Metagenomic dissection of the intestinal microbiome in the giant river prawn Macrobrachium rosenbergii infected with Decapod iridescent virus 1.

Microbiome in the intestines of aquatic invertebrates plays pivotal roles in maintaining intestinal homeostasis, especially when the host is exposed to pathogen invasion. Decapod iridescent virus 1 (DIV1) is a devastating virus seriously affecting the productivity and success of crustacean aquaculture. In this study, a metagenomic analysis was conducted to investigate the genomic sequences, community structure and functional characteristics of the intestinal microbiome in the giant river prawn Macrobrachiumrosenbergii infected with DIV1. The results showed that DIV1 infection could significantly reduce the diversity and richness of intestinal microbiome. Proteobacteria represented the largest taxon at the phylum level, and at the species level, the abundance of Gonapodya prolifera and Solemya velum gill symbiont increased significantly following DIV1 infection. In the infected prawns, four metabolic pathways related to purine metabolism, pyrimidine metabolism, glycerophospholipid metabolism, and pentose phosphate pathway, and five pathways related to nucleotide excision repair, homologous recombination, mismatch repair, base excision repair, and DNA replication were significantly enriched. Moreover, several immune response related pathways, such as shigellosis, bacterial invasion of epithelial cells, Salmonella infection, and Vibrio cholerae infection were repressed, indicating that secondary infection in M. rosenbergii may be inhibited via the suppression of these immune related pathways. DIV1 infection led to the induction of microbial carbohydrate enzymes such as the glycoside hydrolases (GHs), and reduced the abundance and number of antibiotic-resistant ontologies (AROs). A variety of AROs were identified from the microbiota, and mdtF and lrfA appeared as the dominant genes in the detected AROs. In addition, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration were the main antibiotic resistance mechanisms. Collectively, the data would enable a deeper understanding of the molecular response of intestinal microbiota to DIV1, and offer more insights into its roles in prawn resistance to DIVI infection.

Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

OBJECTIVE: We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. METHODS: An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. RESULT: The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. CONCLUSION: These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

Anti-lipopolysaccharide factors regulated by Stat, Dorsal, and Relish are involved in anti-WSSV innate immune defense in Macrobrachium nipponense.

Anti-lipopolysaccharide factors (ALF) is an important antimicrobial peptide and critical effector molecule with a broad spectrum of antimicrobial activities in crustaceans. In addition to the previously reported five ALFs (MnALF1-5), another three ALFs [MnALF1, which is different from MnALF1 (ALF02818) that has been reported; MnALF6; and MnALF7] and an isoform of MnALF4 (MnALF4-isoform2) were newly identified from Macrobrachium nipponense in this study. MnALF6 has 134 amino acids and one single nucleotide polymorphism (SNP) in MnALF6 resulted in the change of 107th amino acid from E to D. Intron 1 retention produced longer transcript of MnALF6. The full length of MnALF7 has 691 bp with a 363 bp ORF encoding 120 amino acid protein. Three SNPs in MnALF2 resulted in the conversion of amino acids at positions 70, 73, and 91 from T70I73P91 to K70L73S91. The deletion of 13 bp in MnALF4 resulted in early termination of ORF, resulting in MnALF4-isoform2 with only 98 amino acids. The gDNAs of MnALF1, MnALF2, MnALF5, and MnALF6 contain three exons and two introns, while those of MnALF3 and MnALF7 contain three exons, one known intron, and one unknown intron. The MnALF1-7 in M. nipponense were widely distributed in multiple tissues. After white spot syndrome virus (WSSV) stimulation, the expression levels of MnALF1-7 changed. Knockdown of MnALF1-7 could evidently increase the expression of the envelope protein VP28 and the copy number of WSSV during viral infection. Further studies found that silencing of three transcription factors (Stat, Dorsal, and Relish) in M. nipponense significantly inhibit the synthesis of MnALF1-7 during the process of WSSV challenge. This study adds to the knowledge about the roles of ALFs in the innate immune responses to WSSV infection in M. nipponense.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.

Silencing of prophenoloxidase (proPO) gene in freshwater prawn, Macrobrachium rosenbergii, makes them susceptible to white spot syndrome virus (WSSV).

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

The Boundary of the Market for Biosecurity Risk.

