Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regar...Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.展开更多
Porcine reproductive and respiratory syndrome(PRRS) is an important infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV), leading to significant economic losses in swine industry wor...Porcine reproductive and respiratory syndrome(PRRS) is an important infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV), leading to significant economic losses in swine industry worldwide. Although several studies have shown that PRRSV can affect the cell cycle of infected cells, it is still unclear how it manipulates the cell cycle to facilitate its proliferation. In this study, we analyzed the mRNA expression profiles of transcription factors in PRRSV-infected 3D4/21 cells by RNA-sequencing. The result shows that the expression of transcription factor DP2(TFDP2) is remarkably upregulated in PRRSV-infected cells. Further studies show that TFDP2 contributes to PRRSV proliferation and the PRRSV nucleocapsid(N) protein induces TFDP2 expression by activating C/EBPb. TFDP2 positively regulates cyclin A expression and triggers a less proportion of cells in the S phase, which contributes to PRRSV proliferation. This study proposes a novel mechanism by which PRRSV utilizes host protein to regulate the cell cycle to favor its infection. Findings from this study will help us for a better understanding of PRRSV pathogenesis.展开更多
Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time.The porcine reproductive and respiratory syn...Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time.The porcine reproductive and respiratory syndrome virus(PRRSV)is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide.A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies.In this study,we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells.Our results indicated that the labeling method had negligible effect on viral infectivity.We also observed that prior to the entry,PRRSV vibrated on the plasma membrane,and entered the cells via endosome mediated cell entry pathway.Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell.Our results also showed that once inside the cell,PRRSV moved along the microtubule,microfilament and vimentin cytoskeletal elements.During the transport process,virus particles also made contacts with non-muscle myosin heavy chainⅡ-A(NMHCⅡ-A),visualized as small spheres in cytoplasm.This study can facilitate the application of QDs in virus infection imaging,especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV) has been mutating and evolving constantly since its emergence in the1980s, which has brought inestimable economic losses to the global swine industry. The vir...Porcine reproductive and respiratory syndrome virus(PRRSV) has been mutating and evolving constantly since its emergence in the1980s, which has brought inestimable economic losses to the global swine industry. The virus has two genotypes, of which genotype 1 PRRSV(PRRSV 1) first broke out in Germany and mainly prevailed in Europe, which can be clustered into four subtypes based on the ORF5 sequence. Al-though few cases of PRRSV 1 have been reported in China, the prevention and control of PRRSV should not be ignored. The origin of PRRSV, ge-netic evolution and pathogenicity of PRRSV 1 were retrospectively analyzed, in order to provide valuable evidences for molecular epidemiology and immune prevention and control of PRRSV 1.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently, vaccination strategies provide limited pr...Porcine reproductive and respiratory syndrome virus(PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently, vaccination strategies provide limited protection against PRRSV infection, and consequently, new antiviral strategies are urgently required. Andrographolide(Andro) and its derivative potassium dehydrographolide succinate(PDS) have been used clinically in China and other Asian countries as therapies for inflammation-related diseases, including bacterial and viral infections, for decades. Here, we demonstrate that Andro and PDS exhibit robust activity against PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages(PAMs). The two compounds exhibited broad-spectrum inhibitory activities in vitro against clinically circulating type 2 PRRSV GD-HD, XH-GD, and NADC30-like HNhx strains in China. The EC_(50)values of Andro against three tested PRRSV strain infections in Marc-145 cells ranged from 11.7 to 15.3 lmol/L, with selectivity indexes ranging from 8.3 to10.8, while the EC_(50)values of PDS ranged from 57.1 to 85.4 lmol/L, with selectivity indexes ranging from 344 to 515.Mechanistically, the anti-PRRSV activity of the two compounds is closely associated with their potent suppression on NFj B activation and enhanced oxidative stress induced by PRRSV infection. Further mechanistic investigations revealed that PDS, but not Andro, is able to directly interact with PRRSV particles. Taken together, our findings suggest that Andro and PDS are promising PRRSV inhibitors in vitro and deserves further in vivo studies in swine.