Biological and genomic characterization of the novel bacteriophage vB_VpM-pA2SJ1, which infects Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease.

Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.

Effects of Microplastic Exposure against White Spot Syndrome Virus Infection in Pacific White Shrimp (Penaeus vannamei).

Plastic waste has emerged as a major environmental concern in recent years. As plastic waste discharged into the marine environment, it undergoes a breakdown process, eventually accumulating in aquatic organisms in the form of microplastics (MPs). To date, reduced food intake, nutritional absorption, and impaired immune system are known adverse effects of MPs-exposed aquatic organisms. This study aims to investigate whether MP exposure accelerated white spot syndrome virus (WSSV) infection in Pacific white shrimp (Penaeus vannamei) via laboratory tests. Briefly, experimental shrimp were divided into four groups; WSSV (group 1); MP (group 2); WSSV + MP (group 3); and Control (group 4). No mortality was observed in group 2, group 4, and even in group 1. However, group 3 showed a cumulative mortality of 50% during the experimental period. The PCR assay results showed no WSSV in the other three groups (groups 1, 2, and 4), but the dead and alive shrimp collected from group 3 were confirmed to be infected with the virus. Histopathological examination revealed normal structures in the hepatopancreas, gill, and muscle tissues of group 4, whereas numerous abnormally shaped nuclei were detected in the gill tissue of group 2. Moreover, group 1 showed minor WSSV-related lesions with few basophilic inclusion bodies in the gills, interestingly, group 3 exhibited severe lesions with numerous basophilic inclusion bodies in the gills. In conclusion, this study confirmed the correlation between the viral disease of shrimp and MPs, which can cause significant economic losses to the shrimp aquaculture industry.

Incidence of mud crab reovirus (MCRV) outbreak in polyculture ponds of Andhra Pradesh, south east coast of India.

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.

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses.

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

Identification and Full Characterisation of Two Novel Crustacean Infecting Members of the Family Nudiviridae Provides Support for Two Subfamilies.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

Metabolic responses of whiteleg shrimp to white spot syndrome virus (WSSV).

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies.

The T cell factor, pangolin, from Litopenaeus vannamei play a positive role in the immune responses against white spot syndrome virus infection.

As a downstream interactor of ß-catenin, Pangolin which is the homologous protein of the T cell factor/lymphoid enhancer factor (TCF/LEF) in vertebrates is less understood in the research field of immunity. In this study, two isoforms of Litopenaeus vannamei Pangolin (LvPangolin1 and LvPangolin2) were identified. Phylogenetic tree analysis revealed that all of the Pangolin proteins from invertebrates were represented the same lineage. The mRNA expression profiles of the LvPangolin1 and LvPangolin2 genes differed across different tissues. The expression of LvPangolin1 and the amount of LvPangolin1and LvPangolin2 combined (LvPangolinComb) were significantly increased in the haemocyte, intestine and gill but reduced in the hepatopancreas after white spot syndrome virus (WSSV) challenge. The inhibition of LvPangolin1 but not LvPangolinComb significantly reduced the survival rates of L. vannamei after WSSV infection, while significantly higher WSSV viral loads in both LvPangolin1-inhibited and LvPangolinComb-inhibited L. vannamei were observed. Knockdown of LvPangolin by RNAi could distinctly decrease the expression of antimicrobial peptide (AMP) genes and their related transcription factors. All of these results indicate that LvPangolin plays a positive role in the response to WSSV infection and that this may be mediated through regulating the immune signalling pathways which control the expression of AMPs with antiviral abilities.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.