Crystal structure of the yellow 1:2 molecular complex lumiflavin–bisnaphthalene-2,3-diol

In the first molecular complex of the physiologically active neutral form of isoalloxazine studied, lumiflavin–bisnaphthalene-2,3-diol, each flavin is sandwiched between two naphthalenediol molecules with extensive overlap but a moderately large (3·44 Å) spacing, indicating at most weak charge-transfer interaction and in agreement with the yellow colour of the complex, nearly the same as that of the parent lumiflavin

Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain

International audienceAbstractStable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p < 0.05) in the control group (7.6 ± 1.7 days) compared to the immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (p < 0.05) of AUC values of nasal shedding (control: 14.6, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (p < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe

The immunity raised by recent European subtype 1 PRRSV strains allows a better replication of East European subtype 3 PRRSV strain Lena than the immunity raised by an older strain -increased risk for spatial expansion of PRRSV Lena-like strains

The spatial distribution of PRRSV-1 subtypes in Europe is quite stable, most probably due to a strong population immunity induced by the local PRRSV strains. In this study, we evaluated the potential of the immunity induced by several West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) to provide a protection against the highly virulent East European subtype 3 PRRSV strain Lena. Eleven-week-old pigs were inoculated with subtype 1 PRRSV strains (07V063, 13V091 or 13V117). Seven weeks later, the pigs were challenged with PRRSV strain Lena. Clinical, virological and serological parameters were monitored upon challenge. Number of fever days was higher (P < 0.05) in the non-immune control group (7.6 ± 1.7 days) compared to animals from immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (P < 0.05) of AUC values of nasal shedding (control: 14.6 ± 5.6, 07V063-immune: 3.4 ± 3.4, 13V091-immune: 8.9 ± 6.1, 13V117-immune: 8.0 ± 6.1) and viremia (control: 28.1 ± 11.0, 07V063-immune: 5.4 ± 4.4, 13V091-immune: 9.0 ± 1.5, 13V117-immune: 8.3 ± 4.8). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (P < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation of animals with subtype 1 PRRSV strains caused a priming of Lena-specific VN antibody response. Upon challenge with PRRSV Lena a serological booster effect was observed for neutralizing antibodies against strains used for the first inoculation. Our results indicate that immunity elicited by inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. We conclude that the lower protection level elicited by recently isolated subtype 1 PRRSV strains may facilitate spatial expansion of subtype 3 strains from East Europe to West Europe

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the ~450-Å-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar α-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface

Fructose-1,6-Bisphosphatase Overexpression in Pancreatic β-Cells Results in Reduced Insulin Secretion : A New Mechanism for Fat-Induced Impairment of β-Cell Function

OBJECTIVE—Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic β-cell lines exposed to high fat. However, whether specific β-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet β-cell FBPase can result in reduced glucose-mediated insulin secretion

Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons

During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles

Nanoscale structure of type I collagen fibrils: Quantitative measurement of D‐spacing

This article details a quantitative method to measure the D‐periodic spacing of type I collagen fibrils using atomic force microscopy coupled with analysis using a two‐dimensional fast fourier transform approach. Instrument calibration, data sampling and data analysis are discussed and comparisons of the data to the complementary methods of electron microscopy and X‐ray scattering are made. Examples of the application of this new approach to the analysis of type I collagen morphology in disease models of estrogen depletion and osteogenesis imperfecta (OI) are provided. We demonstrate that it is the D‐spacing distribution, not the D‐spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage osteoporosis and OI. The ability to quantitatively characterize nanoscale morphological features of type I collagen fibrils will provide important structural information regarding type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knockout studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments. The distribution of Type I collagen fibril D‐spacing provides important morphological information regarding Type I collagen in diseases such as early stages of osteoporosis and osteogenesis Imperfecta . In this article, the authors use Atomic Force Microscopy (AFM) imaging combined with two Dimensional Fast Fourier Transform (2D FFT) analysis to quantitatively assess Type I collagen fibril D‐spacing. This methodology allows imaging and characterization of Type I collagen constituted biological tissues, hydrogels, and other collagen based biomaterials.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94732/1/117_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/94732/2/biot_201200174_sm_suppinfo.pd

A Triple Amino Acid Substitution at Position 88/94/95 in Glycoprotein GP2a of Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV1) Is Responsible for Adaptation to MARC-145 Cells

The Meat Animal Research Center-145 (MARC-145) cell line has been proven to be valuable for viral attenuation regarding vaccine development and production. Cell-adaptation is necessary for the efficient replication of porcine reproductive and respiratory syndrome virus (PRRSV) in these cells. Multiple sequence analysis revealed consistent amino acid substitutions in GP2a (V88F, M94I, F95L) of MARC-145 cell-adapted strains. To investigate the putative effect of these substitutions, mutations at either position 88, 94, 95, and their combinations were introduced into two PRRSV1 (13V091 and IVI-1173) infectious clones followed by the recovery of viable recombinants. When comparing the replication kinetics in MARC-145 cells, a strongly positive effect on the growth characteristics of the 13V091 strain (+2.1 log10) and the IVI-1173 strain (+1.7 log10) compared to wild-type (WT) virus was only observed upon triple amino acid substitution at positions 88 (V88F), 94 (M94I), and 95 (F95L) of GP2a, suggesting that the triple mutation is a determining factor in PRRSV1 adaptation to MARC-145 cells