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Abstract Aquatic viruses are major threats to global aquacultural productivity. While conventional diagnostic methods for disease investigation are laborious, time‐consuming, and often involve the sacrifice of animals, environmental DNA and RNA (eDNA/eRNA) tools have the potential in being non‐invasive alternatives for the effective and early detection of various pathogens simultaneously. In this study, three seawater filtration methods—Sterivex syringe filtration, centrifugal ultrafiltration, and vacuum pump filtration with iron flocculation—were assessed for the recovery rates in co‐detecting fish virus eDNA/eRNA from natural seawater that was spiked with fish red seabream iridovirus (RSIV, DNA virus) and nervous necrosis virus (NNV, RNA virus). The centrifugal ultrafiltration method was the most effective for the capture of small‐sized viruses like NNV with a recovery rate of 63.23%, while the method of vacuum pump filtration with iron flocculation and chloroform disintegration of filter membranes had the highest RSIV recovery rate of 32.61%. We also optimized both automated and manual nucleic acid extraction methods and found comparable eDNA/eRNA extraction efficiencies. Our findings from the systematic comparison of seawater filtration and extraction methods suggest that each seawater filtration/nucleic acid extraction method can cater to different aquatic animal virus surveillance and disease investigation scenarios. These highlight the potential of virus eDNA/eRNA approaches for advancing the field of disease ecology and safeguarding aquatic animal health.

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major , and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N -glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida : E. piscicida and Streptococcus iniae : S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ , and the leukocytes of red sea bream also induced apoptosis and viability reduction. • A cathepsin Z (CTSZ) was identified from red sea bream (Pagrus major). • PmCTSZ share high conserved cysteine proteases active site and three N-glycosylation sites with that of other species. • PmCTSZ mRNA was highly expressed in spleen and gill from healthy condition. • RPmCTSZ inhibited bacterial growth and induced leukocyte apoptosis. [ABSTRACT FROM AUTHOR]

Abstract Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6–53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida , Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection. Highlights • We cloned and identified peptidoglycan recognition protein 2 (PGRP2) gene from rock bream. • RbPGRP2 gene was highly expressed in liver from healthy rock bream. • RbPGRP2 was upregulated in the fish infected with several pathogens. • rRbPGRP2 was interacteds with bacteria and inhibited the growth of bacteria in the presence of Zn2+. [ABSTRACT FROM AUTHOR]

The Megalocytivirus genus includes three genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV), and has caused mass mortalities in various marine and freshwater fish species in East and Southeast Asia. Of the three genotypes, TRBIV-like megalocytivirus is not included in the World Organization for Animal Health (OIE)-reportable virus list because of its geographic restriction and narrow host range. In 2017, 39 cases of suspected iridovirus infection were isolated from fingerlings of giant sea perch (Lates calcarifer) cultured in southern Taiwan during megalocytivirus epizootics. Polymerase chain reaction (PCR) with different specific primer sets was undertaken to identify the causative agent. Our results revealed that 35 out of the 39 giant sea perch iridovirus (GSPIV) isolates were TRBIV-like megalocytiviruses. To further evaluate the genetic variation, the nucleotide sequences of major capsid protein (MCP) gene (1348 bp) from 12 of the 35 TRBIV-like megalocytivirus isolates were compared to those of other known. High nucleotide sequence identity showed that these 12 TRBIV-like GSPIV isolates are the same species. Phylogenetic analysis based on the MCP gene demonstrated that these 12 isolates belong to the clade II of TRBIV megalocytiviruses, and are distinct from RSIV and ISKNV. In conclusion, the GSPIV isolates belonging to TRBIV clade II megalocytiviruses have been introduced into Taiwan and caused a severe impact on the giant sea perch aquaculture industry.

Red Sea bream iridovirus (RSIV) was initially isolated from marine fish, which belongs to Megalocytivirus , Iridoviridae . It can cause great economic losses in fish culture with high morbidity and mortality. In the present study, the pathogenicity and immune response associated with a RSIV genotype megalocytivirus infection were determined in mandarin fish ( Siniperca chuatsi ). Fish challenged showed typical clinical signs of iridovirus infection, including acute haemorrhages and enlarged visceral organs. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the stomach, spleen, kidney and liver of the diseased fish. Blood cells counting and phagocytic assay indicated that the numbers of the red and white blood cells in the peripheral blood of infected fish increased significantly compared to the control group and the phagocytic percentage of leukocytes peaked at day 6 post infection. Quantitive real-time PCR (qRT-PCR) was also undertaken to analyse the host defensive response in mandarin fish challenged with RSIV. The expression level of ten genes including interferon-related factors (IRFs) IRF1 and IRF7 , Mx , Viperin , JAK1 , STAT1 , TCRα , TNFα , IL-1β and IL-8 during experimental infection were monitored at different point of time in liver, spleen and head kidney. Results revealed varying expression profiles and clear transcriptional activation of these immune related genes in different tissues, which will contribute to better understand the pathogenesis and host defensive system in iridovirus invasion. [ABSTRACT FROM AUTHOR]

