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Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×10 5 copies/μL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period., Competing Interests: Declaration of Competing Interest The author declares no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel "mucus sloughing and hemorrhagic septicemia disease" was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R 2 ) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3 %. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Epizootics of Koi herpesvirus (KHV) cause mass mortality in koi carp (Cyprinus rubrofuscus) and common carp (Cyprinus carpio) worldwide. Rapid and accurate virus detection technology is crucial for preventing pathogen spread and minimizing damage. Although several diagnostic assays have been developed for KHV, the analytical and diagnostic performance of the detection methods has not been evaluated. In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/μL and 8.384 copies/μL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectively, and real-time PCR was found to be 100% and 100%, respectively. Therefore, the newly developed CPA-LFA and real-time PCR assays accurately and rapidly detect KHV. CPA-LFA is particularly suitable for point-of-care diagnosis because of its simple diagnostic process, and real-time PCR analysis is most suitable for precise diagnosis because it can detect low viral loads., Competing Interests: Declaration of Competing Interest The author declares no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R 2 = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R 2 = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A serological test for screening of TiLV in Oreochromis niloticus would be useful for the epidemiological investigations. Using polyclonal antisera against TiLV (TiLV-Ab), an indirect enzyme-linked immune sorbent assay (iELISA) was developed for the detection of TiLV antigen in fish tissue and mucus. After a cutoff value was established and antigen and antibody concentrations were optimized, the iELISA's sensitivity and specificity were assessed. We found the ideal dilutions of TiLV-Ab as 1: 4000 and secondary antibody as 1:65,000. High analytical sensitivity and moderate specificity were displayed by the developed iELISA. The Positive and Negative Likelihood Ratio (LR+, LR-) were 1.75 and 0.29, respectively. The estimated Positive and Negative Predictive Values (PPV and NPV) of the test were 76.19% and 65.62%, respectively. The accuracy of the developed iELISA was estimated as 73.28%. An immunological survey was performed using the developed iELISA with samples from the field and 155/195 fishes tested positive, indicating a 79.48% TiLV antigen positives. Among the pooled organs and mucus tested, the highest positive rate of 92.3% (36/39) is observed in mucus compared to other tissues, and least positive rate is found in liver of 46% (18/39). The newly designed iELISA proved sensitive and may be helpful for extensive examinations of TiLV infections and monitoring disease status even from apparently healthy samples using a non-invasive technology by collecting mucus as sample for iELISA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID 50 /ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R 2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (10 0.301 TCID 50 /ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log 10 , 3.94 log 10 and 3.52 log 10 TCID 50 /ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Waterborne viral infections represent a major threat to fish health. For many viruses, understanding the interplay between pathogens, host and environment presents a major hurdle for transmission. Salmonid alphavirus (SAV) can infect and cause pancreas disease (PD) in farmed salmonids in seawater. During infection, SAV is excreted from infected fish to the seawater. We evaluated two types of filters and four different eluents, for concentration of SAV3. One L of seawater was spiked with SAV3, followed by filtration and virus elution from membrane filters. For the negatively charged MF hydrophilic membrane filter (MF-) combined with NucliSENS® lysis buffer the SAV3 recovery was 39.5 ± 1.8 % by RT-ddPCR and 25.9 ± 5.7 % by RT-qPCR. The recovery using the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter (MD+), combined with NucliSENS® lysis buffer was 19.0 ± 0.1 % by RT-ddPCR and 13.3 ± 3.8 % by RT-qPCR. The limits of quantification (LOQ) and detection (LOD) were estimated to be 5.18 × 10 3 and 2.0 × 10 2 SAV3 copies/L of natural seawater, by RT-ddPCR. SAV3 recovery from small volumes of seawater, and the requirement for standard laboratory equipment, suggest the MF-filter combined with NucliSENS® lysis buffer would be a candidate for further validation in experimental trials., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Lates calcarifer herpes virus (LCHV) is a novel virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per μl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Proteins and nucleic acids from ultrasonically ruptured white spot syndrome virus (WSSV) can infect crayfish and cause death as effectively as intact WSSV virions. In this study, ultrasound was used to rupture the virus