Mycolicibacterium cyprinidarum sp. nov., a rapidly growing species isolated from diseased koi carp, Cyprinus carpio.

A rapidly growing nontuberculous mycobacterium was isolated from diseased koi carp in Niigata, Japan, which was identified as representing a novel Mycolicibacterium species through whole genome sequence analysis. The bacterial isolates (NGTWS0302, NGTWS1803T and NGTWSNA01) were found to belong to the genus Mycolicibacterium through phylogenetic analysis using whole genome sequences of mycobacteria species. The bacterial colony was smooth, moist and non-chromogenic on 1% Ogawa medium at 30 °C. In biochemical characteristic tests, the bacterial isolates showed positive reactions for catalase activity, Tween 80 hydrolysis and tellurite reduction. The isolates were sensitive to 2-4 µg ml-1 ampicillin, kanamycin and rifampicin. Based on these results, we propose a novel Mycolicibacterium species, Mycolicibacterium cyprinidarum sp. nov. The type strain is NGTWS1803T (=JCM 35117T=ATCC TSD-289T).

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Rapid Apta-Chromogenic Detection Method for Nitrofuran Metabolite Determination.

Nitrofuran (NF) contamination in food products is a global problem resulting in the banned utilization and importation of nitrofuran contaminated products. A novel chromogenic detection method using a specific DNA aptamer with high affinity and specificity to nitrofurans was developed. Single-stranded DNA aptamers specific to nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), were isolated using magnetic bead-SELEX. The colorimetric detection of nitrofurans using gold nanoparticles (AuNPs) exhibited an AOZ detection range of 0.01-0.06 ppb with a limit of detection (LOD) of 0.03 ppb. At the same time, this system could detect AMOZ and AHD at a range of 0.06 ppb and 10 ppb, respectively. The fast nitrofuran extraction method was optimized for food, such as fish tissues and honey, adjusted to be completed within 3-6 h. This novel apta-chromogenic detection method could detect NF metabolites with a sensitivity below the minimum required performance limit (MPRL). This analysis will be valuable for screening, with a shortened time of detection for aquaculture products such as shrimp and fish muscle tissues.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model.

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6-24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.

Type I interferon induced by polyinosinic-polycytidylic acid does not contribute to the efficacy of a formalin-killed cell vaccine against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus).

Polyinosinic-polycytidylic acid (poly I:C) is a type of pathogen-associated molecular pattern that can strongly induce the expression of type I interferon (I-IFN). Our previous study has demonstrated that the combination of poly I:C with a recombinant protein antigen not only stimulated the expression of I-IFN but also conferred protection against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus). In this study, our aim was to develop a better immunogenic and protective fish vaccine, for which we intraperitoneally coinjected P. olivaceus with poly I:C and formalin-killed cells (FKCs) of E. piscicida and compared the efficiency of protection against E. piscicida infection with that of FKC vaccine alone. Results showed that the expression levels of I-IFN, IFN-γ, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and the interferon-stimulated genes (ISGs) ISG15 and Mx were significantly increased in the spleen of fish inoculated with poly I:C + FKC. The results of ELISA showed that the levels of specific serum antibodies in the FKC and FKC + poly I:C groups were gradually increased until 28 days postvaccination and were significantly higher than those in the PBS and poly I:C groups. At 3 weeks after vaccination in the challenge test, the respective cumulative mortality rates of fish in the PBS, FKC, poly I:C, and poly I:C + FKC groups were 46.7%, 20.0%, 33.3%, and 13.3% under low-concentration challenge and 93.3%, 46.7%, 78.6%, and 53.3% under high-concentration challenge. This study showed that poly I:C may not provide an effective adjuvant effect with FKC vaccine for intracellular bacterial infections.

Genome sequence of Chionoecetes opilio bacilliform virus, a nimavirus infecting the snow crab Chionoecetes opilio.

