首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 500 毫秒
1.
Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation.  相似文献   

2.
Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. Antimicrobial peptides (AMPs) play an important role of innate immunity either against bacterial pathogens or viruses. Up to date, little is known if any AMP could effectively inhibit fish nodaviruses and its mechanism. In this study, the antiviral activities of three antimicrobial peptides (AMPs) against grouper NNV (GNNV) were screened in the fish cell line. Two of the three AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able to agglutinate purified NNV particles into clump, and the clumps were further confirmed to be viral proteins by TEM and Western blot. The NNV solution, separately pre-mixed with AMP (TH 1-5 or cSALF) or deionized-distilled water for 1 h, was used to infect GF-1 cells, and the levels of capsid protein in the GNNV-AMP-infected cells at 1 h post infection were much lower than that in the GNNV-H2O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infected the cells. Treatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression; however, grouper epinecidin-1 (CP643-1) could induce the expression of Mx in the pre-treated cBB cells. This study revealed three AMPs with anti-NNV activity through two different mechanisms, and shed light on the future application in aquaculture.  相似文献   

3.
We obtained a full-length cDNA clone for the Mx gene of barramundi (Lates calcarifer), using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from a barramundi brain cell line cBB. The Mx cDNA of 2.2kb contains an open reading frame (ORF) of 1875 nucleotides encoding a protein of 624 amino acids. The predicted barramundi Mx protein is 71.4 kDa and contains a tripartite guanosinetriphosphate (GTP)-binding motif at the amino terminal and a leucine zipper at the carboxyl terminal, characteristic of all known Mx proteins. Poly I:C-transfection induced the expression of Mx gene in cBB cells, and the induction level at 28 degrees C was higher than that at 20 degrees C. Moreover, Mx gene expression was also induced by viral infection, including fish nodavirus, birnavirus, and iridovirus. Among these, nodavirus was a stronger inducer than the other two viruses. Using an antiviral activity assay, we revealed that poly I:C-transfected cBB cells had antiviral activity against fish nodavirus and birnavirus, but not iridovirus. Furthermore, the replication of nodavirus and birnavirus could be restored after the expression of Mx gene was down-regulated by siRNA. Therefore, these results indicated that the expression of barramundi Mx gene was able to inhibit the proliferation of fish nodavirus and birnavirus.  相似文献   

4.
Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.  相似文献   

5.

Background

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.

Methodology/Principal Findings

Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.

Conclusions/Significance

The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.  相似文献   

6.
干扰素诱导的鱼类Mx蛋白   总被引:2,自引:0,他引:2  
Mx蛋白是干扰素诱导表达的蛋白家族中的成员,当机体和细胞受病毒感染或诱生剂处理时产生。Mx蛋白和其它干扰素诱导蛋白一起构成宿主细胞的抗病毒状态,以达到抗病毒的目的。研究表明,Mx蛋白具有抗病毒活性,还可能与其它基本生命活动如发育或分化,蛋白质分送和生长有关。在鱼类也发现多种Mx蛋白,具有Mx蛋白家族的共有特征;在肽链末端有一个三联ATP/GTP结合区和发动蛋白家族的结构特征序列;在蛋白C端存在使Mx蛋白形成三聚体的Leu拉链结构以及定位信号。但是迄今没有发现鱼类Mx蛋白的抗病毒活性。文章最后对目前鱼类病毒病的防治及利用抗病毒基因进行鱼类基因工程抗病毒育种进行了探讨。  相似文献   

7.
Li B  Fu D  Zhang Y  Xu Q  Ni L  Chang G  Zheng M  Gao B  Sun H  Chen G 《Molecular biology reports》2012,39(8):8415-8424
Conflicting data existed for the antiviral potential of the chicken Mx protein and the importance of the Asn631 polymorphism in determination of the antiviral activity. In this study we modified the chicken Mx cDNA from the Ser631 to Asn631 genotype and transfected them into COS-I cells, chicken embryonic fibroblast (CEF) or NIH 3T3 cells. The Mx protein was mainly located at the cytoplasm. The transfected cell cultures were challenged with newcastle disease virus (NDV) or vesicular stomatitis virus (VSV), cytopathic affect (CPE) inhibition assay showed that the times for development of visible and full CPE were significantly postponed by the Asn631 cDNA transfection at 48 h transfection, but not by the Ser631 cDNA transfection. Viral titration assay showed that the virus titers were significantly reduced before 72 h postinfection. CEF cells was incubated by the cell lysates extracted from the COS-I cells transfected with pcDNA-Mx/Asn631, could resist and delayed NDV infection. These data suggested the importance of the Asn631 polymorphism of the chicken Mx in determination of the antiviral activities against NDV and VSV at early stage of viral infection, which were relatively weak and not sufficient to inhibit the viral replication at late stage of viral infection.  相似文献   

8.
Chen W  Cao W  Zhao H  Hu Q  Qu L  Hu S  Ge J  Wen Z  Wang X  Li H  Huang K  Bu Z 《Cytokine》2011,54(3):324-329
A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-β has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-β fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-β.  相似文献   

