White Spot Syndrome Virus (WSSV) is a virulent disease known to infect many taxa of Crustacea. Originating in Taiwan, tt is especially poignant in the shrimp aquaculture industry where world-wide infection has resulted in devastating economic loss. Previous work has presented a suite of data that show WSSV as an enveloping bacilliform virus with a completely sequenced genome. Previous work has also identified many viral envelope-proteins, known for their association with the primary-infection phase; and has provided the links between 10 genes and their associated enveloping proteins. However, little is known about the underlying circumstances in a WSSV life cycle.
VP24 is the product of the wsv002 gene, identified by Yang et al. 2001. Further investigation into this protein has led to some research eluding to it being a nucleocapsid protein, whilst others believing it to be an enveloping protein. This study aimed at discovering a more precise idea of the location of VP24 within a WSSV viron, in order to assess it's function.
In order to achieve this, the protein was amplified from the wsv002 gene and a recombinant VP24 protein was expressed from E.coli BL21. The protein was then purified according to the QIAexpressionists system.
An antibody was then prepared by using the purified proteins as an antigen. This was injected into mice every 10 days (no indication is given as to how long this went on for). After the Four days after the last injection the antisera was collected from the mice and purified.
WSSV was collected from confirmed infected Penaus japonica and Procambarus clarkii. This inoculum was then purified and the viral envelope (soluble) was separated from the nucleocapsid (insoluble) via centrifugation.
Both the WSSV virons and suspended nucleocapsid were mixed with the mouse antisera and examined under transmission electron microscope. One control was also examined (being without the mouse antisera).
Finally, an in vivo neutralization experiment was conducted upon crayfish (not specified but probably the same as mentioned above). The crayfish were injected with varying mixtures of WSSV and antibody. One positive and negative control were included.
Computer analysis of the VP24 protein showed it to contain a hydrophobic region and that it did reside in the envelope fraction of the viron. This was further justified by a Western Blot.
The extraction of VP24 allowed for it's hydrophobicity to be analysed by mixing it with detergent Triton X-114. Envelope proteins are known to stick with the envelope due to their high hydrophicity and this test showed VP24 to be present in the detergent phase; making it hydrophobic. This showed an association between the two. Further analysis via electron microscopy demonstrated VP24 to be present in the envelope-protein of WSSV.
An interesting by-product was realized due to this paper. By conducting a far-Western experiment an association was found between the VP24 protein and the VP28 protein. The positioning of VP28 in the Western analysis showed that it had been pulled down by VP24 and that there was a form of interaction previously unknown.
The in vivo neutralization experiment showd expected results. The positive control experiment resulted in 100% mortality whereas the negative control showed no mortality. The various mixes of antibody were shown to delay 100% mortality relative to the measure of dose e.g the lower the dose, the less days it took for 100% mortality to be achieved, and the higher the dose, the longer it took to achieved 10% mortality. This was expressed as true neutralization or delay by the antibody.
In summary, the five previously known proteins of WSSV were shown to exist in the nucleocapsid (VP15) and the viral envelope (VP28, VP26 and VP19). The placement of VP24 was hihgly contested, however this paper shows that VP24 is solely present in the viral envelope via non-ionic detergent analysis and is adequately hydrophobic to be associated with the envelope. Despite Xie & Yang's work, little is known about VP24's role in the WSSV life cycle. However, current work can identify 43% amino acids in both VP24 and VP28 and previous work has also eluded to VP28's role as being involved in the entry of the viron into the cytoplasm. The results of this paper's in vivo assay show significant delay of infection in crayfish suggesting VP24 also plays a role in the WSSV infection process. Xie & Yang further hypothesize that VP24 and VP28 act together in a protein-complex.
This paper is important as it takes tentative yet considerable steps in understanding the molecular functions of this highly fatal disease, showing first-time interactions between VP24 and VP28 proteins; and laying a path for future control and diagnosis.
Xie X & Yang F. 2006 White spot syndrome virus VP24 interacts with VP28 and in involved with virus infection, Journal of General Virology. 87 : 1903-1908.