Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

Assembly of African swine fever virus: role of polyprotein pp220.

Polyprotein processing is a common strategy of gene expression in many positive-strand RNA viruses and retroviruses but not in DNA viruses. African swine fever virus (ASFV) is an exception because it encodes a polyprotein, named pp220, to produce several major components of the virus particle, proteins p150, p37, p34, and p14. In this study, we analyzed the assembly pathway of ASFV and the contribution of the polyprotein products to the virus structure. Electron microscopic studies revealed that virions assemble from membranous structures present in the viral factories. Viral membranes became polyhedral immature virions after capsid formation on their convex surface. Beneath the lipid envelope, two distinct domains appeared to assemble consecutively: first a thick protein layer that we refer to as core shell and then an electron-dense nucleoid, which was identified as the DNA-containing domain. Immunofluorescence studies showed that polyprotein pp220 is localized in the viral factories. At the electron microscopic level, antibodies to pp220 labeled all identifiable forms of the virus from the precursor viral membranes onward, thus indicating an early role of the polyprotein pp220 in ASFV assembly. The subviral localization of the polyprotein products, examined on purified virions, was found to be the core shell. In addition, quantitative studies showed that the polyprotein products are present in equimolar amounts in the virus particle and account for about one-fourth of its total protein content. Taken together, these results suggest that polyprotein pp220 may function as an internal protein scaffold which would mediate the interaction between the nucleoid and the outer layers similarly to the matrix proteins of other viruses.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

African Swine Fever Virus Is Enveloped by a Two-Membraned Collapsed Cisterna Derived from the Endoplasmic Reticulum

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.     

Environmental,geographical and time-related impacts on avian malaria infections in native and introduced populations of house sparrows (Passer domesticus), a globally invasive species

Aim

The increasing spread of vector-borne diseases has resulted in severe health concerns for humans, domestic animals and wildlife, with changes in land use and the introduction of invasive species being among the main possible causes for this increase. We explored several ecological drivers potentially affecting the local prevalence and richness of avian malaria parasite lineages in native and introduced house sparrows (Passer domesticus) populations.

Location

Global.

Time period

2002–2019.

Major taxa studied

Avian Plasmodium parasites in house sparrows.

Methods

We analysed data from 2,220 samples from 69 localities across all continents, except Antarctica. The influence of environment (urbanization index and human density), geography (altitude, latitude, hemisphere) and time (bird breeding season and years since introduction) were analysed using generalized additive mixed models (GAMMs) and random forests.

Results

Overall, 670 sparrows (30.2%) were infected with 22 Plasmodium lineages. In native populations, parasite prevalence was positively related to urbanization index, with the highest prevalence values in areas with intermediate urbanization levels. Likewise, in introduced populations, prevalence was positively associated with urbanization index; however, higher infection occurred in areas with either extreme high or low levels of urbanization. In introduced populations, the number of parasite lineages increased with altitude and with the years elapsed since the establishment of sparrows in a new locality. Here, after a decline in the number of parasite lineages in the first 30 years, an increase from 40 years onwards was detected.

Main conclusions

Urbanization was related to parasite prevalence in both native and introduced bird populations. In invaded areas, altitude and time since bird introduction were related to the number of Plasmodium lineages found to be infecting sparrows.     

Fitness consequences of anthropogenic hybridization in wild red-legged partridge (Alectoris rufa, Phasianidae) populations

Hybridization is a widespread phenomenon, which plays crucial roles in the speciation of living beings. However, unnatural mixing of historically isolated taxa due to human-related activities has increased in recent decades, favouring levels of hybridization and introgression that can have important implications for conservation. The wild red-legged partridge (Alectoris rufa, Phasianidae) populations have recently declined and the releases of farm-reared partridges have become a widespread management strategy. The native range of the red-legged is limited to the south-west of Europe (from Italy to Portugal). This species does not breed in sympatry with the chukar partridge (A. chukar), whose range is Eurasian (from Turkey to China). However, red-legged partridges have often been hybridized with chukar partridges to increase the productivity of farmed birds, and game releases may have spread hybrid birds into the wild. In this study, we investigated the fitness (survival and breeding) differences between hybrid and “pure” red-legged partridges in a wild population located in central Spain. Incubation probability was similar in hybrids and “pure” partridges. Hybrid females laid larger clutches than “pure” ones, but hatching success did not differ between hybrid and “pure” partridges. Hybrid birds had lower survival rate than “pure” ones, mainly because of higher predation rates. Our results show that, despite lower survival, hybrid partridges breed in natural populations, so this could increase extinction risk of wild pure partridge populations, through releases of farmed hybrid birds. The consequences of continued releases could be of vital importance for the long term conservation of wild red-legged partridges.     

Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein.

The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V. The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome. The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids. The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12. The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope. Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein. This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence. The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected.