Vanvimon Saksmerprome

Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of IHHNV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other methods used commonly for nested PCR detection of IHHNV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

Additional random, single to multiple genome fragments of Penaeus stylirostris densovirus in the giant tiger shrimp genome have implications for viral disease diagnosis

Scattered reports of viral inserts in shrimp and insect genomes led to the hypothesis that random, autonomous insertion of such sequences occurs in these organisms and leads to specific, heritable immunity. To test the prediction regarding random insertion of viral sequences into the shrimp genome, we examined the giant tiger shrimp for random genomic insertions of Penaeus stylirostris densovirus (also called IHHNV). By PCR analysis using a set of 7 overlapping primer pairs to cover the whole IHHNV genome (4 kb), PCR failure with some pairs indicated sequence gaps that revealed a random pattern of putative viral inserts in the genomes of individual shrimp. Targeting a putative insert from one arbitrarily selected specimen, we used genome walking to reveal a viral insert linked to a host microsattelite-like fragment. This differed from 2 previously reported inserted fragments of IHHNV in P. monodon. In one specimen, 2 slightly different inserts were revealed, probably on paired chromosomes. By design and use of chimeric shrimp/virus primer pairs we proved that similar insertions occurred in several shrimp specimens, including those infected with IHHNV but showing no signs of disease. For the infected specimens, the inserts gave false positive PCR test results using 309F/R primers and a new IQ2000 test protocol currently recommended for detection of infectious IHHNV. This is the first experimental support for the hypothesis-based prediction that a random number and length of sequence fragments from a single virus genome may occur in the shrimp genome. Since some inserts can give false positive results for infectious IHHNV with the recommended methods above, they may have a negative effect on international seafood trade. In addition, discard of domesticated shrimp breeding stocks based on such false positive results might have negative consequences, if such inserts are related to shrimp viral disease tolerance, as also hypothesized.

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells.

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(®), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp

BackgroundRNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.ResultsFor a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.ConclusionThe present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

Biology, Genome Organization, and Evolution of Parvoviruses in Marine Shrimp

As shrimp aquaculture has evolved from a subsistent farming activity to an economically important global industry, viral diseases have also become a serious threat to the sustainable growth and productivity of this industry. Parvoviruses represent an economically important group of viruses that has greatly affected shrimp aquaculture. In the early 1980s, an outbreak of a shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV), led to the collapse of penaeid shrimp farming in the Americas. Since then, considerable progress has been made in characterizing the parvoviruses of shrimp and developing diagnostic methods aimed to preventing the spread of diseases caused by these viruses. To date, four parvoviruses are known that infect shrimp; these include IHHNV, hepatopancreatic parvovirus (HPV), spawner-isolated mortality virus (SMV), and lymphoid organ parvo-like virus. Due to the economic repercussions that IHHNV and HPV outbreaks have caused to shrimp farming over the years, studies have been focused mostly on these two pathogens, while information on SMV and LPV remains limited. IHHNV was the first shrimp virus to be sequenced and the first for which highly sensitive diagnostic methods were developed. IHHNV-resistant lines of shrimp were also developed to mitigate the losses caused by this virus. While the losses due to IHHNV have been largely contained in recent years, reports of HPV-induced mortalities in larval stages in hatchery and losses due to reduced growth have increased. This review presents a comprehensive account of the history and current knowledge on the biology, diagnostics methods, genomic features, mechanisms of evolution, and management strategies of shrimp parvoviruses. We also highlighted areas where research efforts should be focused in order to gain further insight on the mechanisms of parvoviral pathogenicity in shrimp that will help to prevent future losses caused by these viruses.

Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay.

Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop‐mediated isothermal amplification (LAMP) assay with pre‐addition of hydroxynapthol blue (blue‐LAMP) to investigate ISKNV transmission in tilapia.

Applications of Microalgal Biotechnology for Disease Control in Aquaculture

Aquaculture industries, and in particular the farming of fish and crustaceans, are major contributors to the economy of many countries and an increasingly important component in global food supply. However, the severe impact of aquatic microbial diseases on production performance remains a challenge to these industries. This article considers the potential applications of microalgal technology in the control of such diseases. At the simplest level, microalgae offer health-promoting benefits as a nutritional supplement in feed meal because of their digestibility and high content of proteins, lipids and essential nutrients. Furthermore, some microalgal species possess natural anti-microbial compounds or contain biomolecules that can serve as immunostimulants. In addition, emerging genetic engineering technologies in microalgae offer the possibility of producing ‘functional feed additives’ in which novel and specific bioactives, such as fish growth hormones, anti-bacterials, subunit vaccines, and virus-targeted interfering RNAs, are components of the algal supplement. The evaluation of such technologies for farm applications is an important step in the future development of sustainable aquaculture.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Feasibility of dsRNA treatment for post-clearing SPF shrimp stocks of newly discovered viral infections using Laem Singh virus (LSNV) as a model

Using post-larvae derived from specific pathogen free (SPF) stocks in penaeid shrimp farming has led to a dramatic increase in production. At the same time, new pathogens of farmed shrimp are continually being discovered. Sometimes these pathogens are carried by shrimp and other crustaceans as persistent infections without gross signs of disease. Thus it is that a 5-generation stock of Penaeus monodon SPF for several pathogens was found, post-stock-development, to be persistently-infected with newly-discovered Laem Singh virus (LSNV). In this situation, the stock developers were faced with destroying their existing stock (developed over a long period at considerable cost) and starting the whole stock development process anew in order to add LSNV to its SPF list. As an alternative, it was hypothesized that injection of complementary dsRNA into viral-infected broodstock prior to mating might inhibit replication of the target virus sufficiently to reduce or eliminate its transmission to their offspring. Subsequent selection of uninfected offspring would allow for post-clearing of LSNV from the existing stock and for conversion of the stock to LSNV-free status. Testing this hypothesis using the LSNV-infected stock described above, we found that transmission was substantially reduced in several treated broodstock compared to much higher transmission in buffer-injected broodstock. Based on these results, the model is proposed for post-clearing of SPF stocks using dsRNA treatment. The model may also be applicable to post-clearing of exceptional, individual performers from grow-out ponds for return to a nucleus breeding center.

Infectious spleen and kidney necrosis disease (ISKND) outbreaks in farmed barramundi (Lates calcarifer) in Vietnam

Abstract Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large‐scale barramundi farms in Vietnam in 2012–2014 with estimated losses reaching