Friday, 31 January 2014

TTX biosynthesis remains a mystery!

In addition to the TTX studies already summarised here, I have been looking for a paper that tried to identify the metabolic pathways that potential TTX producing bacteria are utilising. Adam’s last entry already focused on the origin of TTX and the same authors have published this paper in order to investigate it further.     
Chau et al. isolated the bacteria in the tissues of the blue-ringed octopus Hapalochlaena sp. and the sea slug Pleurobranchaea maculata which have both been suggested to have associated bacteria that synthesise TTX. Microbial communities of the both specimens were examined by culturing samples from 4 different agars and colonies were subject to genomic DNA extraction. 16S rRNA genes were amplified so that bacteria could be identified and a phylogenetic tree encompassing all isolated bacteria was established for both animals.   
Most notably, the slug and the octopus showed a greater diversity of bacterial species than previously estimated with 22 and 27 unique bacterial strains, respectively and in general, over 60% belonged to Alteromonadales with Pseudoalteromonas and Alteromonas as most representative genera. High abundance of these genera suggested that they play a key role in the hosts’ physiology and perhaps are also involved in the TTX production which acts as a defence mechanism to the host and therefore increases its fitness.

Additionally, all isolates were screened for three gene types (PKS, NRPS and AMT genes) which were assumed to be involved in the biosynthesis of TTX. Some isolates screened positive for any of the targeted genes and in particular two strains of Pseudoalteromonas from the octopus contained all three genes. However, the PKS and NRPS genes identified coded for unique enzymes which have not been found in any characterised toxin biosynthesis pathways and thus the genes could not be confirmed to be involved in TTX production; yet further analyses are needed to reject or confirm these genes responsible for the toxin synthesis.         
AMT gene sequences from isolates of octopus tissues showed high similarity to AMT genes (e.g. cyrA) that are involved in the biosynthesis of cyclindrospermopsin, a toxin which is assumed have similar molecular components as TTX. Therefore, screening suspect microorganisms for AMT genes could be key for investigating the TTX origin.

In general, it is still unknown how exactly TTX is synthesised. However, this paper provides a basis for understanding the microbial diversity of TTX containing organisms. The authors have only examined two animals and analysing more animals of the same species is important so that it can be determined whether microbial diversity differs between individuals of the same species. It has to be determined whether only the associated bacteria are responsible for TTX, or if perhaps metabolic substances produced by the animals are utilised by the bacteria. Moreover, metagenomic analyses and culture independent identification would be more appropriate to get an insight of the whole microbiota since the majority of all bacteria are not culturable, as well as the targeting of more than just three genes suspected to be responsible for the TTX synthesis.

Chau, R., Kalaitzis, J.A., Wood, S.A., Neilan, B.A., 2013. Diversity and Biosynthetic Potential of Culturable Microbes Associated with Toxic Marine Animals. Marine Drugs, 11, pp. 2695 – 2712.

Wednesday, 29 January 2014

Tetrodotoxin Origins

Tetrodotoxin (TTX) is a toxin lethal to humans, and found in 6 phyla of organisms within the Animalia kingdom including the Chordata, Mollusca, Echinodermata, Chaetognatha, Arthropoda and Platyhelminthes. The phylogenetic and geographical diversity of this group, along with the many similar findings of toxin origins previously attributed to macroorganisms but found to originate from microorganisms, points to the widely supported theory that TTX is produced by a bacterium.

TTXs potency has been known for thousands of years, but only recently has it been postulated to be produced from bacteria. There are many different strains of potential TTX producing bacteria in single animals; however investigations into such bacteria in lab cultures have been inconclusive. This is most likely as conditions and triggers are missing from the usual host symbiotic environment, or the strains of bacteria producing TTX cannot be cultured.

TTX has been isolated from many toxin harbouring animals, but not all. The newt Taricha granulosa has been shown to harbour TTX with negligible evidence of an associated bacterium in the digestional tract, though due to its presence in the newt it’s involvement in TTX production cannot be ruled out. The definitive way to link bacteria and TTX would be defining the biosynthetic pathways or associated genes for producing the toxin.

Further studies of Taricha torosa and T. granulosa show that the newts gain toxicity in response to a non TTX diet, which conflict with the envirionmental dietry origins of TTX bacteria symbionts in puffer fish (Takifugu niphobles). Such environmental mechanisms aren’t uncommon in marine animals.

The best way to completely confirm TTXs origin in bacterium is to find its biosynthetic pathway and understand the genetics underpinning it. However due to TTXs unique structure this has proved to be a difficult task. To this point the literature has been inconclusive on the subject. Many different enzymes could be involved in forming its structure, leading to differing and incomparable studies which slow the development of potential molecular tools for unravelling it.

Current Ideas for the pathway look at the many similar (though not so structurally unique) bacterial toxins such as phaseotoxin, cylindrospermotoxin, and the most similar, saxotoxin. Looking into the proposed biosynthetic pathways for these leads to various unproven conclusions, the most popular of which are the genes are likely clustered together on the genome, and there is likely an enzyme analogous to amidinotransferase for its essential guanium moiety, with supporting non-ribisomal peptide isynthetase and Polyketide synthases machinery similar to saxotoxin production. This pool of thought is further supported by a wealth of TTX animals being linked to saxotoxin production.

 Investigation of the pathway for this toxin is important as it will reveal novel biosynthetic reactions and enzymes. Study of the unique carbon skeleton alone will likely give rise to pathways unlike any other investigated to date.

TTXs specificity to voltage gated sodium channels makes it a good tool in neuroscience, with useful developments coming along in anaesthesia and analgesia. Current techniques for collecting TTX from livers of puffer fish or chemically creating it are low yield, expensive and, in the case of the former, damaging to the marine environment. Due to the complexity and costs of producing this molecule surely future corporate demand allow funding for investigation of the biosynthetic pathway so to allow precise genetic engineering in microbes for a cheaper price. However the range of possibilities for the pathway are so great that much work will need to be done to develop a greater foundation of comparable literature to help solve the TTX biosynthesis puzzle.

Chau, R., Kalaitzis, J. A., & Neilan, B. A. (2011). On the origins and biosynthesis of tetrodotoxin. Aquatic toxicology, 104(1), 61-72.

Further evidence in support of bacterial origins for tetrodotoxin accumulation in puffer fish

Further evidence in support of bacterial origins for tetrodotoxin accumulation in puffer fish

And so it seems we’ve all been inspired by toxins!  Continuing the theme and complementing the post by Caroline, this paper by Auwithoothij & Noomhorm (2012) investigated the major (culturable) microbes associated with the internal organs of the puffer fish, Lagocephalus lunaris in relation to tetrodotoxin (TTX) accumulation.

The study was conducted throughout 2010 in the Gulf of Thailand, with an average of 30 samples of L. lunaris collected each month from January through to December.  Aside from all the fish being the same species, all other characteristics (eg; size, sex, weight, developmental stage) appeared to be highly variable.  All samples once shipboard were immediately put on ice and soaked in ethanol once in the laboratory, before aseptically removing organs to minimise any environmental contamination.

TTX accumulation within the organs of the fish was highest over two periods in the year – February to March and August to October, which reportedly coincides with cooler waters of around 25 °C.  Nine dominant bacterial species were isolated from the organs of L. lunaris, however only Vibrio alginolyticus and Shewanella putrefaciens were reportedly found to produce TTX in this study.  A graphical representation showed that increased loads of S. putrefaciens within the organs appeared to coincide with increased levels of TTX, although a correlation would have benefitted here.

The authors went on to culture S. putrefaciens to ascertain the effects of salinity and temperature on bacterial growth and production of TTX.  Results suggest that whilst S. putrefaciens grows more slowly at lower temperatures (ie; 25 °C rather than 30 °C) it produces higher levels of TTX, which corresponds to the higher accumulation of TTX in the puffer fish organs found at lower temperatures. Salinity had no effect on growth of the bacteria but lower salinities (9 – 22 ‰ rather than 32 ‰) also increased production of TTX.  Whilst not mentioned in the paper, this may correlate to the rainy season for the second period of high TTX concentration found in the organs (August to October), although data for the rainfall experienced in 2010 would need to be obtained to consider this further.

As highlighted by the authors, it must be considered that the higher abundance of S. putrefaciens is due to increased environmental abundance and may therefore be accumulated in puffer fish tissues through the food chain.  Water sampling alongside the fish capture would have contributed greatly to this study, allowing comparison of background bacterial compliment against that found within the fish organs.  Whilst this evidence is still circumstantial, it does provide more fuel for the bacterial origin of TTX argument which, whilst controversial, would explain how TTX is produced in such a wide range of taxa – both marine and terrestrial.  What it isn’t able to answer however, is whether the bacteria are living symbiotically within the fish.  The debate continues!

Auawithoothij, W., & Noomhorm, A. (2012). Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)‐accumulation of puffer fish Lagocephalus lunaris. Journal of applied microbiology, 113(2), 459-465.