and the resulting suspension was filtered through a 50 nm membrane. Analysis by PCR and SDS-PAGE showed that both viral genes (VP19, VP26, VP28 and DNA polymerase) and proteins (VP15, VP19, VP26 and VP28) were present in the filtered solution. Electron microscopy showed that there were no intact virions in the filtered solution. When crayfish were injected with the filtered solution or with intact WSSV, the mortality in each group was 100 %. The same result was seen when crayfish were challenged orally with the filtered solution and intact WSSV. The filtered solution of ultrasonically ruptured virus, which contains viral proteins and residual DNA genome, can thus infect the host as effectively as intact virions. When the solution of viral proteins and residual DNA genome was digested with DNase I and then injected into crayfish, the survival rate was 100 %. We also found that, although viral proteins (except VP15) in the solution of ruptured virus were destroyed by treatment with DNase I, DNase I did not destroy the structural proteins of intact virions. A remaining viral protein in the DNase I-treated solution protects the DNA genome from degradation and we concluded that this protein is VP15, which is a DNA-binding protein. Our study highlights the extreme danger in producing vaccines from proteins obtained by ultrasonic rupture of viruses sincethe viral DNA genome is difficult to degrade and, if present, will lead to viral infection., Competing Interests: Declaration of Competing Interest There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the gene policies on sharing data and materials. The authors declare that they have no competing interests. Yongyong Lai declares that he has no conflict of interest. Fei Zhu declares that he has no conflict of interest. Yinglei Xu declares that she has no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, which is characterized by external and internal hemorrhage, is a serious infectious disease affecting grass carp production. Strains of the causative agent, grass carp reovirus (GCRV), are divided into genotypes I, II and III, which are represented by the isolates GCRV-873, GCRV-HZ08 and GCRV-104, respectively. In this study, a real-time reverse-transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed to detect the genotype III grass carp reovirus GCRV-104. The assay was based on the detection of the vp55 gene which encodes the outer fiber protein of the virus. A portable ESE-Quant Tube scanner, with a dimension of 17.4 × 18.8 cm, weighing about 1 kg, and equipped with temperature settings to amplify the DNA isothermally and spectral devices to detect the amplified products using fluorescence, was used to complete the assay. Under the optimal conditions, the assay took approximately 10 min to complete at 37 °C and showed no cross-reactions with other aquatic viruses. Consequently, this rapid real-time RT-RPA assay is a useful method for the simple, rapid and reliable detection of genotype III GCRV strains in resource-limited diagnostic laboratories., Competing Interests: Declaration of Competing Interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nervous necrosis virus (NNV) is a fish virus belonging to family Nodaviridae. In this study, we prepared partially aggregated and monometric NNV particles to determine reproducibility of two different enzyme-linked immunosorbent assays (ELISAs): antigen-immobilized ELISA and sandwich ELISA. Passing ratios of purified NNV particles through ultrafilters with molecular weight cut off (MWCO) of 10 5 , 3 × 10 5 and 10 6 were 0%, 35.2% and 80.3%, respectively, suggesting that purified NNV particles were partially aggregated whereas those in filtrates with MWCO of 3 × 10 5 could be monometric. Both NNV particles were subjected to ELISAs. Reduction ratios of ELISA values by 2-fold dilution of antigens were 50% in sandwich ELISA regardless of aggregation state of NNV particles. In contrast, those in antigen-immobilized ELISA were 42% (partially aggregated NNV) to 43% (monometric NNV), which were lower than the theoretical value (50%). This could be due to changes in aggregation state of NNV particles during dry-immobilization. Sandwich ELISA has excellent reproducibility from five times of experiments, in comparison with antigen-immobilized ELISA. Furthermore, available range of regression lines (R 2  > 0.99) in sandwich ELISA was wider than that in antigen-immobilized ELISA. These results revealed that sandwich ELISA had better quantitativeness, reproducibility and available range of ELISA values than antigen-immobilized ELISA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Scale drop diseases virus (SDDV), a newly characterized virus of farmed Asian sea bass (Lates calcarifer), has been reported in several countries in Southeast Asia. However, no fully validated detection method is publicly available for disease diagnosis and surveillance. Here, we described a newly developed semi-nested PCR (snPCR) method for detection of the virus from field samples. The designed primers targeting a gene encoding ATPase generated amplicons of 738 bp and 412 bp in the first and second step PCR, respectively. The established protocol could detect down to 100 viral copies/μL template and was 100-fold more sensitive