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Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyltransferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we cloned and expressed the entire NS5 and the RdRpD in a heterologous system and used purified preparations for protein characterizations. We determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA and that the RdRp activity is not inhibited by sofosbuvir. To further examine the characteristics of USUV polymerase in a more specifically biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed their subcellular location. NS5 is predominantly found in the cytoplasm; a significant proportion is directed to the nucleus, and this translocation involves nuclear location signals (NLS) located at least between the MTase and RdRpD domains.

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A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.

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AIM: To study whether additional measurements of motility characteristics of spermatozoa by computer assisted semen analysis (CASA) were more sensitive indicators of reduced semen quality than estimates of percentages of motile, rapid or progressive cells. METHODS: Intermittent scrotal insulation was applied to 6 rams for 16 h per day for 21 days or to 2 of these for 12 h per day for 28 days in the following year. Semen was collected and evaluated by CASA immediately and either frozen or stored at 30 degrees Celsius or 5 degrees Celsius before re-evaluation. RESULTS: Intermittent scrotal insulation caused falls in the percentage of motile, progressive and rapid sperm, as did freezing-thawing and storage at 30 degrees Celsius or 5 degrees Celsius. Motility characteristics (amplitude of lateral head displacement, mean path velocity, mean progressive velocity and curvilinear velocity), as determined by CASA fell only when the percentage of motile sperm was already reduced. Freezing and thawing or liquid storage of the semen from insulated rams caused a greater fall in the percentage of motile and rapid sperm than control semen, but only affected the motility characteristics when the percentage of motile sperm was already reduced. CONCLUSION: Intermittent scrotal insulation affected not only the motility of the freshly collected sperm, but also their ability to withstand the additional stress of storage. The additional data on motility characteristics obtained by CASA appeared to be no more a sensitive indicator than the percentage of motile cells of reductions in semen quality.

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Antecedentes: En la hemorragia digestiva alta (HDA), la endoscopia digestiva alta (EDA) constituye un examen de diagnóstico de importancia fundamental. Objetivos: Evaluar en que medida la EDA contribuyó eficazmente a definir conductas y detener la hemorragia, y comparar los resultados de esta experiencia con los de la literatura. Material y Método: Estudio retrospectivo. 100 pacientes consecutivos internados a partir de junio de 1997 por HDA con compromiso hemodinámico. Todos fueron sometidos a una EDA en las primeras 24 hs de la manifestación inicial del sangrado. En los casos sin diagnóstico y en los que el hallazgo endoscópico correspondió a un sangrado activo o reciente, se efectuó una segunda endoscopia dentro de las 48 hs. La evolución de los pacientes se evaluó en función de tres variables: el resangrado, la necesidad de cirugía y la mortalidad. Resultados: En el 32 por ciento de los casos el diagnóstico del primer estudio fue modificado por los estudios ulteriores, y de los 8 pacientes en los que la primera endoscopía no fue diagnóstica, se produjo resangrado en 4 y fallecieron 2. De los 19 pacientes tratados por vía endoscópica por hemorragia no varicosa, se produjo resangrado en 5 (26 por ciento), mientras que en 15 pacientes sin tratamiento endoscópico, el resangrado fue del 33,3 por ciento (p=0,850). En total se produjo resangrado o persistencia en 24 pacientes, se operaron 13 de ellos como urgencia, y de éstos 2 fallecieron (15,2 por ciento). Por el contrario, hubo 11 pacientes con resangrado o persistencia no operados, en ellos la mortalidad fue del 45 por ciento (p=0,122). La mortalidad en los pacientes con diagnóstico endoscópico fue de 6,5 por ciento y la de los que no lo tuvieron fue del 25 por ciento, sin embargo esta diferencia no fue significativa (p=0,243). Conclusiones: No observamos un criterio uniforme para la descripción endoscópica de las lesiones ni para categorizarlas de acuerdo a los criterios de Forrest y se observó una alta incidencia de resangrado en los pacientes que recibieron tratamiento endoscópico. En los pacientes en que la primera endoscopia no es eficaz como método de diagnóstico, el pronóstico es más grave, y entre los pacientes que resangraron, la mortalidad fue menor entre los que recibieron tratamiento quirúrgico que no fueron operados

