Gain of function studies on predicted host receptors for white spot virus.

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from -8.46 to -11.82 cal mol-1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Hepatic microsporidiosis of mudskipper, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae), due to Microgemma sp.

The present study reports a case of hepatic microsporidiosis caused by Microgemma sp. in brackishwater fish, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from the west coast of India. An eight-month study from September 2017 to April 2018 revealed a prevalence of 11.7% for this parasite. The microsporidian showed tissue-specific infection and did not reveal any gross pathology in infected fish. Small whitish cysts containing microspores of size 0.3-0.5 mm were observed in the liver of fish. The range of pyriform microsporidian spore size varied from 2.9-3.77 × 1.85-2.67 µm. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplification of the DNA extracted from the microsporidian-infected liver tissue using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the first report of a microsporidian from B. dussumieri and the first record of the genus Microgemma from India.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.

Molecular characterization and expression analysis of secretory immunoglobulin M (IgM) heavy chain gene in rohu, Labeo rohita.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila. These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5) from Asian seabass, Lates calcarifer.

MDA5 is the pivotal member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and is reported to play a crucial role in type I IFN-mediated responses against pathogen-associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full-length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N-terminal CARD domains, a DExDc domain, a HELICc domain and a C-terminal RIG-1_C-RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time-points following challenge with Gram-negative bacterium, Vibrio alginolyticus, and Gram-positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development.

Haemato-biochemical, non-specific immunity, antioxidant capacity and histopathological changes in Labeo rohita fingerlings fed rubber protein isolate.

A 60-day feeding trial was conducted to evaluate the haemato-biochemical, innate immune response, antioxidant capacity and histopathological changes in Labeo rohita fingerlings fed rubber protein isolates (RPI). One hundred and eighty fingerlings (average weight 4.45 ± 0.01 g) were distributed into five experimental groups in triplicate and fed with isonitrogenous and isocaloric diets. Soybean protein isolate (SPI) served as the reference diet (Control), and the treatment diets were formulated as RPI25, RPI50, RPI75 and RPI100 replacing 25, 50, 75 and 100% of SPI protein, respectively. The growth performance indices like final body weight (9.54-10.27 g), net weight gain (5.09-5.84 g), metabolic growth rate (4.54-5.02) and feed efficiency ratio (0.60-0.65) among the various groups were not significantly different (P > 0.05). All the haematological parameters, except red blood cells, showed no significant differences compared with the control group (P > 0.05). The immuno-biochemical parameters like albumin, globulin, total immunoglobulin, respiratory burst and lysozyme activities among the various groups did not differ significantly (P > 0.05). The stress enzyme such as superoxide dismutase (SOD), catalase (CAT) and oxidative stress marker malondialdehyde (MDA) showed no significant difference (P > 0.05). Histopathological examination of the liver revealed no marked changes. In summary, the results showed that RPI was well utilised by the fish and its inclusion did not generate any oxidative-induced stress, thus, RPI may be suggested as a potential replacement for SPI in fish diets without any detrimental effects. Hence, protein isolation offers a unique opportunity for the utilisation of rubber seed meal.

Report of leucine-rich repeats (LRRs) from Scylla serrata: Ontogeny, molecular cloning, characterization and expression analysis following ligand stimulation, and upon bacterial and viral infections.

Leucine-rich repeat (LRR) proteins are present in all living organisms, and their participation in signal transduction and defense mechanisms has been elucidated in humans and mosquitoes. LRRs possibly involve in protein-protein interactions also and show differential expression pattern upon challenge with pathogens. In the present study, a new LRR gene was identified in mud crab, Scylla serrata. LRR gene mRNA levels in different developmental stages and various tissues of S. serrata were analysed. Further, the response of the gene against different ligands, Gram-negative bacterium, and white spot syndrome virus (WSSV) was investigated in vitro and in vivo. Full-length cDNA sequence of S. serrata LRR (SsLRR) was found to be 2290 nucleotide long with an open reading frame of 1893bp. SsLRR encodes for a protein containing 630 deduced amino acids with 17 conserved LRR domains and exhibits significant similarity with crustacean LRRs so that these could be clustered into a branch in the phylogenetic tree. SsLRR mRNA transcripts were detected in all the developmental stages (egg, Zoea1-5, megalopa and crab instar), haemocytes and various tissues such as, stomach, gill, muscle, hepatopancreas, hematopoietic organ, heart, epithelial layer and testis by reverse-transcriptase PCR. SsLRR transcripts in cultured haemocytes showed a 2-fold increase in expression at 1.5 and 12h upon Poly I:C induction. WSSV challenge resulted in significant early up-regulation at 3h in-vitro and late up-regulation at 72h in-vivo. Peptidoglycan (PGN)-induction resulted in marginal up-regulation of SsLRR at timepoints, 6, 12 and 24h (fold change below 1.5) and no significant change in the expression at early timepoints. LPS-stimulation, on the other hand, showed either down-regulation or normal level of expression at all timepoints. However, a delayed 5-fold up-regulation was observed in vivo against Vibrio parahaemolyticus infection at 72hpi. The constitutive expression of the LRR gene in all the early life-stages, and its response to various ligands and to viral challenge suggest the possible role of the LRR in immune defense in mud crab. The result provides additional information which would help in future studies in understanding the innate immune pathways in crustaceans.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.