than single step PCR. A Specificity test against extracted DNA from ten bacterial pathogens, tissues from viral infected specimens and fish host revealed no cross amplification. The SDDV snPCR method could detect the virus from all clinical samples showing symptoms of scale drop disease (n = 25) and all samples from outbreaks of an unknown disease (n = 6) whereas all clinically healthy fish sea bass (n = 161) and grouper (n = 45) collected from different provinces tested negative. The newly established protocol might be useful for Asian sea bass farming countries to initiate disease diagnosis and surveillance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acipenser iridovirus-European (AcIV-E) is an important pathogen of sturgeons. Two variants differing by single-nucleotide polymorphisms (SNP) in the Major Capsid Protein gene have been described, but without any indication as to their prevalence in farms. To facilitate epidemiological studies, we developed a high-resolution melting (HRM) assay to distinguish between two alleles (var1 and var2) differing by five point substitutions. The HRM assay detected as little as 100 copies of plasmids harboring cloned sequences of var1 and var2, which have melting temperatures (Tm) differing by only 1 °C. The assay was specific of AcIV-E as demonstrated by the absence of signal when testing a related, yet distinct, virus as well as DNA from an AcIV-E-negative sturgeon sample. Experiments with mixtures of two distinct plasmids revealed abnormal melting curve patterns, which showed dips just before the main melting peaks. These dips in the curves were interpreted as the dissociation of heteroduplexes fortuitously created during the PCR step. Screening AciV-E-positive field samples of Russian sturgeons from three farms revealed the presence of var2, based on the Tm. However, for a few samples, the melting curves showed patterns typical of var2 as the dominant viral genome, mixed with another minor variant which proved to be var1. In conclusion, HRM is a simple method to screen for AcIV-E var1 and var2 and can be used on a large scale in Europe to trace these two variants which likely represent two genetic lineages., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl 2 , 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus -1 , -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R 2  = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases., (Copyright © 2018. Published by Elsevier B.V.)

Cyprinid herpesvirus 2 (CyHV-2) infection is a recently discovered viral disease of farmed silver crucian carp (Carassius auratus gibelio) in mainland China that has resulted in massive fish mortality and appears to be spreading cross the world. Various detection methods based on nucleic acids have been developed, but effective immunological diagnostic method has not been reported for CyHV-2. In this study, recombinant ORF92 protein was used as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods. Firstly, the open reading frame of CyHV-2 ORF92 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF92 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot-blot assay. The resulting mAb-1B7 was applied to detect CyHV-2 in infected Ryukin goldfish fin (RyuF-2) cells and fish tissues by western blotting and immunofluorescence assays. The monoclonal antibody could specifically identify CyHV-2 in infected RyuF-2 cells and gill, kidney, and spleen tissues, suggesting it could be applied for CyHV-2 detection in crucian carp., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Grass carp reovirus (GCRV) is the causative agent of a hemorrhagic disease that causes severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Discrimination between wild-type field and vaccine strains of GCRV is crucial for meaningful disease diagnosis and epidemiological investigation, yet current detection methods do not discriminate between these different viruses. This study exploited sequence differences between vaccine viruses and the virulent and field strains in the S6 gene that is present in all GCRV strains to develop a high resolution melting curve assay to differentiate between virus strains. The high resolution melting curve analysis was as analytically sensitive as real-time quantitative-Polymerase Chain Reaction (qPCR) detection and at least 10 times sensitive than the conventional PCR. This one-step assay will facilitate grass carp hemorrhagic disease outbreak responses and control., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Infectious spleen and kidney necrosis virus (ISKNV) has been recognized as the causative agent of the most serious disease in cultured mandarin fish, Siniperca chuatsi, in China. Disease outbreaks have resulted in substantial losses to the aquaculture industry. Currently, reliable laboratory detection and identification methods are available for this virus. However, rapid detection methods applicable for on-site diagnosis of this infectious agent are unavailable. To address this need, a nearly instrument-free, cost-effective and simple detection method was developed and optimized and incorporates cross priming amplification coupled with vertical flow visualization for rapid identification of ISKNV (ISKNV-CPA-VF). Results show that cross circulation amplification targeting the conserved region of the major capsid protein (MCP) regiment of the ISKNV genome had a sensitivity 10 times greater than traditional PCR at 64 °C within 60 min. The optimized concentration of dNTPs and the concentration for Mg 2+ were 1.0 mmol/L and 10 mmol/L, respectively. No cross-reactions with other viruses or bacteria were observed. When combined with the nucleic acid strip detection technology, visual detection of ISKNV amplified products was realized within 3-5 min following amplification. The simplicity and nearly instrument-free method for this ISKNV-CPA-VF assay shows great potential for on-site diagnostics of ISKNV infection in Siniperca chuatsi., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Spring viremia of carp virus (SVCV) is a highly pathogenic agent of several economically important Cyprinidae fish species. Currently, there are no effective vaccines or drugs for this virus, and prevention of the disease mostly relies on prompt diagnosis. Previously, nested RT-PCR and RT-qPCR detection methods based on the glycoprotein gene G have been developed. However, the high genetic diversity of the G gene seriously limits the reliability of those methods. Compared with the G gene, phylogenetic analyses indicate that the nucleoprotein gene N is more conserved. Furthermore, studies in other members of the Rhabdoviridae family reveals that their gene transcription level follows the order N>P>M>G>L, indicating that an N gene based RT-PCR should have higher sensitivity. Therefore, two pairs of primers and two corresponding probes targeting the conserved regions of the N gene were designed. RT-qPCR assays demonstrated all primers and probes could detect phylogenetically distant isolates specifically and efficiently. Moreover, in artificially infected fish, the detected copy numbers of the N gene were much higher than those of the G gene in all tissues, and both the N and G gene copy numbers were highest in the kidney and spleen. Testing in 1100 farm-raised fish also showed that the N-targeting strategy was more reliable than the G-targeting methods. The method developed in this study provides a reliable tool for the rapid diagnosis of SVCV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/μl of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/μl of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In recent years, an epizootic causing severe mortality among Allogynogenetic crucian carp (ACC), designated as haemorrhagic disease of ACC gill, occurred in Yancheng city of Jiangsu province of China. Obvious haemorrhage in the gills of moribund fish and a mortality rate of 100% were observed when ACCs were artificially infected with liver homogenate from diseased fish. A herpes-like virus, with enveloped virions ranged from 170 to 220 nm in diameter, could be observed in the tissues of challenged ACCs by examination with electron microscopy. Specific products representing the polymerase and helicase genes of Cyprinid herpesvirus-2 (CyHV-2) could be amplified from the challenged fish, suggesting that the haemorrhagic disease of ACC gill was caused by infection with CyHV-2. To rapidly diagnose CyHV-2-infected fish, an easy and effective detection assay with loop-mediated isothermal amplification (LAMP) was established. The LAMP assay was more sensitive than conventional PCR and the limit of detection was approximately 100 copies of target DNA. With this LAMP assay, CyHV-2 could be detected in some asymptomatic ACCs from the epidemic area and in eggs from the diseased ACCs, suggesting that CyHV-2 could infect ACCs latently and that the virus may be passed onto offspring by vertical transmission., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cyprinid herpesvirus-3 (CyHV-3) is a highly infectious pathogen that causes fatal disease in common and koi carp Cyprinus carpio L. CyHV-3 detection is usually based on virus propagation or amplification of the viral DNA using the PCR or LAMP techniques. However, due to the limited susceptibility of cells used for propagation, it is not always possible to successfully isolate CyHV-3 even from tissue samples that have high virus titres. All previously described detection methods including PCR-based assays are time consuming, laborious and require specialized equipment. To overcome these limitations, gold nanoparticles (AuNPs) have been explored for direct and sensitive detection of DNA. In this study, a label-free colorimetric nanodiagnostic method for direct detection of unamplified CyHV-3 DNA using gold nanoparticles is introduced. Under appropriate conditions, DNA probes hybridize with their complementary target sequences in the sample DNA, which results in aggregation of the gold nanoparticles and a concomitant colour change from red to blue, whereas test samples with non complementary DNA sequences remain red. In this study, gold nanoparticles were used to develop and evaluate a specific and sensitive hybridization assay for direct and rapid detection of the highly infectious pathogen termed Cyprinid herpesvirus-3., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50-100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 10(2.5)-10(2.85) TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.