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV),a single-stranded RNA virus,mainly infects cells of monocyte/macrophage lineage.Recently,host microRNAs were shown to be capable of modulating PRRSV infection...Porcine reproductive and respiratory syndrome virus(PRRSV),a single-stranded RNA virus,mainly infects cells of monocyte/macrophage lineage.Recently,host microRNAs were shown to be capable of modulating PRRSV infection and replication by multiple ways such as targeting viral genomic RNA,targeting viral receptor and inducing antiviral response.MicroRNAs are small RNAs and have emerged as important regulators of virus-host cell interactions.In this review,we discuss the identified functions of host microRNAs in relation to PRRSV infection and propose that cellular microRNAs may have a substantial effect on cell or tissue tropism of PRRSV.展开更多
Currently, various porcine reproductive and respiratory syndrome virus(PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing the difficulty of PRRS control. In this stu...Currently, various porcine reproductive and respiratory syndrome virus(PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing the difficulty of PRRS control. In this study, a PRRSV strain named HBap4-2018 was isolated from swine herds suffering severe respiratory disease with high morbidity in Hebei Province of China in 2018. The genome of HBap4-2018 is 15,003 nucleotides in length, and compared with NADC30-like PRRSV, nsp2 of HBap4-2018 has an additional continuous deletion of five amino acids. Phylogenetic analysis based on complete genome and ORF5 showed that HBap4-2018 belonged to lineage 8 of PRRSV-2, which was characterized by highly variable genome. However, HBap4-2018 was classified into lineage 1 based on phylogenetic analysis of nsp2,sharing higher amino acid homology(85.3%–85.5%) with NADC30-like PRRSV. Further analysis suggested that HBap4-2018 was a novel natural recombinant PRRSV with three recombinant fragments in the genome, of which highly pathogenic PRRSV(HP-PRRSV) served as the major parental strains, while NADC30-like PRRSV served as the minor parental strains. Five recombination break points were identified in nsp2, nsp3, nsp5, nsp9 and ORF6, respectively,presenting a novel recombinant pattern in the genome. Piglets inoculated with HBap4-2018 presented typical clinical signs with a mortality rate of 60%. High levels of viremia and obvious macroscopic and histopathological lesions in the lungs were observed, revealing the high pathogenicity of HBap4-2018 in piglets.展开更多
Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degrada...Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation,making them difficult to transform into clinical applications.In this article,an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation.This system worked via two interconnected parts:1)the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure;2)the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation.Experimental results based on model protein(Green Fluorescent Protein,GFP)validated that this system could fulfill protein degradation both in vitro(both Hela and 293T cells)and in vivo(by upconversion optogenetic nanosystem),and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time(0–25 h)and the illumination frequency(the illuminating frequency of 0–30 s every 1 min).We further took another functional protein(The Nonstructural Protein 9,NSP9)into experiment.Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)was inhibited by degrading the NSP9 in this light-induced system,and PRRSV proliferation was affected by different light conditions(illumination time varies from 0–24 h).We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.展开更多
基金supported by the Major Program of National Natural Science Foundation of China (31490603, 31572549)the National Key Technology R & D Program of China (2015BAD12B01-2)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.
基金This work was supported by the National Key Research and Development Program of China(2018YFD0500500)the National Natural Science Foundation of China(31272540)。
文摘Porcine reproductive and respiratory syndrome(PRRS) is an important infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV), leading to significant economic losses in swine industry worldwide. Although several studies have shown that PRRSV can affect the cell cycle of infected cells, it is still unclear how it manipulates the cell cycle to facilitate its proliferation. In this study, we analyzed the mRNA expression profiles of transcription factors in PRRSV-infected 3D4/21 cells by RNA-sequencing. The result shows that the expression of transcription factor DP2(TFDP2) is remarkably upregulated in PRRSV-infected cells. Further studies show that TFDP2 contributes to PRRSV proliferation and the PRRSV nucleocapsid(N) protein induces TFDP2 expression by activating C/EBPb. TFDP2 positively regulates cyclin A expression and triggers a less proportion of cells in the S phase, which contributes to PRRSV proliferation. This study proposes a novel mechanism by which PRRSV utilizes host protein to regulate the cell cycle to favor its infection. Findings from this study will help us for a better understanding of PRRSV pathogenesis.
