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Tilapia lake virus (TiLV) is the main tilapia-infecting virus worldwide, causing serious economic losses. However, there is no vaccine for this viral disease. Here, TiLV ORF10 (TiLV-ORF10) encoding a protein with abundant epitopes was constructed into the eukaryotic expression vector pcDNA3.1, and used to evaluate the immune protective effects in Nile tilapia (Oreochromis niloticus). RT-PCR and western blot analyses confirmed vaccine plasmid expression in tilapia muscle tissues. Moreover, the transcription levels of immunoglobulin M, toll-like receptor 2, myeloid differentiation factor 88, interleukin 8, tumor necrosis factor alpha, gamma-IFN, and nuclear factor κB immune-related genes were statistically significantly upregulated in the spleen, liver, and kidney of vaccinated tilapias (P < 0.05). TiLV challenge experiments showed that relative percent survival (RPS) was significantly enhanced in fish by this DNA vaccine. Moreover, RPS was enhanced further when using a higher amount of the DNA vaccine (85.72% RPS at a DNA dose of 45 μg pcDNA3.1–ORF10). Vaccination with pcDNA3.1–ORF10 significantly reduced virus replication, as evidenced by the low amount of virus in the spleen, liver, and kidney of vaccinated tilapias after TiLV challenge. Thus, pcDNA3.1–ORF10 could induce protective immunity in tilapia and may be a potential vaccine candidate for controlling diseases caused by TiLV.
Nai-Tong Yu; Wei-Wei Zeng; Jian-Hua Wang; Yu-Liang Zhang; Xiu-Chun Zhang; Zhi-Xin Liu. A High Efficacy DNA Vaccine Against Tilapia Lake Virus in Nile Tilapia (Oreochromis niloticus). 2021, 1 .
AMA StyleNai-Tong Yu, Wei-Wei Zeng, Jian-Hua Wang, Yu-Liang Zhang, Xiu-Chun Zhang, Zhi-Xin Liu. A High Efficacy DNA Vaccine Against Tilapia Lake Virus in Nile Tilapia (Oreochromis niloticus). . 2021; ():1.
Chicago/Turabian StyleNai-Tong Yu; Wei-Wei Zeng; Jian-Hua Wang; Yu-Liang Zhang; Xiu-Chun Zhang; Zhi-Xin Liu. 2021. "A High Efficacy DNA Vaccine Against Tilapia Lake Virus in Nile Tilapia (Oreochromis niloticus)." , no. : 1.
The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7–98.3% nucleotide identity, a 96.4–100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.
Weiwei Zeng; Sven Bergmannc; Hanxu Dong; Ying Yang; Minglin Wu; Hong Liu; Yanfeng Chen; Hua Li. Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I. Viruses 2021, 13, 807 .
AMA StyleWeiwei Zeng, Sven Bergmannc, Hanxu Dong, Ying Yang, Minglin Wu, Hong Liu, Yanfeng Chen, Hua Li. Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I. Viruses. 2021; 13 (5):807.
Chicago/Turabian StyleWeiwei Zeng; Sven Bergmannc; Hanxu Dong; Ying Yang; Minglin Wu; Hong Liu; Yanfeng Chen; Hua Li. 2021. "Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I." Viruses 13, no. 5: 807.
Grass carp hemorrhagic disease is a fatal disease caused by the grass carp reovirus (GCRV). The aberrant regulation of transcripts has been implicated in many types of diseases. In the present study, we characterized mRNA and miRNA transcriptomes of different virulent GCRVs using RNA sequencing (RNA-Seq). One hundred eighteen miRNAs were identified as being differentially expressed between different virulent viruses in grass carp fibroblasts. Eight miRNAs were selected to verify the RNA-Seq results using RT-PCR and mRNA methods. In total, 996 differentially expressed mRNA genes were identified in grass carp fibroblasts, while 901 miRNA-mRNA target pairs were observed to be inversely regulated in grass carp fibroblasts. Integrated mRNA/miRNA expression profiling analysis results showed that the most influenced processes were the immune response and cell death. Three miRNAs were shown to exhibit the same expression patterns when two different methods were used and had important functions during viral infection. These results provide insights into the miRNA-mediated regulation of mRNA and valuable resources on transcript variation and regulation during GCRV infection, which are potentially useful for mechanistic and drug studies.
