Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses.

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

Viromes of Freshwater Fish with Lacustrine and Diadromous Life Histories Differ in Composition.

Viruses that infect fish are understudied, yet they provide important evolutionary context to the viruses that infect terrestrial vertebrates. We surveyed gill tissue meta-transcriptomes collected from two species of native freshwater fish from Aotearoa New Zealand-Retropinna retropinna and Gobiomorphus cotidianus. A total of 64 fish were used for gill tissue meta-transcriptomic sequencing, from populations with contrasting life histories-landlocked (i.e., lacustrine) and diadromous-on the South Island and Chatham Islands. We observed that both viral richness and taxonomic diversity were significantly associated with life history and host species, with lacustrine R. retropinna characterised by higher viral alpha diversity than diadromous R. retropinna. Additionally, we observed transcripts of fish viruses from 12 vertebrate host-associated virus families, and phylogenetically placed eight novel RNA viruses and three novel DNA viruses in the Astroviridae, Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae, Totiviridae, Poxviridae, Alloherpesviridae, and Adintoviridae in their evolutionary contexts. These results represent an important survey of the viruses that infect two widespread native fish species in New Zealand, and provide insight useful for future fish virus surveys.

Establishment and Characterization of a Novel Gill Cell Line, LG-1, from Atlantic Lumpfish (Cyclopterus lumpus L.).

The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Isolation of a novel adomavirus from cultured American eels, Anguilla rostrata, with haemorrhagic gill necrosis disease.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.

Effects of ploidy and salmonid alphavirus infection on the skin and gill microbiome of Atlantic salmon (Salmo salar).

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.

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.

Seawater transmission and infection dynamics of pilchard orthomyxovirus (POMV) in Atlantic salmon (Salmo salar).

The Tasmanian salmon industry had remained relatively free of major viral diseases until the emergence of pilchard orthomyxovirus (POMV). Originally isolated from wild pilchards, POMV is of concern to the industry as it can cause high mortality in farmed salmon (Salmo salar). Field observations suggest the virus can spread from pen to pen and between farms, but evidence of passive transmission in sea water was unclear. Our aim was to establish whether direct contact between infected and naïve fish was required for transmission, and to examine viral infection dynamics. Atlantic salmon post-smolts were challenged with POMV by either direct exposure via cohabitation or indirect exposure via virus-contaminated sea water. POMV was transmissible in sea water and direct contact between fish was not required for infection. Head kidney and heart presented the highest viral loads in early stages of infection. POMV survivors presented low viral loads in most tissues, but these remained relatively high in gills. A consistent feature was the infiltration of viral-infected melanomacrophages in different tissues, suggesting an important role of these in the immune response to POMV. Understanding POMV transmission and host-pathogen interactions is key for the development of improved surveillance tools, transmission models and ultimately for disease prevention.

First Evidence of Carp Edema Virus Infection of Koi Cyprinus carpio in Chiang Mai Province, Thailand.

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense.

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3).

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/µl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass, Dicentrarchus labrax.

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors.

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Early viral uptake and host-associated immune response in the tissues of seven-band grouper following a bath challenge with nervous necrosis virus.

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.

Effects of freshwater crayfish on influenza A virus persistence in water.