The major capsid protein (MCP) is a main structural protein of iridoviruses, and is used as a marker for the identification, differentiation and classification of ranaviruses. In the present study, six monoclonal antibodies (mAbs) against recombinant MCP of grouper iridovirus (GIV) were produced and characterized. All of the six mAbs were of IgG1 isotype. Among the mAbs, GIV-MCP-mAb-21 showed the highest reactivity in ELISA and was used to further characterize the expression of GIV-MCP during viral replication. RT-PCR and Western blot analyses revealed that GIV-MCP is a late gene during GIV infection. By immunofluorescence assay, the presence of GIV-MCP was observed in not only the cytoplasm but also the nucleus of GIV-infected cells, a surprising finding that might indicate additional role of GIV-MCP. In conclusion, the newly established GIV-MCP-mAbs are a valuable tool for GIV diagnostic and future studies on GIV pathogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Goldfish Haematopoietic Necrosis caused by Cyprinid herpesvirus 2 (CyHV-2) is a severe fish disease with high level of mortality. This is the first study on detection of this disease by loop-mediated isothermal amplification (LAMP). A set of six primers targeting terminase gene (accession no. EU349285.1) was determined after a serial of tests. Detection limit was 1.09 × 10(-4)μg/μL, which was superior to conventional PCR and real-time PCR. No cross reaction with 28 other viruses or bacteria commonly found in fish was observed. The application of commercial kit and instrument for the LAMP assay could reduce the risk of cross contamination, which is suitable for detection of infection under field conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Frequent outbreaks of grass carp hemorrhagic disease, caused by grass carp reovirus (GCRV) infection, pose as serious threats to the production of grass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnostic methods have been shown effective, lack of commercial monoclonal antibody against grass carp IgM has impeded the development of any reliable immunoassays in detection of GCRV infection. The present study describes the preparation and screening of monoclonal antibodies against the constant region of grass carp IgM protein, and the development of a Western blot (WB) protocol for the specific detection of antibodies against outer capsid VP7 protein of GCRV that serves as antibody-capture antigen in the immunoassay. In comparison to a conventional RT-PCR method, validity of the WB is further demonstrated by testing on clinical fish serum samples collected from a grass carp farm in Jiangxi Province during disease pandemic in 2011. In conclusion, the WB technique established in this study could be employed for specific serodiagnosis of GCRV infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp., (Copyright © 2013 Elsevier B.V. All rights reserved.)

RTG-P1 is a transgenic fish cell line producing luciferase under the control of the IFN-induced Mx rainbow trout gene promoter. This cell line was used to measure viraemia of Salmonid alphavirus (SAV), the cause of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Two SAV genotype 1 (SAV1) isolates were used in this study, F93-125 (tissue-culture adapted, from Ireland) and 4640 (from a field case in Scotland). The kinetics and magnitude of luciferase activity were monitored versus the time of infection. During a direct infection experiment, the induction of luciferase significantly increased 16- and 4-fold after incubation for 6 days with F93-125 at 15 and 20°C, respectively. Filtration and heat treatment experiments demonstrated that the luciferase induction in RTG-P1 was dependent on viral replication. Unlike many cell lines used in fish viral diagnostic, RTG-P1 is not sensitive to salmonid serum, therefore, viraemia could be successfully monitored on serum collected from fish during a cohabitation challenge with 4640 isolate. A peak of viraemia could be detected 16 days post IP inoculation of the shedders. This novel cost-effective method to measure viraemia does not rely on development of cytopathic effect (CPE) in culture, is compatible with non-lethal blood collections in fish and can be used to assign emerging diseases to a viral aetiology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Three species of viral-derived RNA (vRNA, cRNA and mRNA) are produced during an infectious salmon anaemia virus (ISAV) infection. Conventional real-time RT-PCR (RT-qPCR) targeting ISAV segment 8 provides a very sensitive method for the detection of ISAV RNA, however it does not differentiate between these three individual RNA species. In this study, strand-specific tagged primers have been utilised in the RT reaction to specifically produce cDNA corresponding to each of the 3 viral RNA types produced from ISAV segment 8 for the subsequent detection by real-time PCR. The RNA species-specific assay was successfully used to specifically distinguish synthetic T7-produced RNA transcripts representing the 3 species of ISAV RNA at levels up to approximately 10(5)-fold higher than the other types. In addition, the method was applied to investigate the production of segment 8 RNA in time-course tissue culture experiments performed at optimal (15°C), sub-optimal (20°C) and inadequate (25°C) temperatures for replication or in the presence of a chemical inhibitor to