基金support from the National Natural Science Foundation of China(Grant Nos.31570151 and 31490601)the Program for Science and Technology Innovation Talents in Universities of Henan Province(Grant No.17HASTIT039)+1 种基金the Key Scientific Research Project of Henan Province Higher Education(16A180044)the Open Research Fund Program of the State Key Laboratory of Virology of China(Grant No.2017KF005)。
文摘Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time.The porcine reproductive and respiratory syndrome virus(PRRSV)is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide.A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies.In this study,we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells.Our results indicated that the labeling method had negligible effect on viral infectivity.We also observed that prior to the entry,PRRSV vibrated on the plasma membrane,and entered the cells via endosome mediated cell entry pathway.Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell.Our results also showed that once inside the cell,PRRSV moved along the microtubule,microfilament and vimentin cytoskeletal elements.During the transport process,virus particles also made contacts with non-muscle myosin heavy chainⅡ-A(NMHCⅡ-A),visualized as small spheres in cytoplasm.This study can facilitate the application of QDs in virus infection imaging,especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.
基金Supported by Youth Fund Project of Natural Science Foundation of Jiangsu Province(BK20201005)School-Enterprise Cooperation Project of Jiangsu Vocational College of Agriculture and Forestry(2020kj021)。
文摘Porcine reproductive and respiratory syndrome virus(PRRSV) has been mutating and evolving constantly since its emergence in the1980s, which has brought inestimable economic losses to the global swine industry. The virus has two genotypes, of which genotype 1 PRRSV(PRRSV 1) first broke out in Germany and mainly prevailed in Europe, which can be clustered into four subtypes based on the ORF5 sequence. Al-though few cases of PRRSV 1 have been reported in China, the prevention and control of PRRSV should not be ignored. The origin of PRRSV, ge-netic evolution and pathogenicity of PRRSV 1 were retrospectively analyzed, in order to provide valuable evidences for molecular epidemiology and immune prevention and control of PRRSV 1.
基金This work was funded by the National Key Research and Development Program of China(Grant 2017YFD0501404)the National Natural Science Foundation of China(Grant 31872521)+1 种基金the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(Grant 2019BT02N054)the Basic Research&Applying Basic Research Foundation of Guangdong Province(Grant 2019B1515210007)。
文摘Porcine reproductive and respiratory syndrome virus(PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently, vaccination strategies provide limited protection against PRRSV infection, and consequently, new antiviral strategies are urgently required. Andrographolide(Andro) and its derivative potassium dehydrographolide succinate(PDS) have been used clinically in China and other Asian countries as therapies for inflammation-related diseases, including bacterial and viral infections, for decades. Here, we demonstrate that Andro and PDS exhibit robust activity against PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages(PAMs). The two compounds exhibited broad-spectrum inhibitory activities in vitro against clinically circulating type 2 PRRSV GD-HD, XH-GD, and NADC30-like HNhx strains in China. The EC_(50)values of Andro against three tested PRRSV strain infections in Marc-145 cells ranged from 11.7 to 15.3 lmol/L, with selectivity indexes ranging from 8.3 to10.8, while the EC_(50)values of PDS ranged from 57.1 to 85.4 lmol/L, with selectivity indexes ranging from 344 to 515.Mechanistically, the anti-PRRSV activity of the two compounds is closely associated with their potent suppression on NFj B activation and enhanced oxidative stress induced by PRRSV infection. Further mechanistic investigations revealed that PDS, but not Andro, is able to directly interact with PRRSV particles. Taken together, our findings suggest that Andro and PDS are promising PRRSV inhibitors in vitro and deserves further in vivo studies in swine.
