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Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms.

[Display omitted] • First incidence of Mud crab reovirus (MCRV) in a polyculture farm (Scylla serrata and Litopenaeus vannamei) from Andhra Pradesh. • This disease has resulted in complete (100%) mass mortalities in farms resulting in heavy economic losses. • Moribund crabs displayed autotomy, loss of appetite, discoloration of carapace, slow movement etc. • TEM showed non-enveloped icosahedral shaped viral particles of 70 nm in diameter. Reovirus designated as Mud crab reovirus (MCRV) is associated with the mass mortalities of mud crabs resulting in significant economic loss to crab and shrimp-mud crab polyculture farmers in the Nagayalanka, Krishna district, Andhra Pradesh. The 100 % chronic mass mortalities have been attributed to the outbreak of Mud crab reovirus (MCRV) in the polyculture farms. The moribund crabs showed autotomy, discoloration of carapace, loss of appetite, slow movement and loose gills. Histopathological observations of the infected mud crabs showed an atrophied hepatopancreas, complete degeneration of tissues along with viral inclusions in hepatopancreas, gills and muscles. Further analysis using Transmission electron microscopy (TEM), showed that the viral particles had a diameter of 70 nm and exhibited a non-enveloped, icosahedral shape arranged in a crystalline manner. The virus mainly infects the connective tissue of hepatopancreas, gills, muscle and develops in the cytoplasm. RT-PCR reconfirmed the presence of reovirus in the hepatopancreas of spontaneously infected mud crab Scylla serrata. The current study shows the importance of monitoring the MCRV prevalence in polyculture farms to minimize its spread and precautionary measures can be taken by screening the brooders from the crab hatchery and stocking of wild crabs without screening should be avoided in order to prevent MCRV outbreak. [ABSTRACT FROM AUTHOR]

The culture of animals in a controlled environment can be a factor in disease transmission. Mud Crab Reovirus (MCRV) and staphylococcus saprophyticus were co-infected in Scylla serrata , in a fattening pen from MGR Thittu, Cuddalore District, Tamil Nadu, Southeast coast of India. Lethargic, sluggish, partially atrophied hepatopancreas, loose gills, and discoloration of the animal characterized the spontaneously infected moribund crab. Viral invasion in the hepatopancreas with basophilic inclusion was evaluated in the histopathological studies. Molecular evidence shows reovirus infection in crab. Scanning electron microscopy reveals the presence of cocci bacteria. The 16 s RNA sequencing and phylogenetic analysis confirm the infected bacteria were Staphylococcus saprophyticus. Ultrastructural studies provided evidence for the coinfection of MCRV and Staphylococcus saprophyticus. This is the inaugural article on the co-infection of reovirus and bacteria in the mud crab Scylla serrata. • Primary study on simultaneous infection of reovirus and bacteria in aquaculture. • Reovirus causes 100% mortality. • The reovirus pathogen was confirmed using histology, transmission electron microscopy, and molecular studies. • Morphological, biochemical, and molecular studies confirmed bacterial infection. [ABSTRACT FROM AUTHOR]

A STAT gene from Scylla paramamosain , named SpSTAT , was cloned and characterized. The full length of SpSTAT mRNA contains a 5′untranslated region (UTR) of 238 bp, an open reading frame (ORF) of 2388 bp and a 3′ UTR of 326 bp. The Sp STAT protein contains four characteristic STAT domains and showed 84% identity (90% similarity) and 44% identity (64% similarity) to Litopenaeus vannamei STAT protein and Human STAT5a/b protein, respectively. The mRNA of SpSTAT was high expressed in the intestine and eyestalk and low expressed in the heart and muscle. Moreover, expression of SpSTAT was significantly responsive to challenge of mud crab reovirus (MCRV), Poly(I:C), LPS and Staphylococcus aureus. Sp STAT could be activated by Poly(I:C) and LPS to translocate to the nucleus of Drosophila Schneider 2 (S2) cells. Sp STAT could be phosphorylated by interaction with JAK of S. paramamosain (Sp JAK) and activated to translocate to the nucleus of S2 cells. Furthermore, Silencing of SpSTAT in vivo resulted in higher mortality rate of MCRV infected mud crab and increased the viral load in tissues, suggesting that SpSTAT could play an important role in defense against MCRV in mud crab. • The full length of Scylla paramamosain STAT (SpSTAT) was cloned and characterized. • SpSTAT was significantly upregulated by mud crab reovirus(MCRV), Poly(I:C) and LPS. • Sp STAT could be phosphorylated by interaction with Sp JAK in S2 cells. • The nuclear shift of Sp STAT was observed by challenge of Poly(I:C), LPS and Sp JAK. • SpSTAT could play an important role in defense against MCRV in mud crab. [ABSTRACT FROM AUTHOR]

JAK/STAT signaling pathways are associated with the innate immune system and play important roles in mediating immune responses to virus infection. In this study, a Janus kinase gene from Scylla paramamosain (Sp JAK) was cloned and characterized. The full length of Sp JAK mRNA contains a 5′ untranslated region (UTR) of 304 bp, an open reading frame of 3300 bp and a 3′ UTR of 302 bp. The Sp JAK protein contains seven characteristic JAK homology domains (JH1 to JH7) and showed 60% identity (78% similarity), 20% identity (35% similarity), and 21% identity (37% similarity) to the Litopenaeus vannamei JAK (Lv JAK) protein, the Drosophila melanogaster hopscotch protein, and the Homo sapiens JAK2 protein, respectively. The mRNA of Sp JAK showed high expression in the brain and nerve but low expression in the hemocyte and muscle. Moreover, the expression of Sp JAK was significantly upregulated by stimulation with mud crab reovirus (MCRV), poly(I:C), and Vibrio parahaemolyticus. Sp JAK significantly activated the STAT of S. paramamosain (Sp STAT) to translocate to the nucleus of Drosophila Schneider 2 cells. Sp JAK significantly enhanced the activity of the promoter of the WSSV wsv069 gene that was activated significantly by Sp STAT by acting on the STAT-binding DNA motif. These results suggest that Sp JAK activates the JAK/STAT pathway. Furthermore, silencing Sp JAK in vivo resulted in the high mortality rate of MCRV-infected mud crabs and increased the viral load in tissues. Hence, Sp JAK could play an important role in defense against MCRV in mud crab. • The full length of Janus kinase gene from Scylla paramamosain (Sp JAK) was cloned and characterized. • The expression of Sp JAK was significantly upregulated by stimulation with mud crab reovirus (MCRV), poly(I:C). • Sp JAK significantly activated Sp STAT to translocate to the nucleus of S2 cells. • Sp JAK could activates the JAK/STAT pathway. • Sp JAK could play an important role in defense against MCRV in mud crab. [ABSTRACT FROM AUTHOR]

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), Sp ccCTL. The cDNA sequence of Sp ccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. Sp ccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant Sp ccCTL (r Sp ccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with r Sp ccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of Sp ccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that Sp ccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that r Sp ccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing Sp ccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that Sp ccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that Sp ccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs. • Sp ccCTL is a novel C-type lectin protein with a coiled-coil domain. • Sp ccCTL recognizes MCRV by binding viral capsid protein VP11. • Sp ccCTL regulates the expression of certain AMPs. • Sp ccCTL acts as a potential PRR participate in anti-MCRV immunity. [ABSTRACT FROM AUTHOR]

Mud crab ( Scylla paramamosain ) is an economically important marine cultured species in China's coastal area. Mud crab reovirus (MCRV) is the most important pathogen of mud crab, resulting in large economic losses in crab farming. In this paper, next-generation sequencing technology and bioinformatics analysis are used to study transcriptome differences between MCRV-infected mud crab and normal control. A total of 104.3 million clean reads were obtained, including 52.7 million and 51.6 million clean reads from MCRV-infected (CA) and controlled (HA) mud crabs respectively. 81,901, 70,059 and 67,279 unigenes were gained respectively from HA reads, CA reads and HA&CA reads. A total of 32,547 unigenes from HA&CA reads called All-Unigenes were matched to at least one database among Nr, Nt, Swiss-prot, COG, GO and KEGG databases. Among these, 13,039, 20,260 and 11,866 unigenes belonged to the 3, 258 and 25 categories of GO, KEGG pathway, and COG databases, respectively. Solexa/Illumina's DGE platform was also used, and about 13,856 differentially expressed genes (DEGs), including 4444 significantly upregulated and 9412 downregulated DEGs were detected in diseased crabs compared with the control. KEGG pathway analysis revealed that DEGs were obviously enriched in the pathways related to different diseases or infections. This transcriptome analysis provided valuable information on gene functions associated with the response to MCRV in mud crab, as well as detail information for identifying novel genes in the absence of the mud crab genome database. [ABSTRACT FROM AUTHOR]

A Dicer2 gene from Scylla paramamosain , named SpDicer2 , was cloned and characterized. The full length of SpDicer2 mRNA contains a 121 bp 5'untranslated region (UTR), an open reading frame (ORF) of 4518 bp and a 3′ UTR of 850 bp. The Sp Dicer2 protein contains seven characteristic Dicer domains and showed 34%–65% identity and 54%–79% similarity to other Dicer protein domains, respectively. The mRNA of SpDicer2 was high expressed in hemocytes, intestine and gill and low expressed in the eyestalk and muscle. Moreover, expression of SpDicer2 was significantly responsive to challenges by mud crab reovirus (MCRV), Poly(I:C), LPS, Staphylococcus aureus and Vibrio parahaemolyticus. Sp Dicer2 was dispersedly presented in the cytoplasm except for a small amount in the nucleus. Sp Dicer2 could activate Sp STAT to translocate from the cytoplasm to the nucleus, and significantly increase the transcription activity of the wsv069 promoter, suggesting that Sp Dicer2 activated the JAK/STAT pathway. Furthermore, silencing of SpDicer2 in vivo increased the mortality of MCRV infected mud crab and the viral load in tissues and down-regulated the expression of multiple components of Toll, IMD and JAK-STAT pathways and almost all the examined immune effector genes. These results suggested that Sp Dicer2 could play an important role in defense against MCRV via activating the JAK/STAT signaling pathways in mud crab. • The full length of a Dicer2 gene from Scylla paramamosain (SpDicer2) was cloned and characterized. • The expression of Sp Dicer2 was significantly upregulated by stimulation with mud crab reovirus (MCRV), poly(I:C) and LPS. • Sp Dicer2 could activate the JAK/STAT pathway in S. paramamosain. • Sp Dicer2 could play an important role in defense against MCRV in mud crab. [ABSTRACT FROM AUTHOR]