Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Chemical composition of a hot water crude extract (HWCE) from Ulva intestinalis and its potential effects on growth performance, immune responses, and resistance to white spot syndrome virus and yellowhead virus in Pacific white shrimp (Litopenaeus vannamei).

In the present study, a hot water crude extract from Ulva intestinalis (Ui-HWCE) was used as a dietary supplement, and the effects on growth, immune responses, and resistance against white spot syndrome virus (WSSV) and yellowhead virus (YHV) infection in Pacific white shrimp (Litopenaeus vannamei) were investigated. Chemical analyses of Ui-HWCE revealed 13.75 ± 0.41% sulfate, 37.86 ± 5.96% uronic acid, and 46.63 ± 5.16% carbohydrate contents. The monosaccharide content of Ui-HWCE contained glucose (6.81 ± 0.94%), xylose (4.15 ± 0.11%), and rhamnose (25.84 ± 0.80%). Functional group analysis of Ui-HWCE by Fourier transform infrared (FTIR) spectroscopy revealed a typical infrared spectrum of ulvan similar to the infrared spectrum of commercially purified ulvan from Ulva armoricana (77.86 ± 2.19% similarity). Ui-HWCE was added to shrimp diets via top-dressing at 0, 1, 5, and 10 g/kg diet. After 28 days, Ui-HWCE supplementation at 5 g/kg diet efficiently improved shrimp growth performance, as indicated by weight gain, average daily growth, specific growth rates, and villus height determined by observing gut morphology. Additionally, Ui-HWCE feed supplementation at 5 g/kg diet significantly increased immune responses against a pathogenic bacterium (Vibrio parahaemolyticus AHPND stain), including phagocytic activity and clearance efficiency. Furthermore, Ui-HWCE feed supplementation upregulated the expression of several immune-related genes in the hemocytes and gills. Ui-HWCE supplementation at 1 and 5 g/kg resulted in effective anti-YHV but not anti-WSSV activity, which significantly decreased the mortality rate and YHV burden in surviving shrimp. It was concluded that Ui-HWCE supplied at 5 g/kg diet exhibits growth-promoting, immune-stimulatory, and antiviral activity that could protect L. vannamei against YHV infection.

ICTV Virus Taxonomy Profile: Roniviridae.

The family Roniviridae includes the genus Okavirus for three species of viruses with enveloped, rod-shaped virions. The monopartite, positive-sense ssRNA genome (26-27 kb) contains five canonical long open reading frames (ORFs). ORF1a encodes polyprotein pp1a containing proteinase domains. ORF1b is expressed as a large polyprotein pp1ab by ribosomal frameshifting from ORF1a and encodes replication enzymes. ORF2 encodes the nucleoprotein. ORF3 encodes two envelope glycoproteins. ORFX encodes a putative double membrane-spanning protein. Roniviruses infect shrimp but only yellow head virus is highly pathogenic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Roniviridae, which is available at ictv.global/report/roniviridae.

Assessment of the function of gonad-specific PmAgo4 in viral replication and spermatogenesis in Penaeus monodon.

Argonaute family is phylogenetically subdivided into Ago and Piwi subfamilies that operate either transcriptional or post-transcriptional regulation in association with particular types of small RNAs. Among the four members of Ago subfamily (PmAgo1-4) found in black tiger shrimp Penaeus monodon, PmAgo4 exhibits gonad-restricted expression and takes part in transposon repression as the Piwi subfamily. While PmAgo1-3 participate in RNA interference (RNAi)-based mechanism, the role of PmAgo4 in RNAi is still mysterious, and was therefore investigated in this study. The results showed that knockdown of PmAgo4 in shrimp testis did not have a significant effect on the potency of PmRab7 silencing by dsPmRab7. In addition, replication of YHV as well as YHV-induced cumulative mortality in PmAgo4-knockdown shrimp are comparable to the control shrimp, suggesting the irrelevant association of PmAgo4 with RNAi-mediated gene silencing and antiviral immunity. Since PmAgo4 did not function in common with the Ago subfamily, its potential function in gametogenesis of male shrimp was further investigated. The reduction of PmAgo4 transcript levels in male shrimp revealed significant defect in testicular maturity as measured by Testicular Index (TI). Moreover, the numbers of mature sperm in spermatophore of PmAgo4-knockdown shrimp were significantly decreased comparing with the control shrimp. Our studies thus suggest a distinctive role of PmAgo4 that is not consistent with a dsRNA-mediate gene regulation and virus replication, but has a key function in controlling spermatogenesis in P. monodon.

Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA).

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

Suppression of argonautes compromises viral infection in Penaeus monodon.

Argonaute (Ago) proteins, the catalytic component of an RNA-induced silencing complex (RISC) in RNA interference pathway, function in diverse processes, especially in antiviral defense and transposon regulation. So far, cDNAs encoding four members of Argonaute were found in Penaeus monodon (PmAgo1-4). Two PmAgo proteins, PmAgo1 and PmAgo3 shared high percentage of amino acid identity to Ago1 and Ago2, respectively in other Penaeid shrimps. Therefore, the possible roles of PmAgo1 and PmAgo3 upon viral infection in shrimp were characterized in this study. The level of PmAgo1 mRNA expression in shrimp hemolymph was stimulated upon YHV challenge, but not with dsRNA administration. Interestingly, silencing of either PmAgo1 or PmAgo3 using sequence-specific dsRNAs impaired the efficiency of PmRab7-dsRNA to knockdown shrimp endogenous PmRab7 expression. Inhibition of yellow head virus (YHV) replication and delayed mortality rate were also observed in both PmAgo1-and PmAgo3-knockdown shrimp. In addition, silencing of PmAgo3 transcript, but not PmAgo1, revealed partial inhibition of white spot syndrome virus (WSSV) infection and delayed mortality rate. Therefore, our study provides insights into PmAgo1and PmAgo3 functions that are involved in a dsRNA-mediated gene silencing pathway and play roles in YHV and WSSV replication in the shrimp.

Silencing of a Kazal-type serine proteinase inhibitor SPIPm2 from Penaeus monodon affects YHV susceptibility and hemocyte homeostasis.

In shrimp, the Kazal-type serine proteinase inhibitors (KPIs) are involved in host innate immune defense system against pathogenic microorganisms. A five-Kazal-domain SPIPm2 is the most abundant KPIs in the black tiger shrimp Penaeus monodon and up-regulated in response to yellow head virus (YHV) infection. In this study, the role of SPIPm2 in YHV infection was investigated. The expression of SPIPm2 in hemocytes, gill and heart from 48-h YHV-infected shrimp was increased. The expression of SPIPm2 in hemocytes was significantly increased after 12 h of infection and gradually increased higher afterwards. Silencing of SPIPm2 by dsRNA interference resulted in the increased expression of different apoptosis-related genes, the increased expression of transcriptional factors of antimicrobial synthesis pathways, the reduction of circulating hemocytes in the shrimp hemolymph, and the increased susceptibility of the silenced shrimp to YHV infection. The activities of caspase-3 and caspase-7 in the hemocytes of SPIPm2-silenced shrimp was also increased by 5.32-fold as compared with those of the control shrimp. The results suggested that the SPIPm2 was involved in the hemocyte homeostasis.

A novel white spot syndrome virus-induced gene (MjVIG1) from Marsupenaeus japonicus hemocytes.

cDNA of a newly recognized white spot syndrome virus (WSSV)-induced gene (MjVIG1) was characterized from Marsupenaeus japonicus hemocytes; this gene encodes a protein that lack similarity to any known characterized protein. To identify this novel gene, we mainly conducted transcript level analysis, immunostaining and flow cytometry after WSSV infection. MjV1G1 transcript levels were also measured after Yellow head virus (YHV) and Vibrio parahaemolyticus infection tests. In non-infected and WSSV-infected shrimp, MjVIG1 was observed in granule-containing hemocytes. In addition, the MjVIG1 transcript level and ratio of MjVIG1-positive hemocytes both significantly increased, and number of MjVIG1-positive hemocytes slightly increased after WSSV infection. In contrast, MjVIG1 transcript level did not change after YHV and V. parahaemolyticus infection. These results indicated that MjVIG1 might be a WSSV-specific induced gene in M. japonicus hemocytes.

Antimicrobial activity of a serine proteinase inhibitor SPIPm5 from the black tiger shrimp Penaeus monodon.