Nimaviridae (class Naldaviricetes) is a family of double-stranded DNA viruses infecting crustaceans, with the only officially recognized representative being white spot syndrome virus (WSSV). Chionoecetes opilio bacilliform virus (CoBV) was isolated as the causative agent of milky hemolymph disease in the snow crab Chionoecetes opilio, an economically important crustacean in the northwestern Pacific. Here, we present the complete genome sequence of CoBV and show that it is unambiguously a nimavirus. The CoBV genome is a 240-kb circular DNA molecule with 40% GC content that encodes 105 proteins, including 76 WSSV orthologs. Phylogenetic analysis based on eight naldaviral core genes established that CoBV is a member of the family Nimaviridae. The availability of the CoBV genome sequence provides a deeper understanding of CoBV pathogenicity and nimavirus evolution.

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/µg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex).

Lactococcosis, caused by members of the genus Lactococcus, represents a devastating disease inducing mass mortalities and economic losses in many fish species worldwide. The present work aimed to compare the whole genome sequences of three different serotypes of Lactococcus garvieae isolated from diseased cultured striped jack (Pseudocaranx dentex) in Ehime prefecture, Japan. The three serotypes showed different virulence in the challenge test using Japanese amberjack (Seriola quinqueradiata). The genome sequencing revealed that two of the strains (serotype I and serotype III) were identified as L. garvieae, while the third strain (serotype II) was identified as L. formosensis. The chromosome sizes of the three serotypes ranged from 1.9 to 2.0 Mb; the GC content ranges were 38.2 to 38.9%; and the numbers of predicted protein-coding sequences (CDSs) were from 1922 to 1959. Only the serotype II harbours two plasmids, sizes of around 14 kb and 9 kb. The detected virulence factors varied among the different serotypes with some shared factors like adherence, anti-phagocytosis, secretion system, toxin (haemolysin), serum resistance, antimicrobial resistance and others. The genomes also contained factors responsible for resistance to toxic compounds. The genome of the serotype III tended to encode more prophage regions than the other serotypes.

Genomic characterization and identification of virulence-related genes in Vibrio nigripulchritudo isolated from white leg shrimp Penaeus vannamei.

Vibrio nigripulchritudo causes vibriosis in penaeid shrimps. Here, we used Illumina and Nanopore sequencing technologies to sequence the genomes of three of its strains (TUMSAT-V. nig1, TUMSAT-V. nig2, and TUMSAT-V. nig3) to explore opportunities for disease management. Putative virulence factors and mobile genetic elements were detected while evaluating the phylogenetic relationship of each isolated strain. The genomes consisted of two circular chromosomes (I and II) plus one or two plasmids. The size of chromosome I ranged from 4.02 to 4.07 Mb with an average GC content of 46%, while the number of predicted CDSs ranged from 3563 to 3644. The size of chromosome II ranged from 2.16 to 2.18 Mb, with an average GC content of 45.5%, and the number of predicted CDSs ranged from 1970 to 1987. Numerous virulence genes were identified related to adherence, antiphagocytosis, chemotaxis, motility, iron uptake, quorum sensing, secretion systems, and toxins in all three genomes. Higher numbers of prophages and genomic islands found in TUMSAT-V. nig1 suggest that the strain has experienced numerous horizontal gene transfer events. The presence of antimicrobial resistance genes suggests that the strains have multidrug resistance. Comparative genomic analysis showed that all three strains belonged to the same clade.

Identification of a rabbit Ig light chain recombinant protein bound to serum immunoglobulins from different marine fish species.

The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column. The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig-specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig. The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library. A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting. The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting. The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins.

Transcriptome profiling reveals the novel immunometabolism-related genes against WSSV infection from Fenneropenaeus merguiensis.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Effect of feeding different types of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis).

ß-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without ß-glucan; the second and the third group were fed with 2 g kg-1 of ß-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of ß-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with ß-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both ß-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with ß-glucans, especially with ß-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with ß-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing ß-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with ß-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg-1 diet can significantly improve their growth performance and immunity.