Imported goods create value in destination countries but also create biosecurity risk. Although widely used in other domains of the economy, risk markets have not been created to manage losses that occur when exotic pests and diseases are introduced with traded goods. In this article we show that not all biosecurity risks are insurable. Losses arising from effort needed to detect and respond to exotic pests and diseases that breach national borders appear to be insurable because entry of these threats and consequent response costs, can be regarded as random events. As pests and diseases establish and spread, however, loss of access to export markets and productivity losses display systematic risk and appear to be uninsurable. Other insurability criteria support this definition of the boundary of biosecurity risk markets. We use the Australian biosecurity system as an example, although the framework described in this study will be applicable to biosecurity systems worldwide. We argue that biosecurity risk insurance could be incorporated into the current biosecurity system but would require legislation mandating importers to purchase insurance. Advantages of actuarial pricing of biosecurity risk are: (i) an increase in economic efficiency to the extent that importers respond to the price of biosecurity risk; (ii) financial sustainability would improve because actuarial pricing creates a structural link between funds available for biosecurity activities and risk exposure; and (iii) equity issues evident in the current biosecurity system could be addressed because risk creators (importers) would fund response activities through the purchase of insurance.

Regulation of Proteolytic Activity to Improve the Recovery of Macrobrachium rosenbergii Nodavirus Capsid Protein.

The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.

Feasibility of vaccination against Macrobrachium rosenbergii nodavirus infection in giant freshwater prawn.

The giant freshwater prawn/giant river prawn, Macrobrachium rosenbergii is one of the high market value crustaceans cultured worldwide. The intensified aquaculture of the species has led to the outbreak of infectious diseases, prominently, the white tail disease (WTD). It is caused by the infection of Macrobrachium rosenbergii nodavirus (MrNV), which was classified in the family of Nodaviridae. To-date, there are no effective prophylactic and therapeutic agents available against MrNV infection. Vaccination is known to be the most effective prophylactic agent in disease prevention. However, vaccine development against virus infection in crustaceans is equivocal. The feasibility of vaccination in conferring immune protection in crustaceans against infectious diseases is disputable. The argument lies in the fact that crustaceans do not possess adaptive immunity, which is the main immune component that functions to establish immunological memory upon vaccination. Nevertheless, an increasing number of literatures has been documented, which concerns the development of vaccines against infectious diseases in crustaceans. The current review deliberates different approaches in vaccine development against MrNV, which were documented in the past years. It is noteworthy that the live-attenuated MrNV vaccine has not been experimented by far. Thus, the potential of live-attenuated MrNV vaccine in conferring long-term immune protection through the establishment of innate immune memory is currently being discussed.

Transcriptomic analysis of Macrobrachium rosenbergii (giant fresh water prawn) post-larvae in response to M. rosenbergii nodavirus (MrNV) infection: de novo assembly and functional annotation.

BACKGROUND: Macrobrachium rosenbergii, is one of a major freshwater prawn species cultured in Southeast Asia. White tail disease (WTD), caused by Macrobrachium rosenbergii nodavirus (MrNV), is a serious problem in farm cultivation and is responsible for up to 100% mortality in the post larvae stage. Molecular data on how M. rosenbergii post-larvae launches an immune response to an infection with MrNV is not currently available. We therefore compared the whole transcriptomic sequence of M. rosenbergii post-larvae before and after MrNV infection. RESULTS: Transcriptome for M. rosenbergii post-larvae demonstrated high completeness (BUSCO Complete: 83.4%, fragmentation: 13%, missing:3.3%, duplication:16.2%; highest ExN50 value: 94%). The assembled transcriptome consists of 96,362 unigenes with N50 of 1308 bp. The assembled transcriptome was successfully annotated against the NCBI non-redundant arthropod database (33.75%), UniProt database (26.73%), Gene Ontology (GO) (18.98%), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG) (20.88%), and Kyoto Encyclopedia of Genes and Genome pathway (KEGG) (20.46%). GO annotations included immune system process, signaling, response to stimulus, and antioxidant activity. Differential abundance analysis using EdgeR showed 2413 significantly up-regulated genes and 3125 significantly down-regulated genes during the infection of MrNV. CONCLUSIONS: This study reported a highly complete transcriptome from the post-larvae stage of giant river prawn, M. rosenbergii. Differential abundant transcripts during MrNV infection were identified and validated by qPCR, many of these differentially abundant transcripts as key players in antiviral immunity. These include known members of the innate immune response with the largest expression change occurring in the M. rosenbergii post-larvae after MrNV infection such as antiviral protein, C-type lectin, prophenol oxidase, caspase, ADP ribosylation factors, and dicer.

Macrobrachium rosenbergii nodavirus (MrNV)-CP-RNA-2 DNA vaccine confers protective immunity in giant freshwater prawn Macrobrachium rosenbergii against MrNV infection.

