Evaluation of protective effects of heat-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine against herpesviral hematopoietic necrosis disease (HVHND) in goldfish (Carassius auratus).

Cyprinid herpesvirus-2 (CyHV-2) is an important virus that causes herpesviral hematopoietic necrosis disease (HVHND) leading to huge economic losses in goldfish (Carassius auratus). However, until now no proper prophylactic measure or treatment is available for CyHV-2 infection in goldfish. Hence, in this experiment, we developed a heat-inactivated CyHV-2 vaccine and evaluated its performance in goldfish. Initially, CyHV-2 was propagated in the fantail goldfish fin (FtGF) cell line and the titer of the viral inoculum was 107.8 TCID50/ml. Subsequently, various temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) were evaluated to achieve the complete inactivation of CyHV-2. Only the viral inoculum inactivated at 80 °C for 1 h did not show any cytopathic effect in the FtGF cell line after five blind passages. Hence the heat-inactivated CyHV-2 vaccine developed at 80 °C was further used for immunization trials in goldfish. The experimental goldfish were intraperitoneally immunized with 300 µL of the heat-inactivated CyHV-2 vaccine. Subsequently, the kidney and spleen tissues were sampled at various time points post-vaccination (6th hr, 2nd day, 4th day, 6th day, 10th day, 16th day, and 30th day) to evaluate the expression of immune genes (IL-12, IL-10, IFN-γ, CD8, and CD4). A significant upregulation of immune genes was observed at various time points in the kidney and spleen tissue of the vaccinated goldfish. Furthermore, in order to study the efficacy of the vaccine, the experimental fish were challenged with CyHV-2 (107.8 TCID50/ml) after the 30th day post-vaccination. The survival of the fish in the vaccine group (86.7%) was significantly higher compared to the non-vaccinated group (20%). Moreover, the relative percentage survival of the vaccinated group was 83.34%. In spite of the single dose, the heat-killed vaccine developed in the present study elicited the immune response and offered better protection in goldfish against CyHV-2. However, further large-scale field performance evaluation studies are necessary to develop this vaccine on a commercial scale.

Study on amyloodiniosis outbreak in captive-bred percula clownfish (Amphiprion percula) and improved control regimens.

The amyloodiniosis outbreak was documented with high mortality of percula clownfish (Amphiprion percula) and subsequently, various therapeutics were evaluated to control the infection. The affected fish exhibited symptoms such as discoloration of the skin and jerky movement with severe respiratory stress. Microscopic examination of the gill and body surface of the infested moribund fish showed the presence of brown coloured spherical shaped trophonts with the size range of 150-300 µM. Copper sulphate (10 ppm) and hydroxychloroquine phosphate (10 ppm) treatment with 14 days of continuous bath showed 76 ± 5.3 and 66 ± 4.3% survival rates. However, formalin (10 ppm) and malachite green (10 ppm) treated groups showed a survival of 41 ± 1.7 and 32.7 ± 1.2% respectively. The present results suggest that, use of copper sulphate to treat amyloodiniosis in clownfish will relatively more effective than other treatment options. The findings will be helpful to mitigate amyloodiniosis in marine finfish aquaculture, particularly marine ornamentals.

Identification of in vivo induced antigens of the malacosporean parasite Tetracapsuloides bryosalmonae (Cnidaria) using in vivo induced antigen technology.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that causes proliferative kidney disease (PKD) in wild and farmed salmonids in Europe and North America. The life cycle of T. bryosalmonae completes between invertebrate bryozoan and vertebrate fish hosts. Inside the fish, virulence factors of T. bryosalmonae are induced during infection or interactions with host cells. T. bryosalmonae genes expressed in vivo are likely to be important in fish pathogenesis. Herein, we identify in vivo induced antigens of T. bryosalmonae during infection in brown trout (Salmo trutta) using in vivo induced antigen technology (IVIAT). Brown trout were exposed to the spores of T. bryosalmonae and were sampled at different time points. The pooled sera were first pre-adsorbed with antigens to remove false positive results. Subsequently, adsorbed sera were used to screen a T. bryosalmonae cDNA phage expression library. Immunoscreening analysis revealed 136 immunogenic T. bryosalmonae proteins induced in brown trout during parasite development. They are involved in signal transduction, transport, metabolism, ion-protein binding, protein folding, and also include hypothetical proteins, of so far unknown functions. The identified in vivo induced antigens will be useful in the understanding of T. bryosalmonae pathogenesis during infection in susceptible hosts. Some of the antigens found may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against T. bryosalmonae in salmonids.

