José Antonio Boga

Etiology of Sporadic Cases of Pediatric Acute Gastroenteritis in Asturias, Spain, and Genotyping and Characterization of Norovirus Strains Involved

ABSTRACT From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.

Horizontal Transmissible Protection against Myxomatosis and Rabbit Hemorrhagic Disease by Using a Recombinant Myxoma Virus

ABSTRACT We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.

Coexpression of MT1 and RORα1 melatonin receptors in the Syrian hamster Harderian gland

Abstract:  Melatonin acts through several specific receptors, including membrane receptors (MT1 and MT2) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT1 and RORα1 in this gland was determined by nested RT‐PCR followed by amplicon sequencing and Western‐blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT1 receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal‐synthesized melatonin. On the contrary, although a RORα1‐immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORα1 on the role of this indoleamine against oxidative stress is discussed.

Role of Metapneumovirus in Viral Respiratory Infections in Young Children

ABSTRACT The contribution of human metapneumovirus (hMPV) relative to that of other respiratory viruses as a cause of respiratory infections in children less than 1 year old has been evaluated. From October 2003 to April 2004, nasopharyngeal samples from 211 children less than 1 year old were analyzed to detect respiratory viruses. Respiratory syncytial virus (RSV) was the predominant virus isolated (96 children [45.5%]), followed by influenza A virus, parainfluenza virus, adenovirus, cytomegalovirus, and herpes simplex virus type 1, which were only occasionally detected. From January 2004 to April 2004, a nested retrotranscription-PCR, using in-house primers directed to the matrix protein gene of hMPV, was carried out on samples in which no other viruses were detected. hMPV was detected in 18 (16.2%) children, indicating that this virus was the second-most-frequent cause of viral respiratory infections in children less than 1 year old. The rate of hospitalization for RSV- and hMPV-infected children was higher than 75%. While RSV had a peak from December to February, hMPV was increasingly detected from January to April. The mean age of hMPV-infected children (6.44 ± 3.64 [mean ± standard deviation] months) was significantly higher than that of RSV-infected children (3.99 ± 2.96 [mean ± standard deviation] months). On the other hand, 64.3% of the RSV-infected children and 12.5% of the hMPV-infected children showed high levels of C-reactive protein. Although several authors have reported that clinical symptoms of hMPV-positive patients mirrored those of RSV-positive patients, differences between the two viruses can be found.

Physiological oxidative stress model: Syrian hamster Harderian gland—sex differences in antioxidant enzymes

The Syrian hamster Harderian gland, a juxtaorbital organ exhibiting marked gender-associated differences in contents of porphyrins and melatonin, was used as a model system for comparing strong (in females) and moderate (in males) physiological oxidative stress. Histological differences showing much higher cell damage in females were studied in conjunction with lipid peroxidation and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Lipid peroxidation and enzyme activities were measured throughout the circadian cycle, revealing the importance of dynamical processes in oxidative stress. Especially in lipid peroxidation and in catalase, short-lasting rises exhibited strongest gender differences. Peaks of lipid peroxidation were about three times higher in females, compared to males. Catalase peaks of females exceeded those in males by several hundred-fold. Average levels of superoxide dismutase and glutathione peroxidase were about three or two times higher in females, respectively. A clear-cut diurnally peaking rhythm was found in glutathione peroxidase of females, which was not apparent in males. Glutathione reductase showed differences in time patterns, but less in average activities. The time courses of lipid peroxidation and of protective enzymes are not explained by circulating melatonin, whereas melatonin formed in the Harderian gland should contribute to differences in average levels. Neither damage nor antioxidative defense simply reflect the illumination cycle and are, therefore, not only a consequence of photoreactions.

The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants

Abstract We have investigated the use of five different promoter-5′UTR leader combinations to develop a potato plant-based expression system for the production of VP60 protein from rabbit haemorrhagic disease virus (RHDV). The relative efficiency of CaMV 35S, modified CaMV 35S, sunflower polyubiquitin and patatin promoters, as well as the addition of the 5′ leader sequence from φ10 gene of T7 phage, on VP60 gene expression in leaf and tuber tissues of plants, cultivated both in vitro and as potted plants, was investigated. Our results indicated that the B33 promoter with the φ10 leader gave the highest level of expression in tubers. The rabbits immunised with tuber plant extracts containing VP60 elicited specific antibody responses and were protected against challenge with virulent RDHV.