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Antecedentes: En la hemorragia digestiva alta (HDA), la endoscopia digestiva alta (EDA) constituye un examen de diagnóstico de importancia fundamental. Objetivos: Evaluar en que medida la EDA contribuyó eficazmente a definir conductas y detener la hemorragia, y comparar los resultados de esta experiencia con los de la literatura. Material y Método: Estudio retrospectivo. 100 pacientes consecutivos internados a partir de junio de 1997 por HDA con compromiso hemodinámico. Todos fueron sometidos a una EDA en las primeras 24 hs de la manifestación inicial del sangrado. En los casos sin diagnóstico y en los que el hallazgo endoscópico correspondió a un sangrado activo o reciente, se efectuó una segunda endoscopia dentro de las 48 hs. La evolución de los pacientes se evaluó en función de tres variables: el resangrado, la necesidad de cirugía y la mortalidad. Resultados: En el 32 por ciento de los casos el diagnóstico del primer estudio fue modificado por los estudios ulteriores, y de los 8 pacientes en los que la primera endoscopía no fue diagnóstica, se produjo resangrado en 4 y fallecieron 2. De los 19 pacientes tratados por vía endoscópica por hemorragia no varicosa, se produjo resangrado en 5 (26 por ciento), mientras que en 15 pacientes sin tratamiento endoscópico, el resangrado fue del 33,3 por ciento (p=0,850). En total se produjo resangrado o persistencia en 24 pacientes, se operaron 13 de ellos como urgencia, y de éstos 2 fallecieron (15,2 por ciento). Por el contrario, hubo 11 pacientes con resangrado o persistencia no operados, en ellos la mortalidad fue del 45 por ciento (p=0,122). La mortalidad en los pacientes con diagnóstico endoscópico fue de 6,5 por ciento y la de los que no lo tuvieron fue del 25 por ciento, sin embargo esta diferencia no fue significativa (p=0,243). Conclusiones: No observamos un criterio uniforme para la descripción endoscópica de las lesiones ni para categorizarlas de acuerdo a los criterios de Forrest y se observó una alta incidencia de resangrado en los pacientes que recibieron tratamiento endoscópico. En los pacientes en que la primera endoscopia no es eficaz como método de diagnóstico, el pronóstico es más grave, y entre los pacientes que resangraron, la mortalidad fue menor entre los que recibieron tratamiento quirúrgico que no fueron operados (AU)

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The genetic bases of sexual isolation between Drosophila melanogaster and D. simulans have been mainly studied in females, and there is little information about the role of the males in interspecific mating discrimination. Using D. simulans synthetic lines with compound chromosomes from a population of the Seychelles Islands (high frequency of interspecific mating) and a multimarker strain (low frequency), we show that D. simulans males play an important role in discriminating D. melanogaster females. The genetics of male discrimination fits well with the inheritance mode of a single locus, dominant for sexual isolation, located in chromosome II near the net mutation (2L-0.0). The heterospecific mating success of the male was not related to his sexual vigor. The specific load of male cuticular hydrocarbons was counted as a possible source of discrimination used by the D. melanogaster female.

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The mechanism of human-to-human transmission of the polyomaviruses JC virus (JCV) and BK virus (BKV) has not been firmly established with regard to possible human exposure. JCV and BKV have been found in sewage samples from different geographical areas in Europe, Africa, and the United States, with average concentrations of 10(2) to 10(3) JCV particles/ml and 10(1) to 10(2) BKV particles/ml. Selected polyomavirus-positive sewage samples were further characterized. The JCV and BKV present in these samples were identified by sequencing of the intergenic region (the region found between the T antigen and VP coding regions) of JCV and the VP1 region of BKV. The regulatory region of the JCV and BKV strains found in sewage samples presented archetypal or archetype-like genetic structures, as described for urine samples. The stability (the time required for a 90% reduction in the virus concentration) of the viral particles in sewage at 20 degrees C was estimated to be 26.7 days for JCV and 53.6 days for BKV. The presence of JCV in 50% of the shellfish samples analyzed confirmed the stability of these viral particles in the environment. BKV and JCV particles were also found to be stable at pH 5; however, treatment at a pH lower than 3 resulted in the detection of free viral DNA. Since most humans are infected with JCV and BKV, these data indicate that the ingestion of contaminated water or food could represent a possible portal of entrance of these viruses or polyomavirus DNA into the human population.