Outbreak of Parasitic Dinoflagellate Piscinoodinium sp. Infection in an Endangered Fish from India: Arulius Barb (Dawkinsia arulius).

Freshwater velvet disease is caused by the dinoflagellate parasite, Piscinoodinium sp. This parasite has been reported in tropical and subtropical fishes, and it can cause devastating losses. Moreover, Piscinoodinium sp. is identified as one of the least studied finfish parasites, and the available molecular information about this parasite is meager. Recently, Piscinoodinium sp. was responsible for the 100% cumulative mortality of the captive-bred F1 generation of Arulius barb (Dawkinsia arulius), an endangered freshwater fish native to India. The trophont stages of the parasite were observed in the skin and gills of the affected fish. The total DNA was extracted from the trophonts collected from the affected Arulius barb and the partial nucleotide sequence of the rDNA complex region (2334 bp) was amplified using PCR. The amplified PCR product exhibited a high sequence identity (97.61%) with Piscinoodinium sp. In the phylogenetic analysis of the SSU rDNA, Piscinoodinium sp. emerged as a separate clade from other dinoflagellate species. This is the first report of the infection of Piscinoodinium sp. in Arulius barb and the molecular information generated from this study can serve as a baseline to study the diversity of the parasite in India. Furthermore, the impact of this parasite among wild fish stock is not known, and this parasite needs further research focus to generate more molecular information and to understand the host-pathogen interaction.

Genome-wide alternative splicing profile in the posterior kidney of brown trout (Salmo trutta) during proliferative kidney disease.

BACKGROUND: The cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD. RESULTS: RNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value < 0.05) were identified from 19,722 alternatively spliced events. Among the DEAS genes, the least and most abundant alternative splicing types were alternative 5' splice site (5.23%) and exon skipping (70.59%), respectively. The DEAS genes were significantly enriched for sodium-potassium transporter activity and ion homeostasis (ahcyl1, atp1a3a, atp1a1a.1, and atp1a1a.5). The protein-protein interaction network analysis enriched two local network clusters namely cation transporting ATPase C-terminus and Sodium/potassium ATPase beta chain cluster, and mixed inclusion of Ion homeostasis and EF-hand domain cluster. Furthermore, the human disease-related salmonella infection pathway was significantly enriched in the protein-protein interaction network. CONCLUSION: This study provides the first baseline information about alternative splicing in brown trout during PKD. The generated data lay a foundation for further functional molecular studies in PKD - brown trout infection model. The information generated from the present study can help to develop therapeutic strategies for PKD in the future.

A natural outbreak of infectious spleen and kidney necrosis virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India.