Macrobrachium rosenbergii Nodavirus (MrNV) causes white tail disease (WTD) in Giant freshwater prawn Macrobrachium rosenbergii which leads to immense economic losses in hatcheries and farms. In the present study, we cloned the capsid protein gene of MrNV-CP-RNA-2 (1146 bp) into a DNA vaccine vector pVAX1 to form MrNV-CP-RNA-2- pVAX1. The bacterial transformant, containing the MrNV-CP gene, was coated on the fish diet pellets and fed to juvenile M. rosenbergii for 40 days. After the vaccine delivery, group of M. rosenbergii were challenged with virulent MrNV on 20 and 40th days post-vaccination (dpv) respectively and monitored for the survival. The non-vaccinated M. rosenbergii succumbed to death (100%) within 5 days, whereas the MrNV-CP-RNA-2- pVAX1 treated groups had the survivals of 60 and 80% in 20 and 40 dpv respectively (P ≤ 0.001). To study the MrNV infection level, double step PCR was performed at different dpv. The results revealed that in 20 dpv group, the infection was decreased to 65% and in 40 dpv group the infection decreased to 69% from control diet fed prawns (P < 0.001). Haematological parameters like coagulation time, total haemocyte count (THC) and oxyhaemocyanin levels were performed for the control and vaccinated prawns. The vaccination helped to decrease the time of coagulation, improved THC and oxyhaemocyanin levels at a significant level (p < 0.001) when compared to the non-vaccinated group. The immunological parameters like prophenol oxidase (ProPO), superoxide anion and intra-agar lysozyme activity were also performed and the results revealed that the level of proPO, superoxide anion and lysozyme activities were significantly (P ≤ 0.05) increased in 20 and 40 dpv groups respectively, when compared with the non-vaccinated groups. Based on the vaccination trials, the DNA vaccine construct MrNV-CP-RNA-2-pVAX1 effectively improved the survival against MrNV challenge, helped to decrease viral load and enhanced the immune system to protect the prawn from MrNV infection. This vaccine construct is highly useful to protect the M. rosenbergii from MrNV infection.

Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection.

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

Antiviral activity of cathelicidin 5, a peptide from Alligator sinensis, against WSSV in caridean shrimp Exopalaemon modestus.

White spot disease caused by white spot syndrome virus (WSSV) is responsible for harming shrimp aquaculture industry and results in a pandemic throughout the world. Cathelicidin 5 treatment enhanced immune parameters including antioxidant enzyme activity and immune-related genes expression in shrimp Exopalaemon modestus. Shrimp treated with cathelicidin 5 and inoculated with white spot syndrome virus (WSSV) exhibited a significantly lower mortality rate and lower viral VP28 amplification and expression than control. This study addresses the role of cathelicidin 5 in immune stimulatory and antiviral activities that could protect E. modestus from WSSV infection.

Peptide inhibitors of Macrobrachium rosenbergii nodavirus.

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant (KDrel) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a KDrel value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a KDrel value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.

ICTV virus taxonomy profile: Sarthroviridae.

The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.

The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China.

White tail disease (WTD), a major disease prevailing in the larval stage of Macrobrachium rosenbergii, caused by Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), led to the economic loss of shrimp industry in China. In order to establish a convenient, sensitive and selective molecular diagnostic method to detect MrNV and XSV for the Chinese shrimp (MrNV/XSV-chin), a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a lateral flow dipstick (LFD) method were developed. A set of four specific primers and a labeled probe were designed according to the six conserved gene sequence regions encoding for the MrNV capsid protein CP43 and the XSV capsid protein CP17. The detection of MrNV and XSV simultaneously by RT-LAMP was performed at 61 °C in a single reaction for 60 min followed by hybridization with an FITC-labeled probe for 5 min and visualized by LFD. The RT-LAMP-LFD assay had a sensitivity of approximately 100-fold higher than conventional PCR. In addition, the assay could detect MrNV/XSV-chin from limited amount of RNA extracts as low as 1.0 pg extracted from Macrobrachium rosenbergii. This assay was simple to use, required little instrumentation, and exhibited excellent specificity for the MrNV/XSV-chin compared with other shrimp viruses. In conclusion, a convenient, sensitive and selective practical molecular diagnostic method was developed with the potential for diagnosis and prevention of WTD.

Molecular biology of Macrobrachium rosenbergii nodavirus infection in giant freshwater prawn.

