Development of real-time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3.

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV).

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

Interferon regulatory factors inhibit TiLV replication by activating interferon-a3 in tilapia (Oreochromis niloticus).

Tilapia lake virus (TiLV) is an emerging virus that seriously threatens the tilapia industries worldwide. Interferon regulatory factors (IRFs), which are the crucial mediators regulating the response of interferon (IFN) to combat invading viruses, have not yet been reported in tilapia during TiLV infection. Here, six IRF (IRF1, IRF2, IRF4, IRF7, IRF8, and IRF9) homologs from tilapia were characterized and analyzed. These IRFs typically shared the conserved domains and phylogenetic relationship with IRF homologs of other species. Tissue distribution analysis showed that all six IRF genes were expressed in various tissues, with the highest expression in immune-related tissues. Furthermore, overexpression of IRFs in tilapia brain (TiB) cells significantly inhibited TiLV propagation, as evidenced by decreased viral segment 8 gene transcripts and copy numbers of viral segment 1. More importantly, all six IRF genes significantly enhanced the promoter activity of type I interferon-a3 (IFNa3) in TiB cells, suggesting that tilapia IRF genes serve as positive regulators in activating IFNa3. Surprisingly, the promoter activity of IFNa3 mediated by IRF genes was markedly inhibited post-TiLV infection, indicating that TiLV antagonized IRF-mediated IFN immune response. Taken together, six IRF genes of tilapia are highly conserved transcription factors that inhibit TiLV infection by activating the promoter of IFNa3, which is in turn restrained by TiLV. These findings broaden our knowledge about the functionality of IRF-mediated antiviral immunity in tilapia against TiLV infection and host-TiLV interaction, which lays a foundation for developing antiviral strategies in tilapia cultural industries.

Coxsackievirus and adenovirus receptor inhibits tilapia lake virus infection via binding to viral segment 8 and 10 encoded protein.

The global aquaculture industry of tilapia (Oreochromis niloticus) has been significantly impacted by the emergence of tilapia lake virus (TiLV). However, effective prevention and control measures are still not available due to a lack of unclear pathogenesis of TiLV. Our previous transcriptome found that coxsackievirus and adenovirus receptor (CAR) was in response to TiLV infection in tilapia. To explore the potential function of OnCAR, the effect of OnCAR on TiLV proliferation was analyzed in this study. The OnCAR open reading frame (ORF) sequence of tilapia was 516 bp in length that encoded 171 amino acids with an Ig-like domain and transmembrane region. The OnCAR gene showed widespread expression in all investigated tissues, with the highest levels in the heart. Moreover, the OnCAR gene in the liver and muscle of tilapia exhibited dynamic expression levels upon TiLV challenge. Subcellular localization analysis indicated that OnCAR protein was mainly localized on the membrane of tilapia brain (TiB) cells. Importantly, the gene transcripts, genome copy number, S8-encoded protein, cytopathic effect, and internalization of TiLV were obviously decreased in the TiB cells overexpressed with OnCAR, indicating that OnCAR could inhibit TiLV replication. Mechanically, OnCAR could interact with viral S8 and S10-encoded protein. To the best of our knowledge, OnCAR is the first potential anti-TiLV cellular surface molecular receptor discovered for inhibiting TiLV infection. This finding is beneficial for better understanding the antiviral mechanism of tilapia and lays a foundation for establishing effective prevention and control strategies against tilapia lake virus disease (TiLVD).

Comparative transcriptional analysis between virulent isolate HN1307 and avirulent isolate GD1108 of grass carp reovirus genotype II.

As a widespread epidemic virus, genotype II of the grass carp reovirus poses a significant threat to the grass carp farming industry in China. Different genotype II isolates cause different degrees of virulence, although the underlying pathogenic mechanisms remain largely unknown. In this work, infections of grass carp with the virulent isolate grass carp reovirus (GCRV)-HN1307 and the avirulent isolate GCRV-GD1108 were performed to reveal a possible mutual transcriptional discrepancy. More differentially expressed genes (DEGs) were identified in the HN1307-infected group, which defined a grossly similar gene ontology (GO) pattern and different pathway landscape as the GD1108-infected group. Gene set enrichment analysis revealed that pathways related to innate immunity and metabolism were reciprocally activated and suppressed, respectively, following infection withHN1307, compared with GD1108. The trend analysis further indicated that immune-related pathways were involved in one of the four statistically significant profiles. Network analysis of transcription factor-gene interactions and protein-protein interactions on the immune-related profile suggested that among the core transcriptional factors (TFs) (UBTF, HCFC1, MAZ, MAX, and NRF1) and the hub proteins (Tlr3, Tlr7, Tlr9, Irf3, and Irf7), the latter were highly enriched in the toll-like receptor signaling pathway. Real-time quantitative PCR performed on the selected mRNAs validated the relative expression. This work will provide insights into the distinct transcriptional signatures from avirulent and virulent isolates of GCRV, which may contribute to the development of products for prevention.