Several investigations have recently assessed the ability of some aquatic invertebrates to act as tools for avian influenza A virus (IAV) surveillance as well as their potential role(s) in IAV ecology. Because of this, as well as the high IAV seroprevalence rates noted in select mesocarnivores that commonly inhabit aquatic and semi-aquatic habitats, we evaluated the effects that freshwater crayfish have on IAV in water at three dose levels and monitored for the presence of IAV in crayfish tissues (gill and green gland) and haemolymph at multiple time points. At relatively high, medium and low (approximately 104 , 103 and 102  EID50 /ml, respectively) doses, mesocosms containing crayfish (Orconectes sp.) had less detectable IAV RNA present when final water samples were assayed (9 days post-contact [DPC]). In general, containers without crayfish present had nearly three-fold greater quantities of viral RNA at 9 DPC. A varying number of RNA positive samples were detected for the three crayfish sample types collected. Gill tissue produced the largest number of positive non-water samples (n = 26), with the highest quantities detected from crayfish sampled on 1 and 4 DPC (103.5  EID50 equivalent/ml). On a few occasions, gill (n = 8) and haemolymph samples (n = 1) produced higher quantities of viral RNA than their respective water samples or water samples collected 1-2 DPC earlier, but these differences were typically minor. Based upon water samples, statistical models indicated that the interaction of dose and crayfish exposure days explained most of the variation in these data. Future efforts should address if crayfish exposed to IAV-laden water have the capacity to successfully transmit IAVs to mammals and birds which frequently prey upon them.

First detection of koi herpesvirus and carp oedema virus in Iraq associated with a mass mortality in common carp (Cyprinus carpio).

At the end of October 2018, a mass fish mortality occurred in Iraq, involving thousands of tons of cultured and wild common carp (Cyprinus carpio) along Euphrates and Tigris rivers. Fish were found dead or moribund along rivers coasts, showing lethargy, dyspnoea and flared gills. At necropsy, discoloration patches were noticed on the gills. Wet preparations showed rare metacercariae and Dactylogyrus spp. Samples were subjected to bacteriological tests and virological investigation through real-time PCR and nested PCR. Both were positive for koi herpesvirus (KHV) and carp oedema virus. Results obtained were confirmed by the OIE reference laboratory of KHV disease (KHVD) at Cefas (UK) and by sequence analysis. This is the first report on the detection of both viruses in Iraq.

A Comparison of Nonlethal and Destructive Methods for Broad-Based Infectious Agent Screening of Chinook Salmon Using High-Throughput qPCR.

Molecular tools, such as high-throughput quantitative polymerase chain reaction (HT-qPCR), are useful for monitoring multiple infectious agents in wild animal populations (i.e., broad-based screening). If destructive tissue samples cannot be obtained due to experimental design requirements (e.g., bio-telemetry; holding with repeated biopsy) or the conservation status of host species, then nonlethally sampled tissues can be substituted. However, infection profiles have been found to differ between nonlethally and destructively sampled tissues. We present a comparative analysis of nonlethal (gill and blood) and destructive (pool of internal and external tissue) approaches for broad-based infectious agent screening of adult Chinook Salmon Oncorhynchus tshawytscha. Of a possible 47 agents, 16 were detected overall by nonlethal and destructive methods. Our results indicated moderate differences in infection profiles among tissues, with limitations of each tissue type dependent on the ecology of each agent. The gill was the most comprehensive screening tissue, as more infectious agents were detected overall in gill (n = 16) than in blood (n = 12) or multi-tissue pools (n = 15). The agreement in the estimated agent prevalence between tissue types ranged from poor to excellent, while overall agent community structure (the combined prevalence of all agents) showed low agreement between tissue types. Two agents occurred at 100% prevalence in all tissue types. Nine agents, including types of bacteria and gill parasites, were more prevalent in gill than in blood, while five agents, including one virus and several microparasites, were more prevalent in blood. Future studies should pair microscopy and histopathology with HT-qPCR to better characterize host health and disease development relative to molecular detection of agents across tissue types.

TaqMan real-time and conventional nested PCR tests specific to yellow head virus genotype 7 (YHV7) identified in giant tiger shrimp in Australia.

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥108) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1-7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia.

Detection of Betanodavirus in experimentally infected European seabass (Dicentrarchus labrax, Linnaeus 1758) using non-lethal sampling methods.

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non-lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50 /ml and 106 TCID50 /ml, respectively), and samples collected 7, 15 and 30 days post-infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non-lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT-qPCR). The presence of virus in non-lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular-infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme-linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus-specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.