Analysis of vp28 gene in white spot syndrome virus infection

Diseases in Asian Aquaculture VI. White spot syndrome has been reported in many shrimp growing countries of the world. Since WSSV has a wide geographic distribution and host range, studies on the comparison of morphology, virulence, genomic composition and protein composition among WSSV isolates have been carried out by various researchers Wongteerasupaya et al.

Analysis of vp28 gene in white spot syndrome virus infection

Demonstrate expression of WSSV structural proteins in Litopenaeus vannamei by injection of replicon particles. Sequences for VP15 and VP19 will have a histidine tag added to facilitate detection of the expressed protein.

Analysis of vp28 gene in white spot syndrome virus infection

RNA will be transcribed and electroporated into Vero cells. Following incubation, the Vero cells will be harvested and lysed. The lysate will be analyzed by Western immunoblotting to demonstrate expression of the desired protein.

Once a replicon has been confirmed to be producing the desired protein, it can be used to produce RP vaccine.

White spot syndrome virus: an overview on an emergent concern

The RP vaccine will be harvested from culture fluids and the potency will be measured by antigen- specific IFA and tested in a cytopathic effect CPE assay to assure the absence of detectable replication-competent virus.

Specific pathogen free stocks of L. Three replicates of this experiment will be utilized. Histological sections and tissues will be evaluated for presence of VP Experimental animals will be challenged by oral gavage 14 days after the initial injection with RP.

Mortality rates and viral copy number of experimental and control groups will be compared to determine if statistical differences exist between those groups.

Whole larvae will be sacrificed and evaluated using epiflourescence microscopy. Control animals will also be used. This study will be used to determine if infective remains intact through the digestive tract, and if RP infects and expresses a foreign protein in PL20 animals.

This will allow for an additional alphavirus to be evaluated for use as an oral WSSV vaccine. A sham control group of will be used.

Analysis of vp28 gene in white spot syndrome virus infection

The surviving juvenile shrimp will be challenged by submersion in WSSV macerate from acutely infected shrimp. At the termination of the experiment pleopod samples taken for PCR testing and determination of viral copy number.

Develop alphavirus replicon particles RP expressing structural proteins of WSSV virus Alphavirus replicon particles expressing structural protein genes of WSSV were created by cloning WSSV sequences synthesized commercially into an existing alphavirus backbone vector.

Once the genes were cloned into the replicon vector, the insert was sequenced to confirm the identity of the construct. Specified mass amounts of the replicon and helper RNAs were mixed and electroporated into Vero cells.

Electroporated cell suspensions were seeded into roller bottles and incubated at 37oC for 18 hours. RP were harvested from culture fluids and the infectious titer of the RP preparation was measured by antigen- specific IFA. Twenty five animals were injected with 50 uL of cell culture media to serve as a control group.

Animals were sedated and euthanized in an ice slurry at 24, 48, 72, 96, and hours post injection. Whole shrimp were fixed in Davidson's Fixative and cut into sections. An immunohistochemistry assay utilizing a monoclonal antibody to VP28 was used to analyze animals inoculated with VP28 expressing RP.

A sham injection group received 50 uL of cell culture media. Experimental animals were then challenged by injection 3 days after the initial injection with RP. Animals were observed for mortality over a 21 day period.white spot syndrome virus (WSSV) and synthesized using wheat germ cell-free technology was investigated in kuruma The WSSV VP28 gene was amplified by PCR using the pEU-VP28 Fw and pEU-VP28 Rv primers (Table 1), which contain restriction enzyme sites for BamHI and SpeI, respectively.

PCR cycling conditions were: 1 cycle of AB Gene. The present study revealed that appearance of VP28 protein is related to infection in hostThe differential transcription of VP28 gene in different hosts has been observed and agrees with the results of the present study that WSSV caused % mortality in freshwater crab In fresh water crab, transcript of VP28 was clearly detected at 2 days p.i.

White spot syndrome virus (WSSV) infection is commonly detected by vp qPCR assay in wild crayfish, Procambarus clarkii, a widespread crustacean species in the aquatic environment in China. The virions of crayfish WSSV have been isolated and purified. White Spot Disease (WSD), caused by the White Spot Syndrome Virus (WSSV), has been the most problematic viral pathogen affecting global shrimp farming since its emergence in WSSV is highly virulent and may result in % mortality in ponds within days of infection [13].

White Spot Syndrome Virus VP28 Gene White Spot Syndrome Virus Infection Viral Protein Synthesis Protein Synthesis Inhibitor Cycloheximide These keywords were added by machine and not by the authors. This process is experimental and the keywords . Abstract. VP33, also termed VP, VP37 or VP36B, is a minor envelope protein of white spot syndrome virus (WSSV).

Because of its low abundance and lack of a transmembrane domain, we hypothesized that VP33 is likely to be attached to the viral envelope by .

Dr. John Thomas | VIT University -