Development of a rapid and sensitive reverse transcription real-time quantitative PCR assay for detection and quantification of grass carp reovirus II.

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.

Recombinant Lactococcus lactis Expressing Grass Carp Reovirus VP6 Induces Mucosal Immunity Against Grass Carp Reovirus Infection.

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery.

Susceptibilities of ten fish cell lines to infection with Tilapia lake virus.

Tilapia lake virus disease (TiLVD) caused by Tilapia lake virus (TiLV) is a great threat to the global tilapia culture industry. Effective prevention and control strategies have not been developed due to limited basic research of pathogenesis of TiLVD. Cell lines from different fish species have been found to be permissive to TiLV infection. In the current study, we comprehensively analyzed TiLV susceptibilities to 10 permanent growing fish cell lines. We found that the highest viral titers were generated onto TiB cells originated from the tilapia species Oreochromis mossambicus, MSF from the largemouth bass Micropterus salmoides, CAMK from the hybrid snakehead Channa argus × Channa maculata and SS derived from the perch species Siniperca chuatsi. Viral copy numbers from these four cell lines ranged from 4 × 107 copies/µL to 4.6 × 108 copies/µL. Confocal immunofluorescent microscopy also indicated that all 10 cell lines can support varying degrees of viral infection and replication. TiLV particles can be observed in cells from randomly selected three fish species using electron microscope. This study will assist in research and development of prevention and control of TiLVD.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for the detection of KHV.

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56 ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.

Establishment of a cell line from swim bladder of the Grass carp (Ctenopharyngodon idellus) for propagation of Grass Carp Reovirus Genotype II.

A cell line was established from swim bladder of the Grass carp (Ctenopharyngodon idellus) (CiSB), which was permissive for infection and propagation of Grass Carp Reovirus (GCRV). CiSB cells displayed optimal growth at 27 °C using M199 medium containing 10% fetal bovine serum and a fibroblastic-like morphology. Karyotype analysis revealed that the average diploid chromosome number was 52 in 58% of cells at passage 60 compared to the wild type Grass carp cells (2n = 48). Infection with GCRV II isolate Hunan1307 was tracked by immunofluorescence and virus titration assay. The virus titer reached 105.2 TCID50/mL on 7th days post infection (dpi). Healthy adult Grass carp that were challenged with the virus propagated onto CiSB cells, displayed the typical symptoms and histopathological changes of Grass carp hemorrhagic disease (GCHD). Therefore, the CiSB cells can be used to propagate GCRV II and serve as a useful tool to study the pathogenesis of GCHD.

Integrated analysis of mRNA-miRNA expression in Tilapia infected with Tilapia lake virus (TiLV) and identifies primarily immuneresponse genes.

We investigated differential gene expression in Tilapia infected with the Tilapia Lake virus (TiLV).We used high-throughput sequencing to identify mRNAs and miRNAs involved in TiLV infection progression We identified 25,359 differentially expressed genes that included 863 new genes. We identified 1770, 4142 and 4947 differently expressed genes comparing non-infected controls with 24 and 120 h infections and between the infected groups, respectively. These genes were enriched to 291 GO terms and 62 KEGG pathways and included immune system progress and virion genes. High-throughput miRNA sequencing identified 316 conserved miRNAs, 525 known miRNAs and 592 novel miRNAs. Furthermore, 138, 198 and 153 differently expressed miRNAs were found between the 3 groups listed above, respectively. Target prediction revealed numerous genes including erythropoietin isoform X2, double-stranded RNA-specific adenosine deaminase isoform X1, bone morphogenetic protein 4 and tapasin-related protein that are involved in immune responsiveness. Moreover, these target genes overlapped with differentially expressed mRNAs obtained from RNA-seq. These target genes were significantly enriched to GO terms and KEGG pathways including immune system progress, virion and Wnt signaling pathways. Expression patterns of differentially expressed mRNA and miRNAs were validated in 20 mRNA and 19 miRNAs by qRT-PCR. We also were able to construct a miRNA-mRNA target network that can further understand the molecular mechanisms on the pathogenesis of TiLV and guide future research in developing effective agents and strategies to combat TiLV infections in Tilapia.

