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The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F 1 ATP synthase β subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson’s correlation coefficients reached to 0.67 ± 0.05 and the Mander’s overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. [ABSTRACT FROM AUTHOR]

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]

Highlights: [•] It is the first real-time PCR assay established based on hepatopancreatic parvovirus Chinese isolate. [•] The assay is specific, sensitive, reproducible and very convenient. [•] Twenty-three of eighty-one samples are HPV positive from seven Chinese provinces. [Copyright &y& Elsevier]

In view of the current demand for rapid detection and identification of pathogens, point-of-care testing (POCT) with fast portability, low consumption, and increased sensitivity and specificity has become more and more popular. The emerging nucleic acid isothermal amplification technology (NAIAT) has shown potential advantages in the development of rapid microbial detection. In this study, a micro-detection slide system was developed based on the NAIAT of various nucleic acids of shrimp pathogens. The system included a micro-detection slide with 48 identical detecting cells precoated with all detection reagents, except the sample template. The process of producing the micro-detection slides mainly combined super-hydrophobic/super-oleophobic and super-hydrophilic materials to obtain separated spaces for detection, and aerosol pollution was eliminated in the form of water-in-oil. The micro-detection slide system was capable of simultaneously detecting 4 groups of samples and 8 important shrimp pathogens and is a relatively low-cost, portable, and high-throughput nucleic acid (RNA and DNA) detection technology. The establishment of this technology will provide key technical support for the construction of biosecurity systems for healthy shrimp culture. We used standard glass slides (75 mm×25 mm) to set up the micro-detection slide system for nucleic acid isothermal amplification. Each slide contains 48 separated super-hydrophilic reaction cells in which the reaction reagent mix is pre-embedded. A super-hydrophobic and oleophobic region surrounds the reaction sites, which are essential in positioning the sealing oil, i.e., dimethicone, over the reaction mixture to prevent evaporation and cross-contamination. Each of 8 reaction cells comprises a group of detection units for one sample. There are 4 groups of detecting cells used for sample detection, and the remaining 2 groups of detecting cells are a negative and positive control, respectively. In this study, each group of detecting cells will detect 8 different pathogens. Image 1 • The micro-detection slide system for multiple pathogens detection is developed. • Four samples and eight pathogens can be tested simultaneously in 55 min. • The cost of the micro-detection slide system is less than $1. [ABSTRACT FROM AUTHOR]

• A TaqMan probe based real-time PCR assay for detection of DIV1 targets the MCP gene. • The real-time PCR assay is highly specific and the detection limit was 1.2 copies. • The assay has DSe and DSp of 97.2% and 98.7%, respectively. Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA. [ABSTRACT FROM AUTHOR]

• The capsid protein IHHNV is expressed in a baculovirus insect cell system. • The capsid protein can self-assemble into virus-like particles (VLPs). • The VLPs encapsulate the pcDNA3.1(+)-EGFP plasmid DNA. • The VLPs introduce the plasmid into shrimp hemocytes. Virus-like particles (VLPs) are potential containers for delivery of therapeutic agents at the nanoscale. In this study, the capsid protein of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was expressed in a baculovirus insect cell system. The 37-kDa recombinant protein containing the hexahistidine residues (His Tag) at N-terminal was purified using immobilized metal affinity chromatography (IMAC) and assembled into VLPs with a diameter of 23 ± 3 nm analyzed by transmission electron microscopy. We also verified that disassembly/reassembly of IHHNV-VLPs was controlled in the presence and absence of DTT. The efficiency of IHHNV-VLPs to encapsulate plasmid DNA was about 48.2%, and the VLPs encapsulating the pcDNA3.1(+)-EGFP plasmid DNA could recognize the primary shrimp hemocytes and deliver the loaded plasmid into cells by detection of expressed enhanced green fluorescent protein (EGFP). These results implied that the IHHNV-VLPs might be a good candidate for packaging and delivery of expressible plasmid DNA, and may produce an antiviral product in shrimp cells for gene therapy. [ABSTRACT FROM AUTHOR]