Qiucheng Yao; Mengdi Zhang; Shaopo Zu; Hong Yang; Weitian Xie; Jinjun Chen; Zhibao Chen; Ye Ge; Weiwei Zeng; Zhihui Zhao. Integrated mRNA and microRNA Transcriptome Sequencing Characterizes Sequence Variants and mRNA-microRNA Regulatory Networks in Grass Carp Fibroblasts Infected with Virulent and Attenuated GCRV. Marine Biotechnology 2021, 23, 342 -355.
AMA StyleQiucheng Yao, Mengdi Zhang, Shaopo Zu, Hong Yang, Weitian Xie, Jinjun Chen, Zhibao Chen, Ye Ge, Weiwei Zeng, Zhihui Zhao. Integrated mRNA and microRNA Transcriptome Sequencing Characterizes Sequence Variants and mRNA-microRNA Regulatory Networks in Grass Carp Fibroblasts Infected with Virulent and Attenuated GCRV. Marine Biotechnology. 2021; 23 (2):342-355.
Chicago/Turabian StyleQiucheng Yao; Mengdi Zhang; Shaopo Zu; Hong Yang; Weitian Xie; Jinjun Chen; Zhibao Chen; Ye Ge; Weiwei Zeng; Zhihui Zhao. 2021. "Integrated mRNA and microRNA Transcriptome Sequencing Characterizes Sequence Variants and mRNA-microRNA Regulatory Networks in Grass Carp Fibroblasts Infected with Virulent and Attenuated GCRV." Marine Biotechnology 23, no. 2: 342-355.
Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Vaccination is the most effective way to prevent and control viral diseases. There is currently no vaccine on the market to control TiLV infections. Recently, we identified that TiLV segment 8 encoded a protein (VP20) with immunogenicity sufficient for use as a vaccine antigen. In this study, the immune responses and protective efficacy elicited by VP20 with different prime-boost vaccination regimens (DNA only, protein only, and DNA plus protein) in tilapia was evaluated. The results indicated that both pV-optiVP20 plasmid and rVP20 protein induced humoral and cellular immune responses. Tilapia in the DNA prime-protein boost group developed significantly higher levels of antibody response compared with those immunized with either the DNA or the protein alone (P < 0.05). Furthermore, the highest mRNA levels of the genes TNF-α, IL-1β, IgM, CD4, MHC-Ia and MHC-II were induced following priming with the pV-optiVP20 and boosting with the rVP20. Additionally, tilapia inoculated with the pV-optiVP20 prime-rVP20 boost regimen had a 72.5% survival rate against challenge with the lethal TiLV 2017A compared with 50% or 52.5% survival using the pV-optiVP20 and the rVP20 vaccinations alone. These findings demonstrated that VP20-based vaccine could elicit both humoral and cellular immune responses and significantly protected the fish against infectious challenge by TiLV. The DNA prime-protein boost immunization strategy showed the potential advantage over a solo vaccination.
Weiwei Zeng; Yingying Wang; Xiaoyu Chen; Qing Wang; Sven M. Bergmann; Ying Yang; Yahui Wang; Bo Li; Yuefeng Lv; Hua Li; Wensheng Lan. Potency and efficacy of VP20-based vaccine against tilapia lake virus using different prime-boost vaccination regimens in tilapia. Aquaculture 2021, 539, 736654 .
AMA StyleWeiwei Zeng, Yingying Wang, Xiaoyu Chen, Qing Wang, Sven M. Bergmann, Ying Yang, Yahui Wang, Bo Li, Yuefeng Lv, Hua Li, Wensheng Lan. Potency and efficacy of VP20-based vaccine against tilapia lake virus using different prime-boost vaccination regimens in tilapia. Aquaculture. 2021; 539 ():736654.
Chicago/Turabian StyleWeiwei Zeng; Yingying Wang; Xiaoyu Chen; Qing Wang; Sven M. Bergmann; Ying Yang; Yahui Wang; Bo Li; Yuefeng Lv; Hua Li; Wensheng Lan. 2021. "Potency and efficacy of VP20-based vaccine against tilapia lake virus using different prime-boost vaccination regimens in tilapia." Aquaculture 539, no. : 736654.