Molecular cloning, characterisation and expression analysis of melanoma differentiation associated gene 5 (MDA5) of green chromide, Etroplus suratensis.

Innate immune system recognises pathogen-associated molecular patterns (PAMPs) by limited number of germline encoded and non-clonally developed pathogen recognition receptors (PRRs). Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are important cytosolic PRRs for sensing viral RNAs. The receptor encoded by melanoma differentiation associated gene 5 (MDA5), an RLR, recognises viral RNA and enhances antiviral response in host cells. The full-length MDA5 cDNA in Etroplus suratensis was cloned and found to have 3673 nucleotides encoding a polypeptide of 978 amino acids. The deduced amino acid sequence contains four main structural domains: two CARD domains in the N-terminal region, a DExDc (DEAH/DEAD box helicase domain), HELICc (C-terminal helicase) domain and a C-terminal regulatory domain (RD). Phylogenetic analysis revealed a close relationship of E. suratensis MDA5 (EsMDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus, both belonging to Cichlidae family. EsMDA5 transcripts were ubiquitously expressed in all the 12 tissues tested in healthy fish. Although, transcript level was found to be the highest in muscle, high expression was also detected in the spleen, head kidney and hindgut. In poly I:C-injected fish, EsMDA5 transcripts showed peak expression in the spleen, intestine and heart at 12h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of EsMDA5 was observed at 6 and 48 hpi, respectively. Further, liver tissue showed an increasing trend in expression profile from 6 to 48 hpi. Interferon promoter stimulator-1 (IPS-1) gene, an adaptor triggering RIG-I- and MDA5-mediated type I interferon induction, also showed up-regulated expression at initial time-points in poly I:C-injected E. suratensis. The constitutive expression and up-regulation of EsMDA5 and the IPS-1 genes in different tissues indicate that EsMDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes.

The Toll-pathway plays key roles in regulating the innate immune response in invertebrates. Myeloid differentiation factor 88 (MyD88) and Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) are key molecules in this signalling pathway. To investigate the role of Toll-pathway in innate immune response of shrimp, Penaeus monodon, MyD88 (PmMyD88) and TRAF6 (PmTRAF6) were identified and characterised. PmMyD88 cDNA is 1716 bp long with an open reading frame (ORF) of 1449 bp encoding a putative protein of 482 amino acids, with a death domain, a TIR domain and C-terminal extension domain. PmTRAF6 cDNA is 2563 bp long with an ORF of 1785 bp (594 amino acids) with an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled region and a MATH domain. In healthy shrimp, PmMyD88, PmTRAF6 and PmToll were detected in 15 tissues with the highest expression in midgut, eyestalk and lymphoid organ, respectively. Responses of these genes to WSSV in experimentally-infected P. monodon as well as in cultured haemocytes and also effect of poly I:C on the gene expression in vitro was investigated at six time-points in seven tissues. PmToll showed significant up-regulation at all time-points of infection in six tissues and until 24 h post-infection in vitro. However, poly I:C-induced haemocytes showed up-regulation of the gene until 48 h post-exposure. WSSV caused significant up-regulation of PmMyD88 in most of the tissues tested. The virus challenge as well as poly I:C induction in vitro also resulted in significant up-regulation of the gene. Up-regulated expression of PmTRAF6 was detected in haemocytes and lymphoid organ at late stage of infection. In vitro virus challenge showed significant up-regulation of PmTRAF6 at almost all time-points whereas no significant change in the expression was observed on poly I:C induction. The responses of these key genes, observed in the present study, suggest that Toll-pathway as a whole may play a crucial role in the immune response against viruses in shrimp.

Nonspecific effect of double-stranded (ds) RNA on prophenoloxidase (proPO) expression in Penaeus monodo n.

RNA interference-mediated silencing is an effective way of controlling white spot syndrome virus (WSSV). However, the effect of RNAi on the innate immune mechanism is not well understood. Prophenoloxidase (proPO) is an important component of the shrimp innate immunity. In the present study, nonspecific effect of two double-stranded (ds)RNA-expressing constructs, one targeting vp28 gene of WSSV (pCMV-VP28-LH) and another targeting green fluorescent protein (GFP) (pCMV-GFP-LH) on proPO2 gene expression, is investigated. mRNA expression levels of proPO2 in hemocytes of DNA construct-injected shrimp were estimated using real-time PCR with elongation factor 1-α as internal control. Empty vector (pcDNA)-injected shrimp were used as experimental control. In pCMV-VP28-LH-injected shrimp, proPO2 showed significant upregulation until 48 h post-injection (p.i.). Similarly, pCMV-GFP-LH-injected animals showed high levels of expression until 72 h p.i. WSSV-challenged animals, compared to pcDNA-injected control group, showed no significant change in expression of the gene until 24 h. However, an increased expression was noticed at 48 h p.i. Our results suggest that neither the plasmids nor the long hairpin RNA expressed by the constructs has any nonspecific silencing effect on the proPO2 expression. On the contrary, the consistent upregulation of proPO2 observed in shrimp injected with dsRNA at early time-points indicates the possibility of nonspecific protection against WSSV infection.

