Astrovirus induces diarrhea in the absence of inflammation and cell death

Astroviruses are a leading cause of infantile viral gastroenteritis worldwide. Very little is known about the mechanisms of astrovirus-induced diarrhea. One reason for this is the lack of a small-animal model. Recently, we isolated a novel strain of astrovirus (TAstV-2) from turkeys with the emerging infectious disease poult enteritis mortality syndrome. In the present studies, we demonstrate that TAstV-2 causes growth depression, decreased thymus size, and enteric infection in infected turkeys. Infectious TAstV-2 can be recovered from multiple tissues, including the blood, suggesting that there is a viremic stage during infection. In spite of the severe diarrhea, histopathologic changes in the intestine were mild and there was a surprising lack of inflammation. This may be due to the increased activation of the potent immunosuppressive cytokine transforming growth factor beta during astrovirus infection. These studies suggest that the turkey will be a useful small-animal model with which to study astrovirus pathogenesis and immunity.     

Characterization of the expression,intracellular localization,and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase

Mouse hepatitis virus (MHV) RNA synthesis is mediated by a viral RNA-dependent RNA polymerase (RdRp) on membrane-bound replication complexes in the host cell cytoplasm. However, it is not known how the putative MHV RdRp (Pol) is targeted to and retained on cellular membranes. In this report, we show that a 100-kDa protein was stably detected by an anti-Pol antiserum as a mature product throughout the virus life cycle. Gradient fractionation and biochemical extraction experiments demonstrated that Pol was not an integral membrane protein but was tightly associated with membranes and coimmunoprecipitated with the replicase proteins 3CLpro, p22, and p12. By immunofluorescence confocal microscopy, Pol colocalized with viral proteins at replication complexes, distinct from sites of virion assembly, over the entire course of infection. To determine if Pol associated with cellular membranes in the absence of other viral factors, the pol domain of gene 1 was cloned and expressed in cells as a fusion with green fluorescent protein, termed Gpol. In Gpol-expressing cells that were infected with MHV, but not in mock-infected cells, Gpol relocalized from a diffuse distribution in the cytoplasm to punctate foci that colocalized with markers for replication complexes. Expression of Gpol deletion mutants established that the conserved enzymatic domains of Pol were dispensable for replication complex association, but a 38-amino-acid domain in the RdRp unique region of Pol was required. This study demonstrates that viral or virus-induced factors are necessary for Pol to associate with membranes of replication complexes, and it identifies a defined region of Pol that may mediate its interactions with those factors.     

Influence of killing method on Lepidoptera DNA barcode recovery

The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large‐scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA‐friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA‐friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA‐based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis.     

Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana

Arabidopsis accessions differ largely in their seed dormancy behavior. To understand the genetic basis of this intraspecific variation we analyzed two accessions: the laboratory strain Landsberg erecta (Ler) with low dormancy and the strong-dormancy accession Cape Verde Islands (Cvi). We used a quantitative trait loci (QTL) mapping approach to identify loci affecting the after-ripening requirement measured as the number of days of seed dry storage required to reach 50% germination. Thus, seven QTL were identified and named delay of germination (DOG) 1-7. To confirm and characterize these loci, we developed 12 near-isogenic lines carrying single and double Cvi introgression fragments in a Ler genetic background. The analysis of these lines for germination in water confirmed four QTL (DOG1, DOG2, DOG3, and DOG6) as showing large additive effects in Ler background. In addition, it was found that DOG1 and DOG3 genetically interact, the strong dormancy determined by DOG1-Cvi alleles depending on DOG3-Ler alleles. These genotypes were further characterized for seed dormancy/germination behavior in five other test conditions, including seed coat removal, gibberellins, and an abscisic acid biosynthesis inhibitor. The role of the Ler/Cvi allelic variation in affecting dormancy is discussed in the context of current knowledge of Arabidopsis germination.     