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.

Melatonin reduces endoplasmic reticulum stress and autophagy in liver of leptin‐deficient mice

The sedentary lifestyle of modern society along with the high intake of energetic food has made obesity a current worldwide health problem. Despite great efforts to study the obesity and its related diseases, the mechanisms underlying the development of these diseases are not well understood. Therefore, identifying novel strategies to slow the progression of these diseases is urgently needed. Experimental observations indicate that melatonin has an important role in energy metabolism and cell signalling; thus, the use of this molecule may counteract the pathologies of obesity. In this study, wild‐type and obese (ob/ob) mice received daily intraperitoneal injections of melatonin at a dose of 500 μg/kg body weight for 4 weeks, and the livers of these mice were used to evaluate the oxidative stress status, proteolytic (autophagy and proteasome) activity, unfolded protein response, inflammation and insulin signalling. Our results show, for the first time, that melatonin could significantly reduce endoplasmic reticulum stress in leptin‐deficient obese animals and ameliorate several symptoms that characterize this disease. Our study supports the potential of melatonin as a therapeutic treatment for the most common type of obesity and its liver‐associated disorders.

Porphyric enzymes in hamster Harderian gland, a model of damage by porphyrins and their precursors. A chronobiological study on the role of sex differences

The Syrian hamster Harderian gland (HG), representing a highly porphyrogenic organ, was used as a model system for studying physiologically occurring damage of biomolecules by porphyrins and their precursors, phenomena associated with from the pathological situation of porphyrias. The species used exhibits the peculiarity of much higher porphyrogenesis in females than in males, offering possibilities for comparison of effects by different porphyrin levels in one species. Since concentrations of free, and therefore, radical-generating porphyric metabolites are difficult to determine in the presence of high amounts of secreted and crystallizing porphyrins, which are, moreover, mainly surface-reactive, and since indications existed for temporal changes in the oxidative stress caused by these molecules, the following approach was chosen: in HGs of both females and males, activities of the relevant porphyric enzymes, delta-aminolevulinate synthase (ALA-S), delta-aminolevulinate dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), were determined throughout the circadian cycle. Results were compared with the temporal patterns of lipid peroxidation and protein damage in the same glands. In females, a strong correspondence was observed between protein carbonyl and lipid peroxidation, peaking at the end of both photophase and scotophase; maximal activities of the three porphyric enzymes ALA-S, ALA-D, and PBG-D either coincided or slightly preceded the peaks of oxidative damage. In males, lower enzyme activities, especially in PBG-D, were associated with weakly expressed rhythmicity. Correspondingly, lipid peroxidation was lower and exhibited a smaller rhythm amplitude; protein carbonyl of males showed a temporal pattern differing from that of females, with regard to amplitude and phasing. These data are in agreement with morphological observations demonstrating particularly severe cell damage in the female HG under normal conditions.

Androgen regulation of gene expression in the Syrian hamster Harderian gland

The androgenic control of sexual dimorphism has been studied in the Harderian gland from Syrian hamster and compared to rat Harderian gland, a system without dimorphism. Hybridization in situ with a rat cDNA clone has revealed the presence of androgen receptor mRNA in all secretory cells from male and female hamster glands. Testosterone or 5-alpha-dihydrotestosterone administration to females both caused a 60% decrease in the levels of 5-aminolevulinate synthase mRNA after 1 day of treatment, but the resulting patterns of in vitro translation using RNA from glands treated with the two androgens are different. Testosterone alters the mRNA levels for androgen receptor and 5-aminolevulinate synthase in the glands only 6 h after its implantation in females, and the action is maintained up to 10 days of treatment. Finally, androgen administration to females or deprivation in males alter androgen receptor but not 5-aminolevulinate synthase mRNA levels in rat Harderian glands. Our results suggest that the androgen receptor from Harderian glands is responsible for the sexual dimorphism found in Syrian hamsters, whereas the lack of sexual dimorphism in rat seems to be due to a restricted effect of androgens in the glands.