Pathogen recognition receptors in channel catfish: II. Identification, phylogeny and expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs).

Vertebrates including teleost fish have evolved an array of pathogen recognition receptors (PRRs) for detecting and responding to various pathogen-associated molecular patterns (PAMPs), including Toll-like receptors (TLRs), nucleotide-binding domain, leucine-rich repeat containing receptors (NLRs), and the retinoic acid inducible gene I (RIG-I) like receptors (RLRs). As a part of the series of studies targeted to characterize catfish PRRs, we described 22 NLR receptors in the sister contribution. Here in this study, we focused on cytosolic PRRs recognizing nucleotide pathogen-associated molecular patterns (PAMPs) of invading viruses, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors). Three RLRs with DExD/H domain containing RNA helicases, retinoic acid inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), were identified from channel catfish, Ictalurus punctatus. The catfish RIG-I encodes 937 amino acids that contains two CARDs, a DExDc, a HELICc and a RD domains. MDA5 encodes 1005 amino acids with all the domains identified for RIG-I. LGP2 encodes 677 amino acids that contain other domains but not the CARD domain at the N-terminus. Phylogenetic analyses of the three genes of catfish showed close clustering with their counterparts from other teleost fish. All the genes were found to be constitutively expressed in various tissues of catfish with minor variations. Channel catfish ovarian cells when infected with channel catfish virus showed significant increase in the transcript abundance of all the three genes. Further, RLR genes showed significant increases in expression in the liver tissue collected at different time-points after bacterial infection as well. The results indicate that the catfish RLRs may play important roles in antiviral and anti-bacterial immune responses.

Monodon baculovirus of shrimp.

Among the viruses infecting penaeid shrimp, monodon-type baculovirus (MBV) otherwise known as Penaeus monodon singly enveloped nuclear polyhedrosis virus (PmSNPV), is one of the widely reported and well described viruses. It is a rod-shaped, enveloped, double-stranded DNA virus, and considered till recently, as the type A baculovirus. Besides MBV, two strains of SNPV are reported-plebejus baculovirus and bennettae baculovirus. MBV was reported to be originated from Taiwan and has wide geographic distribution and is reported to be enzootic in wild penaeids of the Indo-pacific coasts of Asia. The virus also has diverse host-range including a variety of cultured and captured shrimp species and freshwater prawn, Macrobrachium rosenbergii. MBV has been reported in all life stages of P. monodon with late larval, postlarval and young juvenile as the most susceptible stages/ages. However, MBV has not been documented in early larval stages. Although MBV has been reported to be tolerated well by shrimp, the infection has been attributed to decreased productivity. The target organs or tissues of MBV are the hepatopancreatic tubules and duct epithelium of postlarvae, juveniles and adults, and the anterior midgut epithelium of very young postlarvae. The prominent clinical sign of infection is the presence of multiple spherical inclusion bodies in the hepatopancreas and midgut epithelial cells. The major mode of transmission of the virus is horizontal through oral exposure to occlusion bodies, contaminated tissues or fomites. Minor morphometric variation of the virus has been reported among different isolates. The rod-shaped enveloped virus particles range from 265-324 nm in length and 42-77 nm in diameter. Although complete genome sequence of MBV is not available, nucleic acid of MBV is circular, double-stranded DNA with a genome size ranging from 80 to 160 kbp. The virus codes for a 53 kDa major polyhedrin polypeptide and two minor 47 and 49 kDa polypeptides. A variety of diagnostic tools have been reported for this virus including real-time PCR and LAMP-based detection. Taxonomic position is still uncertain and International Committee on Taxonomy of Viruses lists MBV as a tentative species named PemoNPV in the genus Nucleopolyhedrovirus. However, according to the latest genomic information on the virus, it has been suggested to create a new group of non-occluded bacilliform viruses called nudiviruses with MBV as one of the members. The aim of the current work is to describe the knowledge on the status, distribution and host-range, pathology, transmission, virus structure and morphogenesis, genomic characteristics, diagnosis and the latest taxonomic position of MBV.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.

Cardiac myxosporiosis of pearl-spot, Etroplus suratensis (Bloch), due to Myxobolus etropli sp. nov.

A new myxosporean, Myxobolus etropli sp. nov., was found to infect the bulbus arteriosus of Etroplus suratensis (Bloch) from brackishwater lagoons of Muttukkadu, Chennai coast, India. A survey from May 1993 to October 1994 revealed a prevalence rate of 33.7% of this parasite. Macroscopic discoloured foci/cysts were seen in the bulbus arteriosus of the fish. The parasite showed strict host and site specificity. Histopathology showed that the infection was restricted to the bulbus. This is the first report of a myxosporean from E. suratensis.