Oral Vaccination of Baculovirus-Expressed VP28 Displays Enhanced Protection against White Spot Syndrome Virus in Penaeus monodon

Overview:

WSSV, of the genus Whispovirus, is one of the most threatening infectious pathogens to the shrimp culture industry, meaning that any means of treating of vaccinating against this virus would be incredibly well received. WSSV usually has a mortality rate of 100% over a period of 2 to 5 days. Currently, no effective vaccines or adequate treatments are available. It is known to be difficult to vaccinate invertebrates due to the lack of a true adaptive immune response. Inverts rely solely on an innate immune response. It has been proved that a quasi-immune response exists which survives after exposure to the WSSV virus, creating some hope in the field.VP28 is one of five major structural proteins found in WSSV and studies show this protein is associated with the virion envelope. It is thought to be responsible for the initial viral infection. The authors of this experiment (Musthaq and Kwang), by inserting VP28 into a baculovirus, managed to get a VP28 protein expressed on the surface and use this as a method of VP28-based recombinant vaccination. This recombinant vaccine was named Bac-VP28. A construct that did not have the VP28 gene was termed Bac-wt. The amount of VP28 present in Bac-VP28 was measured as 65.3micrograms per ml and considered as an abundant quantity.

Oral Vaccination Results:

Shrimp were orally administered Bac-VP28, Bac-wt and PBS respectively (groups 1-3). Bac-VP28 and Bac-wt coated feed was administered continuously for 7 days before the shrimp were subsequently challenged with the WSSV. Batch 1 and 2 were treated identically, except batch 1 received a WSSV dose of 3dpv and batch 2 received a dose of 15dpv.

In all cases, the positive control group (3) showed 100% mortality, as did group 2, treated with the Bac-wt. The group (1) treated with Bac-VP28 however showed only an 18.3% (batch1) and 23.3% (batch2) mortality rate, which is significantly reduced.

A negative control group (without the virus) showed no mortality.

Immersion Vaccination Results:

As with the oral vaccination, there were the 3 groups, Bac-VP28 (1), Bac-wt (2) and PBS (3). Instead of through feed, they were vaccinated through immersion in a solution. Again, all shrimps in batch 1 were challenged with WSSV at 3dpv and those in batch 2 challenge with WSSV at 15dpv.

All of group 2 (Bac-wt) and 3 (PBS) showed 100% mortality, yet those treated in group 1 (Bac-VP28) showed a mortality rate of only 25% (batch1) and 31.6% (batch2). This was a significant decrease in mortality rate.

Summary

The use of baculovirus as a vector for vaccination has been shown to be effective when present in oral and immersion vaccination, yet more so when administered orally. Further tests need to be carried out to ensure that this test creates an immune memory response, but the results have presented avenues down which further trials can be created.


Syed MS, Kwang J (2011) Oral vaccination of baculovirus-expressed VP28 displays enhanced protection against White Spot Syndrome Virus in Penaeus monodon. PLOS ONE 6: e26428.10.1371/journal.pone.0026428 PubMed: 22069450.

TRANSCRIPTOMIC PROFILE OF SHRIMPS DYING FROM FUNGAL & VIRAL INFECTIONS

I wrote a post (and published it on the blog on 25^th October 2013) describing evolutionary arms race between bacteria and their phages. In that context, there are also similar interactions between invertebrates and their pathogens. Red Queen Hypothesis proposes that pathogens are continuously trying to evade immune system of their hosts and on the other side immune system of the host is trying to improve its defence system against the pathogens. And perhaps, there is equilibrium between host’s defence and virulence of pathogens; when this equilibrium moves to one side, the result is either disease epidemic or disease resistance shown by the host. 

Viral diseases are the major problem in shrimp farming worldwide with White Spot Syndrome Virus (WSSV) being the most serious pathogen infecting farmed shrimps as well as wild crustaceans. Like diseases of corals, under stressful conditions, opportunistic microbes can become pathogenic. This could lead to emergence of new diseases.

Considering the economic impact of shrimp diseases, research into understanding host-microbe interaction of shrimp diseases has been encouraged. This study explored transcriptomic profile of shrimps during experimentally-induced viral (WSSV) and fungal (Fusarium solani) infection and mortality events. Gene expression profiles in hemocytes from diseased shrimps were studied. High throughput microfluidic RT-qPCR analyses were used for identifying pathogen-specific gene expression signatures.