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.
Yingying Li; Qing Wang; Feng Hu; Yingying Wang; Sven M. Bergmann; Weiwei Zeng; Jiyuan Yin; Cunbin Shi. Development of a double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) for the detection of KHV. Journal of Fish Diseases 2021, 44, 913 -921.
AMA StyleYingying Li, Qing Wang, Feng Hu, Yingying Wang, Sven M. Bergmann, Weiwei Zeng, Jiyuan Yin, Cunbin Shi. Development of a double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) for the detection of KHV. Journal of Fish Diseases. 2021; 44 (7):913-921.
Chicago/Turabian StyleYingying Li; Qing Wang; Feng Hu; Yingying Wang; Sven M. Bergmann; Weiwei Zeng; Jiyuan Yin; Cunbin Shi. 2021. "Development of a double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) for the detection of KHV." Journal of Fish Diseases 44, no. 7: 913-921.
Genotype II of grass carp reovirus (GCRV) is an epidemic strain that is devastating to the grass carp industry in China. Infections of grass carp caused with virulent GCRV-HuNan1307 and avirulent GCRV-GD1108 isolates were carried out to determine the underlying pathogenic mechanisms. The differential host protein response was examined after infection with both virus isolates using an iTRAQ-based comparative quantitative proteomics. Differentially expressed proteins for GD1108 (34) and HuNan1307 (222) infections were identified and determined canonical pathways and functional networks involved in both GCRV infections. Infection with HuNan1307 activated the RIG-I pathway leading to antiviral response as well as the Jak-STAT pathway that governs general metabolic functions. These pathways were not activated by infection with the avirulent isolate. Infection with HuNan1307 also induced an abbreviated an intense immune response and lead to major disorders of the metabolism. In contrast, the GD1108 infection induced a mild and moderate immune response leaving basic metabolism unchanged. This study provides a comprehensive view at the protein level of the underlying pathogenic mechanisms used by a virulent GCRV type II isolate. Genotype II of grass carp reovirus (GCRV) is an epidemic strain that is devastating to the grass carp industry in China. The unclear etiological characters, genetic variation and pathogenesis of GCRV genotype II blocked the development of techniques as well as products on prevention and control of grass carp haemorrhagic disease (GCHD). Here, iTRAQ strategy was firstly used to compare the proteomes of grass carp infected with virulent and avirulent isolates of GCRV genotype II. The obtained data showed that abbreviated and intense immune response and disorders of the metabolism were reasons for the mortality of fish infected with virulent GCRV isolate. The results would provide a train of thought to promote the development of techniques as well as products on prevention and control of GCHD.
Zhishen Huang; Yingying Wang; Siyu Wu; Jiyuan Yin; Wenli Zhou; Ting Gao; Yingying Li; Sven M. Bergmann; Caixia Gao; Yahui Wang; Weiwei Zeng; Qing Wang. An iTRAQ-based comparative proteomic analysis of grass carp infected with virulent and avirulent isolates of grass carp reovirus genotype II. Aquaculture 2021, 535, 736426 .
AMA StyleZhishen Huang, Yingying Wang, Siyu Wu, Jiyuan Yin, Wenli Zhou, Ting Gao, Yingying Li, Sven M. Bergmann, Caixia Gao, Yahui Wang, Weiwei Zeng, Qing Wang. An iTRAQ-based comparative proteomic analysis of grass carp infected with virulent and avirulent isolates of grass carp reovirus genotype II. Aquaculture. 2021; 535 ():736426.
Chicago/Turabian StyleZhishen Huang; Yingying Wang; Siyu Wu; Jiyuan Yin; Wenli Zhou; Ting Gao; Yingying Li; Sven M. Bergmann; Caixia Gao; Yahui Wang; Weiwei Zeng; Qing Wang. 2021. "An iTRAQ-based comparative proteomic analysis of grass carp infected with virulent and avirulent isolates of grass carp reovirus genotype II." Aquaculture 535, no. : 736426.
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.
