Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for effi...Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection.In this study,the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus(PRRSV) infection of MARC-145 cells was investigated.Pretreatment of MARC-145 cells with methyl-β-cyclodextrin(MβCD),a drug used to deplete cholesterol from cellular membrane,significantly reduced PRRSV infection in a dose-dependent manner.This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment,suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol.Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry,especially virus attachment and release.These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.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)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 virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promot...Porcine reproductive and respiratory syndrome virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5(MDA5),a receptor that senses viral RNA.In this study,the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed,and the detailed mechanisms were explored.We found that the interaction between P62 and MDA5 is enhanced due to two factors:the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2αand the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells.As a result of these modifications,the classic P62-mediated autophagy is triggered.Additionally,porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2(CCT2),which is enhanced by PRRSV nsp3.This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination.In summary,enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways:the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2,leading to intense innate immune suppression.The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.展开更多
Porcine reproductive and respiratory syndrome（PRRS） caused by porcine reproductive and respiratory syndrome virus（PRRSV） is one of the most infectious diseases in the swine industry worldwide, causing big eco- nom...Porcine reproductive and respiratory syndrome（PRRS） caused by porcine reproductive and respiratory syndrome virus（PRRSV） is one of the most infectious diseases in the swine industry worldwide, causing big eco- nomic losses. Vaccines are major weapons against PRRSV, however, current available vaccines have several limita- tions. Developing chemical drugs as alternatives is required. On the basis of traditional medical knowledge, we pur- posely selected 15 natural products originated from Chinese herbs with anti-infectious effects. Their antiviral activi- ties were evaluated by PRRSV-indueed cytopathic effect（CPE） on MARC-145 cells and reverse transcription poly- merase chain reaction（RT-PCR） assay. Compounds ethoxysanguinarine（EOSG） and atractylodinol were found to be the hits which could significantly reduce PRRSV-associated CPE with 50% inhibited concentration（ICso） values of 7.9 and 39.4 ~tmol/L, respectively. Meanwhile, compounds ethoxysanguinarine and atractylodinol significantly de- creased mRNA expression of ORF7 gene in a dose-dependent manner. Study results suggest that compounds ethoxy- sanguinarine and atractylodinol may be useful anti-PRRSV drugs for swine industry or the hits for further lead opti- mization.展开更多
In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics...In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2(Nsp2) and glycoprotein 5(GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates – HH08, DY, and YN-2011 – were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.展开更多
The nonstructural protein 10（nsp10） of porcine reproductive and respiratory syndrome virus（PRRSV） encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10...The nonstructural protein 10（nsp10） of porcine reproductive and respiratory syndrome virus（PRRSV） encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.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(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
Porcine reproductive and respiratory syndrome(PRRS)caused by PRRS virus(PRRSV)has been regarded as a persistent challenge for the swine farms worldwide.microRNAs(miRNAs)play key roles in regulating almost every import...Porcine reproductive and respiratory syndrome(PRRS)caused by PRRS virus(PRRSV)has been regarded as a persistent challenge for the swine farms worldwide.microRNAs(miRNAs)play key roles in regulating almost every important biological process,including virus-host interaction.In this study,we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection.Subsequently,we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages(PAMs).Through bioinformatic analysis,we found that there existed a potential binding site of miR-204 on the 30UTR of microtubule associated protein 1 light chain 3B(MAP1LC3B,LC3B),a hallmark of autophagy.Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation(CLIP)assay,we demonstrated that miR-204 directly targeted LC3B,thereby downregulating autophagy.Meanwhile,we investigated the interplay between autophagy and PRRSV replication in PAMs,confirming that PRRSV infection induces autophagy,which in turn facilitates viral replication.Overall,we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs.These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.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
Porcine reproductive and respiratory syndrome(PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ(IFNγ)-mediated type Ⅰ cell-mediated immune response plays an important role in pr...Porcine reproductive and respiratory syndrome(PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ(IFNγ)-mediated type Ⅰ cell-mediated immune response plays an important role in protection from,and clearance of, PRRS virus(PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and cd T lymphocytes were previously reported to produce IFNc during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNc producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine(PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV...Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV GP4 protein monoclonal antibody(Mab)5F12 was successfully prepared.It was identified as IgG2b subclass and had better stability and specificity,which not only responded with recombinant PRRSV GP4 protein,but also with PRRSV.Phage display technique had varieties of applications,in particular,the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines.In this study,Mab-5F12 was used as the target for biopanning a 12-mer phage random peptide library.After four rounds of biopanning,two phage-displayed peptides,named P-A and P-G(AKFEVCSPVVLG and GVNQENMLHFSF)were identified that recognized Mab-5F12 specifically.Sequence analysis showed that one or more of the peptides exhibited partial sequence similarity to the native GP4 protein sequence,which corresponded to 69-80 and 84-95 aa segments of the HP-PRRSV GP4 protein.Furthermore,real-time quantitative RT-PCR and indirect immunofluorescence assay indicated consistently the abilities of P-A and P-G to block viral infection in Marc-145 cells and they could function as antiviral agents for PRRSV.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),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
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 National Natural Science Foundation of China (Grant No. 30770082)National Basic Research Program of China (Grant No. 2005CB523200)
文摘Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection.In this study,the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus(PRRSV) infection of MARC-145 cells was investigated.Pretreatment of MARC-145 cells with methyl-β-cyclodextrin(MβCD),a drug used to deplete cholesterol from cellular membrane,significantly reduced PRRSV infection in a dose-dependent manner.This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment,suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol.Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry,especially virus attachment and release.These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.
