Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.     

Phenotypically Different Microalgal Morphospecies with Identical Ribosomal DNA: A Case of Rapid Adaptive Evolution?

The agents driving the divergence and speciation of free-living microbial populations are still largely unknown. We investigated the dinoflagellate morphospecies Scrippsiella hangoei and Peridinium aciculiferum, which abound in the Baltic Sea and in northern temperate lakes, respectively. Electron microscopy analyses showed significant interspecific differences in the external cellular morphology, but a similar plate pattern in the characteristic dinoflagellate armor. Experimentally, S. hangoei grew in a wide range of salinities (0–30), whereas P. aciculiferum only grew in low salinities (0–3). Despite these phenotypic differences and the habitat segregation, molecular analyses showed identical ribosomal DNA sequences (ITS1, ITS2, 5.8S, SSU, and partial LSU) for both morphospecies. Yet, a strong interspecific genetic isolation was indicated by amplified fragment length polymorphism (F _ST = 0.76) and cytochrome b (cob) sequence divergence (∼1.90%). Phylogenetic reconstructions based on ribosomal (SSU, LSU) and mitochondrial (cob) DNA indicated a recent marine ancestor for P. aciculiferum. In conclusion, we suggest that the lacustrine P. aciculiferum and the marine-brackish S. hangoei diverged very recently, after a marine–freshwater transition that exposed the ancestral populations to different selective pressures. This hypothetical scenario agrees with mounting data indicating a significant role of natural selection in the divergence of free-living microbes, despite their virtually unrestricted dispersal capabilities. Finally, our results indicate that identical ITS rDNA sequences do not necessarily imply the same microbial species, as commonly assumed.     

Richness and Composition of Vascular Plants and Cryptogams along a High Elevational Gradient on Buddha Mountain, Central Tibet

We explored patterns of plant species richness and composition along an elevational gradient (4,985?C5,685?m a.s.l.) on Buddha Mountain, 100?km northwest of Lhasa, Tibet. We recorded the presence of plants and lichens in 1-m² quadrats separated by 25-m elevational intervals (174 quadrats in 29 elevational bands) along a vertical transect with a SE aspect. We recorded 143 total species, including 107 angiosperms, 2 gymnosperms, 27 lichens, and 7 mosses. We measured stone cover in each quadrat, and soil pH, C, N and C/N ratio from two randomly located samples collected from 10-cm depth within each band. C, N and C/N decreased with elevation, stoniness increased and soil pH did not change with altitude. We employed detrended correspondence analysis (DCA), canonical correspondence analysis (CCA) and generalized linear models (GLMs) to assess the relationships of species richness and species composition to the environment. The first two axes of the CCA biplot explained 87.7% of total variation in the species-environment relationship, and 27.7% of total variance of species data. The first CCA axis is associated with elevation, while the second axis is related to soil pH and stone cover. We also compared patterns in species richness against expectations from species pools interpolated from the literature. Total species richness was relatively constant between 4,985 and 5,400?m a.s.l. and declined continuously above 5,400?m a.s.l. Similar declining patterns were observed for forbs and graminoids. Cushion plants and lichens abundance exhibited a unimodal relationship with altitude while shrubs declined monotonically. Except for lichens, models derived from our observations and the literature were quite similar in shape. The proportion of the species pool represented in each elevational band increased as a function of elevation for non-vascular plants, but decreased markedly for vascular plants. Thus, vascular plants are more likely to be constrained by dispersal at higher elevations, resulting in more local endemism, while the relatively easily-dispersed high-elevation cryptogams have little local differentiation. Our comparative approach demonstrates that complex scale-dependent differences between life forms may underlie the apparent simplicity of elevational gradients. Furthermore, elevational gradients summarized from distributional notes cannot be assumed to be proxies for elevational gradients on individual mountain slopes.     

Transthoracic echocardiography for imaging of the different coronary artery segments: a feasibility study

Background

Cardiac resynchronisation therapy (CRT) improves mortality and morbidity in heart failure patients with wide QRS. Observational studies suggest that patients having more left ventricular dyssynchrony pre-implantation obtain greater benefit on ventricular function and symptoms with CRT.

Aim

To provide an analysis of the prevalence and type of dyssynchrony in patients included in the CARE-HF trial.

Methods

100 patients 67 (58 to 71) years were examined with echocardiography including tissue doppler imaging before receiving a CRT-pacemaker. Atrio-ventricular dyssynchrony (LVFT/RR) was defined as left ventricular filling time <40% of the RR-interval. Inter-ventricular mechanical delay (IVMD) was measured as the difference in onset of Doppler-flow in the pulmonary and aortic outflow tracts >40 ms. Intra-ventricular (regional) dyssynchrony in a 16-segment model was expressed either as a delayed longitudinal contraction (DLC) during the postsystolic phase or by tissue synchronisation imaging (TSI) with a predefined time-difference in systolic maximal velocities >85 ms.

Results

LVFT/RR was present in 34% and IVMD in 60% of patients while intra-ventricular dyssynchrony was present in 85% (DLC) and 86% (TSI) with a high agreement between the measures (Kappascore 0.86–1.00), indicating the methods being interchangeable. Patients with cardiomyopathy (53%) were more likely to have LVFT/RR <40% (45% vs. 21% (p= 0.02)) and more segments affected by intra-ventricular dyssynchrony 4(3, 5) vs. 3(1, 4), p = 0.002, compared to patients with ischemic heart disease.

Conclusion

The prevalence of intra-ventricular dyssynchrony is high in patients with heart failure, wide QRS and depressed systolic function. Most important, TSI appears to be a fast and reliable method to identify patients with intra-ventricular dyssynchrony likely to benefit from CRT.     

Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q₂₆₆→L₂₆₆ substitution or insertion next to the putative cleavage site (R₂₆₇) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L₂₆₆ codons in screening samples and Q₂₆₆ codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.