vary the RNA populations and investigate its effectiveness. Variation in RNA production was observed between the optimal and sub-optimal temperatures and in the presence of the chemical inhibitor. Production of all RNA species was completely inhibited at 25°C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Polymerase chain reaction (PCR) assays allow for the rapid and accurate detection of infectious agents through identification of nucleic acid sequences. However, contamination of samples with positive DNA can lead to false-positive results. In this study, positive control plasmids were developed to minimize false-positive reactions due to PCR contamination during detection of SVCV by semi-nested reverse-transcription PCR. An ampicillin resistance gene was truncated by PCR amplification, and the fragments were inserted into pGEM-T Easy vectors; the resulting plasmids were named SVCV chimeric plasmid-1 and chimeric plasmid-2, respectively. Through a series of semi-nested PCRs, the use of SVCV chimeric plasmids-1 and -2 was shown to ensure correct diagnoses, free from PCR contamination. The results of this study show that PCR positive controls can be created without use of viral nucleic acids or pathogen-infected tissues. The technique can be applied to quarantined material and can be used to detect other pathogens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Koi Herpes Virus (KHV) has been classified recently as a member of the Alloherpesviridae within the Herpesvirales order (Waltzek et al., 2005). Although one of the unique features of Herpesviridae, the sister family of Herpesvirales, is latent infection, it has not been demonstrated consistently that KHV of Alloherpesviridae can cause latent infection and be reactivated from latency. To investigate if KHV genomic DNA is present in koi exposed to KHV infection, 10 healthy fish were investigated from a koi population with a history of a KHV outbreak. No gross lesions or microscopic changes were observed at necropsy or by histological examination. No infectious virus was isolated from either the blood plasma or tissues. However, KHV DNA was detected in the white blood cells of nine of the ten fish by real-time PCR and PCR-Southern blot. KHV DNA was also detected in the brain, eye, spleen, gills hematopoietic kidney, trunk kidney, and intestine of nine of the ten fish by PCR-Southern blot. Interestingly, KHV DNA was also detected in the intestinal contents from seven of ten koi. Portions of major capsid gene DNA, amplified from two of the ten koi WBCs, were found to be identical to KHV-U. This study demonstrated that KHV genomic DNA can be detected in normal koi exposed previously to KHV and suggests that KHV becomes latent in fish., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)

An improved simple, rapid and sensitive method for detecting grass carp reovirus (GCRV) based on RT-PCR was developed by combining an advanced RNA extraction technique and targeting segment 10 as a template. The results indicate that highly efficient RT-PCR amplification of GCRV genome segments can be obtained using column-extracted RNA as a template, which is suitable not only for full-length gene amplification up to a size of 1.5 kb, but also for partial genome detection. Moreover, by targeting the highly divergent segment 10, the sensitivity of RT-PCR detection is improved significantly; as little as 1.0 fg of the 515 bp S10 dsRNA can be detected by one-step RT-PCR amplification. Furthermore, this method exhibits good reproducibility and specificity, and no amplicons were observed when RNA fragments other than those from GCRV were used as templates. The entire detection process can be completed within 4-5h from RNA extraction, much faster than methods reported previously. Overall, the improved detection technique may be applied for rapid diagnosis of GCRV or other dsRNA viruses., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A RT-qPCR assay that was developed and optimised for detection of betanodaviruses was validated for use as a diagnostic test for viral nervous necrosis disease of fish. Four betanodavirus genotypes were detected but the sensitivity was greatest for redspotted grouper nervous necrosis virus (RGNNV). The analytical sensitivity was 10-1000-fold greater than that of a nested RT-PCR assay and the limit of detection was <0.4 TCID(50) units per reaction. The assay was highly repeatable (standard deviation of estimated log(10)(viral copies) 0.10+/-0.08) and reproducible (standard deviation of estimated log(10)(viral copies) 0.08+/-0.06). Diagnostic accuracy was assessed in 2193 samples comprising tissue homogenates from Australian bass (Macquaria novemaculeata) and barramundi (Lates calcarifer), and also in SSN-1 tissue culture supernatants, using virus isolation in striped snake head (SSN-1) cell culture as the gold standard. Diagnostic sensitivity and specificity were 100% when the assay was applied to Australian bass tissue and SSN-1 tissue culture supernatants, but for barramundi tissue were 99.1% and 92.8%, respectively. The apparent imperfect specificity was shown by specific amplification of alternate regions of the betanodavirus genome to be due to the lower sensitivity of virus isolation. This is the first study to report the diagnostic performance of a RT-qPCR assay for detection of betanodavirus., (2009 Elsevier B.V. All rights reserved.)

