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Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP AHPND ), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R 2  = 0.998) in the range of 4 × 10 9 to 4 × 10 0 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei , Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA) −1 . Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load. [ABSTRACT FROM AUTHOR]

A disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in China and several countries of Southeast Asia, leading to serious losses in production. Litopenaeus vannamei (also known as Pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. A new nodavirus, termed covert mortality nodavirus (CMNV), has been shown to be the etiological agent. In this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and quantitative detection of CMNV. The optimal conditions for this newly developed RT-LAMP reaction were found to be 6 mM MgCl 2 and 1.6 mM dNTPs, an incubation temperature of 65 °C and a reaction time of 50 min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3 pg total RNA of CMNV-infected shrimp and 27 copies of the target plasmid. The diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription PCR (RT-PCR) assay was 96.4% and 94.4%, respectively. The reaction products were detected by visual inspection after staining with an in-tube DNA fluorescent dye, a measure taken to eliminate the risk of contamination. The quantitative RT-LAMP assay for CMNV showed high correlation coefficient ( r 2 = 0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5 × 10 2 to 6.7 × 10 6 copies per mg of cephalothorax tissue. Thus, the CMNV RT-LAMP assay is a sensitive and specific new tool for the field detection and quantification of CMNV in the diagnosis and surveillance of covert mortality disease. [ABSTRACT FROM AUTHOR]

WSSV is one of the most harmful pathogeny in the pacific white shrimp, and genetic variations caused the strains of different virulence. MicroRNAs (miRNAs) involved in the regulation of virus defense. To understand the different virulence of WSSV on miRNA expression in Litopeneaus vannamei , the deep sequencing was performed to compare two small RNA libraries prepared from hepatopancreas of Litopeneaus vannamei infected with normal-virulence or low-virulence WSSV. Approximately 29,398,623 raw reads from normal-virulence library and 35,291,803 raw reads from low-virulence library were obtained. There were about 37 miRNAs homologs identified. Sixteen miRNAs were significantly up-regulated and twenty-one miRNAs were significantly down-regulated in normal-virulence infection library compared with low-virulence infection library. Of these, Igi-miR-1175-3p was the most significant different miRNA, followed by bmo-miR-1175-3p and ipu-miR-26b, respectively. The putative target genes for differentially expressed miRNAs were concerned with biological processes, signal meditated, cell differentiation and apoptosis, immune recognition and other more functions. The results will help to understand the miRNAs response to different virulence WSSV infection. [ABSTRACT FROM AUTHOR]

This study conducted a 30-day feeding trial and a subsequent 20-day anti-virus infection trial to determine the effects of probiotic Bacillus PC465 on the growth, health status, and disease resistance of Litopenaeus vannamei . Shrimp samples were fed with three practical diets prepared from shrimp feed containing varying probiotic doses [0 (control), 10 7 , and 10 9 CFU g −1 ]. Probiotic supplementation significantly increased the weight gain and survival of L. vannamei ( p < 0.05). The effect of 10 9 CFU g −1 on the growth rate was higher than that of 10 7 CFU g −1 . Compared with those in the control group, the activities of digestive enzymes, such as amylase, protease, and lipase, in the shrimp mid-gut significantly increased in the probiotic-fed groups on days 15 and 30, except lipase on day 30. The influence of 10 9 CFU g −1 on enzyme activities was also greater than that of 10 7 CFU g −1 . Scanning electron microscopy revealed folds and large ravines across the interior surface of the mid-gut, and the number of these folds and ravines increased significantly after the probiotic was administered. The probiotic treatment significantly ( p < 0.05) enhanced the transcription of penaeidin 3 a (Pen-3 a ), peroxinectin, C-type lectin 3 (Lec-3), and thioredoxin (Trx) in the hemocytes of L. vannamei . Likewise, probiotic treatment increased the transcription of hemocyanin in the hepatopancreas of L. vannamei . The probiotic treatment also significantly increased the transcription of prophenoloxidase (proPO) but decreased the transcription of crustin in hemocytes. By contrast, the same treatment failed to increase the transcription of Ras-related protein (Rab-6) in hemocytes. The number of species and biomass of Bacillus in the mid-gut were higher in the probiotic-fed group than in the control group. The total biomass of microbes was higher in the shrimp fed with 10 7 CFU g −1 than in the shrimp fed with 10 9 CFU g −1 and the control group on days 15 and 30 post-feeding. In two white spot syndrome virus (WSSV) infections, the weight gain, survival, and WSSV copies within the gills of the probiotic-treated shrimp significantly differed ( p < 0.05) from those of the control group. Relatively efficient protection was associated with probiotic feeding. Results suggested that Bacillus PC465 feeding improves the growth performance, survival, digestion, and nutrient absorption of L. vannamei . Probiotic treatment also enhances the microbial structures in the gut, promotes the immune status of shrimp, and provides protection against viral infection. The supplementation with 10 9 CFU g −1 can also improve the growth and survival of L. vannamei . [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is the main pathogen of shrimp culture, and has brought great losses of the shrimp aquaculture industry every year since it has been found. However, the specific mechanism of the virus into the cell is not very clear. Recent research suggests that clathrin-mediated endocytosis is involved in WSSV infection. By sequence analysis, clathrin coat AP17 is an σ subunit of AP-2 complex which is involved in clathrin-mediated endocytosis. To obtain the full-length sequence of Clathrin coat AP17 of Litopenaeus vannamei ( LvCCAP17 ), the rapid amplification of cDNA ends (RACE) was performed to get the sequence of 3'and 5′ end and splicing by DNAMAN. The full-length sequence of LvCCAP17 is 842 bp and expected to encoding 142 amino acids, and the amino acid sequence was analyzed by online software. The mRNA expression of LvCCAP17 in different tissues was carried out with quantitative real-time PCR and the LvCCAP17 was detected in all tested tissues of Litopenaeus vannamei . The transcriptional expression level of LvCCAP17 in epithelium and hepatopancreas was significantly up-regulated after WSSV infection. Far-Western blotting and ELISA assay showed that LvCCAP17 interacted with rVP26 and rVP37. Silencing of LvCCAP17 gene by double-strand RNA (dsRNA) interference significantly delay of cumulative mortality rate in WSSV infected shrimp and reduced the expression level of immediate early gene 1 ( ie1 ) and vp28 . These results indicated that clathrin-meated endocytosis is responsible for WSSV infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] White spot syndrome virus VP12 interacts with LvANT with dose-dependent manner. [•] LvANT was commonly found in all the tissues detected. [•] LvANT was colocalized with mitochondrion in shrimp hemocytes. [•] Injection of LvANT antiserum decreased the cumulative mortality of shrimp challenged by WSSV. [Copyright &y& Elsevier]