细胞自噬与病毒复制

细胞自噬是真核生物中一种高度保守的细胞内容物降解过程,在维持细胞的内环境稳定中起着重要作用。同时,自噬参与固有免疫系统对病原微生物的识别,以帮助吞噬细胞进行有效的吞噬作用并清除细胞内外的病原体。而病毒,尤其是RNA病毒,具有快速进化以应对宿主细胞中的变化的能力,能通过利用或抑制宿主细胞的自噬作用来为自身的复制服务。因此,针对自噬途径的药物筛选和治疗策略越来越成为抗病毒研究的热点。     

Lipoprotein Inhibitor of Newcastle Disease Virus from Chicken Lung

A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 mug/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Protein Synthesis in Newcastle Disease Virus-Infected Chicken Embryo Cells

A double-isotopic label difference analysis of polyacrylamide gels has been used to distinguish between cellular and viral protein accumulation in infected cells and to quantify the kinetics of accumulation of viral polypeptides. This technique, coupled with the determination of total radioactive amino acid incorporation in infected cultures, has revealed the following kinetic patterns. Viral polypeptides are first detected in infected cultures 2.0 to 2.5 h postinfection. The rate of accumulation of radioactive amino acids in viral polypeptides increases to a maximum (30 to 35% of the rate of accumulation in uninfected control cultures), whereas the rate of accumulation of radioactive amino acids in host-cell protein decreases to a minimum (20% of the rate of accumulation in uninfected control cultures) by 5 to 6 h postinfection. All of the viral polypeptides detected late in infection are also present at the earlier times, and the major virion structural polypeptides are present in approximately the same (N/G-2, 53K) or slightly increasing (L, G-1, M) relative amounts. One peak area containing a nonstructural glycopeptide with an apparent molecular weight of 66,000 shows significant alterations in rates of accumulation during infection. Inhibition in the rate of radioactive amino acid incorporation into both trichloroacetic acid-soluble and acid-precipitable material during infection has been demonstrated. However, these two inhibition phenomena can be uncoupled temporally by incubating infected cultures at 36 C instead of the usual 40 C, suggesting that they may not be directly related.     

West Nile Virus (WNV) Replication Is Independent of Autophagy in Mammalian Cells

Autophagy is a homeostatic process responsible for recycling cytosolic proteins and organelles. Moreover, this pathway contributes to the cell’s intrinsic innate defenses. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. Here, we have investigated the role of autophagy in West Nile virus (WNV) replication. Experiments in cell lines derived from a variety of sources, including the kidney, liver, skin, and brain, indicated that WNV replication does not upregulate the autophagy pathway. Furthermore, WNV infection did not inhibit rapamycin-induced autophagy, suggesting that WNV does not disrupt the authophagy signaling cascade. Perturbation of the autophagy pathway by depletion of the major autophagy factors Atg5 or Atg7 had no effect on WNV infectious particle production, indicating that WNV does not require a functional autophagy pathway for replication. Taken together, the results of our study provide evidence that WNV, unlike several other viruses of the family Flaviviridae, does not significantly interact with the conventional autophagy pathway in mammalian cells.     

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.Virulent strains of Newcastle disease virus (NDV) infect domestic poultry and other birds, causing a rapidly spreading viral disease that affects the alimentary and respiratory tracts as well as the central nervous system (55). In humans, however, NDV is well tolerated (17, 18). Other than mild fever for a day, only a few adverse effects have been reported. NDV, also known as avian paramyxovirus 1, is an enveloped virus containing a negative-sense, single-stranded RNA genome which codes for six proteins in the order (from 3′ to 5′) of nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large polymerase protein (19). There are many different strains of NDV, classified as either lytic or nonlytic for different types of cells. Lytic and nonlytic NDV strains both replicate much more efficiently in human cancer cells than they do in most normal human cells (43). Viruses of both strain types have been investigated as potential anticancer agents (30, 49, 52). The NDV strains that have been evaluated most widely for the treatment of cancer are 73-T, MTH-68, and Ulster (1, 7, 11, 17, 18, 53, 54, 56, 71).Initial binding of NDV to a host cell takes place through the interaction of HN molecules in the virus coat with sialic acid-containing molecules on the cell surface (31). NDV neuraminidase has strict specificity for the hydrolysis of the NeuAc-α2,3-Gal linkage, with no hydrolysis of the NeuAc-α2,6-Gal linkage (41).NDV infection of tumor cells not only improves T-cell responses (53, 58, 68), but has also been reported to vigorously stimulate innate immune responses. In the course of NDV infection, large amounts of alpha interferon (IFN-α) are released (68) and in turn activate dendritic cells and NK cells and polarize, in concert with interleukin-12 (IL-12), toward a Th1 T-cell response (33, 44, 47). In addition, NDV induces antitumor cytotoxicity in murine macrophages which produce increased amounts of tumor necrosis factor alpha (TNF-α) and nitric oxide (51, 60) and in human monocytes through the induction of TRAIL (64). Little is known about the NDV-mediated activation of NK cells. The coincubation of peripheral blood mononuclear cells with NDV was shown previously to stimulate NK-mediated cytotoxicity (70). Enhanced cytotoxicity correlates with the induction of IFN-α (70). It is not known, however, whether NDV-infected cells can directly activate NK cells and, if so, which molecular interactions are involved.The cytolytic activity of NK cells against virus-infected or tumor cells is regulated by the engagement of activating or inhibitory NK cell surface receptors, the actions of cytokines, and cross talk with other immune cells (32, 39). Most inhibitory receptors recognize particular major histocompatibility complex (MHC) class I alleles and thereby ensure the tolerance of NK cells against self antigens (38). Activating receptors on human NK cells include CD16; NKG2D; the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46; as well as NKp80; DNAM-1; and various stimulatory coreceptors (32).NCR are important activating receptors for the antitumor and antiviral activities of NK cells (5, 32, 37). Heparan sulfate has been discussed previously as a cellular ligand for NKp46, NKp44, and NKp30 (9, 26, 27), and nuclear factor BAT3, which can be released from tumor cells under stress conditions, has been described as a cellular ligand for NKp30 (42). Ligands for NKp30 and NKp44 can be detected on the surfaces and in the intracellular compartments of several kinds of tumor cells (10). Moreover, a number of pathogen-derived NCR ligands have been reported. The hemagglutinin protein of influenza virus and the HN of Sendai virus can bind to NKp46 and NKp44 and activate NK cells (3, 24, 34). The pp65 protein of human cytomegalovirus has been shown to bind NKp30 and inhibit its function (4). Human immunodeficiency virus, vaccinia virus, and herpes simplex virus have also been shown to upregulate the expression of cellular NCR ligands in infected cells (13, 14, 62). The Plasmodium falciparum erythrocyte membrane protein 1 is involved in the NCR-mediated NK cell attack against infected erythrocytes (36). Furthermore, NKp46 recognizes cells infected with mycobacteria (22, 61), and NKp44 was recently reported to directly bind to the surfaces of mycobacteria and other bacteria (21).In this study, we investigated the expression of ligand structures for NCR and NKG2D on NDV-infected cells. We demonstrate that NDV HN proteins which are strongly expressed on NDV-infected tumor cells function as activating ligand structures for NKp44 and NKp46 but that cellular ligands for NKG2D are partially downregulated during NDV infection.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Effects of Naturally Occurring Six- and Twelve-Nucleotide Inserts on Newcastle Disease Virus Replication and Pathogenesis

Newcastle disease virus (NDV) isolates contain genomes of 15,186, 15,192 or 15,198 nucleotides (nt). The length differences reflect a 6-nt insert in the 5′ (downstream) non-translated region (NTR) of the N gene (15,192-nt genome) or a 12-nt insert in the ORF encoding the P and V proteins (causing a 4-amino acid insert; 15,198-nt genome). We evaluated the role of these inserts in the N and P genes on viral replication and pathogenicity by inserting them into genomes of two NDV strains that have natural genome lengths of 15,186 nt and represent two different pathotypes, namely the mesogenic strain Beaudette C (BC) and the velogenic strain GB Texas (GBT). Our results showed that the 6-nt and 12-nt inserts did not detectably affect N gene expression or P protein function. The inserts had no effect on the replication or virulence of the highly virulent GBT strain but showed modest degree of attenuation in mesogenic strain BC. We also deleted a naturally-occurring 6-nt insertion in the N gene from a highly virulent 15,192-nt genome-length virus, strain Banjarmasin. This resulted in reduced replication in vitro and reduced virulence in vivo. Thus, although these inserts had no evident effect on gene expression, protein function, or replication in vivo, they did affect virulence in two of the three tested strains.     

Hemolytic Interaction of Newcastle Disease Virus and Chicken Erythrocytes. II. Determining Factors

Standard procedures have been described for measuring paramyxovirus-induced hemolysis. The choice of these procedures is based on the analysis of the behavior of eight different strains of Newcastle disease virus. Significant strain-specific differences in hemolytic activity have been found. The presence of at least two kinds of inhibitors of hemolysis in virus preparations necessitates the use of purified virus when comparisons of hemolytic activities are to be made. In addition, it has been stressed that both hemagglutination titers and hemolysis determinations provide only relative values. Thus, quantitative comparisons can be made only with results obtained on the same day and with the same erythrocyte preparation.     

