A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the Infectious Spleen and Kidney Necrosis Virus.

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

Molecular characterization of lymphocystis disease virus in Indian glass fish: first report from the Andaman Islands.

Here, we report the first detection of lymphocystis disease virus (LCDV) in Indian glass fish in the Andaman Islands, India. Microscopic examination revealed the presence of whitish clusters of nodules on the fish's skin, fins, and eyes. The histopathology of the nodules revealed typical hypertrophied fibroblasts. Molecular characterization of the major capsid protein (MCP) gene of the virus showed a significant resemblance to known LCDV sequences from Korea and Iran, with 98.92% and 97.85% sequence identity, respectively. Phylogenetic analysis confirmed that the MCP gene sequence of the virus belonged to genotype V. This study represents the first documented case of LCDV in finfish from the Andaman Islands, emphasizing the necessity for continued monitoring and research on the health of aquatic species in this fragile ecosystem.

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.

Development and comparison of qPCR and qLAMP for rapid detection of the decapod iridescent virus 1 (DIV1).

Decapod iridescent virus 1 (DIV1) is a new virus discovered in recent years that infects farmed shrimp. DIV1 is highly infectious and causes substantial economic loss to the aquaculture industry of China. To prevent and control the spread and outbreak of DIV1 in a timely manner, it is necessary to establish an efficient method for DIV1 diagnosis. In this study, quantitative real-time polymerase chain reaction (qPCR) and quantitative real-time loop-mediated isothermal amplification (qLAMP) detection methods were established based on the specific sequence of the viral ATPase gene. The results indicated that the minimum detection limits of qPCR and qLAMP were 1.9 × 101 copies/µL and 1.9 × 102 copies/µL, respectively; the designed primer had good specificity for DIV1 and did not react with 13 other viruses, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), infectious hypodermal and haematopoietic necrosis virus (IHHNV), etc. A total of 43 clinical samples suspected of DIV1 infection were diagnosed by qPCR and qLAMP. Our qPCR demonstrated results consistent with a qPCR assay published previously, and the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of qLAMP were 85.71% and 100%, respectively. This result indicates that qPCR and qLAMP have good accuracy in the detection of DIVI in clinical samples. As established in this study, qPCR and qLAMP combined with a comprehensive comparative analysis can provide effective new solutions for the detection of DIV1.

Infectious spleen and kidney necrosis virus-associated large-scale mortality in farmed giant gourami, Osphronemus goramy, in India.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

Development of cross-priming amplification (CPA) combined with colorimetric and lateral flow dipstick visualization for scale drop disease virus (SDDV) detection.

Scale drop disease virus (SDDV) is one of the most important pathogens that causes scale drop disease (SDD) in Asian sea bass (Lates calcarifer). The outbreaks of this disease are one of the factors causing substantial losses in Asian sea bass aquaculture. In this study, the uracil-DNA glycosylase (UDG)-supplemented cross-priming amplification (UCPA) combined with a colorimetric detection method using the hydroxynaphthol blue (HNB) and lateral flow dipstick (LFD) for detection of SDDV was developed. The UDG was utilized to prevent carryover contamination, and the CPA reactions can be readily observed by HNB and LFD. The CPA primers and probe were designed to target the major capsid protein (MCP) gene of the SDDV. The optimized UCPA conditions were performed at the temperature of 61°C for 60 min. The UCPA assays demonstrated specificity to SDDV without cross-reaction to other tested viruses including red-spotted grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV) and Lates calcarifer herpes virus (LCHV), and other bacterial species commonly found in aquatic animals. The sensitivity of the UCPA-HNB and UCPA-LFD was 100 viral copies/µl and 10 pg of extracted total DNA, which was 10-fold more sensitive than that of conventional PCR. The UCPA-HNB and UCPA-LFD assays could be used to detect the SDDV infection in all 25 confirmed SDDV-infected fish samples. Therefore, the UCPA coupled with HNB and LFD was rapid, simple and effective and might be applied for diagnosis of SDDV infection.

Detection of scale drop disease virus from non-destructive samples and ectoparasites of Asian sea bass, Lates calcarifer.