基金The support of the State Key Laboratory of Agrobiotechnology(2010SKLAB06-1 and 2012SKLAB01-6)the Research Fund for the Doctoral Program of Higher Education of China(20130008110028)is gratefully acknowledged.
文摘Porcine reproductive and respiratory syndrome virus(PRRSV),a single-stranded RNA virus,mainly infects cells of monocyte/macrophage lineage.Recently,host microRNAs were shown to be capable of modulating PRRSV infection and replication by multiple ways such as targeting viral genomic RNA,targeting viral receptor and inducing antiviral response.MicroRNAs are small RNAs and have emerged as important regulators of virus-host cell interactions.In this review,we discuss the identified functions of host microRNAs in relation to PRRSV infection and propose that cellular microRNAs may have a substantial effect on cell or tissue tropism of PRRSV.
基金The siudy was supported by the Shanghai Municipal Agriculture Science and Technology Project(2020-02-0800-08-F01465)the National Natural Science Foundation of China(32072861)+1 种基金the Natural Science Foundation of Shanghai(20ZR1469600)the earmarked fund for Modern Agro-industry Technology Research System of China(CARS-35)。
文摘Currently, various porcine reproductive and respiratory syndrome virus(PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing the difficulty of PRRS control. In this study, a PRRSV strain named HBap4-2018 was isolated from swine herds suffering severe respiratory disease with high morbidity in Hebei Province of China in 2018. The genome of HBap4-2018 is 15,003 nucleotides in length, and compared with NADC30-like PRRSV, nsp2 of HBap4-2018 has an additional continuous deletion of five amino acids. Phylogenetic analysis based on complete genome and ORF5 showed that HBap4-2018 belonged to lineage 8 of PRRSV-2, which was characterized by highly variable genome. However, HBap4-2018 was classified into lineage 1 based on phylogenetic analysis of nsp2,sharing higher amino acid homology(85.3%–85.5%) with NADC30-like PRRSV. Further analysis suggested that HBap4-2018 was a novel natural recombinant PRRSV with three recombinant fragments in the genome, of which highly pathogenic PRRSV(HP-PRRSV) served as the major parental strains, while NADC30-like PRRSV served as the minor parental strains. Five recombination break points were identified in nsp2, nsp3, nsp5, nsp9 and ORF6, respectively,presenting a novel recombinant pattern in the genome. Piglets inoculated with HBap4-2018 presented typical clinical signs with a mortality rate of 60%. High levels of viremia and obvious macroscopic and histopathological lesions in the lungs were observed, revealing the high pathogenicity of HBap4-2018 in piglets.
基金This work was sponsored by the National Key Research and Development Program of China(Nos.2019YFA0906500 and 2017YFA0205104)the National Natural Science Foundation of China(Nos.31971300,817719709,51873150 and 51573128)Tianjin Natural Science Foundation(No.19JCYBJC28800)and Young Elite Scientists Sponsorship Program by Tianjin.
文摘Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation,making them difficult to transform into clinical applications.In this article,an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation.This system worked via two interconnected parts:1)the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure;2)the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation.Experimental results based on model protein(Green Fluorescent Protein,GFP)validated that this system could fulfill protein degradation both in vitro(both Hela and 293T cells)and in vivo(by upconversion optogenetic nanosystem),and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time(0–25 h)and the illumination frequency(the illuminating frequency of 0–30 s every 1 min).We further took another functional protein(The Nonstructural Protein 9,NSP9)into experiment.Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)was inhibited by degrading the NSP9 in this light-induced system,and PRRSV proliferation was affected by different light conditions(illumination time varies from 0–24 h).We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.
