First generic one step real-time Taqman RT-PCR targeting the RNA1 of betanodaviruses.

The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50-100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 10(2.5)-10(2.85) TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.

An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus.

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 10(0) 50% egg infectious dose (EID50)/mL and 4.2 gene copies/µl. The linear ranges of the assay span within 10(6)-10(0) EID50/mL and 10(6)-10(0) gene copies/µl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a 'double check' approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

Influence of a cement industry on the fine and ultrafine particles composition in a rural area.

The cement industry of this work is located in the Fumane valley, in the north of Verona. The environmental impact of the air emissions from the plant was studied using different methods: the characterisation of the raw materials utilised in the production process and of the emissions from the chimney of the clinker kiln; the sampling of the air particles on filter in the region around the plant; the biomonitoring using transplanted mosses; the study of the air pollution dispersion using a model.

[Avoidable mortality in the Italian regions, 1980-1990]. / Le "morti evitabili" nelle regioni Italiane, 1980-90.

In Italy, between 1890-85 and 1986-90, rates for most "avoidable deaths" decreased: the decline ranged between 20% and 50%. For ages 5-64 years, total "avoidable mortality" declined in both sexes at a greater percentage (-26/27%) than mortality for all causes (-12%). In both periods, often the higher mortality rates (for causes of death selected as "avoidable" and for total avoidable mortality") were observed in the regions of Southern Italy. The "avoidable mortality" summary score showed higher values in 5 regions of Southern Italy (Compania, Sicily, Puglia, Calabria and Basilicata, the last in the period 1986-90, and in 2 regions of Northern Italy (Piedmont and Valle d'Aosta). The regions with higher values of the "avoidable mortality" summary score had also SMRs for total "avoidable mortality" significantly higher than the standard population (whole Italy of the same period). Hypertensive and cerebrovascular diseases, all respiratory diseases (age 1.4 years), perinatal mortality, and, only in the first period, the chronic rheumatic heart disease and all respiratory diseases (age 5-14 years) showed the highest degree of regional variations. The SMRs significantly higher than the standard population were more frequent in females (also after exclusion of sex-specific causes of death). The number of SMRs significantly higher than the standard population and the range of the "avoidable mortality" summary scores were smaller in the second period.