Characterisation of a maize-infecting potyvirus from Spain

In order to characterise and classify an unknown maize-infecting potyvirus isolated from fields in northeast Spain, the entire coat protein gene and the C-terminal twothirds of the large nuclear inclusion protein (NIb) gene were cloned and sequenced. Protein sequencing enabled the cleavage site between the two proteins to be deduced and also revealed that on storage the viral coat protein undergoes a specific degradation in which the N-terminal 39 amino acids are removed. Comparison of the nucleotide sequence of the 3 non-coding region of the viral RNA and the predicted amino acid sequence of the coat protein with the equivalent regions of other members of the potyvirus group revealed that the Spanish virus is closely related to maize dwarf mosaic virus strain A.     

Hepatitis E virus in Cuba: A cross-sectional serological and virological study in pigs and people occupationally exposed to pigs

Surveillance of hepatitis E virus (HEV) in risk groups is an important strategy to monitor its circulation pattern and to timely detect changes thereof. The aims of this cross-sectional study were to estimate the prevalence of HEV infections in pigs and humans from different regions of the country, to identify risk factors for increasing anti-HEV IgG prevalence and to characterize HEV strains. The presence of anti-HEV antibodies was assessed by commercial ELISA in serum samples from the general population, farm and slaughterhouse employees, as well as pigs sampled in the three regions of Cuba from February to September 2016. Overall, individuals with occupational exposure to swine or swine products (70/248, 28.2%) were 4 times more likely to be seropositive compared to the general population (25/285, 8.7%; OR: 4.18; p < .001). Within the risk group, risk factors included age, number of years working in a professional activity with direct exposure to swine, geographic region and distance between residence and closest professional swine setting, while wearing gloves had a protective effect. Prevalence of total anti-HEV antibodies in swine was 88.2% (165/187) and HEV RNA was detected by real-time RT-PCR in 9.2% (16/173) swine stools. All HEV strains sequenced clustered within genotype 3. Some strains clearly belonged to subtype 3a, while another group of strains was related with subtypes 3b and 3 k but partial HEV sequences did not allow unequivocal subtype assignment. These findings suggest that the high HEV exposure in Cuban individuals with swine-related occupations could be due to enzootic HEV in certain regions, direct contact with infectious animals or their products as well as environmental contamination.     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

Subcellular localization and isoenzyme pattern of peroxidase and polyphenol oxidase in beet root (Beta vulgaris L.)

The two enzymes involved in enzymatic browning reactions, polyphenol oxidase (PPO) and peroxidase (PO), have been partially purified and extracted from different fractions of beet root. PPO is mainly located in the membrane fraction, and it was also found in the soluble fraction. In both cases PPO was in its latent state. However, PO activity was higher in the soluble fraction than in the membrane fraction. Nevertheless, the highest values of specific activity for PO were obtained from the solubilized enzyme from acetone powders. Under native isoelectric focusing (IEF), several PPO isoenzymes were present in the pH range of 4.8-5.8. All of these isoenzymes shared a single band with a similar apparent mass under sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PO was also analyzed by IEF, showing a complex isoenzyme pattern in all fractions. The characteristic basic PO isoenzyme of high pI found in both the soluble fraction and the solubilized enzyme from acetone powders was not detected in the membrane fraction. The kinetic characterization of PPO and PO from all fractions was carried out.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Effect of 4-amino-6-methyl-3-phenylamino-1,2,4-triazin-5(4H)-one on the integrity of chloroplasts from barley leaves

4-Amino-6-methyl-3-phenylamino-1,2,4-triazin-5(4H)-one alters the integrity of chloroplasts in barley (Hordeum vulgare L. var. Hassan) leaves as judged by their sedimentation profiles in linear gradients of sucrose. This effect may be related to the inhibition caused by 4-amino-6-methyl-3-phenylamino-1,2,4-triazine-5(4H)-one of both ascorbate peroxidase and superoxide dismutase activities, and could thus be explained by an accumulation of toxic reduced-oxygen species in illuminated chloroplasts.     