Tuesday, 28 January 2014

Effects of the dinoflagellate Alexandrium minutum and its toxin (saxitoxin) on the functional activity and gene expression of Crassostrea gigas hemocytes

Paralyzing people, but what about the shellfish?
So we know the economic implications of paralytic shellfish poisoning (PSP) caused by harmful algal blooms (HABs) as well as the effects in human health (i.e. paralysis) but what effects does the toxin have on one of it's phycotoxin vectors, the Pacific oyster?

Mello et al., (2013)⁠ set out to compare the effects of a saxitoxin (STX) producing algae, Alexandrium minutum and the STX itself on in vitro haemocytes of Crassostrea gigas. Haemocytes are the primary component of molluscan immunity so any changes in their behaviour could have catastrophic effects on whole populations. In vitro also provides a much more controlled environment with less inter-individual variability then using whole organisms, especially where pooled haemolymph samples are used as was in this case. A. minutum and STX both caused significant reductions in phagocytic ability of haemocytes though only A. minutum alone caused swelling of haemocytes. This is indicative of the oyster mounting an immune response with the cells increasing in size ready for phagocytosis, however there seems to be a mechanism relating to the algae and toxin that reduces their ability to do this. Furthermore both treatments had negative impacts on the oxidative burst, a mechanism by which the haemocytes produces reactive oxygen species (ROS) to damage invading pathogens. This was most pronounced in the STX only treatment with between 72 and 80% reductions. As well a looking at functional biomarkers the authors used the quantitative polymerase reaction (qPCR) to determine expression of immune and stress related genes was modulated. Of the genes studied, four showed some level of up or down regulation relating to STX and one was up-regulated by the presence of A. minutum. This gene was an interleukin (IL-17), which are usually related to inflammation, so this could signify the preparation of an immune response in relation to algal infection. In contrast, the STX treatment caused a reduction in expression of IL-17. STX seems to inhibit the interleukin's production and the difference is likely due to differences in the bioavailability of the toxin between treatments, as A. minutum cultures generally have a low extracellular toxin level until they are 30 days old (6 days old in this experiment). However the mechanism for reduction is unclear. The heat shock protein (HSP) gene HSP70 was up-regulated by STX implying that the toxin has a denaturing affect as this HSP is a chaperone that acts against misfolded proteins. Similarly defensin, an antimicrobial peptide (AMP), has increased expression under STX treatment. AMPs are poorly understood relating to xenobiotics but it could be due to the oyster recognising STX as a pathogenic product and preparing for infection, this is however wild speculation on my part and would need to be studied further. The only other change in expression was relating to biotransformation. The down regulation of CYP356A1 by STX could leave oysters vulnerable to other harmful chemicals though further research into the synergistic effects would need to be carried out.

So from this research it appears that the immune system of the Pacific oyster is indeed compromised by STX and Alexandrium. The implication of this is potential infection by other pathogens which would be devastating for fisheries already impacted by HABs. However the immune response is very complicated and just looking at 11 genes is not enough. There are many isoforms of CYPs and HSPs in oysters and these may be activated/deactivated as part of the response. Ideally transcriptome wide expression would be carried out for the two treatments and then all differences could be observed. This is unlikely at the current price but if HABs continue to increase in frequency it might be an avenue worth investigating in the future.

Mello, D. F., Silva, P. M. da, Barracco, M. A., Soudant, P., & Hégaret, H. (2013). Effects of the dinoflagellate Alexandrium minutum and its toxin (saxitoxin) on the functional activity and gene expression of Crassostrea gigas hemocytes. Harmful Algae, 26, 45–51.

Poisonous puffer fish or bamboozling bacteria? Who is to blame for TTX production?

Following from this Mondays lecture I thought I’d find out if there was any definite proof that bacteria are the cause of TTX in Puffer fish, and I managed to find a relatively recent paper (2011) which had ID’ed a new TTX-producing bacterial species found in puffer fish intestines.

TTX (as we learnt in Colins lecture) is seen in a wide variety of animals (from the Blue Ringed Octopus to several species of puffer fish.) The amount of TTX in puffer fish is species specific, and varies in concentration with regards to different organs and different seasons.  In general the ovary and liver are believed to be the most toxic organs as they have the highest TTX levels  - however for this species, Takifugu niphobles the toxicity of the intestines are comparable to their ovaries and liver – and is far more potent than other organs.
The origin of TTX in puffer fish has been extensively studied (see the attached Table, which shows previous studies which have documented TTX-producing bacteria isolated from puffer fish.) It is interesting to note that once again Vibrio returns! My paper mentioned that not only was Vibro alginolyticus the first identified TTX-producing bacterium, but most of the reported TTX-producing strains belong to the genus Vibrio.) We saw before that cultivating puffer fish above the sea bed/in an enclosed water system leads to the production of puffer fish who do not produce TTX – but these puffer fish do become toxic when fed with toxic puffer fish liver or returned to open water. This; alongside the fact that so many organism throughout the world produce TTX, points to the idea that TTX has an exogenous microbial origin.

