Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper ne...Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.展开更多
The origin of T cells in the teleost's brain is unclear.While viewing the central nervous system(CNS)as immune privileged has been widely accepted,previous studies suggest that T cells residing in the thymus but n...The origin of T cells in the teleost's brain is unclear.While viewing the central nervous system(CNS)as immune privileged has been widely accepted,previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs.Here,we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper(Epinephelus coioices)through single-cell RNA-sequencing analysis.After viral CNS infection with red-spotted grouper nervous necrosis virus(RGNNV),the number of slc43a2^(+)T cells synchronously increased in the spleen and brain.During the infection tests in asplenic zebrafish(tlx1^▲zebrafish model),no increase in the number of slc43a2^(+)T cells was observed in the brain.Single-cell transcriptomic analysis indicated that slc43a2^(+)T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response.This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish.展开更多
C-myc is a proto-oncogene that plays an important role in a variety of diseases.There were a lot of research on the correlation between C-myc and human viruses.However,the study about C-myc related to aquatic species ...C-myc is a proto-oncogene that plays an important role in a variety of diseases.There were a lot of research on the correlation between C-myc and human viruses.However,the study about C-myc related to aquatic species virus is very limited.In the present study,the qRT-PCR,cellular immunofluorescence and western blotting determination data reported that C-myc and glutaminase(GLS)genes were significantly upregulated when grouper fin cells(GF-1)were infected with red grouper nervous necrosis virus(RGNNV).After knocking down the C-myc gene,the mRNA and protein levels of GLS,capsid protein(CP)and RNA polymerase(RdRp)of RGNNV were significantly reduced in RGNNV-infected GF-1 cells and the overexpression of the C-myc gene remarkably promoted these genes,which indicated that the replication of the virus and GLS gene were positively regulated by C-myc in RGNNV-infected GF-1 cells.In addition,supplementation of exogenous ATP can partially restore viral replication when RGNNV-infected GF-1 cells were cultured in glutamine-free medium,which confirmed that the glutamine was decomposed into ATP to provide energy for viral replication.Further studies confirmed that overexpression of C-myc can increase the content of ATP in normal cells.To sum up,these data suggested that activation of C-myc gene affected viral replication by regulating GLS expression to drive glutamine dissolution.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
Autophagosome generation,development and maturation are largely dependent on Atg9a,an important member of the autophagy-related protein family.However,the potential role of fish Atg9a in viral infections is poorly und...Autophagosome generation,development and maturation are largely dependent on Atg9a,an important member of the autophagy-related protein family.However,the potential role of fish Atg9a in viral infections is poorly understood.This research involved cloning and characterizing an Atg9a homologue from the orange-spotted grouper(Epinephelus coioides),known as EcAtg9a.The ORF(open reading frame)of EcAtg9a is composed of 2583 nucleotides and encodes 860 amino acids.EcAtg9a was detected in every tissue that was analyzed,and was particularly abundant in liver,kidney,head and blood.EcAtg9a expression levels in grouper spleen(GS)cells increased following infection with SGIV and RGNNV.EcAtg9a was uniformly present in the cytoplasm,while found to co-exist with the endoplasmic reticulum(ER),Golgi apparatus,mitochondria and lysosomes.EcAtg9a suppressed the expression of interferon-related genes and factors linked to inflammation while promoting the replication of SGIV as well as RGNNV in GS cells.In addition,EcAtg9a could interact with the key molecules of the cGAS-STING pathway including EccGAS,EcTBK1,EcSTING,and even EcIRF3.EcAtg9a facilitated the upregulation of Beclin1 and LC3-Ⅱ synthesis within cells,resulting in an augmented formation of LC3 fluorescent aggregates.The level of eIF2αphosphorylation(S51)was increased in EcAtg9a-overexpressing cells,and the p53 protein moved from the cytoplasm into the nucleus.Findings may offer insight into how grouper's innate immunity works against viral infections.展开更多
基金supported by the National Natural Science Foundation of China (No. 31972834)the National Key Research and Development Program of China (No. 2018YFD0900505)
文摘Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.
