larger study (D. B. Herbst, unpublished data). From this larger set of candidate sites, sites were chosen to stratify a gradient in human land-use intensity and minimize differences in gradient and stream size. With two exceptions, sites were located on different streams. Elevations in this coastal watershed range from 979 m to sea level where the San Lorenzo River enters the Monterey Bay. The climate is Mediterranean, with 76-153 cm rain yr −1 . Tributaries drain steep soils of weathering granite, schists, marble, and marine deposits consisting of sandstones, shales and mudstones The San Lorenzo watershed has a history of excess anthropogenic nutrient inputs Field study Each site consisted of a riffle-pool sequence ranging from 40 to 60 m in length. Sites encompassed an anthropogenic gradient of the watershed which ranged from locations with little anthropogenic influence to locations with higher levels of anthropogenic influence such as road crossing and rural development. We used catchment road density (km km −2 ) as an index of human land-use intensity for each site. At each site, we collected primary producers (periphyton), and consumers (benthic invertebrates; rainbow trout, Oncorhynchus mykiss; signal crayfish, Pacifasticus leniusculus) for stable isotope analyses and water samples to obtain nutrient concentrations. All sampling was conducted in June 20-26, 2009 at near base stream flow conditions. Primary producer biomass was characterized by algae (periphyton) scraped from cobbles collected from both a region of slowand fast-water within the sampling site. Previous work has found that water velocity can influence algal stable isotope signatures At each site benthic invertebrates were collected by a Surber sample (0.5 mm mesh; sampling to a depth of 10 cm) in both a region of fast and slow stream flow. Samples were preserved in 70% ethanol. We note that preservation in ethanol can slightly alter isotope signatures (shifting ␦ 13 C approximately 1‰ and ␦ 15 N approximately 0.4 ‰; Venturra and Jeppesen, 2009). We did not adjust for this shift because it is likely relatively consistent within invertebrates that have fairly constrained stoichiometry (as opposed to across taxonomic groups with vastly different stoichiometry). Prior to preparation for stable isotope analysis, invertebrates were sorted and identified to family and functional feeding group (filterer, detritivore, herbivore, or predator) according to Fish and crayfish were collected by three-pass depletion electrofishing. Block nets at the upper and lower extent of each site prevented movement in or out of the site during surveys. Signal crayfish (Pacifastacus leniusculus; n = 35, approximately three per site) and rainbow trout (O. mykiss; n = 65, approximately five per site) were selected as focal species as they were the most abundant top aquatic consumers present across the different sampling sites. Orbital carapace length (crayfish) or total fork length (trout) and wet weight (to the nearest 0.1 g) were measured on-site for each sampled organism. Crayfish muscle tissue and rainbow trout caudal fin clips Algae and benthic invertebrates were oven dried until they reached a constant weight (approximately 48 h at 60 • C), whereas crayfish and trout samples were freeze-dried. To remove 13 Cdepleted lipids, all consumer samples were flushed with three cycles of petroleum ether at 1200 psi in a Dionex ASE 200 Accelerated Solvent Extraction System. Algae and crayfish tissue samples were ground into a homogenous powder with an agate mortar and pestle. Larger benthic invertebrates were also ground into a fine powder, whereas multiple individuals of the same species were aggregated into one sample for smaller invertebrates. Trout fin clips were left intact. Samples were weighed into 5 mm × 9 mm (algae, mean ± standard deviation = 4800 ± 31 g) or 3 mm × 5 mm (benthic invertebrates, 589 ± 53 g; crayfish muscle, 698 ± 30 g; fish fins, 673 ± 78 g) tin capsules (Costech Analytical Technologies). Stable isotope analyses Sample ␦ 13 C and ␦ 15 N were measured on a Carlo Erba 1108 elemental analyzer coupled to a ThermoFinnigan Delta Plus XP isotope ratio mass spectrometer (University of California, Santa Cruz Stable Isotope Laboratory). Sample isotope ratios were corrected relative to working standards with C:N ratios similar to the samples. For internationally calibrated in-house standards, analytical precision of ␦ 13 C and ␦ 15 N was less than 0.2‰. Water nutrients We collected duplicate water samples to measure total nitrate and phosphate concentrations at each site. Unfiltered water samples were collected just under the water surface in acid-rinsed high-density polyethylene (HDPE) bottles and were kept frozen at −20 • C until analysis. Frozen water samples were thawed, filtered through 0.7 m GF/F filters (Whatman), and analyzed for nitrate and phosphate concentrations using a QuikChem 8000 Flow Injection analyzer (Lachat Instruments) at the University of California, Santa Cruz Marine Analytical Labs. Isotopes and mixing models To address our question of how human land-use alters stream N cycling, we used a Bayesian information theoretic approach to estimate models and parameters. Bayesian mixing models allow the use of prior information for parameters and the propagation of uncertainty into estimates of posterior probability distributions of contributions to isotopic mixtures (e.g., where the stable isotope signature of the mixture (␦ 15 N mix ) was a function of proportional contributions of the two sources, anthropogenic sources (f a ) and background watershed sources (f b ), and their respective isotope signatures (␦ 15 N a and ␦ 15 N b ) and where f a + f b = 1. While ␦ 15 N a and ␦ 15 N b were not measured in this study, previous studies have indicated that ␦ 15 N a are generally enriched (prior probability distribution was uniformly distributed between 5‰ and 18‰) and ␦ 15 N b are generally depleted (prior probability distribution uniformly distributed between −2‰ and 2‰) (e.g., where N t was the measured concentration of nitrate at the site and N b was the unknown amount of that nitrate that was from background watershed sources. The prior probability distribution for N b was uniformly distributed between 0 and 10 M NO 3 − ; we did not have direct or published measurements of this prior and thus used a wide and uninformative prior. Through assuming that background watershed nitrate was constant but unknown across sites and estimating this parameter, we estimated the contribution of anthropogenic nitrate as N t − N b . It is important to note that these analyses were focused on the N that was available for algae to uptake and transfer into their stable isotope signatures. Given that algae have relatively fast turnover times, we think that our single measurements of nitrate during low summer baseflow are likely good approximations of N available to algae during this summer baseflow period. Therefore, our period of inference was restricted to this time frame. Furthermore, it is possible that other processes are contributing to observed isotopic patterns which would suggest a potentially different model structure (see section "Discussion"). Bayesian parameter estimation We estimated the posterior probability distributions of parameters and fit models using a Bayesian approach. Specifically, we estimated posterior probability distributions for N b , ␦ 15 N a , and ␦ 15 N b based on the data and our prior specifications. Bayesian modeling is based on the premise that the probability of a given combination of parameter values is defined by the likelihood of the data given the model and the prior belief in the parameter set. Markov chain Monte Carlo (MCMC) sampling was performed in JAGS in R Results The streams sampled in this survey of the San Lorenzo watershed varied in their land-use intensity. Specifically, the road density of the stream basins ranged from 0.89 to 6.13 road km km −2 . Sites with higher human land-use generally had higher nutrient concentrations N isotopes of algae showed a strong positive saturating relationship with nitrate concentratio