A novel grouper immune gene, EcVig was identified in orange-spotted grouper ( Epinephelus coioides ). We recently determined that EcVig expression can be induced by infection with nervous necrosis virus (NNV, an RNA virus), whereas NNV replication may be suppressed when EcVig was overexpressed. Although EcVig appeared to be involved in grouper antiviral activity, its immune effects have not been well characterized. In the present study, two PAMPs (pathogen-associated molecular patterns; lipopolysaccharides [LPS] and synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid [poly(I:C)]), as well as fish DNA virus (red sea bream iridovirus, RSIV; grouper iridovirus, GIV), were used to study EcVig responses in orange-spotted grouper. In addition, groupers were given recombinant type I interferon to determine whether EcVig expression was induced. Poly(I:C) rapidly induced substantial expression of EcVig, whereas LPS stimulation did not appear to have any effect in grouper intestine. Expression levels of total EcVig and other IFN-stimulated genes (ISGs) were all significantly increased after RSIV and GIV infection. Furthermore, stimulation of recombinant type I IFN also increased EcVig expression. We conclude that EcVig may be a novel IFN-stimulated gene that demonstrates an antiviral immune response. [ABSTRACT FROM AUTHOR]

Abstract: Megalocytivirus is a major viral agent that affects fresh and marine fish, and causes serious economic losses in aquaculture in Asian countries. As filter-feeding organisms, bivalve molluscs may act as viral transmitters following the capture of a released virus into seawater from infected fish. We identified megalocytivirus, rock bream iridovirus (RBIV) type in Pacific oysters (Crassostrea gigas) and blue mussels (Mytilus edulis) inhabiting the areas around rock bream farms in Korea. In artificial viral-depuration analysis, the concentration of iridovirus Sachun (IVS) particles, which is a major RBIV strain in Korea, decreased more rapidly in seawater than in the digestive glands of mussels. Cohabitation of mussels with IVS-infected rock bream (Oplegnathus fasciatus) resulted in the presence of viral particles in the gill, mantle and digestive gland of the mussels. In addition, the virus was retained in the digestive gland for 28days, regardless of water temperature. Infection with IVS derived from the digestive gland of cohabitating mussels also led to 100% cumulative mortality in rock breams. In this study, we investigated megalocytivirus carried by bivalve molluscs inhabiting the vicinity of rock bream farms, and revealed the potential risk posed by these viral particles by modelling the cohabitation of mussels with RBIV-infected rock bream. [Copyright &y& Elsevier]

The Megalocytivirus genus includes three genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV), and has caused mass mortalities in various marine and freshwater fish species in East and Southeast Asia. Of the three genotypes, TRBIV-like megalocytivirus is not included in the World Organization for Animal Health (OIE)-reportable virus list because of its geographic restriction and narrow host range. In 2017, 39 cases of suspected iridovirus infection were isolated from fingerlings of giant sea perch (Lates calcarifer) cultured in southern Taiwan during megalocytivirus epizootics. Polymerase chain reaction (PCR) with different specific primer sets was undertaken to identify the causative agent. Our results revealed that 35 out of the 39 giant sea perch iridovirus (GSPIV) isolates were TRBIV-like megalocytiviruses. To further evaluate the genetic variation, the nucleotide sequences of major capsid protein (MCP) gene (1348 bp) from 12 of the 35 TRBIV-like megalocytivirus isolates were compared to those of other known. High nucleotide sequence identity showed that these 12 TRBIV-like GSPIV isolates are the same species. Phylogenetic analysis based on the MCP gene demonstrated that these 12 isolates belong to the clade II of TRBIV megalocytiviruses, and are distinct from RSIV and ISKNV. In conclusion, the GSPIV isolates belonging to TRBIV clade II megalocytiviruses have been introduced into Taiwan and caused a severe impact on the giant sea perch aquaculture industry. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) is a member of the pentraxin family and is an acute-phase response to plasma protein; its level in blood increases rapidly in response to trauma, inflammation, and infection. In the present study, we analysed the molecular characteristics of the Oplegnathus fasciatus CRP (RbCRP) gene sequence using multiple alignments and phylogenetic analyses of the deduced amino acids. In addition, we also examined RbCRP gene expression in rock bream infected with the pathogens Edwardsiella piscicida ( E. piscicida ), Streptococcus iniae ( S. iniae ) or red sea bream iridovirus (RSIV) and in healthy rock bream individuals. In healthy individuals, RbCRP was ubiquitously expressed in all 14 tested tissues, mainly in the trunk kidney and head kidney. Expression of RbCRP was notably upregulated in the spleen and whole kidney after RSIV infection. This study can provide basic data on the innate immune system of the rock bream to viral and microbial infections., (© 2019 The Author(s).)

The interferon-related developmental regulator 1 (IFRD1) protein is expected to play a role in the regulation of inflammatory responses in adult mice, since it is known to repress transcription of NF-κB in myoblasts that regenerate skeletal muscle after traumatic injury Micheli et al., 2011. The IFRD2 gene is expressed in many tissues including skeletal muscle, kidney, heart, brain, lung, placenta and liver in adult humans and is highly expressed in adult human skeletal muscle and heart. In mice, interferon-related developmental regulator 2 (IFRD2) may be associated with early haematopoiesis after gastrulation and in the hepatic primordium Buanne et al., 1998. In this study, we analysed the molecular characteristics of the IFRD2 gene identified from Pagrus major (PmIFRD2) and performed multiple alignments and phylogenetic analyses of the protein sequence. In addition, we examined the expression pattern of IFRD2 in healthy red sea bream tissues and the temporal expression pattern after challenging with various pathogens [ Edwardsiella piscicida ( E. piscicida ), Streptococcus iniae ( S. iniae ) and red sea bream iridovirus (RSIV)]. This study characterises the non-specific immune response of the red sea bream after viral and microbial infections.