A two-domain Kazal-type serine proteinase inhibitor, SPIPm5, from Penaeus monodon was studied. Its transcript was expressed in all tissues tested including the hemocytes, stomach, gill, lymphoid organ, muscle, intestine and heart albeit less in hepatopancreas and eyestalk. The expression of SPIPm5 gene was also up-regulated by heat stress, white spot syndrome virus (WSSV) infection and yellow head virus (YHV) infection. Injection of recombinant rSPIPm5 protein into normal shrimp to mimic heat stress condition did not have or had little stimulating effect on the expression of other immune genes: crustinPm1, penaeidin3, penaeidin5, Hsp70, SPIPm2 and SPIPm5. Like some other proteinase inhibitors, the rSPIPm5 could inhibit the hemolymph proPO activity. In survival experiments, the rSPIPm5 could prolong the life of WSSV-infected shrimp similar to the effect of heat stress. The rSPIPm5 also helped the YHV-, Vibrio harveyi- and V. parahaemolyticus-infected shrimp survive longer. The increased endurance against microbial infection was due to the inhibitory effects presumably activated by rSPIPm5 on viral replication and bacterial growth but not the expression of antimicrobial peptides. Therefore, the SPIPm5 plays an important role in shrimp innate immunity against the viral and bacterial infection.

Immunostimulation and yellow head virus (YHV) disease resistance induced by a lignin-based pulping by-product in black tiger shrimp (Penaeus monodon Linn.).

Yellow head virus (YHV) is classified as one of the most serious pathogens causing a harmful disease in many penaeids, especially black tiger shrimp (Penaeus monodon), with high economic loss. To determine a potent and practical prophylactic strategy for controlling this disease, the toxicity of the by-product kraft lignin and its ability to control severe YHV infection were investigated in juvenile black tiger shrimp (15.9 ± 1.2 g body weight). The median lethal dosage at 96 h (96-hrs LD50) of lignin in shrimp was 297 mg/L. Lignin was further added to shrimp diets via top-dressing to assess its ability to elicit immune stimulation activity. At 14 days after feeding, shrimp fed 1, 3, 5 and 10 g of lignin/kg of diet exhibited significantly higher levels of phagocytic activity (PA) than the control group (P < 0.05). However, differences in total hemocyte count among treatments were not significant during the experimental period (P > 0.05). Additionally, lignin supplementation at 1-10 g/kg for 14 days failed to protect experimental shrimp against YHV infection. The antiviral activity of lignin against YHV in black tiger shrimp was notable in vitro because compared to control shrimp (96.7 ± 5.8%; P < 0.05), shrimp injected with a pre-incubated solution of YHV and lignin at 1, 5, 10 and 20 mg/L exhibited significantly lower mortality rates, 23.3 ± 5.8, 16.7 ± 5.8, 23.3 ± 5.8, and 20.0 ± 0.0%, respectively, after a lethal dose of YHV at 14-20 days after injection. These potent effects were clearly supported and confirmed by histopathological and RT-PCR analyses. Based on these results, the pulping by-product kraft lignin efficiently inhibits YHV infection in black tiger shrimp. This information will facilitate the development of practical methods to control yellow head disease in the marine shrimp culture industry.

Suppression of PmRab11 inhibits YHV infection in Penaeus monodon.

Yellow head virus (YHV) is one of the most serious pathogens that causes worldwide shrimp production loss. It enters the cells via clathrin-mediated endocytosis and utilizes small GTPase Rab proteins such as PmRab5 and PmRab7 for intracellular trafficking. In this study, molecular cloning and functional analysis of Rab11 during YHV infection were investigated. PmRab11 cDNA was cloned by Rapid amplification of cDNA ends (RACEs). It contained two forms of sizes 1200 and 1050 bp distinct at the 5' UTR. The coding region of PmRab11 was 645 bp, encoding 214 amino acids. It also demonstrated the characteristics of Rab11 proteins containing five GTP-binding domains, five Rab family domains, four Rab subfamily domains and a prenylation site at the C-terminus. Suppression of PmRab11 using dsRNA-PmRab11 either before or after YHV-challenge resulted in significant inhibition of YHV levels in the hemocytes and viral release in the supernatant in both mRNA and protein levels. In addition, the silencing effect of PmRab11 in YHV-infected shrimps resulted in a delay in shrimp mortality for at least 2 days. Immunofluorescence study showed co-localization between PmRab11 and YHV at 24-72 h post YHV-challenge. In contrast, the co-localization signals were absence in the PmRab11 knockdown hemocytes and the YHV signals accumulated at the perinuclear region at 24 h post YHV-challenge. Then, accumulation of YHV was hardly observed after 48-72 h. These results suggested that PmRab11 is required for YHV infection in shrimp.