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As a rule, progeny of crosses between Drosophila simulans females and D. melanogaster males are formed by sterile males, because females die as embryos. However, populations of these species have been found that produce a certain frequency of viable hybrid females. We have found that 94% of the females of a D. simulans population from Tel Aviv gave hybrid progenies with both sexes. The segregation of phenotypes with different rescue success adjusts to the action of a single, dominant, zygotic-acting gene involved in hybrid female viability. This gene, which we named 'Simulans hybrid females rescue' (Shfr), is temperature-sensitive, showing a much smaller effect as temperature increases. Reciprocal crosses between Tel Aviv and a nonrescue population indicate some influence of cytoplasm or maternal effect in rescue. Using a chromosome substitution analysis we have located Shfr on the second chromosome. Using synthetic lines with this chromosome having different segments from Tel Aviv and from a multimarker strain we have mapped Shfr between black (2 L-43. 0) and pearly (2 R-74.0).

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Recent research on attachment theory as a model for understanding adult relationships and for psychotherapy suggests that the attachment organization of therapists could influence their approach to therapeutic work. A postal questionnaire including two measures of current adult attachment and one of early loss experience, together with measures of current clinical practice, was completed by 196 British clinical psychologists (40.4% return rate). Seventy per cent of the sample rated themselves as secure (a greater proportion than in the general population). The sample as a whole scored higher on the attachment pattern of 'compulsive care-giving' than on any of the other insecure patterns of attachment. A number of significant differences were found between the secure and insecure groups in approach to work, interest in working with different clients and difficulty experienced in therapeutic practice. A relationship was found between the amount of early loss experienced and current attachment style, therapeutic orientation and experience of therapy. Implications for the training and development of psychologists are considered and directions for future research are suggested.

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The sexual isolation among the related species Drosophila melanogaster, D. simulans and D. mauritiana is asymmetrical. While D. mauritiana males mate well with both D. melanogaster and D. simulans females, females of D. mauritiana discriminate strongly against males of these two species. Similarly, D. simulans males mate with D. melanogaster females but the reciprocal cross is difficult. Interspecific crosses between several populations of the three species were performed to determine if (i) males and females of the same species share a common sexual isolation genetic system, and (ii) males (or females) use the same genetic system to discriminate against females (or males) of the other two species. Results indicate that although differences in male and female isolation depend on the populations tested, the isolation behaviour between a pair of species is highly correlated despite the variations. However, the rank order of the isolation level along the populations was not correlated in both sexes, which suggests that different genes act in male and female sexual isolation. Neither for males nor for females, the isolation behaviour of one species was paralleled in the other two species, which indicates that the genetic systems involved in this trait are species-pair specific. The implications of these results are discussed.

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The genetic factors involved in the sexual isolation between Drosophila simulans females and D. melanogaster males are hardly known. We present a study of female mating propensity of D. simulans from different geographic origins with D. melanogaster males. Our results reveal the existence of genetic variability in D. simulans for this trait. Female discrimination is dominant against high female interspecific mating, which differs from that detected in other Drosophila species crosses.

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Drosophila simulans and D. melanogaster are sibling cosmopolitan species with imperfect ethological isolation. Hybridization is easy between D. melanogaster females and D. simulans males, but the reciprocal cross has been traditionally considered as very scarce and little is known about the environmental and genetic factors that affect it. We used classical genetic analyses to determine the influence of each major chromosome on the breakdown of sexual isolation between females of D. simulans and D. melanogaster males. In addition, we have made a first attempt to locate the genetic systems involved in this process. At least two genes, or two groups of genes, are responsible for hybridization, located in the X chromosome and in the left arm of chromosome II. The inheritance mode of both genetic systems is different. The genes in the X chromosome show dominance for high levels of hybridization, whereas those in chromosome II show dominance for low levels. These results contrast with other investigations on the melanogaster subgroup, suggesting independent evolutionary events events during the speciation process in each species.