Analysis of transcriptome-wide expression revealed mortality-related gene expression signatures in shrimps dying either because of viral or fungal infection. This paper grabbed my attention especially because it also demonstrates RNAi mechanisms acting in WSSV infected cells. LvDcr2 which is involved in RNA interference pathway was found to be up-regulated, two days after the infection of WSSV whereas fungal infection did not alter LvDcr2 expression at all. Authors reported that most of the analysed antiviral genes stimulate RNAi mechanisms against viral infection. Similarly, authors also found up-regulation of other anti-viral genes (not related with RNAi) which could lead to other defence mechanisms against viruses. All in all, as one can expect, WSSV-specific gene expression profile contained up-regulation of many anti-viral genes. Nevertheless, all these up-regulated antiviral defence mechanisms could not rescue shrimp during lethal viral infection. I am wondering if animals naturally have such an elaborate mechanisms of fighting viral infections (RNAi in particular) although, why we see so many incidents of viral outbreaks, not only in aquaculture but also in other animals, including humans? Does the viral infection-induced death mean failure of these mechanisms or mutated better viruses or both? Perhaps, infection of mutated viruses is essential to update these antiviral mechanisms.

Shrimps dying because of lethal fungal infections showed characteristic drop in transcription. Expression of more genes (68%) was halted in shrimps dying from fungal infection compared to viral infection (WSSV) which halted only 26% of infection-modulated genes. This means fungal infection had stronger effect on suppression of analyzed genes. Expression of some of the genes was not at all affected by either of the infection. The authors also noted that some transcriptomic response was similar for both viral and fungal infection. For example, both the infections affected expression of protease inhibitor and prophenoloxidase (proPO) both of which have been shown to be associated with diseases. Authors reported that defence mechanisms related to proPO cascade and melanin production might be impaired in viral and fungal -infected cells. Thus, these pathogens could evade immune system of shrimp by interfering with proPO related defence mechanisms of shrimp.

In summary, this is the first study demonstrating immune related transcriptomic response of shrimps which are about to die from the infection. Such gene expression signatures could be used for diagnostic purposes. As mentioned before, this paper has reinforced RNAi related pathways against viral infection. This is noteworthy in relation to the talk by Dr Leigh Owens (on 10^th January 2014) and his work on using RNAi in crustacean aquaculture.  

Goncalves, P., Guertler, C., Bachère, E., de Souza, C. R., Rosa, R. D., & Perazzolo, L. M. (2014). Molecular signatures at imminent death: Hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections. Developmental & Comparative Immunology, 42(2), 294-301.

Coral holobiont analysis to try and identify pathogens?

Bacterial profiling of White Plague Disease in a comparative coral species framework

Corals are now understood to be so much more than simple cnidarians. The coral holobiont is now widely accepted to incorporate all of the micro-organisms associated with corals as well as the polyps themselves. This makes characterization of disease very hard especially in discerning between a single responsible pathogen and a group of contributing micro-organisms. Diseases are usually grouped together by the displayed response. However we don't know if similar responses are caused by the same pathogen or if the same pathogens cause similar symptoms in different coral species. To try and answer some of these questions Roder et al., (2014) set out to compare the microbial community patterns of two white plague disease (WPD) infected coral species, Porites lutea and Pavona duerdeni, with their healthy counterparts. All samples were collected from the same reef in Thailand.

Using both Phylochip microarray and 16S rRNA sequence analysis they found 14,213 operational taxonomic units (OTU) across the coral species. Diseased coral samples had roughly 30% more OTUs then their healthy counterparts in both species indicating that a less diverse more stable community is beneficial. Between species there were 1003 OTUs that differed in abundance, which emphasizes the difficulty of isolating a single pathogen as there are many species contributing to the holobiont. Even within one geographic location these coral species have differences in bacterial community structure. There we 629 OTUs that differed in abundance between healthy and diseased corals of which two thirds were more abundant in the diseased individuals. Healthy coral had higher abundances of Comamonadaceae, Enterobacteriaceae and Streptococcaceae while Colwelliaceae, Pseudomonadaceae, Rhizobiaceae, Oceanospirillaceae, Vibrionaceae and Rhodobacteraceae were more abundant in the diseased corals. This implies some relationship between these families and the disease however there is no evidence for this and it is equally likely that these are opportunistic microbes that make use of the corals weakened state for colonization and growth.