A large-scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam, an artificial tropical lake made for irrigation and drinking water supply in Kerala, India during 2018. This dam is located in the premises of Western Ghats, recognized as one of the biodiversity hotspots of the world. The objective of this study was to identify the aetiological agent of this large-scale mortality of E. suratensis by systematic diagnostic investigation and identification of the pathogen. Virus isolation was carried out on a species-specific pearlspot fin (PSF) cell line. Infected PSF cells showed cytopathic effects (CPEs) like cell shrinkage, rounding, enlargement, clustering, and subsequent detachment of cells with a high viral titre of 106⋅95 TCID50 ml-1 at 8 days post-inoculation (dpi). Histopathological examination of the fish showed the presence of numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclusions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed the presence of large numbers of 125-132 nm viral particles in the spleen tissues. PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene sequence confirmed that the causative agent was infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7 ± 2.7% mortality in the E. suratensis (body weight 11.01 ± 2.7 g; body length 8.01 ± 2.23 cm) injected with 1 × 104⋅25 TCID50 ml-1 ISKNV per fish. Our detailed investigation provided definitive diagnosis of ISKNV in the severe mass mortality event in wild E. suratensis in Peechi Dam, India, adding one more species to expanding host range of ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the perilous nature of this disease, particularly among the wild fish population.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine.

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 µl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

Co-Infection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in the Wild Crustaceans of Andaman and Nicobar Archipelago, India.

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.

Infectious spleen and kidney necrosis virus-associated large-scale mortality in farmed giant gourami, Osphronemus goramy, in India.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish.

The accuracy of quantitative real time PCR (RTqPCR) can be attained only when a suitable reference gene is used. The gene expression for a particular gene may vary within different cells at different conditions. Hence, the suitability and stability of various potential reference genes have to be determined for expression studies. In this study, we have examined the potential of four different reference genes including ß-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3P-dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1AA) in seven different tissues including gill, liver, kidney, spleen, heart, muscle and intestine of goldfish (Carassius auratus). The housekeeping genes were analyzed from healthy fish and in CyHV-2 challenged fish. Based upon the real time PCR results the gene expression varied among the genes and in tissues. The expression levels of the housekeeping genes were then compared and evaluated with the RefFinder web tool which analyses results using four different algorithms - BestKeeper, delta Ct, geNorm and NormFinder. EF1AA was ranked to be the best gene in healthy fish by BestKeeper and geNorm analysis. The delta Ct and NormFinder algorithm have found 18S to be a stable gene in healthy fish but 18S was given to be least expressed in challenged fish. ACTB was also given as a stable gene by geNorm analysis in both healthy and challenged fish. Also, in CyHV-2 challenged fish, EF1AA was identified as the best gene by all the three analysis except by BestKeeper analysis, where it has ranked GADPH as the best housekeeping gene. Expression of the four candidate reference genes differed across all tissue types tested, inferring that a thorough study of the reference genes is necessary for cross tissue comparison. These results can be further used in the immune gene response study of goldfish infected with any viral pathogen to develop better health strategies in the disease management of goldfish aquaculture.

Identification and Expression Profiling of Toll-Like Receptors of Brown Trout (Salmo trutta) during Proliferative Kidney Disease.

Proliferative kidney disease is an emerging disease among salmonids in Europe and North America caused by the myxozoan parasite Tetracapsuloides bryosalmonae. The decline of endemic brown trout (Salmo trutta) in the Alpine streams of Europe is fostered by T. bryosalmonae infection. Toll-like receptors (TLRs) are a family of pattern recognition receptors that acts as sentinels of the immune system against the invading pathogens. However, little is known about the TLRs' response in salmonids against the myxozoan infection. In the present study, we identified and evaluated TLR1, TLR19, and TLR13-like genes of brown trout using data-mining and phylogenetic analysis. The expression pattern of TLRs was examined in the posterior kidney of brown trout infected with T. bryosalmonae at various time points. Typical Toll/interleukin-1 receptor protein domain was found in all tested TLRs. However, TLR13-like chr2 had a short amino acid sequence with no LRR domain. Phylogenetic analysis illustrated that TLR orthologs are conserved across vertebrates. Similarly, a conserved synteny gene block arrangement was observed in the case of TLR1 and TLR19 across fish species. Interestingly, all tested TLRs showed their maximal relative expression from 6 to 10 weeks post-exposure to the parasite. Our results suggest that these TLRs may play an important role in the innate defense mechanism of brown trout against the invading T. bryosalmonae.

