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Aim: To evaluate the gene expression changes that occur soon after the active infection of two susceptible cell types with human herpesvirus 8 (HHV-8). Materials & methods: The expression profile of 282 human genes involved in the inflammatory process was investigated in HHV-8 A1 or C3 subtype-infected and mock-infected human epithelial cells and lymphoid cells. Results: The HHV-8-induced transcriptional profiles in the epithelial and lymphoid cells were very different. A robust increase in the expression was found in genes belonging to different categories, especially the categories of inflammation response and signal transduction. Conclusion: These results indicate that during early infection, HHV-8 induces a variety of cell type-specific processes, thus providing infection signatures useful as potential targets for therapeutic intervention.

Viral infections have been associated with autoimmune connective tissue diseases. To evaluate whether active infection by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV)-6, -7, -8, as well as parvovirus B19 (B19V) occur in patients with autoimmune connective tissue diseases, viral DNA loads were assessed in paired samples of serum and peripheral blood mononuclear cells (PBMCs) of 115 patients affected by different disorders, including systemic sclerosis, systemic, and discoid lupus erythematosus, rheumatoid arthritis, and dermatomyositis. Two additional groups, patients affected by inflammatory diseases (n=51) and healthy subjects (n=58) were studied as controls. The titers of anti-HHV-6 and anti-EBV antibodies were also evaluated. Cell-free HHV-6 serum viremia was detected in a significantly higher proportion of connective tissue diseases patients compared to controls (P

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (γ = 0.62 and γ = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample

The demogenetic structure of the Amiata donkey, an endangered breed from Central Italy, was investigated using information from pedigrees. Genealogical data of 602 donkeys reared in Tuscany were recorded in a database and analysed by the computer package ENDOG. Population size increased from 89 subjects in 1995 to 503 (129 males and 374 females) in 2005. Animals were distributed among 152 herds, but the effective number of herds was 21, suggesting that a small number of herds provided stallions for the entire breed. The maximum number of traced generation was 4, the mean maximum generation was 1.14, the mean com- plete generation was 0.53, and the mean equivalent generation was 0.78. The average relatedness coeffi- cient (AR) in the 503 alive animals was 0.94% while the mean F was 0.29% so the effective population size was 172.41. Among 24 animals with a 4-generation history, 3 (12.5%) were 25% inbred. Although the incompleteness of genealogical information did not permit accurate inference of the current values of popu- lation genetic parameters, the present work represents a first step towards an efficient management of the breed.

Liposomes containing Distamycin A (DA) may be clinically useful in the treatment of ocular HSV infections, especially in acyclovir-resistant HSV keratitis. This study evaluated the in vitro and in vivo performance of a topical controlled release liposomal formulation containing DA (DA-Lipo) aimed at reducing the toxicity of the encapsulated active agent and improving drug uptake by ocular tissues. The bioavailability of DA in the tear fluid and the DA uptake into the cornea were increased after instillation of DA-Lipo in rabbits, reaching the DA corneal concentration corresponding to IC50 values against HSV without any sign of transcorneal permeation of drug. DA-Lipo was definitely less cytotoxic then plain DA in rabbit corneal epithelial cells. These results provide new insights into the correlation between the in vitro data and the drug kinetics following ocular applications of liposomal vesicles.

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

Objective(s): Herpes simplex virus (HSV) infections in immunocompromised individuals may require prolonged antiviral therapy resulting in the emergence of viral strains resistant to the currently employed antiviral drugs. Distamycin A (DA), a basic antibiotic belonging to the lexitropsin DNA minor groove binding drugs, exhibits antiviral properties. In this study we evaluated the in vitro cytotoxicity and antiviral activity of DA against HSV type 1 and HSV type 2 clinical isolates from transplanted patients and compared them with those of acyclovir (ACV) in search of alternative antiviral drugs. Methods: Viral detection and typing was performed by multiplex PCR and immunofluorescence assay; the in vitro cytotoxicity of DA and the antiviral activity of ACV and DA was evaluated respectively by neutral red uptake assay and plaque reduction assay for HSV2 isolates and fluorescence reduction assay for HSV1 isolates. Results: Tissue culture 50% cytotoxic concentration of DA was 58 µM. Tissue culture 50% inhibitory concentration values ranged from 0.16 to 7.4 µM for the ACV-sensitive and from 5.4 to 32 µM for the ACV-resistant viral strains. Conclusions: In spite of the lower activity against ACV-resistant strains, DA may be used as an antiherpetic drug.