Both Genome Segments Contribute to the Pathogenicity of Very Virulent Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.     

Mise à jour taxonomique et répartition des puces du genre Ctenophthalmus Kolenati 1856 en région paléarctique occidentale (Insecta : Siphonaptera : Ctenophthalmidae)

Summary. Taxonomic update and geographic distribution of fleas of the genus Ctenophthalmus Kolenati 1856 in the Western Palearctic Region (Insecta: Siphonaptera: Ctenophthalmidae). Among fleas (Siphonaptera), the genus Ctenophthalmus is the one that comprises the largest number of taxa and is also characterized by a large geographical range. Here, we present a taxonomic revision of the Western Paleartic subgenera, groups, species and subspecies. We recognized a total of 143 taxa (57 species and 86 subspecies). These taxa are clustered into 23 groups of species, which fall into seven of the 16 subgenera of the genus Ctenophthalmus. According to Hopkins & Rothschild (1966), the subgenus Ctenophthalmus would only include the agyrtes group, itself divided into subgroups. We decided to raise these subgroups to group status to clarify taxonomic relationships within the subgenus Ctenophthalmus. Within this subgenus, the arvernus group is renamed baeticus, the fransmiti group is confirmed, and the egregius group is created. For each taxon, we provided information on geographical distribution, mammalian hosts, and host specificity.     

Development of an analytical approach for identification and quantification of 5-α-androst-16-en-3-one in human milk

A method was developed for the quantification of 5-α-androst-16-en-3-one in human breast milk based on application of a stable isotope dilution assay using 5α-androst-16-en-3-one-6, 6-d₂. The procedure includes extraction of the human milk by hexane with subsequent clean-up of the obtained extract by gel permeation and silica gel column chromatography. The extracted samples were analyzed by gas chromatography–mass spectrometry. Using this method 5-α-androst-16-en-3-one could be identified and for the first time quantified in a concentration range of 26–155 ng/kg in human milk.     

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by >90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.     

Alternative development in Polystoma gallieni (Platyhelminthes,Monogenea) and life cycle evolution

Considering the addition of intermediate transmission steps during life cycle evolution, developmental plasticity, canalization forces and inherited parental effect must be invoked to explain new host colonization. Unfortunately, there is a lack of experimental procedures and relevant models to explore the adaptive value of alternative developmental phenotypes during life cycle evolution. However, within the monogeneans that are characterized by a direct life cycle, an extension of the transmission strategy of amphibian parasites has been reported within species of Polystoma and Metapolystoma (Polyopisthocotylea; Polystomatidae). In this study, we tested whether the infection success of Polystoma gallieni within tadpoles of its specific host, the Stripeless Tree Frog Hyla meridionalis, differs depending on the parental origin of the oncomiracidium. An increase in the infection success of the parasitic larvae when exposed to the same experimental conditions as their parents was expected as an adaptive pattern of non-genetic inherited information. Twice as many parasites were actually recorded from tadpoles infected with oncomiracidia hatching from eggs of the bladder parental phenotype (1.63 ± 0.82 parasites per host) than from tadpoles infected with oncomiracidia hatching from eggs of the branchial parental phenotype (0.83 ± 0.64 parasites per host). Because in natural environments the alternation of the two phenotypes is likely to occur due to the ecology of its host, the differential infection success within young tadpoles could have an adaptive value that favors the parasite transmission over time.     

NMR localization of the O-mycoloylation on PorH,a channel forming peptide from Corynebacterium glutamicum

PorH and PorA are two small peptides that, in complex, form a voltage-dependent ion channel in the outer membrane of Corynebacterium glutamicum. Specific post-translational modifications on PorA and PorH are required for the formation of a functional ion channel. The assignment of PorH proton NMR chemical shifts in DMSO, allowed identifying unambiguously the exact position of the PorH O-mycoloylation on Ser 56 side chain. This was further confirmed by site directed mutagenesis and mass spectrometry. Together with the previously published localization of PorA mycoloylation, this provides the complete primary structure characterization of this outer membrane porin.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.