Previous and new PCRs for KHV detection were compared by estimation of their sensitivity in recognizing KHV DNA in plasmids, cell culture extracted KHV DNA and total DNA obtained from field tissue samples. A modified real-time PCR (Gilad et al., 2004), combined with an internal control system (IC2, Hoffmann et al., 2006) in a duplex assay, was used as a "gold standard". The lowest reliably determined virus concentration between, 5 and 10 KHV DNA genomic equivalents, was found by real-time PCR (Gilad et al., 2004), nested PCR (Bergmann et al., 2006) and one-tube semi-nested PCR. All other published and unpublished PCRs, as well as the commercial Loopamp, recognized KHV DNA at higher concentrations only. Additionally, KHV variants, newly adapted to European conditions, which could not be detected by PCR according to Bercovier et al. (2005) were found in two field samples from carp and koi from different regions of Germany. A negative influence of sample pooling was shown with field samples tested by real-time PCR., (2009 Elsevier B.V. All rights reserved.)

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection., (2009 Elsevier B.V. All rights reserved.)

Red-spotted grouper nervous necrosis virus (RGNNV) causes high mortality in marine fish larvae cultured in China. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on loop-mediated isothermal amplification (LAMP) was developed. A set of four primers, two outer and two inner, was designed from RGNNV genome RNA. The LAMP reaction mix was optimized. The method was specific as no cross-reaction was observed between white spot syndrome virus, koi herpesvirus, infectious spleen and kidney necrosis virus, mud crab reovirus, and grass carp hemorrhage virus. The sensitivity of LAMP was 100-fold higher than the nested PCR in detecting the presence of RGNNV. RGNNV was detected in the brain of Trachinotus ovatus that showed typical symptoms of NNV infection, with the standardized LAMP procedure.

Loop mediated isothermal amplification (LAMP) assay is used for rapid diagnosis of Cyprinid herpesvirus-3, formerly designated koi herpesvirus (KHV), with comparable sensitivity to PCR. To reduce the time required for the LAMP assay, an immunocapture (IC) and direct binding (DB) techniques were developed to exclude the DNA extraction step in molecular diagnostic procedures of the virus. Both techniques were evaluated by using PCR and CyHV-3-LAMP assays. The DB-LAMP/PCR assays were more sensitive (detecting 0.1 virus particles/ml) than the IC-LAMP/PCR assays (detecting 1 virus particle/ml). By using the SYBR Green I stain and the DB/LAMP assay the complete CyHV-3 diagnostic process can be achieved within 90 min compared to more than 5 h for the routine PCR assay. Both assays (IC/DB) could amplify successfully CyHV-3 from clinical samples which prove its application to diagnostic tests. The DB-LAMP assay is a simple, rapid, sensitive technique and applicable to the diagnosis of CyHV-3 in the field.

Viral haemorrhagic septicaemia virus (VHSV), a member of the Rhabdoviridae family, is a major viral pathogen of cultured salmonid fish, and also infects a wide range of marine fish species. In the present study, two real time PCR protocols (based on SYBR Green and TaqMan) were developed for the detection of strains belonging to all known genotypes of VHSV. Validation of the procedure, in terms of sensitivity, specificity and repeatability/reproducibility (R&R), was also performed. For this purpose, several pairs of primer amplifying regions corresponding to viral G and N genes were assayed. In the SYBR Green-based real time PCR, these primers failed to detect strains from some of the genotypes and/or showed low R&R. In order to improve the detection capacity, a multiplex procedure was designed, which enabled detection of all strains, with high R&R. The sensitivity of the procedure was measured, and a detection limit of 1 fg/microl of viral RNA or 10 copies of cloned plasmid was established. On the other hand, the TaqMan probe-based multiplex real time PCR detected all European strains, with similar levels of sensitivity and R&R, but failed to detect the American types.

The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 degrees C for 60min. The detection limit of RT-LAMP is 10(-6) NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers.