Engineering a replication-incompetent viral vector for the delivery of therapeutic RNA in crustaceans.

Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, Macrobrachium rosenbergii nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV(ΔRdRp) carrying the GFP RNA cargo resulting in MrNV(ΔRdRp)-GFP. Upon incorporating MrNV(ΔRdRp)-GFP in the diet of the marine Pacific white shrimp (Penaeus vannamei), MrNV(ΔRdRp) particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within 8 days postadministration of the viral vector, which indicated a lack of MrNV(ΔRdRp)-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.

Identification of a Novel Solinvivirus with Nuclear Localization Associated with Mass Mortalities in Cultured Whiteleg Shrimp (Penaeus vannamei).

The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family Solinviviridae. The novel virus has been tentatively termed Penaeus vannamei solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other (Nylan deria fulva virus 1) solinvivirus. A unique feature of this virus that differs from other members of the Solinviviridae is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family Solinviviridae.

Current status of infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) in the Peruvian and Ecuadorian shrimp industry.

Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3-100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.

Experimental reproduction of White Feces Syndrome in whiteleg shrimp, Penaeus vannamei.

White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp (Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.

A Hint of Primitive Mucosal Immunity in Shrimp through Marsupenaeus japonicus Gill C-Type Lectin.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is Marsupenaeus japonicus gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from M. japonicus displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and Vibrio counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus.

Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub-populations. In this study, we separated hemocytes into two sub-populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May-Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA-seq analysis by Miseq, which revealed 16 differentially-expressed transcripts between the two sub-populations. In the upper-layer, the highly expressed transcripts that were homologous to immune-related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower-layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower-layer also provides insight on the mechanism of the anti-microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper-layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers.

Extracellular trap formation in kuruma shrimp (Marsupenaeus japonicus) hemocytes is coupled with c-type lysozyme.

In invertebrates, hemocytes play an important role in immune responses. Recently, a novel form of innate immune mechanism called extracellular traps (ETs) was identified in shrimps, where DNA and antimicrobial peptides form complex structure to entrap the invading microbes. In this study, we detected the formation of ETs from hemocytes of kuruma shrimp in response to various stimulations, including phorbol myristate acetate (PMA), lipopolysaccharide (LPS), peptidoglycan (PGN) and Escherichia coli. E. coli cells were also found to be trapped by ET fibers. Fluorescence imaging revealed that c-type lysozyme proteins were released around the ET complex after E. coli stimulation, suggesting the presence of a coupled antimicrobial immune response involving ET formation and AMP release.

Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii.

White Spot Syndrome Virus (WSSV) remains the most widespread and devastating infectious agent that hit the shrimp aquaculture industry worldwide. To date, there are no known effective strategies yet to combat WSSV infection. Hence, functional studies on genes critical for viral infection is essential in elucidating shrimp-virus interaction. Here we report the function of a gene from WSSV coding for a non-structural protein, VP9, utilizing RNA interference. Silencing of VP9 gene also effectively suppressed other gene region in the WSSV genome (wsv168 gene) as early as day 1 post infection (dpi). Three set-ups using Macrobrachium rosenbergii shrimp were prepared for treatment using VP9-dsRNA, GFP-dsRNA, and PBS. Each shrimp was challenge with WSSV, and survival rate was recorded. VP9- and GFP-dsRNA injected shrimps showed a significant survival rate of 80% and 70%, respectively, in contrast to 0% of the PBS injected shrimps at 25dpi. Re-infection of shrimp survivors using a higher viral titer concentration, concurrent with the infection of new shrimp samples for the PBS control group, resulted in a significant 67% survival rate for VP9-dsRNA compared to 0% with that of GFP-dsRNA and PBS group. Challenge test on two more species, Penaeus monodon and Marsupenaeus japonicus, also significantly increased survival after VP9-dsRNA treatment. Our results provided evidence that VP9 gene plays an essential role in WSSV replication and it can be a potent target gene in the development of RNAi therapeutics for shrimps.