T cells cause acute immunopathology and are required for long-term survival in mouse adenovirus type 1-induced encephalomyelitis

Infection of adult C57BL/6 (B6) mice with mouse adenovirus type 1 (MAV-1) results in dose-dependent encephalomyelitis. Utilizing immunodeficient mice, we analyzed the roles of T cells, T-cell subsets, and T-cell-related functions in MAV-1-induced encephalomyelitis. T cells, major histocompatibility complex (MHC) class I, and perforin contributed to acute disease signs at 8 days postinfection (p.i.). Acute MAV-1-induced encephalomyelitis was absent in mice lacking T cells and in mice lacking perforin. Mice lacking alpha/beta T cells had higher levels of infectious MAV-1 at 8 days, 21 days, and 12 weeks p.i., and these mice succumbed to MAV-1-induced encephalomyelitis at 9 to 16 weeks p.i. Thus, alpha/beta T cells were required for clearance of MAV-1. MAV-1 was cleared in mice lacking perforin, MHC class I or II, CD4+ T cells, or CD8+ T cells. Our results are consistent with a model in which either CD8+ or CD4+ T cells are sufficient for clearance of MAV-1. Furthermore, perforin contributed to MAV-1 disease but not viral clearance. We have established two critical roles for T cells in MAV-1-induced encephalomyelitis. T cells caused acute immunopathology and were required for long-term host survival of MAV-1 infection.     

Studies of decarboxylation in photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds

Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate. Direct measurements of product formation confirmed that release via this pathway is faster for CNB-caged compounds than for related caged compounds without an alpha-carboxylate substituent and a rationale for the faster release rate is proposed. In a second pathway, photodecarboxylation of the starting material occurs: this pathway leads only to a slow, minor release of the photoprotected species. The extent to which the latter pathway contributes is affected by the nature of buffer salts in the irradiated solution. It was more prominent in an amine-based buffer (MOPS) than in phosphate buffer.     

The influence of uncouplers on facilitated diffusion of sorbose in Saccharomyces cerevisiae

Sorbose uptake in Saccharomyces cerevisiae, strain Delft 1, proceeds via mediated passive transport. In the cell sorbose is distributed in at least two compartments. Efflux studies showed that sorbose uptake in one of these compartments is not readily reversible. Uncouplers of oxidative phosphorylation inhibit both transport velocity and steady-state uptake level. It could be shown that these two effects are caused by different modes of action of the uncouplers. None of these two effects could be ascribed to changes of the electrochemical H⁺ gradient or of the intracellular pH. It is suggested that the inhibition of uptake velocity is caused by binding of the uncoupler to the sorbose translocator, thus lowering the transport activity. The uncoupler binding site is probably located at the intracellular fragment of the carrier. The second effect, reduction of the steady-state uptake level, is probably due to blocking of sorbose influx into the compartment that exhibits poor reversibility.     

The relative influence of competition and prey defences on the trophic structure of animalivorous bat ensembles

Deterministic filters such as competition and prey defences should have a strong influence on the community structure of animals like animalivorous bats which have life histories characterized by low fecundity, low predation risk, long life expectancy and stable populations. We investigated the relative influence of these two deterministic filters on the trophic structure of animalivorous bat assemblages in South Africa. We used null models to test if patterns of dietary overlap were significantly different from patterns expected by chance and multivariate analyses to test the correlations between diet and phenotype (body size, wing morphology and echolocation). We found little evidence that competition structured the trophic niche of coexisting bats. Contrary to predictions from competition, dietary overlap between bats of ensembles and functional groups (open-air, clutter-edge, and clutter foragers) were significantly higher than expected by chance. Instead, we found support for the predictions of the allotonic frequency hypothesis: there were significant relationships between peak echolocation frequency and the proportion of moths in the diets of bats at local and regional scales, and peak echolocation frequency was the best predictor of diet even after we controlled for the influence of body size and phylogeny. These results suggest that echolocation frequency and prey hearing exert more influence on the trophic structure of sympatric animalivorous bats than competition. Nonetheless, differential habitat use and sensory bias may also be major determinants of trophic structure because these are also correlated with frequencies of bat calls.