Yuru Yang; Yingying Wang; Qing Wang; Weiwei Zeng; Yingying Li; Jiyuan Yin; Siyu Wu; Cunbin Shi. Establishment of a cell line from swim bladder of the Grass carp (Ctenopharyngodon idellus) for propagation of Grass Carp Reovirus Genotype II. Microbial Pathogenesis 2021, 151, 104739 .
AMA StyleYuru Yang, Yingying Wang, Qing Wang, Weiwei Zeng, Yingying Li, Jiyuan Yin, Siyu Wu, Cunbin Shi. Establishment of a cell line from swim bladder of the Grass carp (Ctenopharyngodon idellus) for propagation of Grass Carp Reovirus Genotype II. Microbial Pathogenesis. 2021; 151 ():104739.
Chicago/Turabian StyleYuru Yang; Yingying Wang; Qing Wang; Weiwei Zeng; Yingying Li; Jiyuan Yin; Siyu Wu; Cunbin Shi. 2021. "Establishment of a cell line from swim bladder of the Grass carp (Ctenopharyngodon idellus) for propagation of Grass Carp Reovirus Genotype II." Microbial Pathogenesis 151, no. : 104739.
The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP) causes retardation of shrimp growth, leading to significant financial losses in shrimp aquaculture. Therefore, the development of an efficient and sensitive detection method will be conducive to the prevention and control of the shrimp parasite. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) method that can detect EHP within 15 min at a constant temperature of 38.5 °C. The detection limit of this EHP RPA was 10 copies/μL of DNA molecules per reaction. The specificity of EHP RPA was tested, and the assay did not cross-react with white spot syndrome virus (WSSV), shrimp hemocyte iridescent virus (SHIV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), or Vibrio parahaemolyticus. Field and clinical applicability of this assay was evaluated using 61 field samples. The coincidence rate of the detection results for the clinical samples between RPA and qPCR was 95.1 %. In summary, the real-time RPA analysis provides an efficient and sensitive detection method for EHP.
Gen Li; Feng Cong; Weiyou Cai; Jinhui Li; Miaoli Wu; Li Xiao; Xiaoliang Hu; Weiwei Zeng; Dongsheng He. Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of enterocytozoon hepatopenaei (EHP). Aquaculture Reports 2021, 19, 100584 .
AMA StyleGen Li, Feng Cong, Weiyou Cai, Jinhui Li, Miaoli Wu, Li Xiao, Xiaoliang Hu, Weiwei Zeng, Dongsheng He. Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of enterocytozoon hepatopenaei (EHP). Aquaculture Reports. 2021; 19 ():100584.
Chicago/Turabian StyleGen Li; Feng Cong; Weiyou Cai; Jinhui Li; Miaoli Wu; Li Xiao; Xiaoliang Hu; Weiwei Zeng; Dongsheng He. 2021. "Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of enterocytozoon hepatopenaei (EHP)." Aquaculture Reports 19, no. : 100584.
Grass carp haemorrhagic disease (GCHD) is one of the most important diseases affecting grass carp aquaculture. The rare minnow (Gobiocypris rarus) is a small experimental fish that has been proven to be sensitive to GCRV infection. However, few studies have definitively shown that rare minnow can be used as a disease model for GCRV-II, a recently emerging dominant genotype. In the present study, we investigated clinical characteristics, tissue tropism, histopathology, and relative expression of immune-related genes in rare minnow infected with GCRV-II virulent strain HuNan1307. Hemorrhage of rare minnow was induced by the GCRV-II, which could proliferate in the fishes, and the viral load peaked on the day 7. The histopathologic changes in rare minnow were primarily vasodilation and hyperaemia in multiple organs, cell degeneration and necrosis, as well as loose intercellular in spleen, kidney, brain and heart. Electron microscopic observation in the kidney revealed virus similar in shape and size to those GCRV-II infections in grass carp. Genes encoding IL-8, Mx, TLR5, IgM and IFNα were relatively high up-regulated in different tissues, while multiple genes in liver and spleen were significantly up-regulated, which was consistent with GCRV-II infection in grass carp (P < 0.05). Our study suggested that rare minnow can be used as a disease model of GCRV-II as a tool to study pathogenesis and develop vaccine against GCRV-II.