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A chromosome substitution analysis was carried out in Drosophila melanogaster to elucidate the contribution of the X, II and III chromosomes to female receptivity, and to sexual isolation with its sibling species D. simulans. Two lines from the same base population with extreme phenotypes, the one showing high receptivity and weak isolation and the other showing low receptivity and strong isolation, were used. Genes involved in receptivity were located in chromosome III whereas those affecting sexual isolation were located in all the three chromosomes. These results indicate that the direct relationship between female receptivity and sexual isolation previously detected in D. melanogaster could be because of the occurrence of genes common to the two traits in the chromosome III.

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From a critical review of the literature on mating speed in Drosophila, the importance of fast mating in male fitness is questioned. The genetic architecture of male mating speed (MMS) has been evaluated in D. melanogaster through a populational analysis and a full 5 x 5 diallel cross between inbred lines. The results emphasize the fundamental role of the female genotype in both the absolute and the relative MMS performances. Somewhat different genetic architectures for MMS are revealed according to the female used in the tests. It is suggested that different parts of the complex genetic system involved in the male's "behavioral sexual phenotype" are relevant depending on the female's characteristics, thus causing the heterogeneity in the MMS genetic architecture. An overall picture reveals a genetic system characterized by additivity, dominance for fast mating, and no influence of the X chromosome. There results do not support strong natural selection favoring fast mating in Drosophila males.

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Genetic variation has been found in males of a D. simulans population for their eagerness to hybridize with D. melanogaster females. In a search for traits involved in this hybridization, males of D. simulans were tested for mating speed and sexual vigour. Between-male differences were detected in both sexual traits, but no relationship was noticed between them, nor with the frequency of hybridization. Thus male mating propensities appear to be unrelated to the breakdown of sexual isolation between these sibling species.

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A wild population of Drosophila melanogaster was subjected to selection for high and low female receptivity. Selection was only applied in females on the basis of the mating times observed in a 30-min period. We applied a selection paradigm that allowed us to conclude that females unmated after 30 min correspond to the truncated upper tail of the distribution underlying mating times. Selection was successful, with mean mating times of about 5 min for the high lines, 35 min for the low lines, and 12 min for the control. Receptivity of the selection lines remained steady after 42 generations of relaxed selection, suggesting a lack of natural selection on receptivity in laboratory conditions. We also found a clear relationship between high or low receptivity and high or low frequency of hybridization with D. simulans males, emphasizing the role of female receptivity in maintaining isolation with its sibling species D. simulans.

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Receptivity of sexually mature, virgin females, measured as the time to copulation, has been investigated. Two independent 5 x 5 diallel crosses were carried out, each one with a different male tester genotype. Interactions between male and female genotypes were detected because some lines appeared as dominant in one diallel and recessive in the other. The overall picture emerging from the analyses shows a genetic system for female receptivity characterized by additive genes, with dominance for high receptivity in one of the diallel crosses. This remarkable additive component does not support the action of natural selection favouring females with extreme values of receptivity, rather, the lack of intrasexual female competition for mates is suggested.

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Females of Drosophila melanogaster and males of D. simulans hybridizing in a nonchoice condition were artificially selected for 12 generations. The frequency of hybridization increased from 10% to 79%. Response to selection occurred in both species, particularly in D. melanogaster. Female receptivity was the primary sexual trait that accounted for breaking up sexual isolation in these species, but it remained unclear which elements of the D. simulans male courtship were involved.

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Fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 x 10(6) frozen spermatozoa from four control rams and from four rams submitted to a moderate (1.4-2.2 degrees C), but repeated, intermittent (16 h/day for 21 consecutive days) increase in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe from concentration of progesterone in blood plasma at 17 days and by ultrasound at 65 days after insemination. No differences were observed in the pregnancy rate at 17 days between ewes inseminated with semen collected from control rams (56.0, 65.2, 66.7 and 60.3%) and from heated rams (60.6, 71.8, 63.6 and 48.2%) before or after 4, 15 and 21 days of heating, respectively. In contrast, the rate of embryonic mortality between 17 and 65 days after insemination was significantly higher at days 4, 15 and 21 in the heated rams (78.7, 78.6 and 93%) than in the control rams (55, 59 and 65.7%). These results indicate that an intermittent slight, but repeated, increase in the subcutaneous scrotal temperature could induce a significant increase in the embryonic mortality rate. As these changes were apparent on day 4 of heating, an effect must have occurred on sperm stored in the epididymis.

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