YHV-responsive gene expression under the influence of PmRelish regulation.

In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression were obtained. Nucleotide sequences of 252 and 99 cDNA clones from the forward and reverse libraries, respectively, were obtained and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium Vibrio harveyi infection. Together with the results using YHV infection previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.

A novel gonad-specific Argonaute 4 serves as a defense against transposons in the black tiger shrimp Penaeus monodon.

Argonaute is a key protein of the small-RNA guided gene regulation process. The Argonaute family is generally divided into two subfamilies; AGO and PIWI. In this study, a cDNA encoding a novel type of Argonaute (PmAgo4) in the black tiger shrimp Penaeus monodon was identified and characterized. PmAgo4 cDNA contained an open reading frame of 2433 nucleotides that can be translated into a deduced amino acid with the conserved PAZ and PIWI domains. PmAgo4 was phylogenetically clustered with the AGO subfamily while exhibited a gonad-specific expression pattern similar to that of proteins in the PIWI subfamily. The expression of PmAgo4 did not change significantly in response to either double-stranded RNA or yellow head virus injection suggesting that PmAgo4 may not be the main AGO proteins that play a role in dsRNA-mediated gene silencing or antiviral defense. Interestingly, PmAgo4 appeared to participate in the control of transposons since the activation of both DNA transposon and retrotransposon was detected in the testis of PmAgo4-knockdown shrimp. Our study thus provided the first evidence for an unusual type of the AGO proteins that was predominantly expressed in shrimp gonad and implication of its role in protecting the shrimp genome against an invasion of transposons.

Activation of PmRelish from Penaeus monodon by yellow head virus.

Humoral innate immune response against pathogenic infection is partly responsible by the Imd pathway in which a transcription factor Relish relays the infection signals to the nuclei for the expression of antimicrobial proteins. A PmRelish gene which encoded a protein of 1195 amino acids was cloned. The PmRelish was constitutively expressed in all tissues tested and mostly up-regulated upon YHV infection. In hemocytes, the PmRelish expression was up-regulated upon Vibrio harveyi, yellow head virus (YHV) and white spot syndrome virus (WSSV) challenges. Using dsRNA silencing of PmRelish gene, it was shown that the expression of penaeidin5 but not anti-lipopolysaccharide factor ALFPm3, crustinPm1 and penaeidin3 was under the regulation of Imd pathway. Under PmRelish silencing, the shrimp were more susceptible to infection by YHV with the 50% survival rate reduced from about 72 h to 42 h. The PmRelish was detected in the cytoplasm of all the hemocytes from both uninfected and YHV-infected shrimp. The accumulation of activated PmRelish in the nuclei was not clearly observed but the activated PmRelish was detected in the YHV-infected hemocytes by Western blot analysis. Thus, the PmRelish and, hence, the Imd pathway respond to the YHV infection.

Silencing of PmYPR65 receptor prevents yellow head virus infection in Penaeus monodon.

The suppression of viral replication by double-stranded RNAs (dsRNA) specific to mRNAs of either virus or host genes has been widely investigated as a possible shrimp disease therapy. PmYRP65, a yellow head virus (YHV) receptor, was previously identified and characterized in the black tiger shrimp, Penaeus monodon. In our previous study, entry of YHV into cells of the Oka organ of P. monodon required the host receptor PmYRP65 and silencing of PmYRP65 in vitro led to a complete suppression of YHV replication in the cells. In this study, PmYRP65 was shown to be in vivo suppressed by dsRNA specific for PmYRP65, leading to inhibition of YHV replication and almost complete abolition of shrimp mortality following YHV challenge. Targeting PmYRP65 could be an effective YHV antiviral shrimp strategy.

Protection of yellow head virus infection in shrimp by feeding of bacteria expressing dsRNAs.