Whole-genome analysis of red sea bream iridovirus spread in 2021 in Japan provided epidemiological and viral traits insight.

Red sea bream iridovirus (RSIV) is the pathogen that causes red sea bream iridoviral disease. It causes a huge loss to the Japanese aquaculture industry. In 2021, outbreaks of red sea bream iridovirus occurred in South Japan. This study analysed nine whole-genome sequences of RSIV isolated in Oita and Ehime Prefectures in 2021 using a short-read next-generation sequencer. Nine isolates had highly uniform sequences, and there was no variant depending on locations or host species. Phylogenetic analyses with other reported megalocytivirus isolates showed that RSIV isolated in 2021 was genetically different from RSIV previously isolated in Oita and Ehime Prefectures in 2017-2019. These results suggest that RSIV isolated in Oita and Ehime Prefectures in 2021 might spread from a common ancestor different from the recent one. Additionally, it was found that RSIV isolated in 2021 had sequence mutations on protein-coding sequences that may be involved in viral pathogenicity and infectivity.

Molecular evidence for homologous strains of infectious spleen and kidney necrosis virus (ISKNV) genotype I infecting inland freshwater cultured Asian sea bass (Lates calcarifer) in Thailand.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

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.

Development of single nucleotide polymorphism (SNP) application for detection and genotyping of RSIV-type megalocytiviruses.

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.

Crustacean Genome Exploration Reveals the Evolutionary Origin of White Spot Syndrome Virus.

White spot syndrome virus (WSSV) is a crustacean-infecting, double-stranded DNA virus and is the most serious viral pathogen in the global shrimp industry. WSSV is the sole recognized member of the family Nimaviridae, and the lack of genomic data on other nimaviruses has obscured the evolutionary history of WSSV. Here, we investigated the evolutionary history of WSSV by characterizing WSSV relatives hidden in host genomic data. We surveyed 14 host crustacean genomes and identified five novel nimaviral genomes. Comparative genomic analysis of Nimaviridae identified 28 "core genes" that are ubiquitously conserved in Nimaviridae; unexpected conservation of 13 uncharacterized proteins highlighted yet-unknown essential functions underlying the nimavirus replication cycle. The ancestral Nimaviridae gene set contained five baculoviral per os infectivity factor homologs and a sulfhydryl oxidase homolog, suggesting a shared phylogenetic origin of Nimaviridae and insect-associated double-stranded DNA viruses. Moreover, we show that novel gene acquisition and subsequent amplification reinforced the unique accessory gene repertoire of WSSV. Expansion of unique envelope protein and nonstructural virulence-associated genes may have been the key genomic event that made WSSV such a deadly pathogen.IMPORTANCE WSSV is the deadliest viral pathogen threatening global shrimp aquaculture. The evolutionary history of WSSV has remained a mystery, because few WSSV relatives, or nimaviruses, had been reported. Our aim was to trace the history of WSSV using the genomes of novel nimaviruses hidden in host genome data. We demonstrate that WSSV emerged from a diverse family of crustacean-infecting large DNA viruses. By comparing the genomes of WSSV and its relatives, we show that WSSV possesses an expanded set of unique host-virus interaction-related genes. This extensive gene gain may have been the key genomic event that made WSSV such a deadly pathogen. Moreover, conservation of insect-infecting virus protein homologs suggests a common phylogenetic origin of crustacean-infecting Nimaviridae and other insect-infecting DNA viruses. Our work redefines the previously poorly characterized crustacean virus family and reveals the ancient genomic events that preordained the emergence of a devastating shrimp pathogen.

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells.

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.

Comparative genomics inferred two distinct populations of piscine pathogenic Streptococcus agalactiae, serotype Ia ST7 and serotype III ST283, in Thailand and Vietnam.

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008-2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.