In this paper
Five bacterial strains were isolated from the intestines of Takifuga niphobles (a puffer fish collected from coastal Hong Kong waters.) A mouse bioassay was used to reveal one of the strains: gutB01, caused typical symptoms of TTX intoxication (colvusions and dyspnea, with all mice bioassays killed within 10-15minutes.)
An ELISA method was also used to further demonstrate there was TTX production from the strain gutB01. Both of these methods were used to procure a toxicity value (how much TTX was produced from 24h cultivation of gutB01). This was much higher in the mouse bioassay (7.7µg/L) than the ELISA (4.3µg/L) – this could be due to the presence of some unidentified toxic components (possible TTX derivative) in the mouse bioassay, or due to the fact the cells had been in different growth phases.
Mass spectrometry (in this paper, the MALDI-TOF) was also used to confirm the chemical identity of the toxin produced by gutB01 (which matched a TTX standard.)
MIDI analysis was used to identify gutB01 as Raoultella terrigena (this method is apparently very common for microbial identification, and works via matching the fatty acid profile of an unknown species to the fatty acid profiles of various references microbial species in a database.) This ID was confirmed with the sequence obtained from a 16S-23S rDNA ITA.

In conclusion: this paper revealed a novel TTX-producing bacteria species (Raoultella terrigena) associates and lives in the intestines of the puffer fish (Takifugu niphobles.) The TTX found in puffer fish is most likely to be due to this bacterium however (as always) the papers advises caution as this study did not exclude the possibility that other bacteria in this species of puffer fish could also be contributing to TTX production.

Yu, V. C. H., Yu, P. H. F., Ho, K. C., & Lee, F. W. F. (2011). Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles. Marine drugs,9(11), 2384-2396.

Monday, 27 January 2014

A red algae secondary metabolite confers resistance to white spot syndrome virus and enhanced immunity in shrimp

Following an observation made by Thai shrimp farmers that shrimp cultured in ponds containing Gracilaria fisheri (a red algae) had a greater survival rate than those grown in ponds without, this team of researches isolated sulphated galactans from G. fisheri to investigate their immunostimulatory and anti-viral effects.
After extracting the sulphated galactan, its sulphate and carbohydrate content was analysed, its molecular mass determined and it was analysed by NMR and FT-IR spectroscopy. All with the aim of identifying the molecules present. These analyses revealed that the sulphated galactan of G. fisheri is a partially pyruvated and methylated agar-obiose structure with a backbone of alternating units of two sugars,  3-linked b-D-galactopyranose (G) and 4-linked 3,6-anhydro-a-L-galactopyranose.
Having identified the compound, they moved on to investigating its effects on the immune system of the shrimp Penaeus monodon. Firstly, they bioencapsulated SG in Artemia salina by storing them in two concentrations, 100µg/ml and 200µg/ml of a saline solution containing the SG. They then separated a population of shrimp into three treatments, those fed on normal A.salina, those fed on 100µg/ml SG A.salina and those fed on 200µg/ml SG A.salina. They kept the three treatments on their different diets for one week and then extracted haemolymph to compare several immune parameters: total haemocyte count, phenoloxidase activity, superoxide dismutase activity and superoxide anion production.
In SG treated shrimp, all immune parameters showed significant increase, an increase that was dose dependent. The results were as follows:
·         Total haemocyte count increased 141% with 100µg/ml and 186% with 200µg/ml
·         Phenoloxidase activity increased 360% with 100µg/ml and 1300% with 200µg/ml
·         Superoxide dismutase activity increased 555% with 100µg/ml and 635% with 200µg/ml
·         Superoxide anion production increased 204% with 100µg/ml and 341% with 100µg/ml
Having shown the immunostimulatory effect of SG, they proceeded to investigate its antiviral effects. For this they challenged each treatment of shrimp with White Spot Syndrome Virus and added a control group that underwent the same injection procedure but with an inert saline solution rather than WSSV. Over a 14 day period they measured mortality, viral load, expression of viral protein VP 28 and immune parameters. Control shrimp showed 100% mortality by day 10, whereas 100µg/ml SG treated shrimp showed 63.5% mortality at the end of the 14 days, and 200µg/ml showed just 39.5% mortality. Decreased mortality in SG treated shrimp correlated with significant increases in all immune parameters measured. At day 2, all WSSV inoculated shrimp showed VP28 gene amplification. Haemocytes collected on day 5 showed relatively low VP28 gene expression in 200mg/ml SG treated shrimp. By day 10 all inoculated controls were dead and 200mg/ml showed no VP28 gene amplification.
The mechanism of antiviral action is thought to involve an interaction between the SG’s sulphate groups and surface receptors on haemocytes. SG from G.fisheri contains two sulphate groups per disaccharide repeating unit, a relatively large amount compared to other plant or seaweed derived secondary metabolites used for antiviral functions, making it a more efficient antiviral compound. SGs from other red seaweeds have been reported to have antiviral activity against herpes simplex virus, cytomegalo virus, dengue virus and respiratory syncytial virus in humans.
Although this study only challenged SG treated shrimp with WSSV, the resulting increase in many immune parameters such as those that combat oxidative stress suggests that G.fisheri SG has the potential to be used as a non-specific immunostimulant and antiviral compound that could help protect shrimp from a range of diseases and stressors. The authors highly recommend it as a feed supplement to be used in shrimp aquaculture.