基金supported by the National Key Research and Development Program of China(2022YFD2400502)the National Natural Science Foundation of China(42176103,41825013,42230409,42276127)+2 种基金the Guangdong Provincial Natural Science Foundation(2022A1515012505)the Key-Area Research and Development Program of Guangdong Province(2021B0202040002)China Agricultural Research System(CARS-47-G16)。
文摘The origin of T cells in the teleost's brain is unclear.While viewing the central nervous system(CNS)as immune privileged has been widely accepted,previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs.Here,we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper(Epinephelus coioices)through single-cell RNA-sequencing analysis.After viral CNS infection with red-spotted grouper nervous necrosis virus(RGNNV),the number of slc43a2^(+)T cells synchronously increased in the spleen and brain.During the infection tests in asplenic zebrafish(tlx1^▲zebrafish model),no increase in the number of slc43a2^(+)T cells was observed in the brain.Single-cell transcriptomic analysis indicated that slc43a2^(+)T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response.This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish.
基金jointly supported by the National Natural Science Foundation of China(31872606,31902409,31572657,U1701233).
文摘C-myc is a proto-oncogene that plays an important role in a variety of diseases.There were a lot of research on the correlation between C-myc and human viruses.However,the study about C-myc related to aquatic species virus is very limited.In the present study,the qRT-PCR,cellular immunofluorescence and western blotting determination data reported that C-myc and glutaminase(GLS)genes were significantly upregulated when grouper fin cells(GF-1)were infected with red grouper nervous necrosis virus(RGNNV).After knocking down the C-myc gene,the mRNA and protein levels of GLS,capsid protein(CP)and RNA polymerase(RdRp)of RGNNV were significantly reduced in RGNNV-infected GF-1 cells and the overexpression of the C-myc gene remarkably promoted these genes,which indicated that the replication of the virus and GLS gene were positively regulated by C-myc in RGNNV-infected GF-1 cells.In addition,supplementation of exogenous ATP can partially restore viral replication when RGNNV-infected GF-1 cells were cultured in glutamine-free medium,which confirmed that the glutamine was decomposed into ATP to provide energy for viral replication.Further studies confirmed that overexpression of C-myc can increase the content of ATP in normal cells.To sum up,these data suggested that activation of C-myc gene affected viral replication by regulating GLS expression to drive glutamine dissolution.
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funded by the National Key Research and Development Program of China(2023YFC2812100 and 2022YFD2400501)the Key-Area Research and Development Program of Guangdong Province(2021B0202040002)+2 种基金Science and Technology Project of Guangzhou(2023E04J0137)the China Agriculture Research System of MOF and MARA(CARS-47-G16)Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory(Zhuhai)(311021006).
文摘Autophagosome generation,development and maturation are largely dependent on Atg9a,an important member of the autophagy-related protein family.However,the potential role of fish Atg9a in viral infections is poorly understood.This research involved cloning and characterizing an Atg9a homologue from the orange-spotted grouper(Epinephelus coioides),known as EcAtg9a.The ORF(open reading frame)of EcAtg9a is composed of 2583 nucleotides and encodes 860 amino acids.EcAtg9a was detected in every tissue that was analyzed,and was particularly abundant in liver,kidney,head and blood.EcAtg9a expression levels in grouper spleen(GS)cells increased following infection with SGIV and RGNNV.EcAtg9a was uniformly present in the cytoplasm,while found to co-exist with the endoplasmic reticulum(ER),Golgi apparatus,mitochondria and lysosomes.EcAtg9a suppressed the expression of interferon-related genes and factors linked to inflammation while promoting the replication of SGIV as well as RGNNV in GS cells.In addition,EcAtg9a could interact with the key molecules of the cGAS-STING pathway including EccGAS,EcTBK1,EcSTING,and even EcIRF3.EcAtg9a facilitated the upregulation of Beclin1 and LC3-Ⅱ synthesis within cells,resulting in an augmented formation of LC3 fluorescent aggregates.The level of eIF2αphosphorylation(S51)was increased in EcAtg9a-overexpressing cells,and the p53 protein moved from the cytoplasm into the nucleus.Findings may offer insight into how grouper's innate immunity works against viral infections.