Jiaming Chen; Ouqin Chang; Yingying Li; Yingying Wang; Chao Liu; Jiyuan Yin; Sven M. Bergmann; Weiwei Zeng; Qing Wang. Establishment of a rare minnow (Gobiocypris rarus) disease model for grass carp reovirus genotype II. Aquaculture 2020, 533, 736133 .
AMA StyleJiaming Chen, Ouqin Chang, Yingying Li, Yingying Wang, Chao Liu, Jiyuan Yin, Sven M. Bergmann, Weiwei Zeng, Qing Wang. Establishment of a rare minnow (Gobiocypris rarus) disease model for grass carp reovirus genotype II. Aquaculture. 2020; 533 ():736133.
Chicago/Turabian StyleJiaming Chen; Ouqin Chang; Yingying Li; Yingying Wang; Chao Liu; Jiyuan Yin; Sven M. Bergmann; Weiwei Zeng; Qing Wang. 2020. "Establishment of a rare minnow (Gobiocypris rarus) disease model for grass carp reovirus genotype II." Aquaculture 533, no. : 736133.
Aeromonas hydrophila is ubiquitous in the aquaculture industry and a constant cause of severe disease and economic losses. The early diagnosis of these infections is crucial for disease surveillance and prevention. We developed a real‐time recombinase polymerase amplification (real‐time RPA) assay for detection of A. hydrophila using the haemolysin gene. The assay was performed at 37°C for 20 min and was highly specific with no cross‐reaction with other fish pathogens or with other Aeromonas species. The assay detection limit was 102 copies of the Aeromonas hydrophila per reaction. Compared with traditional culture‐based method or real‐time PCR, the diagnostic sensitivity and specificity of the real‐time RPA were 73.7 and 100%, as well as 64.7 and 93%. Our newly developed real‐time RPA was specific and sensitive and can be used in large‐scale and point‐of‐care field investigations of A. hydrophila infections to enable earlier diagnoses.
Yang Qu; Qing Wang; Yingying Li; Yingying Wang; Jiyuan Yin; Yan Ren; Chun Liu; Xiaofang Liu; Yahui Wang; Weiwei Zeng. Development of a real‐time recombinase polymerase amplification assay for rapid detection of Aeromonas hydrophila. Journal of Fish Diseases 2020, 44, 469 -477.
AMA StyleYang Qu, Qing Wang, Yingying Li, Yingying Wang, Jiyuan Yin, Yan Ren, Chun Liu, Xiaofang Liu, Yahui Wang, Weiwei Zeng. Development of a real‐time recombinase polymerase amplification assay for rapid detection of Aeromonas hydrophila. Journal of Fish Diseases. 2020; 44 (4):469-477.
Chicago/Turabian StyleYang Qu; Qing Wang; Yingying Li; Yingying Wang; Jiyuan Yin; Yan Ren; Chun Liu; Xiaofang Liu; Yahui Wang; Weiwei Zeng. 2020. "Development of a real‐time recombinase polymerase amplification assay for rapid detection of Aeromonas hydrophila." Journal of Fish Diseases 44, no. 4: 469-477.
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.
Yingying Wang; Qing Wang; Yingying Li; Jiyuan Yin; Yan Ren; Cunbin Shi; Sven M. Bergmann; Xinping Zhu; Weiwei Zeng. Integrated analysis of mRNA-miRNA expression in Tilapia infected with Tilapia lake virus (TiLV) and identifies primarily immuneresponse genes. Fish & Shellfish Immunology 2020, 99, 208 -226.
AMA StyleYingying Wang, Qing Wang, Yingying Li, Jiyuan Yin, Yan Ren, Cunbin Shi, Sven M. Bergmann, Xinping Zhu, Weiwei Zeng. Integrated analysis of mRNA-miRNA expression in Tilapia infected with Tilapia lake virus (TiLV) and identifies primarily immuneresponse genes. Fish & Shellfish Immunology. 2020; 99 ():208-226.
Chicago/Turabian StyleYingying Wang; Qing Wang; Yingying Li; Jiyuan Yin; Yan Ren; Cunbin Shi; Sven M. Bergmann; Xinping Zhu; Weiwei Zeng. 2020. "Integrated analysis of mRNA-miRNA expression in Tilapia infected with Tilapia lake virus (TiLV) and identifies primarily immuneresponse genes." Fish & Shellfish Immunology 99, no. : 208-226.
Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.
Feng Cong; Fanwen Zeng; Miaoli Wu; Jingjing Wang; Bihong Huang; Yingying Wang; Qing Wang; Shouquan Zhang; Lei Ma; Pengju Guo; Weiwei Zeng. Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus. Molecular and Cellular Probes 2019, 50, 101494 .
AMA StyleFeng Cong, Fanwen Zeng, Miaoli Wu, Jingjing Wang, Bihong Huang, Yingying Wang, Qing Wang, Shouquan Zhang, Lei Ma, Pengju Guo, Weiwei Zeng. Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus. Molecular and Cellular Probes. 2019; 50 ():101494.
Chicago/Turabian StyleFeng Cong; Fanwen Zeng; Miaoli Wu; Jingjing Wang; Bihong Huang; Yingying Wang; Qing Wang; Shouquan Zhang; Lei Ma; Pengju Guo; Weiwei Zeng. 2019. "Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus." Molecular and Cellular Probes 50, no. : 101494.
Serological assays for indirect screening of tilapia lake virus (TiLV) are currently not available. We examined the potential use of an antigenic protein encoded by genome segment 8 of TiLV, tentatively called S8 protein, as a coating antigen for development of indirect enzyme-linked immune-sorbent assay (iELISA) for the detection of antibody against TiLV. After optimization of antigen and antibody concentration, determination of a cutoff value, reproducibility, sensitivity and specificity of the iELISA were evaluated. We found that the optimal concentration of the coating S8 protein antigen was 1.0 μg/mL with a secondary antibody dilution of 1:10.000, and the cut-off value was defined to be 0.258 OD units. The iELISA also showed high analytic specificity and was able to distinguish between antibodies against TiLV and other pathogens. It was more sensitive than the current IFA technique for detecting TiLV. In addition, for detection of artificial infection samples, the results showed that the diagnostic sensitivity and specificity of the iELISA compared with IFA was 100% and 92.6%, respectively; while the diagnostic sensitivity and specificity of the iELISA versus semi-nested PCR was 80.8% and 95.6%, respectively. A serological survey was performed using the iELISA with tilapia sera samples from the field compared with an unknown status regarding TiLV, 13/73 serum samples tested positive by iELISA, indicating a 17.8% TiLV antibody positives. In conclusion, our newly developed iELISA was specific and sensitive, and it may be useful for large-scale investigations of TiLV infections and monitoring trials of future vaccination protocols.
Huzi Hu; Weiwei Zeng; Yingying Wang; Qing Wang; Sven M. Bergmann; Jiyuan Yin; Yingying Li; Xiaoyu Chen; Caixia Gao; Defeng Zhang; Chun Liu; Yan Ren; Cunbin Shi. Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia. Aquaculture 2019, 520, 734756 .
AMA StyleHuzi Hu, Weiwei Zeng, Yingying Wang, Qing Wang, Sven M. Bergmann, Jiyuan Yin, Yingying Li, Xiaoyu Chen, Caixia Gao, Defeng Zhang, Chun Liu, Yan Ren, Cunbin Shi. Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia. Aquaculture. 2019; 520 ():734756.
Chicago/Turabian StyleHuzi Hu; Weiwei Zeng; Yingying Wang; Qing Wang; Sven M. Bergmann; Jiyuan Yin; Yingying Li; Xiaoyu Chen; Caixia Gao; Defeng Zhang; Chun Liu; Yan Ren; Cunbin Shi. 2019. "Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia." Aquaculture 520, no. : 734756.
Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis.
Yafang Tang; Weiwei Zeng; Yingying Wang; Qing Wang; Jiyuan Yin; Yingying Li; Chengbao Wang; Sven M. Bergmann; Caixia Gao; Huzi Hu. Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus. Microbial Pathogenesis 2019, 139, 103859 .
AMA StyleYafang Tang, Weiwei Zeng, Yingying Wang, Qing Wang, Jiyuan Yin, Yingying Li, Chengbao Wang, Sven M. Bergmann, Caixia Gao, Huzi Hu. Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus. Microbial Pathogenesis. 2019; 139 ():103859.
Chicago/Turabian StyleYafang Tang; Weiwei Zeng; Yingying Wang; Qing Wang; Jiyuan Yin; Yingying Li; Chengbao Wang; Sven M. Bergmann; Caixia Gao; Huzi Hu. 2019. "Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus." Microbial Pathogenesis 139, no. : 103859.
A brain cell line (CAMB) derived from hybrid snakehead (Channa argus (♂) × Channa maculata (♀)) was established by trypsin and collagenase combined digestion. The culturing conditions and cell biological characteristics were systematically studied. For growth of the cells, M199 medium containing 10% fetal bovine serum was used and at 27 °C incubated. Based on morphological analysis, CAMB cells were confirmed to be epithelial. The cell line has been subcultured more than 80 times since its initial primary culture. Chromosome analysis revealed that CAMB cells had an abnormal chromosome number 2n = 64, whereas the chromosome number in the hybrid snakehead was 45. The suitability of CAMB for tilapia lake virus (TiLV) was demonstrated. A CPE was observed after infection with TiLV-2017A. The highest TiLV titer was observed after 12 days post infection (dpi) and reached 107.2 TCID50/mL. The virus replication was confirmed by electron microscopic observations. Additionally, immunofluorescence assay confirmed the presence of TiLV-2017A after infection of CAMB. Therefore, CAMB cells can be a useful tool for the investigation of the pathogenesis of the TiLV induced disease in tilapia.
Yingying Wang; Zhili Li; Qing Wang; Weiwei Zeng; Yingying Li; Jiyuan Yin; Sven M. Bergmann; Xinping Zhu. Establishment of a brain cell line obtained from hybrids of Channa argus ×Channa maculata for the detection of tilapia lake virus. Microbial Pathogenesis 2019, 138, 103810 .
AMA StyleYingying Wang, Zhili Li, Qing Wang, Weiwei Zeng, Yingying Li, Jiyuan Yin, Sven M. Bergmann, Xinping Zhu. Establishment of a brain cell line obtained from hybrids of Channa argus ×Channa maculata for the detection of tilapia lake virus. Microbial Pathogenesis. 2019; 138 ():103810.
Chicago/Turabian StyleYingying Wang; Zhili Li; Qing Wang; Weiwei Zeng; Yingying Li; Jiyuan Yin; Sven M. Bergmann; Xinping Zhu. 2019. "Establishment of a brain cell line obtained from hybrids of Channa argus ×Channa maculata for the detection of tilapia lake virus." Microbial Pathogenesis 138, no. : 103810.
Recently, substantial mortality of farmed and wild tilapia caused by tilapia lake virus (TiLV) infection has been observed worldwide. However, sensitive and reliable diagnostic method is limited. A reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay has been applied for the detection of TiLV nucleotide sequence. Six primers targeting two locations on the target gene based on a highly conserved sequence in the segment 1 (S1) region of the TiLV genome have been designed. The optimized RT‐LAMP reaction was maintained at the isothermal condition of 63°C for 45 min. And the amplifications could be verified by turbidity or a colour change with the addition of SYBR Green I. Subsequently, RT‐LAMP products could be observed by a ladder pattern following gel electrophoresis. The species‐specific assay showed that the method was sensitive enough to detect as low as 1.6 copies of viral particle, and the assay was highly specific because no cross‐reactivity was observed with other pathogens, and had a diagnostic sensitivity and specificity of 100% when TiLV‐positive samples and non‐target virus were tested. In summary, all the results demonstrate that this RT‐LAMP is a rapid, effective and sensitive method for TiLV detection in tilapia aquaculture.
Jiyuan Yin; Qing Wang; Yingying Wang; Yingying Li; Weiwei Zeng; Jiexing Wu; Yan Ren; Yafang Tang; Caixia Gao; Huzi Hu; Sven M. Bergmann. Development of a simple and rapid reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus. Journal of Fish Diseases 2019, 42, 817 -824.
AMA StyleJiyuan Yin, Qing Wang, Yingying Wang, Yingying Li, Weiwei Zeng, Jiexing Wu, Yan Ren, Yafang Tang, Caixia Gao, Huzi Hu, Sven M. Bergmann. Development of a simple and rapid reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus. Journal of Fish Diseases. 2019; 42 (6):817-824.
Chicago/Turabian StyleJiyuan Yin; Qing Wang; Yingying Wang; Yingying Li; Weiwei Zeng; Jiexing Wu; Yan Ren; Yafang Tang; Caixia Gao; Huzi Hu; Sven M. Bergmann. 2019. "Development of a simple and rapid reverse transcription–loopmediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus." Journal of Fish Diseases 42, no. 6: 817-824.
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.
Yingying Wang; Qing Wang; Weiwei Zeng; Jiyuan Yin; Yingying Li; Yan Ren; Cunbin Shi; Sven M. Bergmann; Xinping Zhu. Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus. Journal of Fish Diseases 2018, 41, 1803 -1809.
AMA StyleYingying Wang, Qing Wang, Weiwei Zeng, Jiyuan Yin, Yingying Li, Yan Ren, Cunbin Shi, Sven M. Bergmann, Xinping Zhu. Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus. Journal of Fish Diseases. 2018; 41 (12):1803-1809.
Chicago/Turabian StyleYingying Wang; Qing Wang; Weiwei Zeng; Jiyuan Yin; Yingying Li; Yan Ren; Cunbin Shi; Sven M. Bergmann; Xinping Zhu. 2018. "Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus." Journal of Fish Diseases 41, no. 12: 1803-1809.
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.
Weiwei Zeng; Yingying Wang; Yanmin Guo; Sven M. Bergmann; Jiyuan Yin; Yingying Li; Yan Ren; Cunbin Shi; Qing Wang. 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). Journal of Fish Diseases 2018, 41, 1811 -1819.
AMA StyleWeiwei Zeng, Yingying Wang, Yanmin Guo, Sven M. Bergmann, Jiyuan Yin, Yingying Li, Yan Ren, Cunbin Shi, Qing Wang. 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). Journal of Fish Diseases. 2018; 41 (12):1811-1819.
Chicago/Turabian StyleWeiwei Zeng; Yingying Wang; Yanmin Guo; Sven M. Bergmann; Jiyuan Yin; Yingying Li; Yan Ren; Cunbin Shi; Qing Wang. 2018. "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)." Journal of Fish Diseases 41, no. 12: 1811-1819.
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.
Yanmin Guo; Weiwei Zeng; Qing Wang; Yingying Wang; Yingying Li; Jiyuan Yin; Yan Ren; Cunbin Shi. Use of high-resolution melting curve analysis to differentiate vaccine and wild type strains of grass carp reovirus genotype II. Journal of Virological Methods 2018, 256, 111 -115.
AMA StyleYanmin Guo, Weiwei Zeng, Qing Wang, Yingying Wang, Yingying Li, Jiyuan Yin, Yan Ren, Cunbin Shi. Use of high-resolution melting curve analysis to differentiate vaccine and wild type strains of grass carp reovirus genotype II. Journal of Virological Methods. 2018; 256 ():111-115.
Chicago/Turabian StyleYanmin Guo; Weiwei Zeng; Qing Wang; Yingying Wang; Yingying Li; Jiyuan Yin; Yan Ren; Cunbin Shi. 2018. "Use of high-resolution melting curve analysis to differentiate vaccine and wild type strains of grass carp reovirus genotype II." Journal of Virological Methods 256, no. : 111-115.
Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID assay combined IFA was conducted. The results indicated that TCID of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis.
Qing Wang; Hualiang Xie; Weiwei Zeng; Linchuan Wang; Chun Liu; Jiexing Wu; Yingying Wang; Yingying Li; Sven M. Bergmann. Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells. Microbial Pathogenesis 2018, 114, 68 -74.
AMA StyleQing Wang, Hualiang Xie, Weiwei Zeng, Linchuan Wang, Chun Liu, Jiexing Wu, Yingying Wang, Yingying Li, Sven M. Bergmann. Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells. Microbial Pathogenesis. 2018; 114 ():68-74.
Chicago/Turabian StyleQing Wang; Hualiang Xie; Weiwei Zeng; Linchuan Wang; Chun Liu; Jiexing Wu; Yingying Wang; Yingying Li; Sven M. Bergmann. 2018. "Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells." Microbial Pathogenesis 114, no. : 68-74.