Shrimp Viruses Research Articles

An enhanced immuno-dot blot assay for the detection of white spot syndrome virus in shrimp using antibody conjugated gold nanoparticles probe

White Spot Syndrome (WSS) is a worldwide ailment of penaeid shrimp, a lethal and contagious disease in shrimp caused by the White Spot Syndrome Virus (WSSV). It is essential to control; handle early findings and routine screenings under field conditions. Though PCR affirmed sensing of the causative viral nucleic acid has many advantages, for its routine use custom necessitate an advanced equipment and technical expertise. In comparison, protein-based immuno detecting methods are easier to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this study, we focused on a sensitive immuno detection method which developed by gold nanoparticles coupled with alkaline phosphatase conjugated secondary antibody in turn recognized primary anti-serum raised against WSSV. We successfully enhanced the sensitivity of immuno-dot blot assay by 80 fold over the conventional method and visually it can be detected up to 1 ng/mL of purified WSSV. The approach described in this study is a prototype for the development of simple and inexpensive diagnostic tool will provide the routine screening of WSS in shrimp farms.

Immunomodulation by DNA vaccination against white spot syndrome virus (WSSV)

Vaccines (subunit and DNA) targeting major envelope proteins VP19 and/or VP28 of white spot syndrome virus (WSSV) in penaeid shrimp were developed and elicited good protection against white spot disease (WSD). However, the immune responses in shrimp after administration of these vaccines are not well understood. In this study, we developed a DNA vaccine encoding the VP28 envelope protein in kuruma shrimp (Marsupenaeus japonicus) and confirmed the potentiality of protection against WSSV infection. The efficacy of the DNA vaccine against WSSV infection was confirmed by WSSV artificial challenge at 7 days post vaccination in kuruma shrimp. However, the efficacy of the vaccine did not last 30 days post vaccination. The transcript of VP28 gene derived from expression vector in tissues of vaccinated shrimp was analyzed by RT-PCR. The transcript of VP28 gene was detected in various tissues including muscle, hemolymph, gill, intestine, stomach, heart, hepatopancreas and lymphoid organ tested at 1, 3 and 7 days post vaccination. Subsequently, the expression of innate immunerelated genes in intestine and lymphoid organ was analyzed at 1, 3 and 7 days post vaccination. The expression of innate immune-related genes such as Rab7, penaeidin, lysozyme, and crustin was up-regulated upon DNA vaccination. These results suggest that DNA vaccination induces significant protection against WSSV by stimulating innate immune responses in kuruma shrimp.

Cardiomyopathy Syndrome of Atlantic Salmon (Salmo salar L.) Is Caused by a Double-Stranded RNA Virus of the Totiviridae Family

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease.

Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV

Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus ( MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests.

Complete nucleic acid sequence of Penaeus stylirostris densovirus ( PstDNV) from India

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus ( PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan ( AY355307), and two isolates ( AY362547 and AY102034) from Thailand.

Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

Prevalence of three shrimp viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

Assessment of the Roles of Copepod Apocyclops royi and Bivalve Mollusk Meretrix lusoria in White Spot Syndrome Virus Transmission

Here, we investigate the roles of copepods and bivalve mollusks in the transmission of white spot syndrome virus (WSSV), which is the causative pathogen of an acute, contagious disease that causes severe mortalities in cultured shrimp. Copepods are common components in seawater ponds and are often eaten as live food by shrimp post-larvae. WSSV has been detected in these animals, but it is unknown whether this was due to contamination or infection. Meanwhile, the bivalve mollusk Meretrix lusoria is often used as live food for brooders, and in Taiwan, this hard clam is sometimes co-cultured with shrimp in farming ponds. However, mollusks' ability to accumulate, or allow the replication of, shrimp viruses has not previously been studied. In this study, WSSV, the copepod Apocyclops royi and bivalve mollusk M. lusoria were experimentally challenged with WSSV and then assayed for both the presence of the virus and for viral gene expression. Results showed that the WSSV genome could be detected and that the viral loads were increased in a time-dependent manner after challenge both in A. royi and M. lusoria. Reverse transcriptase PCR monitoring of WSSV gene expression showed that WSSV could replicate in A. royi but not in M. lusoria, which suggested that WSSV, while could infect A. royi, was only accumulated in M. lusoria. A bioassay further showed that the WSSV accumulated in M. lusoria could be transmitted to Litopenaeus vannamei and cause severe infection.

Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes

Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10 2 copies of the target DNA to less than 70 min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei shrimp cultured in South Korea

In October 2010, cuticular deformities with abnormal growth suppression were detected among cultured Litopenaeus vannamei shrimp in one farm located on the Taean peninsula along the western coast of Korea. We detected infectious hypodermal and haematopoietic necrosis virus (IHHNV) from the moribund L. vannamei shrimp and its pond water via PCR assays. The IHHNV strain isolated from the shrimps was designated K1 and the strain isolated from the pond water was designated K2, and the capsid genes were sequenced for genetic comparison and phylogenetic analysis with others obtained from different geographical regions. The results demonstrated that the Korean strains were similar to infectious IHHNV and were closely related to the Type 1 strains. This is the first report, to the best of our knowledge, of the detection of IHHNV from L. vannamei in Korean cultured shrimp.

Efficacy of an Inactivated Vaccine and Nutritional Additives Against White Spot Syndrome Virus (WSSV) in Shrimp (Penaeus monodon)

Although the immune system of shrimps is not comparable to that of vertebrates, shrimps can acquire protection against pathogenic challenge by building up immunity. In this study, formalin-inactivated virus (FIV) was administered by injection, bath-immersion, or orally to determine levels of vaccination-mediated protection against the pathogenic white spot syndrome virus (WSSV). Diets supplemented with alfalfa, methyl sulfonyl methane (MSM), or wheat grass were provided with or without FIV. Shrimp injected with FIV and challenged 3, 15, or 30 days after vaccination had cumulative and relative survivals of 83%, 67%, and 33%, respectively. Survival of shrimp challenged by bath-immersion 3-45 days after vaccination by immersion was significantly higher than in the unvaccinated control. Orally vaccinated shrimp challenged by bath-immersion were partially protected up to 45 days after vaccination (cumulative survival 63.7%, relative 61.7%) but not til 60 days after vaccination (cumulative 8%, relative 3.2%). Survival of unvaccinated shrimp challenged by bath-immersion improved when shrimp were fed a diet supplemented with wheat grass or MSM, but not alfalfa. Survival was further enhanced when FIV was provided together with diets supplemented with wheat grass (cumulative 72.7%, relative 94.8%) or MSM (cumulative 73.3%, relative 96.3%).

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp

A multiplex LAMP (mLAMP) assay was developed for the simultaneous detection of two penaeid shrimp viruses, namely, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV), which are the major viral pathogens of penaeid shrimp worldwide. Two sets of the LAMP primers were designed for VP28 gene of WSSV and non-structural protein 1 (NS1) gene of IHHNV, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers, to construct the mLAMP assay by combining these two sets totaling eight primers. The mLAMP method was distinguishable between WSSV and IHHNV due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 102 and 103 times higher on the detection limits for WSSV and IHHNV, respectively, than those of the nested-PCR (nPCR) and classical PCR methods. No positive results were displayed when nucleic acids from other shrimp pathogen DNAs were used as mLAMP templates. Uracil-N-glycosylase (UNG) was used to prevent carry-over contamination of mLAMP products. In addition to its high specificity and sensitivity, the developed multiplex LAMP assay offers an efficient, convenient and rapid tool for screening penaeid shrimp viruses since both WSSV and IHHNV can be diagnosed in a single reaction, and will be useful for the control of these viruses in shrimp.

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 ( ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes ( WSSV- DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.

Development of multiplex real-time PCR and high-resolution melt analysis for detection of three viruses in penaeid shrimp

Development of multiplex real-time PCR and high-resolution melt analysis for detection of three viruses in penaeid shrimp

Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously

The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.