Although prevention of shrimp mortality from yellow head virus (YHV) infection via dsRNA injection has been well demonstrated for many years, it has not yet been applied in a farm culture because of its impracticality. Hence, oral administration of dsRNA becomes an alternative and desirable approach. This study is the first to demonstrate that oral feeding of Escherichia coli expressing shrimp Rab7 gene (dsRab7) or YHV protease gene (dsYHV) could inhibit YHV replication and lowered shrimp mortality. E. coli HT115 expressing dsRab7 or dsYHV or a combination of these dsRNAs were embedded in agar and used to feed vannamei shrimp at early juvenile stage before YHV challenge. After 4 days of continuous feeding of dsRNAs, strong inhibitory effect on shrimp mortality was observed in which dsRab7 gave the highest effect (70% reduction from the control) whereas dsYHV showed a 40% reduction. Our results reveal the potential of anti-YHV strategy via orally delivered dsRNA for application in the shrimp farm industry.

The essential role of clathrin-mediated endocytosis in yellow head virus propagation in the black tiger shrimp Penaeus monodon.

Yellow head virus (YHV) is one of the most widespread viruses seriously affecting black tiger shrimp (Penaeus monodon) cultivation. A previous microarray study demonstrated that clathrin coat assembly protein 17 (AP17) was significantly up-regulated after YHV infection (Pongsomboon et al., 2011). Clathrin coat AP17 is a part of the assembly protein σ2 (AP-2) complex which is involved in clathrin-mediated endocytosis. Quantitative RT-PCR (qRT-PCR) revealed that the clathrin coat AP17 gene was up-regulated 3-fold at 12 h post YHV infection. In addition, immunofluorescence microscopy showed that clathrin coat AP17 was highly expressed in the cytoplasm of the YHV-infected hemocytes. Knockdown of the clathrin coat AP17 gene dramatically reduced YHV replicativity by 32-fold. Interestingly, shrimp pre-treated with chlorpromazine, a commercial drug that inhibits clathrin-dependent endocytosis, exhibited significantly low levels of YHV infection. Taken together, these results suggest that clathrin-mediated endocytosis is involved in YHV propagation in P. monodon.

N-Linked glycosylation is essential for the yellow head virus replication cycle.

Yellow head virus (YHV) particles contain a nucleocapsid protein (p20) and two envelope glycoproteins (gp116 and gp64). The glycans attached to the two glycoproteins are N-linked and are complex and high mannose types, respectively. Here, we show that treatment with the N-linked glycosylation inhibitor tunicamycin in YHV-infected black tiger shrimp (Penaeus monodon) resulted in less severe yellow head disease and reduced mortality when compared with untreated control shrimp. Quantitative real-time reverse transcription PCR analysis also revealed lower YHV copy numbers in the haemolymph of treated than control shrimp. This was concurrent with less intense immuno-reactions in tissues of treated versus untreated shrimp using mAbs against all three YHV structural proteins. In addition, transmission electron microscopy of lymphoid organ tissue of the treated and untreated shrimp [eight collected at 36 h and eight at 48 h post-infection (p.i.)] revealed only unenveloped nucleocapsids in all but one of the treated shrimp (collected at 48 h p.i.). By contrast, all the untreated shrimp showed a mixture of many unenveloped and enveloped virions. These results were supported by purification of YHV from the cell-free haemolymph of treated and untreated shrimp followed by YHV structural protein analysis by SDS-PAGE. It revealed three expected structural protein bands (116, 64 and 20 kDa) from the untreated shrimp but no structural protein bands from the tunicamycin-treated shrimp (confirmed by Western blot analysis). Overall, the results indicated that blocking glycosylation with tunicamycin inhibited the formation of mature YHV virions and their subsequent release into shrimp haemolymph, reducing the severity of 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.

Shrimp molecular responses to viral pathogens.

From almost negligible amounts in 1970, the quantity of cultivated shrimp (~3 million metric tons in 2007) has risen to approach that of the capture fishery and it constitutes a vital source of export income for many countries. Despite this success, viral diseases along the way have caused billions of dollars of losses for shrimp farmers. Desire to reduce the losses to white spot syndrome virus in particular, has stimulated much research since 2000 on the shrimp response to viral pathogens at the molecular level. The objective of the work is to develop novel, practical methods for improved disease control. This review covers the background and limitations of the current work, baseline studies and studies on humoral responses, on binding between shrimp and viral structural proteins and on intracellular responses. It also includes discussion of several important phenomena (i.e., the quasi immune response, viral co-infections, viral sequences in the shrimp genome and persistent viral infections) for which little or no molecular information is currently available, but is much needed.