The Malacosporean Myxozoan Parasite Tetracapsuloides bryosalmonae: A Threat to Wild Salmonids.

Tetracapsuloides bryosalmonae is a myxozoan parasite responsible for proliferative kidney disease (PKD) in a wide range of salmonids. PKD, characterized by high mortality and morbidity, is well known for affecting aquaculture operations and wild salmonid populations across Europe and North America. The life cycle of T. bryosalmonae revolves around freshwater bryozoan and salmonid fish hosts. In recent years, T. bryosalmonae has been reported among wild salmonids from the European countries where it has not been reported previously. T. bryosalmonae is believed to be a possible reason for the diminishing wild salmonid populations in the natural water bodies of many European countries. Climate crisis driven rising water temperature can further accelerate the distribution of T. bryosalmonae. Expansion of the geographical distribution of T. bryosalmonae may further advocate the decline of wild salmonid populations, especially brown trout (Salmo trutta) in their habitats. Mathematical models are used to understand the pattern and distribution of T. bryosalmonae among the host in the natural water bodies. The present manuscript not only summarizes the incidences of T. bryosalmonae among the wild salmonid populations, but also discusses the contemporary understanding about the development of T. bryosalmonae in its hosts and the influences of various factors in the spread of the disease in the wild.

Transcriptome profiling of posterior kidney of brown trout, Salmo trutta, during proliferative kidney disease.

BACKGROUND: Tetracapsuloides bryosalmonae is a myxozoan parasite which causes economically important and emerging proliferative kidney disease (PKD) in salmonids. Brown trout, Salmo trutta is a native fish species of Europe, which acts as asymptomatic carriers for T. bryosalmonae. There is only limited information on the molecular mechanism involved in the kidney of brown trout during T. bryosalmonae development. We employed RNA sequencing (RNA-seq) to investigate the global transcriptome changes in the posterior kidney of brown trout during T. bryosalmonae development. METHODS: Brown trout were exposed to the spores of T. bryosalmonae and posterior kidneys were collected from both exposed and unexposed control fish. cDNA libraries were prepared from the posterior kidney and sequenced. Bioinformatics analysis was performed using standard pipeline of quality control, reference mapping, differential expression analysis, gene ontology, and pathway analysis. Quantitative real time PCR was performed to validate the transcriptional regulation of differentially expressed genes, and their correlation with RNA-seq data was statistically analyzed. RESULTS: Transcriptome analysis identified 1169 differentially expressed genes in the posterior kidney of brown trout, out of which 864 genes (74%) were upregulated and 305 genes (26%) were downregulated. The upregulated genes were associated with the regulation of immune system process, vesicle-mediated transport, leucocyte activation, and transport, whereas the downregulated genes were associated with endopeptidase regulatory activity, phosphatidylcholine biosynthetic process, connective tissue development, and collagen catabolic process. CONCLUSION: To our knowledge, this is the first RNA-seq based transcriptome study performed in the posterior kidney of brown trout during active T. bryosalmonae development. Most of the upregulated genes were associated with the immune system process, whereas the downregulated genes were associated with other metabolic functions. The findings of this study provide insights on the immune responses mounted by the brown trout on the developing parasite, and the host molecular machineries modulated by the parasite for its successful multiplication and release.

Kinetics of local and systemic immune cell responses in whirling disease infection and resistance in rainbow trout.

BACKGROUND: Whirling disease (WD), caused by the myxozoan parasite Myxobolus cerebralis, is responsible for high mortalities in rainbow trout hatcheries and natural populations. To elucidate how resistant and susceptible rainbow trout strains respond to early invasion, a well-established model of WD was used to demonstrate the kinetics of local and systemic immune responses in two rainbow trout strains, the susceptible American Trout Lodge (TL) and the more resistant German Hofer strain (HO). METHODS: Parasite load and cellular immune responses were compared across several time points after M. cerebralis exposure to elucidate the kinetics of immune cells in resistant and susceptible rainbow trout in response to early invasion. In the course of the 20 days following exposure, leukocyte kinetics was monitored by flow cytometry in the caudal fin (CF), head kidney (HK) and spleen (SP). For the analysis of the leukocyte composition, cells were stained using a set of monoclonal antibodies with known specificity for distinct subpopulations of rainbow trout leukocytes. RESULTS: Experiments indicated general increases of CF, HK and SP myeloid cells, while decreases of B cells and T cells in the SP and HK were observed at several time points in the TL strain. On the other hand, in the HO strain, increases of T cells were dominant in CF, HK and SP at multiple time points. The differences between HO and TL were most distinct at 2, 4, 12 and 48 hours post-exposure (hpe) as well as at 4 days post-exposure (dpe), with the vast majority of innate immune response cells having higher values in the susceptible TL strain. Alteration of the leukocyte populations with augmented local cellular responses and excessive immune reactions likely lead to subsequent host tissue damage and supports parasite invasion and development in TL. CONCLUSIONS: The findings of this study highlight the significance of effective local and systemic immune reaction and indicate proper activation of T lymphocytes critical for host resistance during M. cerebralis infection. The present study provides insights into the cellular basis of protective immune responses against M. cerebralis and can help us to elucidate the mechanisms underlying the variation in resistance to WD.

Transcriptome Analysis Based on RNA-Seq in Understanding Pathogenic Mechanisms of Diseases and the Immune System of Fish: A Comprehensive Review.

In recent years, with the advent of next-generation sequencing along with the development of various bioinformatics tools, RNA sequencing (RNA-Seq)-based transcriptome analysis has become much more affordable in the field of biological research. This technique has even opened up avenues to explore the transcriptome of non-model organisms for which a reference genome is not available. This has made fish health researchers march towards this technology to understand pathogenic processes and immune reactions in fish during the event of infection. Recent studies using this technology have altered and updated the previous understanding of many diseases in fish. RNA-Seq has been employed in the understanding of fish pathogens like bacteria, virus, parasites, and oomycetes. Also, it has been helpful in unraveling the immune mechanisms in fish. Additionally, RNA-Seq technology has made its way for future works, such as genetic linkage mapping, quantitative trait analysis, disease-resistant strain or broodstock selection, and the development of effective vaccines and therapies. Until now, there are no reviews that comprehensively summarize the studies which made use of RNA-Seq to explore the mechanisms of infection of pathogens and the defense strategies of fish hosts. This review aims to summarize the contemporary understanding and findings with regard to infectious pathogens and the immune system of fish that have been achieved through RNA-Seq technology.

Expression Profile of Penaeus monodon Ubiquitin Conjugating Enzyme (PmUbc) at Protein Level in White spot syndrome virus Challenged Shrimp.

White spot syndrome virus (WSSV) is one of the major pathogens in shrimp aquaculture. Four proteins of WSSV are predicted to encode a RING H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimps can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, expression profile of Penaeus monodon Ubiquitin conjugating enzyme (PmUbc) was studied at protein level in WSSV challenged shrimp. A time point analysis of the expression of PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge in P. monodon. Recombinant PmUbc (rPmUbc) was produced in prokaryotic expression vector, BL21 (DE3) pLys S. The PmUbc expression pattern was studied by ELISA with rPmUbc antibodies raised in rabbit. A significant increase in PmUbc expression at 24 h post infection (hpi) was observed followed by a decline till 72 hpi. Since the up-regulation and a tremendous decline of PmUbc protein expression was observed at 24 and in 72 hpi respectively in ELISA, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. Many findings have shown that viral infection can up-regulate expression of ubiquitin and that the ubiquitin system plays a key role in the course of viral infection. The present study reveals the expression patterns of PmUbc at protein level in WSSV infected P. monodon. However, further studies are to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this major culprit in shrimp culture industry.