Biological characteristics of maize dwarf mosaic potyvirus from Spain

Two isolates of maize dwarf mosaic virus (MDMV-Sp and MDMV-Spl) obtained from maize in the northeast of Spain were studied. Both isolates induced similar reactions on 6 sorghum cultivars, johnsongrass and oat (cv. Clintland), with the exception of MDMV-Sp which produced a different reaction on one sorghum cultivar. Thirty-three grass species were tested as possible hosts (16 previously untested) and 18 were found to be susceptible. Among those, eight were previously unidentified hosts for MDMV:Aegilops ovata, A. ventricosa, Avena barbata, Bromus alopecurus, B. diandrus, B. fasciculatus, Echinaria capitata andLolium rigidum. Both isolates were transmitted from maize to maize nonpersistently byRhopalosiphum maidis, R. padi, Schizaphis graminum andSitobium avenae. The virus was not seed-transmitted in Mo17 and B73 maize inbred lines. Pinwheels, scrolls and short curved laminated aggregates were observed in the cytoplasm of maize cells infected by MDMV-Sp or MDMV-Sp1. In addition, laminated aggregates were observed in cells infected by MDMV-Sp1.     

Renal handling of calcium and phosphorus in experimental renal hyperparathyroidism in dogs

Twenty-four hour urinary excretion, fractional excretion and the filtered load of calcium and phosphorus were monitored as hyperparathyroidism evolved in a model of progressive canine renal failure. Thirteen beagles of both sexes aged four and a half months were used. Nine of them were subjected to a renal damaging schedule (neomycine, 60 mg/kg/48 h, IM, 32 weeks) in order to induce chronic renal failure leading to secondary hyperparathyroidism (2HPT group). The remaining four were kept as the control group. The experiment was conducted over 32 weeks. Blood and 24 h urine were collected every four weeks. Calcium, phosphorus and creatinine were analyzed. Plasma parathormone and calcitonin were determined at weeks 0, 12, 24 and 32. The level of renal function in the 2HPT animals was reduced to 25% of that of the controls (endogenous creatinine clearance was 0.45 +/- 0.22 mL/min/kg as opposed to 1.81 +/- 0.54 mL/min/kg). Hyperparathyroidism was confirmed by a progressive increase in the levels of the parathyroid hormone. Calcitonin levels were not modified. A tendency to hypocalcaemia was observed, reaching statistically significant levels from the twenty-eighth week of the study, when hyperphosphataemia also became significant. Daily urinary excretion of calcium and phosphorus remained at values considered normal throughout the experiment with no alteration imputable to the impaired renal function. This is explained by the decrease in the filtered load of these elements (in both cases statistically significant from the 24th week on) being associated with an increase in their fractional excretion. Thus, calcium and phosphorus urinary excretion values could be maintained in a normal range up to the end of the experiment, showing that renal calcium handling in dogs with experimentally induced renal failure seems to differ from that observed in human patients.     

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.)

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.     

Radiographic features of the limbs of juvenile and subadult loggerhead sea turtles (Caretta caretta)

This study aimed to provide the normal radiographic anatomic appearance of the limbs of the loggerhead sea turtle, Caretta caretta. Dorsopalmar and dorsoplantar radiographs were taken of the forelimbs and hindlimbs of 15 juvenile and 15 subadult loggerhead sea turtles, 17 alive and 13 dead. For comparison, computed tomographic, gross anatomic, osteologic, and histologic studies were performed on the limbs of 5 of the sea turtles. Bones from the distal part of the fore and hind flippers were seen in detail with a mammographic film-screen combination. The pectoral and pelvic girdles, superimposed by the carapace, were better seen on standard radiographs with the use of rare-earth intensifying screens. Mammographic radiographs of the manus of 5 small juvenile turtles showed active growth zones. Visualization of bone contours in the distal part of the limbs was clearer than in mammals owing to the very few superimpositions. The presence of a substantial amount of cartilage in the epiphyses produced better visibility of limb ends. We conclude that use of a mammography film-screen combination is the best way to evaluate the bony and joint structures of the limbs of sea turtles. Radiography provides reliable images for clinical purposes. Considering the low cost and logistics of this technique, it is a practical ancillary test for marine animal rehabilitation centers to use.