Non-destructive sampling methods offer practical advantages to detection and monitoring of viral pathogens in economically important farmed fish and broodstock. Here, we investigated whether blood, mucus and fin can be used as non-lethal sample sources for detection of scale drop disease virus (SDDV) in farmed Asian sea bass, Lates calcarifer. Detection of SDDV was performed in parallel from three non-destructive and seven destructive sample types, collected from both clinically sick fish and subclinical fish obtained from an affected farm. The results showed that SDDV was detectable in all 10 sample types with the percentage ranging from 20% to 100%. Blood was the best non-destructive sample source exhibited by the fact that it yielded 100% SDDV-positive tests from both sick (n = 12, 95% CI: 69.9-99.2) and clinically healthy fish (n = 4, 95% CI: 39.6%-97.4%) and is considered a "sterile" sample. This study also revealed concurrent infection of SDDV and two ectoparasites Lernanthropus sp. and Diplectanum sp., in all affected fish (n = 8, 95% CI: 46.7-99.3) during the disease outbreak. These ectoparasites also tested positive for SDDV by PCR, indicating that they were potential sample sources for PCR-based detection of SDDV and possibly other viruses infecting Asian sea bass.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Complete genome sequence and analysis of a novel lymphocystivirus detected in whitemouth croaker (Micropogonias furnieri): lymphocystis disease virus 4.

A novel lymphocystivirus causing typical signs of lymphocystis virus disease in whitemouth croaker (Micropogonias furnieri) on the coast of Uruguay was detected and described recently. Based on genetic analysis of some partially sequenced core genes, the virus seemed to differ from previously described members of the genus Lymphocystivirus. In this study, using next-generation sequencing, the whole genome of this virus was sequenced and analysed. The complete genome was found to be 211,086 bp in size, containing 148 predicted protein-coding regions, including the 26 core genes that seem to have a homologue in every iridovirus genome sequenced to date. Considering the current species demarcation criteria for the family Iridoviridae (genome organization, G+C content, amino acid sequence similarity, and phylogenetic relatedness of the core genes), the establishment of a novel species ("Lymphocystis disease virus 4") in the genus Lymphocystivirus is suggested.

A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1.

Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA.

First report of megalocytivirus (iridoviridae) in cultured bluegill sunfish, Lepomis macrochirus, in China.

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.

Phylogenomic characterization of red seabream iridovirus from Florida pompano Trachinotus carolinus maricultured in the Caribbean Sea.

Between 2010 and 2016, six mortality events were observed in Florida pompano (Trachinotus carolinus) maricultured in the Dominican Republic. Histopathological examination and conventional PCR confirmed a megalocytivirus (MCV) infection in each case. Subsequently, next-generation sequencing and phylogenomic analyses confirmed that MCV DNA was present in the infected pompano tissue samples from 2010, 2014, and 2016, and each was determined to be red seabream iridovirus (RSIV). Annotation of the RSIV genome sequences identified 121 open reading frames, and BLASTN analysis revealed the highest nucleotide sequence identity (> 99%) to a RSIV clade 1 MCV isolated from a moribund red seabream (Pagrus major) maricultured in Japan. These cases represent the first fully sequenced RSIV genomes detected outside of Asia and are the earliest reports of MCV infections in Florida pompano. This recent geographical expansion of RSIV warrants further attention to determine its potential economic and ecological impact.

Molecular detection and phylogenetic analysis of megalocytivirus in Brazilian ornamental fish.

Megalocytiviruses have a worldwide distribution, causing serious economic loss to the global aquaculture industry. They also present a threat to ornamental fish trade because megalocytiviral infections have unspecified symptoms, making early diagnosis difficult. In this study, 100 ornamental fish from 24 different species were tested by PCR for megalocytivirus, with a 47% positive rate being identified. Phylogenetic reconstruction, based on the major capsid protein (MCP) gene, clustered all Brazilian samples into a single clade, showing identity values ranging from 99% to 100% when compared to each other. This is the first report of megalocytivirus infection in some ornamental fish species in Brazil.

Complete genome sequence and phylogenetic analysis of megalocytivirus RSIV-Ku: A natural recombination infectious spleen and kidney necrosis virus.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981-66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.

Detection and quantification of shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR.

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R2 = 0.998) in the range of 4 × 109 to 4 × 100 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei, Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (µg DNA)-1. Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

Genomic characterization of a novel iridovirus from redclaw crayfish Cherax quadricarinatus: evidence for a new genus within the family Iridoviridae.

A novel iridovirus, Cherax quadricarinatus iridovirus (CQIV), was identified from diseased C. quadricarinatus in 2014. This virus is considered as a new threat to crustacean aquaculture because it is lethal to both peneaid shrimp and crayfish. Here, we determined the complete genome sequence of CQIV. The double-stranded DNA genome is 165 695 bp in length with a G+C content of 34.6 %. A total of 178 open reading frames (ORFs) have been predicted, encoding hypothetical proteins ranging from 50 to 1327 amino acids. Forty-seven of these exhibit similarities to proteins of known functions. Phylogenetic analysis based on multiple alignments of conserved proteins shows that CQIV clusters with the members of the family Iridoviridae, but is placed in a distinct clade from all the five known genera. It indicates that CQIV may represent a new genus in the family Iridoviridae, for which we propose the name Cheraxvirus based on the host organism.

ICTV Virus Taxonomy Profile: Iridoviridae.

The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

UNLABELLED: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.