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Background The latency-associated transcript ( LAT) gene of herpes simplex virus ( HSV)-2 is the only detectable viral gene expressed during latent infection in neurons. LAT inhibits apoptosis and maintains latency by promoting the survival of infected neurons. However, whether LAT functions during HSV-2 infection via its encoded RNAs or via its encoded proteins remain unknown. Increasing evidence has indicated that LAT is likely to functionally promote the establishment of latent infection via LAT-encoded micro RNAs (mi RNAs). Aim To explore whether the RL1 fragment of the five adjacent mi RNAs has an effect on cell apoptosis, then provide supporting evidence to elucidate the potential role of these mi RNAs and to aid screening of their cellular targets. Methods A number of techniques, including MTT assay, flow cytometry and DNA ladder analysis, were used to verify the role of the RL1 fragment and the contribution of the individual mi RNAs to the anti-apoptotic effect. Results Five mi RNAs (miR-H3, miR-H4 -3p, miR-H4 -5p, miR-H24 and miR-H19) were detected by quantitative PCR in PC12 cells stably expressing RL1 after pEGFP- RL1 plasmid transfection in vitro. The data indicated that expression of HSV-2 LAT RL1 seems to provide protection against apoptosis of PC12 cells induced by ActD. Antisense mi RNAs specifically inhibiting these five mi RNAs could efficiently reduce their expression. Transfection of antisense-miR-H3, antisense-miR-H4 -5p and antisense-miR-H19 into PC12 cells stably expressing RL1 were able to partly reverse the anti-apoptotic effect of these mi RNAs. Conclusions These findings indicate that the apoptotic role of the RL1 fragment is likely to be related to overexpression of miR-H3, miR-H4 -5p and miR-H19 in PC12 cells. [ABSTRACT FROM AUTHOR]

A new genotype of yellow head virus (YHV), designated as YHV-8, was found in farmed shrimp Fenneropenaeus chinensis suffering suspectedly from EMS/AHPNS (early mortality disease/acute hepatopancreatic necrosis disease) in China in 2012. In this study, a one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) assay was developed for better detection of both genotypes of YHV-1 and YHV-8. A set of six specific primers was successfully designed targeting a conserved region of the YHV genome. The LAMP reaction was optimized to contain 8 mmol l−1 Mg2+ and 1·4 mmol l−1 dNTPs, and to be performed at 58°C for 60 min. The detection sensitivity of the rRT-LAMP method was as low as 7 × 100 copies per reaction. The specificity of the method was validated by the absence of any cross-reaction with the RNA samples extracted from other shrimp viruses (Taura syndrome virus, white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus) and specific pathogen-free (SPF) shrimp. The resulting standard curves showed high correlation coefficient values. Furthermore, the test of farm samples showed that YHV was detected in three of 111 Litopenaeus vannamei, six of eight Fenneropenaeus chinensis, five of 19 Macrobrachium rosenbergii and none of the nine Marsupenaeus japonicus. These results suggest that this assay is applicable widely as a new rapid and sensitive detection method in the research of YHV. Significance and Impact of the Study In this study, we designate a new genotype of yellow head virus (YHV) as YHV genotype 8 (YHV-8) which was detected in diseased shrimp in China. A rapid, sensitive and specific rRT-LAMP detecting method for both YHV-8 and YHV-1 has been established. It is anticipated that this novel assay will be instrumental for diagnosis and surveillance of the virulent genotypes of YHV. [ABSTRACT FROM AUTHOR]

White spot syndrome virus ( WSSV) is one of the main pathogens seriously threatening penaeid shrimp farming in the world. This study designed and synthesized five specific primers based on the conserved sequences of WSSV genome, optimized the reaction system and conditions, and finally established a cross-priming amplification ( CPA)-based detection method for WSSV ( WSSV- CPA). The results indicated that the optimized reaction temperature and time of WSSV- CPA were 63°C and 60 min, respectively. The detection limit of the WSSV- CPA assay was as low as 10 copies μl−1 and shared same sensitivity with the WSSV- qPCR assay. Due to dispensing with expensive thermal cycler, time- and cost-saving, and ease of use in field, it is anticipated that the WSSV- CPA method developed in this study will be instrumental for the diagnosis and surveillance of WSSV. Significance and Impact of the Study White spot syndrome, caused by white spot syndrome virus ( WSSV), is the major disease threatening the shrimp aquaculture industry and leads to tens of billion dollars of economic losses in the world each year. This study established a CPA-based method for detecting WSSV, which is rapid, sensitive and specific. It is anticipated that this novel assay will be instrumental for diagnosis and surveillance of WSSV. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the diversity of dominant autochthonous microbiota along the digestive tract of juvenile Epinephelus coioides following the dietary administration of probiotic Bacillus pumilus for 60 days. Methods and Results: Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) with subsequently sequencing analysis was used to assess the gut microbiota . Generally similar DGGE patterns were observed in the foregut, midgut and hindgut of E. coioides, while the similarity dendrogram clearly revealed three different clusters depending on the three compartments of the GI tract. Dietary administration of B. pumilus stimulated its colonization in each compartment of the digestive tract. Samples collected from the probiotic group and the control group showed similar DGGE patterns, and no significant difference in the total number of bands and the Shannon index were detected between the probiotic group and the control group, suggested that B. pumilus exert no significant effect on the gut microbiota. However, various potentially beneficial bacteria, such as uncultured Bacillus sp. clone QJNY94-like, Nitratireductor sp. YCSC5-like, Methylobacterium hispanicum-like and Microbacterium sp. YACS1-like bacteria were stimulated by probiotic B. pumilus, while the potential harmful Staphylococcus saprophyticus-like bacterium was depressed. Conclusions: Autochthonous gut microbiota of E. coioides was modulated to some degree, not significant, by probiotic B. pumilus, various potentially beneficial bacteria were selectively stimulated, while one potential harmful species was depressed. Significance and Impact of the Study: This work represents the first report that dietary administration of probiotic B. pumilus modulated the gut microbiota of E. coioides. These findings broaden our understanding of probiotic effects at the gut level, which is helpful in understanding the mechanisms that underpin host benefits. [ABSTRACT FROM AUTHOR]

p.r. rajan, j.h.-y. lin, m.-s. ho and h.-l. yang. 2003. To detect Photobacterium damselae ssp. piscicida using the PCR technique and plating method. Two strains of P. damselae ssp. piscicida were isolated from cultured cobia ( Rachycentron canadum) at two different fish farms in Taiwan. A pair of primers was designed to detect the capsular polysaccharide gene of P. damselae ssp. piscicida by PCR. Reference strains of different genus and different clinical strains were used for this study. The expected product (410 bp) was obtained from both P. damselae ssp. piscicida and P. damselae ssp. damselae, and they were differentiated by culturing on thiosulphate citrate bile salts–sucrose agar (TCBS-1). Photobacterium damselae ssp. damselae grew on TCBS-1 producing green colonies whereas P. damselae ssp. piscicida did not grow. The methods used are cost and labour effective when compared with the other methods and commercially available kits. This work provides an integrated set of methods to identify the species P. damselae and to differentiate P. damselae ssp. piscicida from P. damselae ssp. damselae. [ABSTRACT FROM AUTHOR]