9.
10.
O Haller  M Frese  D Rost  P A Nuttall    G Kochs 《Journal of virology》1995,69(4):2596-2601
We show that tick-transmitted Thogoto virus is sensitive to interferon-induced nuclear Mx1 protein, which is known for its specific antiviral action against orthomyxoviruses. Influenza virus-susceptible BALB/c mice (lacking a functional Mx1 gene) developed severe disease symptoms and died within days after intracerebral or intraperitoneal infection with a lethal challenge dose of Thogoto virus. In contrast, Mx1-positive congenic, influenza virus-resistant BALB.A2G-Mx1 mice remained healthy and survived. Likewise, A2G, congenic B6.A2G-Mx1 and CBA.T9-Mx1 mice (derived from influenza virus-resistant wild mice) as well as Mx1-transgenic 979 mice proved to be resistant. Peritoneal macrophages and interferon-treated embryo cells from resistant mice exhibited the same resistance phenotype in vitro. Moreover, stable lines of transfected mouse 3T3 cells that constitutively express Mx1 protein showed increased resistance to Thogoto virus infection. We conclude that an Mx1-sensitive step has been conserved during evolution of orthomyxoviruses and suggest that the Mx1 gene in rodents may serve to combat infections by influenza virus-like arboviruses.  相似文献   

11.
12.
Interferon (IFN) plays crucial roles in innate immune responses against viral infections. In the present study, we report cloning and characterization of the IFN gene from the sevenband grouper (Epinephelus septemfasciatus), and the anti-viral effects of its recombinant IFN protein in vivo. The isolated cDNA from sevenband grouper IFN encoded a protein consisting of 178 amino acids, and its first 22 amino acids represented a putative signal peptide. We named the identified sevenband grouper IFN gene as SgIFNa1 based on the result from phylogenetic analysis that categorized the deduced protein sequence into fish IFNa family. The expression of SgIFNa1 mRNA in the head kidney cells was induced by synthetic Poly(I:C), which is known as an inducer of IFN. It has also been confirmed that injection of recombinant SgIFNa1 protein (rSgIFNa1) upregulates expression of the Mx gene, which is known as an IFN-responsive gene, in head kidney cells. Moreover, we observed that preliminarily injection of rSgIFNa1 provided significant protection against a lethal challenge of nervous necrosis virus (NNV), which is a serious disease of sevenband grouper. These results demonstrate that SgIFNa1 has anti-viral activity and the administration of rSgIFNa1 to sevenband grouper is effective in preventing severe symptom development after NNV infection.  相似文献   

13.
Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.  相似文献   

14.
Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.  相似文献   

15.
Influenza A virus and various single-stranded RNA viruses have been reported to be blocked by IFN-stimulated Mx protein. Here we present a mathematical model of regulation of mouse Mx1 protein induction and action under influenza infection. Parameter estimates are derived from published experimental data. Numerical solutions of the model equations completely correspond to experimental data. The model is used to analyse the role of virus- and interferon-mediated expression of Mx1 in maintenance of antiviral state. The study suggests that virus- and IFN-induced Mx1 proteins act on different stages of intracellular ontogenesis of influenza virus and these actions result in different efficacy of cell protection. The model demonstrates that the synergistic action of inteferon and virus in regulation of Mx1 gene expression is the important factor of antiviral resistance. The results of simulation permit to assume that the active form of Mx1 protein is trimer.  相似文献   

16.
Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.  相似文献   

17.
Ebola hemorrhagic fever is a rapidly progressing acute febrile illness characterized by high virus replication, severe immunosuppression, and case fatalities of ca. 80%. Inhibition of phosphorylation of interferon regulatory factor 3 (IRF-3) by the Ebola VP35 protein may block the host innate immune response and play an important role in the severity of disease. We used two precisely defined reverse genetics-generated Ebola viruses to investigate global host cell responses resulting from the inhibition of IRF-3 phosphorylation. The two viruses encoded either wild-type (WT) VP35 protein (recEbo-VP35/WT) or VP35 with an arginine (R)-to-alanine (A) amino acid substitution at position 312 (recEbo-VP35/R312A) within a previously defined IRF-3 inhibitory domain. When sucrose-gradient purified virus was used for infection, host cell whole-genome expression profiling revealed striking differences in human liver cell responses to these viruses differing by a single amino acid. The inhibition of host innate immune responses by WT Ebola virus was so potent that little difference in interferon and antiviral gene expression could be discerned between cells infected with purified WT, inactivated virus, or mock-infected cells. However, infection with recEbo-VP35/R312A virus resulted in a strong innate immune response including increased expression of MDA-5, RIG-I, RANTES, MCP-1, ISG-15, ISG-54, ISG-56, ISG-60, STAT1, IRF-9, OAS, and Mx1. The clear gene expression differences were obscured if unpurified virus stocks were used to initiate infection, presumably due to soluble factors present in virus-infected cell supernatant preparations. Ebola virus VP35 protein clearly plays a pivotal role in the potent inhibition of the host innate immune responses, and the present study indicates that VP35 has a wider effect on host cell responses than previously shown. The ability to eliminate this inhibitory effect with a single amino acid change in VP35 demonstrates the critical role this protein must play in the severe aspects this highly fatal disease.  相似文献   

18.
19.
20.
Over sub-culturing a cell line generates a selective pressure which can result in key cellular functions being altered such as gene and protein expression. The present study set out to determine whether serial sub-culturing affects the antiviral state of the Salmon Head Kidney (SHK-1) cell line. Cells were cultured under constant conditions and real-time PCR was performed to measure the level of interferon (IFN) and Mx gene expression over different passage numbers. A significant increase in the basal level of IFN and Mx gene expression was recorded at passage number 58 (3 and 14-fold increase versus passage number 53), suggesting a sub-culturing effect on the type I IFN response in SHK-1 cells. Passage dependent variations in morphology and cell sub-populations have been previously observed in SHK-1 cells. Such variations in cell sub-types were suspected to be responsible for the fluctuations in IFN and Mx gene expression recorded in this study.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号