Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through posttranscriptional mechanisms. Here, we have used real-time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into the lytic cycle. During latency, BHRF1 miRNAs were seen only in cells with detectable Cp-and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However, this variation could not be explained by differential miRNA turnover, as all EBV miRNAs appeared to be remarkably stable. Following entry into the virus lytic cycle, miR-BHRF1-2 and -1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally, we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady-state levels of EBV miRNAs.Epstein-Barr virus (EBV), a B lymphotropic gammaherpesvirus with potent growth-transforming properties, is etiologically linked to a number of malignancies of lymphoid and epithelial cell origin, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), and nasopharyngeal carcinoma (NPC) (52). As illustrated in Fig. 1A, these different tumor settings can be distinguished by alternative patterns of EBV latent gene expression. Thus, EBV-driven PTLD lesions and growth-transformed lymphoblastoid cell lines (LCLs) display a latency III form of infection, characterized by the expression of six EBV nuclear antigens transcribed from one of two alternative promoters (Wp and Cp), and three latent membrane proteins (50, 63); in addition, a recent study (36) reported that LCLs also weakly express the viral Bcl2 homologue BHRF1 as a latent antigen. In contrast, most BL tumor cell lines which retain the original BL tumor phenotype in vitro show a more restricted pattern of latent antigen expression (termed latency I), in which the Cp, Wp, and LMP promoters are silent and a single nuclear antigen EBNA1 is transcribed from a novel promoter, Qp (46,54). However, a subset of BL lines display a third form of latency (termed Wp-restricted latency), in which Wp-initiated transcripts give rise to EBNA1, -3A, -3B, -3C, and BHRF1 (35,36,38). In addition to the above-mentioned latent antigens, two sets of RNAs are also expressed in all forms of EBV infection. These are ...

Several hepatitis A virus (HAV) and human norovirus (HuNoV) outbreaks due to consumption of contaminated berries and vegetables have recently been reported. Model experiments were performed to determine the effectiveness of freeze-drying, freeze-drying combined with heating, and steam blanching for inactivation of enteric viruses that might be present on the surface of berries and herbs. Inactivation of HAV and inactivation of feline calicivirus, a surrogate for HuNoV, were assessed by viral culturing and quantitative reverse transcription PCR (RT-PCR), whereas HuNoV survival was determined only by quantitative RT-PCR. While freeze-drying barely reduced (<1.3 log 10 units) the amount of HAV RNA detected in frozen produce, a greater decline in HAV infectivity was observed. The resistance of HuNoV genogroup I (GI) to freeze-drying was significantly higher than that of HuNoV GII on berries. Addition of a terminal dry heat treatment at 120°C after freeze-drying enhanced virus inactivation by at least 2 log 10 units, except for HuNoV GII. The results suggest that steam blanching at 95°C for 2.5 min effectively inactivated infectious enteric viruses if they were present in herbs. Our results provide data for adjusting food processing technologies if viral contamination of raw materials is suspected.

Human noroviruses (NoVs) are the most common viruses causing acute gastroenteritis in humans. Performance characteristics of two commercial quantitative NoV RT-PCR assays, the Norovirus real-time RT-PCR Kit (AnDiaTec) and the Type I and Type II kits (Generon), and the international assay as selected by the CEN/TC/WG6/TAG4 group were evaluated for the specific detection and quantitation of 59 NoV samples, including different subtypes of NoV genogroup I and II. The results showed that the method proposed by the CEN/TC/WG6/TAG4 group was 100% specific since it was able to detect all samples tested. The commercialized kits evaluated failed to detect a vast majority of NoV GI strains. Additionally the Generon kit did not succeed to detect strains from GII.3, GII.5, GII.6, GII.7, GII.8, GII.12 and GII.17. In addition, the detection limit using the most prevalent strain, NoV GII.4, was 2.5 PCRU per reaction using both commercial kits. Despite this good sensitivity for NoV GII.4 detection it is concluded that both commercial assays are not suitable for the detection and quantitation of most NoV subtypes. Therefore the method proposed by the CEN/TC/WG6/TAG4 group is recommended for epidemiological studies and outbreaks investigations.