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).

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.

GroEL, a novel vaccine candidate of piscine Streptococcus agalactiae identified by immunoproteome.

Streptococcus agalactiae is the major etiological agent of streptococcosis, which is responsible for huge economic losses in fishery, particularly in tilapia (Oreochromis niloticus) aquaculture. A research priority to control streptococcosis is to develop vaccines, so we sought to figure out the immunogenic proteins of S. agalactiae and screen the vaccine candidates for streptococcosis in the present study. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting and mass spectrometry (MS), was employed to investigate the immunogenic proteins of S. agalactiae THN0901. Whole-cell soluble proteins were separated using 2-DE, and the immunogenic proteins were detected by western blotting using rabbit anti-S. agalactiae sera. A total of 17 immunoreactive spots on the soluble protein profile, corresponding to 15 different proteins, were identified by MALDI-TOF/TOF MS. Among the immunogenic proteins, GroEL attracted our attention as it was demonstrated to be immunogenic and protective against other streptococci. Nevertheless, to date, there have been no published reports on the immunogenicity and protective efficacy of GroEL against piscine S. agalactiae. Therefore, recombinant GroEL (rGroEL) was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Immunization of tilapia with rGroEL resulted in an increase in antibody titers and conferred protection against S. agalactiae, with the relative percentage survival of 68.61 ±â€¯7.39%. The immunoproteome in the present study narrows the scope of vaccine candidates, and the evaluation of GroEL immunogenicity and protective efficacy shows that GroEL forms an ideal candidate molecule in subunit vaccine against S. agalactiae.

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.

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.

Identification of IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent NF-κB activation.

Interleukin 1 (IL-1) receptor-associated kinase (IRAK) family members are crucial signal transducer in the Toll-like receptor/IL-1R signal pathway, which mediates downstream signal cascades involved in the innate and adaptive immune responses. In this study, we identified an IRAK-4 protein (EcIRAK-4) in the orange-spotted grouper (Epinephelus coioides), with an N-terminal death domain, a proST domain, and a central kinase domain, similar to that of other fishes and mammals. A sequence alignment and phylogenic analysis demonstrated that full-length EcIRAK-4 shares a high degree of sequence identity with those of other fishes, especially the roughskin sculpin, and their death domains and kinase domains share greater identity than their proST domains. A conservation analysis indicated that most of the functional sites in mammalian IRAK-4 are conserved in IRAK-4 of the grouper and other fishes, with the exception of the sites of interaction with IRAK-2 and one autophosphorylation site within the activation loop. EcIRAK-4 is broadly expressed in all the tissues examined, with highest expression in the head kidney and liver. After infection with Cryptocaryon irritans, EcIRAK-4 expression was significantly upregulated, especially in the skin, which suggests that this molecule is involved in the host's defense against parasitic infection. Surprisingly, after cotransfection with grouper MyD88, EcIRAK-4 significantly impaired the NF-κB activity induced by MyD88. EcIRAK-4 was uniformly distributed throughout the cytoplasm in HeLa cells. These findings suggest that although IRAK-4 is evolutionarily conserved between fish and mammals, its signal transduction function is markedly different.