Comparison of RNA Polymerase Associated With Newcastle Disease Virus and a Temperature-Sensitive Mutant of Newcastle Disease Virus Isolated from Persistently Infected L Cells

An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo) and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.     

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress,Autophagy, and Apoptosis

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.     

Hemolytic Interaction of Newcastle Disease Virus and Chicken Erythrocytes. I. Quantitative Comparison Procedure

The extent to which erythrocytes are hemolyzed by Newcastle disease virus is a function of the relative concentrations of both virus and erythrocytes. Under proper conditions, the interaction of a single virus particle with an erythrocyte is sufficient to cause lysis. The extent of hemolysis is directly proportional to virus concentration only when the virus-erythrocyte ratio is very low. At the higher virus-erythrocyte ratios usually employed in hemolysis experiments, the extent of hemolysis is proportional to the logarithm of the virus concentration. Thus, quantitative comparisons of hemolytic activities of different virus preparations cannot be made by directly comparing the extent of hemolysis. Relative hemolytic activities must be determined by comparing virus concentrations which yield equivalent amounts of hemolysis (the quantitative comparison procedure).     

Use-Dilution Test and Newcastle Disease Virus

The use-dilution test for evaluating the effectiveness of disinfectants against bacteria was modified to determine the effectiveness of disinfectants against a group of viruses. Modifications were kept to a minimum to retain the general principles of the test and thereby retain the test's familiarity among testing laboratory personnel. Modifications included the use of a standard allantoic fluid suspension of Newcastle disease virus instead of a standard bacterial culture. The only other modification was the inoculation of six embryonated chicken eggs (10 to 12 days old) with 0.1 ml of nutrient broth into which a carrier ring was transferred after a standard period in diluted disinfectant. The death or survival of 60 embryos, then, is the criterion by which a disinfectant can be judged effective at use-dilution. Experiments are described which establish the validity of the modified test procedure. The effectiveness of nine common disinfectants against Newcastle disease virus as judged by this test procedure is reported.     

新城疫病毒7793 HN蛋白在昆虫SF9细胞中的表达研究

本实验对新城疫病毒(newcastle disease virus,NDV) 7793 HN蛋白在杆状病毒表达系统中表达进行研究。提取病毒RNA并将其逆转录成cDNA,经PCR同义点突变在HN基因片段中加入NcoⅠ/XhoⅠ酶切位点,通过该酶切位点将HN基因克隆至穿梭载体p FastBac1质粒以构建重组质粒pFastBac1HTB-HN,然后用脂质体转染pFastBac1HTB-HN杆粒至昆虫SF9细胞。28℃无菌培养含pFastBac1HTB-HN杆粒的SF9细胞48 h,收集细胞培养上清液中的第一代病毒,感染SF9细胞,置28℃无菌培养60 h,收集细胞培养上清液,离心去除细胞碎片,取上清液中的第二代病毒,继续感染SF9细胞,置28℃无菌培养72 h,收集SF9细胞,用SDSPAGE和Western blotting验证HN蛋白的表达。SDS-PAGE和Western blotting均显示HN杆粒感染的SF9细胞成功表达了HN蛋白。本研究结果为进一步研究NDV7793-HN蛋白的抗肿瘤作用提供可靠试验依据。     