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supported by the Tianjin Synthetic Biotechnology Innovation Capability Improvement Project in China(TSBICIP-KJGG-014)the key underprop project of Tianjin Science and Technology Bureau in China(20YFZCSN00340)to Jinhai Huang。
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is a major economically devastating pathogen that has evolved various strategies to evade innate immunity.Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5(MDA5),a receptor that senses viral RNA.In this study,the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed,and the detailed mechanisms were explored.We found that the interaction between P62 and MDA5 is enhanced due to two factors:the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2αand the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells.As a result of these modifications,the classic P62-mediated autophagy is triggered.Additionally,porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2(CCT2),which is enhanced by PRRSV nsp3.This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination.In summary,enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways:the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2,leading to intense innate immune suppression.The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.
文摘Porcine reproductive and respiratory syndrome（PRRS） caused by porcine reproductive and respiratory syndrome virus（PRRSV） is one of the most infectious diseases in the swine industry worldwide, causing big eco- nomic losses. Vaccines are major weapons against PRRSV, however, current available vaccines have several limita- tions. Developing chemical drugs as alternatives is required. On the basis of traditional medical knowledge, we pur- posely selected 15 natural products originated from Chinese herbs with anti-infectious effects. Their antiviral activi- ties were evaluated by PRRSV-indueed cytopathic effect（CPE） on MARC-145 cells and reverse transcription poly- merase chain reaction（RT-PCR） assay. Compounds ethoxysanguinarine（EOSG） and atractylodinol were found to be the hits which could significantly reduce PRRSV-associated CPE with 50% inhibited concentration（ICso） values of 7.9 and 39.4 ~tmol/L, respectively. Meanwhile, compounds ethoxysanguinarine and atractylodinol significantly de- creased mRNA expression of ORF7 gene in a dose-dependent manner. Study results suggest that compounds ethoxy- sanguinarine and atractylodinol may be useful anti-PRRSV drugs for swine industry or the hits for further lead opti- mization.
基金supported by the Jilin Province Science and Technology Development Project(No.20140101123JC)the Fundamental Research Fund of Jilin Universitythe Program for Changjiang Scholars and Innovative Research Team in University(No.IRT1248)
文摘In the present study, 89 porcine reproductive and respiratory syndrome virus(PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-struc-tural protein 2(Nsp2) and glycoprotein 5(GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discon-tinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natu-ral recombination events occurred between strains. Three isolates – HH08, DY, and YN-2011 – were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolu-tionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.
基金supported by the National Key Technology R&D Program of China (2015BAD12B01-2)the Major Program of National Natural Science Foundation of China (31490603)the earmarked fund for Modern Agroindustry Technology Research System of China (CARS-36)
文摘The nonstructural protein 10（nsp10） of porcine reproductive and respiratory syndrome virus（PRRSV） encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.
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This study was supported by the National Natural Science Foundation of China(Grant No.31630076),Chinathe National Major Special Project on New Varieties Cultivation for Transgenic Organisms(grant no.2016ZX08009-003-006),China.
文摘Porcine reproductive and respiratory syndrome(PRRS)caused by PRRS virus(PRRSV)has been regarded as a persistent challenge for the swine farms worldwide.microRNAs(miRNAs)play key roles in regulating almost every important biological process,including virus-host interaction.In this study,we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection.Subsequently,we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages(PAMs).Through bioinformatic analysis,we found that there existed a potential binding site of miR-204 on the 30UTR of microtubule associated protein 1 light chain 3B(MAP1LC3B,LC3B),a hallmark of autophagy.Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation(CLIP)assay,we demonstrated that miR-204 directly targeted LC3B,thereby downregulating autophagy.Meanwhile,we investigated the interplay between autophagy and PRRSV replication in PAMs,confirming that PRRSV infection induces autophagy,which in turn facilitates viral replication.Overall,we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs.These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.
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supported by Grant from The National Natural Science Foundation of China (NSFC, Grant No. 31490601)National Key Research and Development Program (Grant No. 2016YFD0500703)+1 种基金Major Science and Technology Projects in Henan Province (Grant No. 171100110200)Luoyang Heluo Talent Plan (Dr. Kegong Tian)
文摘Porcine reproductive and respiratory syndrome(PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ(IFNγ)-mediated type Ⅰ cell-mediated immune response plays an important role in protection from,and clearance of, PRRS virus(PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and cd T lymphocytes were previously reported to produce IFNc during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNc producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine(PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests.
基金Supported by the National Natural Science Foundation of China(31372438,31200122)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)GP4 protein was prokaryotically expressed,and used as an antigen to immunize six-week-old BALB/c female mice.With conventional cell fusion method,an anti-PRRSV GP4 protein monoclonal antibody(Mab)5F12 was successfully prepared.It was identified as IgG2b subclass and had better stability and specificity,which not only responded with recombinant PRRSV GP4 protein,but also with PRRSV.Phage display technique had varieties of applications,in particular,the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines.In this study,Mab-5F12 was used as the target for biopanning a 12-mer phage random peptide library.After four rounds of biopanning,two phage-displayed peptides,named P-A and P-G(AKFEVCSPVVLG and GVNQENMLHFSF)were identified that recognized Mab-5F12 specifically.Sequence analysis showed that one or more of the peptides exhibited partial sequence similarity to the native GP4 protein sequence,which corresponded to 69-80 and 84-95 aa segments of the HP-PRRSV GP4 protein.Furthermore,real-time quantitative RT-PCR and indirect immunofluorescence assay indicated consistently the abilities of P-A and P-G to block viral infection in Marc-145 cells and they could function as antiviral agents for PRRSV.
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.