Gill-associated virus and recombinant protein vaccination in Penaeus monodon

Recent studies in penaeid shrimp and various crustaceans showing specific antiviral responses to the DNA based White spot syndrome virus (WSSV) induced by pre-exposure to viral proteins are of particular research interest as invertebrates lack the classical adaptive immune response characterised in vertebrate animals. However, this protein-induced defence response has not been reported for any other invertebrate virus, and the mechanisms remain unknown. Here we investigated the generality of this defence response by challenging Penaeus monodon (Black Tiger shrimp) with the RNA based Gill-associated virus (GAV) following pre-exposure to fragments of the GAV gp116 envelope glycoprotein or to the p20 nucleocapsid protein. Shrimp were injected twice with recombinant GAV protein fragments expressed and purified from bacteria before challenge with GAV. Protein vaccination did not protect the shrimp, with mortality rates in shrimp injected with any of the GAV proteins not differing from shrimp either not injected with protein or injected with a non-specific protein. In addition, there was no significant difference in GAV RNA loads amongst the virus challenged shrimp groups. The data suggest that the efficacy of protection following protein-based vaccination may vary amongst different shrimp viruses.

Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam

Over a period of 30 months, wild black tiger shrimp ( Penaeus monodon) were collected from the South China Sea in South-east Asia, in an effort to establish specific pathogen free (SPF) stocks of P. monodon in Brunei Darussalam. The viral status of the broodstock was assessed by real-time and conventional polymerase chain reaction (PCR) using DNA and reverse transcribed RNA extracted from the gill, hepatopancreas and pleopods. Histopathology was performed to confirm PCR results. Over 270 P. monodon were collected, screened and spawned. Of the nine viruses assessed, infectious hypodermal and hematopoietic necrosis virus (IHHNV) was most commonly detected (19.6%), followed by monodon baculovirus (MBV) (7.4%), hepatopancreatic parvovirus (HPV) (3.8%), and Mourilyan virus (MoV) (0.9%). The only multiple viral infections found were a combination of IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, white spot syndrome virus (WSSV) and yellowhead virus (YHV) were not detected in any shrimp. It is concluded that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, which has permitted the development of SPF stocks as a foundation population for selective breeding.

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24 h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp.

Application of high resolution melt (HRM) analysis for duplex detection of Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) in shrimp

In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysis for the simultaneous detection of Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) infection in the freshwater prawn Macrobrachium rosenbergii. This first application of HRM multiplex RT-PCR in shrimp reveals a new potential for rapid and sensitive detection of multiple pathogens. In addition, sequence variation in XSV could be observed from the high resolution melt peaks, as confirmed by sequence analysis. In 19 field samples of the freshwater prawn M. rosenbergii the technique revealed samples negative for both viruses, positive for both viruses or positive for MrNV alone. No sample was found positive for XSV alone. Comparison of these results to those obtained using the same samples in analysis by traditional nested RT-PCR combined with gel electrophoresis revealed that HRM multiplex RT-PCR was more sensitive. Thus, the latter technique allows for rapid and sensitive, simultaneous detection of MrNV and XSV and also has the potential to be adapted for simultaneous detection of other mixed viral infections in shrimp.

Glycosylation of gp116 and gp64 envelope proteins of yellow head virus of Penaeus monodon shrimp

Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp that is classified in the genus Okavirus, family Roniviridae, in the order Nidovirales. Separation of virion proteins treated with peptide-N-glycosidase-F (PNGase-F) in SDS-polyacrylamide gels and the use of glycoprotein-specific staining methods indicated that the gp116 and gp64 envelope glycoproteins possess N-linked rather than O-linked glycans. Competitive binding inhibition of lectins with various oligosaccharide specificities indicated that glycans linked to gp64 are mannose-rich, whilst glycans linked to gp116 possess terminal N-acetylgalactosamine and N-acetylglucosamine in addition to terminal mannose-type sugars. Mass spectrometry analyses of peptides generated from YHV proteins before and after deglycosylation with PNGase-F, using combinations of the endoproteinases trypsin, Asp-N and Lys-C, confirmed occupancy of six of the seven potential N-linked glycosylation sites in gp116 and three of the four potential sites in gp64.