These results differ from a previous study investigating WPD in the Caribbean though differences should be expected as the species investigated were different as well as the reefs being in different oceans. This might indicate that the diseased phenotype is actually caused by many different pathogens or pathogenic communities depending on coral species or location though many further studies will have to be carried out before any responsible microbe(s) can be identified. Aurantimonas coralicida a previously proposed pathogen was not identified in this study. This was thought to be responsible for WPD in the Caribbean though this could not be confirmed and it's absence here at least implies multiple geographic variants of the disease's pathogen if not providing evidence against Aurantimonas coralicida's pathogenicity. In future this Phylochip technique could prove useful in categorizing diseases and, as the genetic databases grow, will provide a more and more comprehensive view of the microbes that constitute the coral holobiont. I think it would be particularly interesting to see how microbial communities differ within the same coral species but at different geographic locations as we may get some insights into how much the coral influences it's own community make up compared to the influence of the surrounding water column.

(Roder et al., 2014)

Roder, C., Arif, C., Bayer, T., Aranda, M., Daniels, C., Shibl, A., … Voolstra, C. R. (2014). Bacterial profiling of White Plague Disease in a comparative coral species framework. The ISME journal, 8(1), 31–9. 

RNA interference offers promising antiviral methods to minimise nodovirus outbreaks in aquaculture

RNA interference offers promising antiviral methods to minimise nodovirus outbreaks in aquaculture

Following on from the talk by Leigh Owens and the recent complementary lecture by Colin, this research used interfering RNA (RNAi) to assess its effectiveness of reducing mortality from white tail disease (WTD) in giant freshwater prawns, Macrobrachium rosenbergii, using redclaw crayfish, Cherax quadricarinatus as a model organism.

Global demand for seafood is increasing and with intensive farming comes disease.  WTD has lead to mass mortalities in prawn hatcheries the world over, causing economic problems and loss in production.  M. rosenbergii nodavirus (MrNV) has been identified as the causative agent of WTD and has caused 100% mortality in hatcheries within one week of the first clinical signs in the hosts.

RNAi is the sequence-specific degradation of complementary mRNA using small, functional pieces of double stranded RNA (dsRNA) to target specific genes.  It has been used as an antiviral mechanism, making it possible to knock-down specific viral genes and limiting any off-target effects.   This experiment targeted protein B2 of MrNV, which inhibits the degradation of viral RNA that would normally occur in the host cells.  Sequence specific RNAi was therefore used to silence the production of protein B2 by degrading viral mRNA – basically an arms race between virus and host cell.

MrNV from infected M. rosenbergii was homogenised with phosphate buffered saline (PBS) and injected into select C. quadricarinatus.  Sequence specific RNAi (targeting protein B2), along with control RNAi (non-specific), was designed and injected into select animals in a cross-over experimental design, also controlling for exposure to MrNV and RNAi by using placebo treatments and PBS.  All experimental animals, unless mortality occurred during the trial, were sacrificed at the end of 60 days.  RNA was extracted from C. quadricarinatus muscle for qPCR analyses and tissue sections prepared for histopathological analysis.

Invertebrates have no acquired immunity and RNAi is important for antiviral innate immunity. Only specific dsRNA to protein B2 was effective in affording increased antiviral defence against MrNV infection in focal C. quadricarinatus (10% mortality) compared with high mortality (60%) and clinical signs consistent with MrNV infection reported in the control RNAi group (see Fig. 1).  

 

The authors note that injection of RNAi + MrNV did not reduce the average viral titre in comparison to the control RNAi + MrNV group, contrasting their results to a similar study in crickets that reported a 10-fold reduction in viral titres (La Fauce & Owens, 2009).  An alternative method of delivering sequence specific dsRNA may yield lower viral counts, for example including it in feeds via a bacterial plasmid.  This may also allow viable scale up for commercial use and offer an alternative to phage therapy (see Sanket’s recent posts).  In Leigh’s talk, he stated that his group has had difficulty in scaling up quantities for phage therapy to be effective outside of laboratory conditions and considers RNAi a more promising viral control technique.  Inclusion of plasmid contained sequence specific dsRNA in commercial feeds may still offer antiviral protection to the prawns, whilst maintaining its select target (eg. the virus) and minimising other undesirable, off target effects.  It will be interesting to see where this promising technique leads, although further long-term, transgenerational research would benefit here, to gain a better understanding of potential long-term impacts this may have.

Hayakijkosol, O., & Owens, L. (2012). B2 or not B2: RNA interference reduces (Macrobrachium rosenbergii) nodavirus replication in redclaw crayfish (Cherax quadricarinatus). Aquaculture, 326, 40-45.