Analysis of VP28 Gene in White Spot Syndrome Virus Infection

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Analysis of VP28 Gene in White Spot Syndrome Virus Infection

Temporal analysis of VP28 gene in White spot syndrome virus infected fresh water crabs

Chiin Nei Chinga Mansi Parihara R Sudhakaranb aSchool of Bioscience and Technology VIT University Vellore 632 014 Tamil Nadu India bAssistant professor SBST VIT University Vellore 632 014 Tamil Nadu India

ABSTRACT

Aquaculture is the farming of aquatic organisms such as fish crustaceans molluscs and aquatic plants Disease emergence is a concern in wild fisheries due to environmental pressures the direct impact of human activities and the risk of pathogens spread from aquaculture Common viruses are Taura syndrome virus TSV white spot syndrome virus WSSV and the necrotizing hepato pancreatitis bacterium NHPB The White Spot Syndrome Virus is the most economically devastating viral pathogen to global shrimp aquaculture production and has been proposed to be capable of infecting all decapod crustaceans WSSV is an enveloped ellipsoid virus which belongs to the genus Whispovirus of the family Nimaviridae VP28 is one of its major envelope proteins and plays a crucial role in viral infection In this study the proteins of the infected crab were purified using SDS page and then Western Blotting was performed to extract that particular protein The VP28 protein will appear as specific bands in the blot

Keywords

Aquaculture White spot syndrome virus VP28 SDSPage Western blotting
INTRODUCTION

White spot syndrome virus WSSV is one of the major shrimp pathogen that causes a high mortality rate of 90100 within 310 days of infection Lightner 1996 Natural WSSV infections have been found in captured and cultured specimens of the mud crab WSSV is an enveloped ellipsoid virus which belongs to the genus Whispovirus of the family Nimaviridae In addition WSSV can also infect a wide range of hosts including both decapod and nondecapod animals with more than hundred species described to date So far the genome from three different WSSV isolates has been sequenced Sequence analysis showed that WSSV contains approximately 500 putative open reading frames ORFs most of which have no homology with any known genes or proteins in public databases Till now more than 50 structural and nonstructural proteins were identified Tools such as polymerase chain reaction PCR and nested PCR originally developed by Lo etal have been widely used and recommended by the Office of International Epizootics OIE to be used as standard diagnostic methods for the detection of WSSV Despite their excellence in specificity and sensitivity these methods were not suited in some circumstances due to their complications the requirement of thermal cycler timeconsuming and laborintensive Moreover the classical agarose gel electrophoresis with ethidium bromide staining following the visualization under the ultraviolet UV transilluminator required to analyze the result of PCR products Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis The blot is a membrane of nitrocellulose or PVDF polyvinylidene fluoride The gel is placed next to the membrane and application of an electrical current induces the proteins in the gel to move to the membrane where they adhere The membrane is then a replica of the gels protein pattern and is subsequently stained with an antibody Therefore these features could be limited their applications particularly in the resourcelimited areas and nonlaboratory environments such as at the pond or station sites

2 MATERIALS AND METHODS

21 Tissue homogenate preparation

Gills muscle hepato pancreas and Head soft tissue from the crab infected with WSSV were homogenized in 110 suspension with NTE Buffer It was then freeze and thaw for three times then centrifuged at 5000 rpm for 5min The supernatant were collected separately in a tube and stored at 20C This supernatant was used for protein analysis

22 Protein estimation

Lowrys method was performed for the estimation

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