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection.

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

An effective live attenuated vaccine against Streptococcus agalactiae infection in farmed Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is an important pathogen associated with various aquatic animals, especially tilapia. Streptococcosis has greatly limited the healthy development of tilapia aquaculture in recent times. The development of novel effective vaccines is important for the prevention and control of streptococcosis in fish. We previously constructed a non-encapsulated S. agalactiae strain △cps by the in-frame deletion method. Here, we evaluated whether this mutant △cps is safe for tilapia and suitable for protection against streptococcosis. We observed that the △cps strain was non-pathogenic to tilapia, and there was no reversion of virulence when it was passaged in tilapia. Moreover, the △cps strain survived for at least 11 d in the main immune organs of tilapia. The tilapia vaccinated via intraperitoneal (IP) injection with △cps strain induced a high antibody titer, and the IgM antibody levels were significantly higher in the vaccinated group than in the control group. The vaccination protected tilapia against the S. agalactiae challenge with a relative percent survival of 90.47%. In addition, tilapia immunized with the △cps strain showed significantly higher expression level of IFN-γ, IL-1ß, MyD88, IgM, and MHC-Iα in the head kidney than those in the control during the entire observation period. The expression of MHC-IIß was inhibited during 1-7 d of immunization. These results revealed that the △cps strain is able to induce humoral and cell-mediated immune response in tilapia. Therefore, the strain △cps has a broad application prospect as a target for attenuation in vaccine development.

Carbon nanotube-based DNA vaccine against koi herpesvirus given by intramuscular injection.

Koi herpesvirus (KHV) also named Cyprinid Herpesvirus 3 (CyHV-3) is one of the most threatening pathogens affecting common carp production as well as the valued ornamental koi carp. The current commercial vaccines available are costly and potentially cause severe stress caused by live virus. KHV ORF149 gene has been proved encoding one of the main immunogenic proteins for KHV. In this study, we coupled a plasmid expression vector for ORF149 to single walled carbon nanotubes (SWCNTs) for an anti-KHV vaccine. The vaccine conferred an 81.9% protection against intraperitoneal challenge with KHV. Importantly, SWCNTs as a promising vehicle can enhanced the protective effects 33.9% over that of the naked DNA vaccine at the same dose. The protection was longer and serum antibody production, enzyme activities and immune-related gene expression were all induced in fish vaccinated with the nanotube-DNA vaccine compared with the DNA alone. Thereby, this study demonstrates that the ORF149 DNA vaccine loaded onto SWCNTs as a novel vaccine might provide an effective method of coping with KHV disease using intra-muscular vaccination.

Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is a major pathogen causing streptococcosis. To prevent and control this bacterial disease, antagonistic bacteria have become a new research hotspot. This study evaluated the probiotic potential of Bacillus velezensis LF01 strain, which is antagonistic to S. agalactiae. The active compounds produced by LF01 showed antimicrobial activity against a broad spectrum of fish pathogens, including S. agalactiae, Streptococcus iniae, Aeromonas hydrophila, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei, and Vibrio harveyi. The antimicrobial compounds were heat stable, pH stable, UV stable, resistant to proteases, and could be stored for a long time. To evaluate the probiotic function of LF01 in Nile tilapia, juveniles were divided into three treatment groups: a control group, an interval feeding group, and a continuous feeding group. Tilapia fed with LF01-supplemented diets (1.0 × 109 CFU/g) showed significantly better growth performances than those of the control group (P < 0.05). Tilapia fed with LF01-supplemented diets significantly increased lysozyme (LZY) and superoxide dismutase (SOD) activities. The expression of three immune-related genes (C3, lyzc, and MHC-IIß) was higher in the intestine, head kidney, and gill of tilapia from the continuous feeding group than in those from the control group (P < 0.05). Tilapia fed with LF01-supplemented diets showed remarkably improved survival rates after S. agalactiae infection, and analysis of their intestinal tract pathogens revealed that the abundance of Edwardsiella and Plesiomonas had significantly decreased compared with the control group. Our findings demonstrate that LF01 is an effective antagonist against various fish pathogens and has potential for controlling infections by Streptococcus spp. and other pathogens in tilapia.

Berberine Influences Blood Glucose via Modulating the Gut Microbiome in Grass Carp.

Berberine (BBR), an isoquinoline alkaloid, is a major pharmacological component of the Chinese herb Coptis chinensis, which has been listed in the Chinese Fisheries Pharmacopeia as a common drug for the control of bacterial fish diseases. However, BBR is poorly absorbed into the systemic circulation but is significantly accumulated in the intestine. It is difficult to explain the mechanism of clinical effects of BBR based on systemic genes and pathways; it has been proved that the function of BBR in mammals is associated with the host metabolic phenotypes mediated by the structural modulation of gut microbiota. The mechanism of pharmacological effects of BBR in fish remains unclear. Here, we fed grass carp (Ctenopharyngodon idellus) a diet supplemented with BBR at a dose of 30 mg/Kg body weight daily and compared them with grass carp fed a regular fish feed diet. Biochemical analysis revealed that fish fed BBR had significantly reduced serum glucose, total cholesterol (TC), and triglyceride (TG) levels, and increased TC (p < 0.05) and TG (p < 0.01) levels in the liver. Deep amplicon sequencing of the V4 region of 16S rRNA genes of the gut microbiota revealed: (i) the composition of gut microbiota after BBR feeding was more diverse than that in the control group; (ii)before fish were fed BBR, the enriched operational taxonomic units (OTUs) mainly belonged to Firmicutes while most enriched OTUs came from Proteobacteria, Planctomycetes, Bacteroidetes, and Firmicutes during BBR feeding and after BBR feeding stopped; (iii) the ratio of Firmicutes to Bacteroidetes was significantly decreased in fish fed BBR. Spearman's rank correlation showed that 32 berberine-OTUs were significantly negative correlated with glucose (p < 0.05). It indicates that BBR may affect the levels of serum glucose by the structural modulation of gut microbiota. Our results provide insight into the effect of BBR on fish metabolism and gut microbiomes, which would be beneficial for the fish welfare.

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real-time quantitative PCR.

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish-farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A. schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A. schubertii. A high correlation coefficient (R2  = 0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18 copies/µl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A. schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A. schubertii showed that the gills and intestines may be the entry for A. schubertii, and the spleen and kidney are major sites for A. schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A. schubertii.

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection.

Infections with koi herpesvirus (KHV) in carp are still a severe problem worldwide. Detection and elimination of infected fish are necessary for control of the Koi herpesvirus disease (KHVD). Serum is an excellent specimen for KHV testing because of high survivability of KHV in serum and ease of collection, storage, and handling. The direct detection of fish viruses based on the sandwich ELISA has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against purified KHV. By using hybridoma-monoclonal antibody technology, two hybridoma cell lines secreting MAbs against the KHV were established. By Western blot and IFAT analysis, the secreted MAbs from cell line IB7IB4 and cell line 7C72F7 recognized proteins of KHV. The result demonstrated that the MAbs were highly specific and sensitive to the KHV, and can be used for monitoring the virus quantification of carp, for example, the direct KHV diagnosis by sandwich enzyme-linked immunosorbent assay(ELISA). An antigen sandwich ELISA applying the biotin-avidin system was established using the biotinylated MAb IB7IB4 and 7C72F7 to detect virus in koi sera. These MAbs did not react with any of the tested other viruses by ELISA except KHV. The detection limit of the test was 3.923ng/ml KHV. Thus, this antigen sandwich ELISA is suitable for recognition of KHV.

Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus.

Tilapia lake virus (TiLV) is an emerging disease threatening tilapia culture in many parts of the world. A cell line from the brain of tilapia, which was named TiB, was established, characterized and subcultured with more than 100 passages. The TiB cell line was optimally maintained at 27°C using medium 199 (M199) supplemented with 10% foetal bovine serum (FBS). Chromosome analysis revealed that 60% of TiB cells at passage 5 maintained the modal chromosome number 2n = 44, while at passage 60, there were 43% of TiB cells with the diploid chromosome number 2n = 50. A significant cytopathic effect was observed in TiB cells after infection with tilapia lake virus (TiLV-2017A), and the viral replication in the cells was confirmed by transmission electron microscopy, immunofluorescence assays and viral titres, indicating the susceptibility of TiB cells to TiLV-2017A. The viral titres of TiLV-2017A in TiB cells reached 107.43 TCID50 /ml within 10 days. The stable growth and susceptibility to fish viruses make TiB cells a useful tool for fish virus-host cell interaction and for immune response of fish.

Development of a VP38 recombinant protein-based indirect ELISA for detection of antibodies against grass carp reovirus genotype II (iELISA for detection of antibodies against GCRV II).

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization.