Viral hemorrhagic septicemia virus(VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Cur...Viral hemorrhagic septicemia virus(VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Currently, no effective vaccines, Food and Drug Administration-approved inhibitors, or other therapeutic intervention options are available against VHSV. α-Lipoic Acid(LA), a potent antioxidant, has been proposed to have antiviral effects against different viruses. In this study, LA(CC_(50)= 472.6 lmol/L) was repurposed to exhibit antiviral activity against VHSV. In fathead minnow cells,LA significantly increased the cell viability post-VHSV infection(EC_(50)= 42.7 lmol/L), and exerted a dose-dependent inhibitory effect on VHSV induced-plaque, cytopathic effects, and VHSV glycoprotein expression. The time-of-addition assay suggested that the antiviral activity of LA occurred at viral replication stage. Survival assay revealed that LA could significantly upregulated the survival rate of VHSV-infected largemouth bass in both co-injection(38.095% vs. 1.887%,P < 0.01) and post-injection manner(38.813% vs. 8.696%, P < 0.01) compared with the control group. Additional comparative transcriptome and q RT-PCR analysis revealed LA treatment upregulated the expression of several antiviral genes, such as IRF7, Viperin, and ISG15. Moreover, LA treatment reduced VHSV-induced reactive oxygen species production in addition to Nrf2 and SOD1 expression. Taken together, these data demonstrated that LA suppressed VHSV replication by inducing antiviral genes expression and reducing VHSV-induced oxidative stress. These results suggest a new direction in the development of potential antiviral candidate drugs against VHSV infection.展开更多
[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyro...[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.展开更多
Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterize...Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterized a new cell line derived from the fin tissue of leopard coral grouper(PLF).The PLF cells were cultured for more than 55 passages.Cytochrome B gene sequencing confirmed the origin of the PLF cells is P.leopardus.Immunostaining against cytokeratin indicated that the PLF cells predominantly consist of epithelial cells.The chromosome number of PLF was 48.The cells grew well in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%–20%fetal bovine serum(FBS)at temperature between 20–28℃,with the highest growth rate at28℃.Transfection with pEGFP-N3 plasmid showed the transfection efficiency was about 35%.Virus susceptibility tests revealed that PLF cells are susceptible to red-spotted grouper nervous necrosis virus(NNV)and viral hemorrhagic septicemia virus(VHSV),and viral proliferation was confirmed by qRT-PCR and western blot.The altered expressions of immune-related genes TBK1,IRF3,and Mx after NNV and VHSV infections suggested that PLF cells can mount an immune response to fish viruses.Thus the PLF cells can be employed for studying virus-host interactions and developing antiviral strategies.展开更多
Viral hemorrhagic septicemia virus（VHSV） and marine birnavirus（MABV） are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economi...Viral hemorrhagic septicemia virus（VHSV） and marine birnavirus（MABV） are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification（LAMP） of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish, Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.展开更多
基金This work was supported by the Pearl River S&T Nova Program of Guangzhou(201806010047)the National Natural Science Foundation of China(31771587)Fundamental Research Funds for the Central Universities。
文摘Viral hemorrhagic septicemia virus(VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Currently, no effective vaccines, Food and Drug Administration-approved inhibitors, or other therapeutic intervention options are available against VHSV. α-Lipoic Acid(LA), a potent antioxidant, has been proposed to have antiviral effects against different viruses. In this study, LA(CC_(50)= 472.6 lmol/L) was repurposed to exhibit antiviral activity against VHSV. In fathead minnow cells,LA significantly increased the cell viability post-VHSV infection(EC_(50)= 42.7 lmol/L), and exerted a dose-dependent inhibitory effect on VHSV induced-plaque, cytopathic effects, and VHSV glycoprotein expression. The time-of-addition assay suggested that the antiviral activity of LA occurred at viral replication stage. Survival assay revealed that LA could significantly upregulated the survival rate of VHSV-infected largemouth bass in both co-injection(38.095% vs. 1.887%,P < 0.01) and post-injection manner(38.813% vs. 8.696%, P < 0.01) compared with the control group. Additional comparative transcriptome and q RT-PCR analysis revealed LA treatment upregulated the expression of several antiviral genes, such as IRF7, Viperin, and ISG15. Moreover, LA treatment reduced VHSV-induced reactive oxygen species production in addition to Nrf2 and SOD1 expression. Taken together, these data demonstrated that LA suppressed VHSV replication by inducing antiviral genes expression and reducing VHSV-induced oxidative stress. These results suggest a new direction in the development of potential antiviral candidate drugs against VHSV infection.
基金Supported by the Twelfth Five-Year Support Project of the Ministry of Science and Technology(2013BAD12B02)Science and Technology Project of State General Administration of the People’s Republic of China for Quality Supervision and Inspection and Quarantine(2015IK195)
文摘[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.
基金the National Natural Science Foundation of China(Nos.32473189,32173001,32273115)the Guangdong Province Special Support Plan Youth Top Talent Project(No.NIQN2024002)+2 种基金the Scientific and Technological Planning Project of Guangzhou City(No.2023B03J1267)the Natural Science Foundation of Guangxi(No.2021GXNSFDA075015)the Natural Science Foundation of Guangdong Province(Nos.2023B1515120074,2024A1515010880)。
文摘Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterized a new cell line derived from the fin tissue of leopard coral grouper(PLF).The PLF cells were cultured for more than 55 passages.Cytochrome B gene sequencing confirmed the origin of the PLF cells is P.leopardus.Immunostaining against cytokeratin indicated that the PLF cells predominantly consist of epithelial cells.The chromosome number of PLF was 48.The cells grew well in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%–20%fetal bovine serum(FBS)at temperature between 20–28℃,with the highest growth rate at28℃.Transfection with pEGFP-N3 plasmid showed the transfection efficiency was about 35%.Virus susceptibility tests revealed that PLF cells are susceptible to red-spotted grouper nervous necrosis virus(NNV)and viral hemorrhagic septicemia virus(VHSV),and viral proliferation was confirmed by qRT-PCR and western blot.The altered expressions of immune-related genes TBK1,IRF3,and Mx after NNV and VHSV infections suggested that PLF cells can mount an immune response to fish viruses.Thus the PLF cells can be employed for studying virus-host interactions and developing antiviral strategies.
基金The grants from the Korea Institute of Ocean Science and Technology under contract No.PE99315
文摘Viral hemorrhagic septicemia virus（VHSV） and marine birnavirus（MABV） are the causative pathogens for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economic losses in aquaculture. Rapid molecular detection for surveillance or diagnosis has been a critical component in reducing the prevalence of pathogen infection. The loop-mediated isothermal amplification（LAMP） of DNA is currently one of the most commonly used molecular diagnostic tools, as it is simple, quick, and easy to amplify target DNA under isothermal conditions. In the present study, a novel and highly specific LAMP assay for the sensitive and rapid detection of VHSV and MABV infection in fish was developed. Using a set of synthesized primers matching a specific region of the genome, the efficiency and specificity of the LAMP assay were optimized in terms of the reaction temperature and DNA polymerase concentration, as they are the main determinants of the sensitivity and specificity of the LAMP assay. In particular, we demonstrated that our assay could be applied to efficient detection of VHSV and MABV infection in the wild fish, Paralichthys olivaceus. Our results demonstrate the simplicity and convenience of this method for the detection of viral infection in aquatic organisms.