Macrobrachium rosenbergii nodavirus (MrNV) has been threatening the giant freshwater prawn aquaculture since 1997, causing white tail disease in the prawn species that leads to 100% lethality of the infected postlarvae. Comprehension of the viral infectivity and pathogenesis at molecular biology level has recently resolved the viral capsid protein and evidenced the significant difference in the viral structural protein compared to other nodaviruses that infect fish and insect. Cumulative researches have remarked the proposal to assert MrNV as a member of new genus, gammanodavirus to the Nodaviridae family. The significance of molecular biology in MrNV infection is being highlighted in this current review, revolving the viral life cycle from virus binding and entry into host, virus replication in host cell, to virus assembly and release. The current review also highlights the emerging aptamers technology that is also known as synthetic antibody, its application in disease diagnosis, and its prophylactic and therapeutic properties. The future perspective of synthetic virology technology in understanding viral pathogenesis, as well as its potential in viral vaccine development, is also discussed.

Experimental vertical transmission of covert mortality nodavirus in Exopalaemon carinicauda.

Viral covert mortality disease (VCMD) has caused serious losses to shrimp aquaculture in China in recent years and the ridgetail white prawn Exopalaemon carinicauda has been suspected to be one important factor in perpetuating the high prevalence of covert mortality nodavirus (CMNV) infections due to its perennial presence in shrimp farming ponds and water from natural habitats. Experiments were carried out to determine the possibility of vertical transmission of CMNV in E. carinicauda in this study. CMNV infection in gonads, fertilized eggs and larvae was investigated by using the methods of reverse transcription nested PCR (nRT-PCR), in situ hybrization (ISH) and transmission electron microscopy (TEM). The ovarian tissue and testis tissue of artificially infected parental E. carinicauda were proved to be CMNV-positive by nRT-PCR. Fertilized eggs were also found to be CMNV-positive by nRT-PCR whether the fertilized eggs originated from the CMNV-positive female broodstock mated with the CMNV-negative male broodstock, or they originated from the CMNV-negative female broodstock mated with the CMNV-positive males. The results of ISH indicated that the positive signals were evident in the oocytes within ovarian tissue and nauplii. By TEM analysis, CMNV virions were observed in oogonia, oocytes, spermatocytes, fertilized eggs and nauplii. The presence of CMNV in fertilized eggs and larvae indicates that CMNV can transmit vertically via sperm and oocytes in E. carinicauda, which highlights the high probability of vertical transmission of CMNV in the main species of cultured shrimp and prawns.

Experimental inoculation of oriental river prawn Macrobrachium nipponense with white spot syndrome virus (WSSV).

The oriental river prawn Macrobrachium nipponense is an economically important species that is widely farmed in China. White spot syndrome virus (WSSV) is one of the most devastating pathogens of the cultured shrimp Litopenaeus vannamei, responsible for massive loss of its commercial products worldwide. We investigated the infectivity and pathogenicity of WSSV in adult M. nipponense using standardized conditions for L. vannamei. The median lethal dose of WSSV in adult M. nipponense was 103.84±0.06 copies g-1, which was about 1000-fold higher than in L. vannamei (100.59±0.22 copies g-1). WSSV was detected by 2-step PCR in the gills, hepatopancreas, muscle, stomach, heart, gut, nerve, integument, pereopod, eyestalk, testis, and ovary of experimentally infected dead M. nipponense. Lesions were observed histologically following WSSV injection, showing basophilic intranuclear inclusion bodies in the hepatopancreas and subsequently in the gills. The clearance of WSSV was observed in hepatopancreas and gills at 48 and 96 h post-inoculation, respectively. No histological lesions were detected in muscle from 0-96 h post-injection. The results show that the oriental river prawn M. nipponense can be infected by WSSV and the infections are self limiting over time; therefore, M. nipponense may serve as a useful model for studying resistance to WSSV.

Molecular importance of prawn large heat shock proteins 60, 70 and 90.

Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2(-ΔΔCT) method. MrHSP60 contains a long chaperonin 60 domain at 46-547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21-624 and MrHSP90 carries a HSP90 domain at 188-719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P < 0.05) expression of MrHSP60, 70 and 90 in haemocyte, gill and hepatopancreas, respectively. Further, the expression level was up-regulated upon bacterial (Aeromonas hydrophilla and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodo virus (MrNV)] infections during various time periods. The gene expression results exhibited the potential involvement of these three HSPs in the immune system of prawn. The study indicated the potentiality of these molecules, thereby protecting cells against pathogens as well as severe cellular and environmental stresses in crustaceans.