Wongprasert, K., Rudtanatip, T., & Praiboon, J. (2014). Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp. Fish & shellfish immunology, 36(1), 52-60.

Bacterial strains as sources of antioxidant, antimicrobial and antiplasmodial compounds in the marine sponge, Phorbas tenacior

Marine sponges are recognised as a rich source of biologically active compounds. Already, some of these compounds have been used as therapeutics and anticancer agents, another one hundred are currently under preclinical evaluation.

More than 30 different phyla of sponge-associated organisms have been recognised and can equate to as much as 37% of the sponges biomass. However, currently only 1% can be cultured in laboratory conditions. These species-specific micro-organisms may play a role in nutrient uptake, stability of the sponge skeleton, chemical defence or antibiofouling agents but these advantages remain poorly understood. It is thought that these bacteria provide a potential renewable source of biomedical agents. However, there are concerns about difficulties in ensuring production at an industrial level.

Transmission electron microscopy and scanning electron microscopy were used to assess the localisation of the bacteria within the sponge. Individual strains were isolated using serial dilutions cultured on various media. The bacterial community was determined by 16S rRNA gene sequencing then BLASTed against a database. Several assays were used to determine antiplasmodial, antioxidant and antimicrobial properties.

A great variety of bioactive molecules have been reported from the genus Phorbas, but this is the first report to describe antioxidant and antiplasmodial activities associated with the P. tenacior sponge.

Micro-organisms were found to be extracellular in the mesohyl of all different shapes and sizes. The two most common phyla associated with this sponge are Proteobacteria and Actinobacteria, including the genera Vibrio and Shewanella. Both of these genera have been shown to have antifouling, antioxidative or antibacterial properties.

It was also discovered that the sponge itself or its associated microbes can produce antioxidant compounds which prevent protein or lipid damage, ensuring the protection of the bacteria when the sponge host produces oxidative compounds. Vertical transmission of Pseudovibrio spp. bacterial larvae to the sponge has provided evidence of a possible symbiotic relationship.

Antiplasmodial activity from Vibrio strains was first reported in this study, with 45% inhibition of Plasmodium falciparum at 100 µg ml-1. No antimicrobial activity was detected against pathogenic environmental strains such as Vibrio harveyi and V. parahaemolyticus. A Citricoccus alkalitolerans strain was also first reported to be associated with a marine sponge. These microbes showed a moderate antimicrobial activity against many environmental stressors including V. parahaemolyticus, therefore could help in host defence against pathogens.

Bioactive compounds produced by the bacterial community of the marine sponge, P. tenacior, could be extremely beneficial to human health and marine organisms, depending on industrial production. Benefits for seafood industries could be a possibility, minimizing the outbreaks of harmful environmental pathogens.

 I thought this was an interesting new development and a good starting point for further research, acting as a wide overview of the bacterial community and its capabilities found within this sponge. Focusing on individual areas could provide a more in-depth knowledge and provide potential uses of these bioactive compounds in biomedical agents.

Dupont, S., Carre-Mlouka, A., Descarrega, F., Ereskovsky, A., Longeon, A., Mouray, E., Florent, I. and Bourguet-Kondracki, M.L. (2013) Diversity and biological activities of the bacterial community associated with the marine sponge Phorbas tenacior (Porifera, Demospongiae). Letters in Applied Microbiology. 58: 42-52

Friday, 24 January 2014


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.