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.
Rachel - Having previously looked at a paper which tests the ability of shrimp to develop an immune response to VP28 (WSSV) through oral exposure, I find this to be an obvious prior test that would have allowed for the research continuation.
ReplyDeleteI question why in my paper they only tested VP28 when clearly, this paper draws conclusions between both VP28 and VP24, perhaps it would be interesting to see a study testing the combination of these two proteins, or even the full 5 proteins known to exist in the nucleocapsid? What do you think?
In addition to your point Ethan, it would be really interesting to see whether combination of both VP24 & VP28 may actually have a synergistic effect - enhancing performance of either protein on its own.
ReplyDeleteI agree, looking at interactions of proteins as a possible factor for virulence would be interesting.
ReplyDeleteSynergism is interesting when approaching the roles proteins may have in infection, however on my previous point (the paper by Syed and Kwang) it has come to my attention that combinations of proteins when creating a vaccine would perhaps be unnecessary.