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.Newcastle disease virus (NDV) is an avian pathogen of the genus Avulavirus in the family Paramyxoviridae (10). The envelope of NDV contains two surface glycoproteins, the fusion (F) protein and the HN (hemagglutinin-neuraminidase [NA]) protein. The F protein mediates viral penetration and requires cleavage-activation by host protease. Cleavability of the F protein is a major determinant of virulence. However, other viral proteins, including HN, also contribute to virulence (5). HN is a multifunctional glycoprotein. It recognizes sialic acid-containing receptors on cell surfaces; promotes the fusion activity of F protein, thereby allowing the virus to penetrate the cell surface; and acts as an NA that removes sialic acid from progeny virus particles to prevent viral self-aggregation (9).HN is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains (14). The ectodomain of the HN protein consists of a 95-amino-acid stalk region supporting a 428-amino-acid terminal globular head. Although mutations in the transmembrane and stalk regions of the HN protein can affect the structure and activities of the protein (11, 15), the antigenic, receptor recognition, and NA active sites are all localized in the globular head (12, 16). The X-ray crystal structure of the globular head of the NDV HN protein has identified residues that appear to contribute to receptor recognition, NA, and fusion activities (4). Previous studies have proposed that conserved residues R174, I175, D198, K236, R416, R498, Y526, and E547 are important in receptor recognition and NA activities and that residues R174 and E547 influence the fusion promotion activity of the HN protein (3, 4, 6). Although transfection studies using plasmids expressing HN mutants of NDV have highlighted the importance of these residues in different biological functions of the HN protein, their contribution to NDV biology and pathogenesis in the context of the complete virus was not known.In this study, we examined the roles of three of the above-named conserved residues, R416, R498, and Y526 (all located near the sialic acid binding site), in the biological activities and pathogenesis of the HN protein of NDV in the context of infectious virus. In addition, comparison of the HN protein sequence between the avirulent strain LaSota and the moderately virulent strain Beaudette C (BC) identified 12 amino acid differences in the globular head region of the HN protein (H203, T214, I219, S228, L269, A271, E293, G310, S494, E495, T502, and N568, named according to the BC amino acid assignment). We also examined five of these nonconserved residues, T214, I219, S494, E495, and N568, located in close proximity to residues identified earlier by crystal structure studies, to determine whether these might affect HN function and contribute to the difference in pathogenicity between the LaSota and BC strains (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Three-dimensional structure of the NDV HN protein showing the positions of amino acid residues that were substituted in the present study. The residues are shown in space-filling mode and represented in different colors. The MacPymol (DeLano Scientific) software was used to generate the model of the globular domain of the NDV HN monomer. The structure was derived from the crystal structure of the NDV HN protein reported by Crennell et al. (4).We used site-directed mutagenesis (2) to introduce individual amino acid substitutions into a cDNA of the HN gene of strain BC. For the conserved residues, we changed arginine at positions 416 and 498 and tyrosine at position 526 to polar glutamine. For the nonconserved residues, the assignments T214, I219, S494, E495, and N568 of strain BC were altered to the corresponding assignments of strain LaSota: S214, V219, G494, V495, and D568, respectively. Each mutagenized HN gene was then inserted into a full-length cDNA clone of the BC antigenome. These clones were transfected into HEp2 cells, and mutant viruses were recovered as previously described (8). These viruses were designated according to the substitutions introduced: T214S, I219V, R416Q, S494G, E495V, R498Q, Y526Q, and N568D. The HN genes from recovered viruses were sequenced. This confirmed the presence of each introduced mutation and the lack of adventitious mutations in the HN gene. To determine the stability of each HN mutation, the recovered viruses were passaged five times in 9-day-old embryonated chicken eggs and five times in chicken embryo fibroblast DF-1 cells. Sequence analysis of the HN gene of the mutant viruses at each passage showed that the introduced mutations were unaltered (data not shown). To rule out the possibility that change in the HN protein sequence could be compensated for by a mutation in the F protein, the F gene from each recovered virus was sequenced. No compensatory mutations in the F gene were observed (data not shown). The HN protein content of each mutant virus, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining, was very similar to that of the parental BC virus (pBC) (Table ​(Table1).1). The multicycle growth kinetics of the recombinant HN mutant viruses in DF-1 cells (Fig. ​(Fig.2)2) showed that the replication kinetics of all of the HN mutant viruses were similar to those of pBC, with the exception of the Y526Q mutant, which showed delayed growth and had a lower virus yield (1.5 to 2.0 log₁₀ PFU/ml) than the parental and other mutant viruses. In addition, the Y526Q mutant produced syncytia at 72 h, whereas the parental and other mutant viruses initiated syncytia at 24 h postinfection. These studies showed the importance of amino acid residue Y526 at the active site of the HN protein of NDV.Open in a separate windowFIG. 2.Multicycle growth kinetics of HN mutants of NDV in chicken embryo fibroblast (DF-1) cells. Cells were infected with the indicated parental or mutant virus at an multiplicity of infection of 0.01. Supernatant samples were collected at 8-h intervals until 64 h postinfection, and virus titers were determined at different time points by plaque assay. Values are averages from three independent experiments.

TABLE 1.

Biological activities of HN mutants of NDV

兔出血症病毒复制子的构建及其在RK-13细胞中的复制

为研究兔出血症病毒(RHDV)的复制机制、病毒与宿主之间的相互作用以及致病机制等,创建一个安全、有效的技术平台,在前期构建的RHDV侵染性克隆基础上,将病毒的衣壳蛋白编码区删除,保留了RHDV复制必需的所有蛋白酶基因和两端的非编码区,构建了RHDV复制子。试验结果证明,将该复制子RNA导入RK13细胞中后,能够进行高水平的复制和表达。     

Temperature-Sensitive Mutants Isolated from L Cells Persistently Infected with Newcastle Disease Virus

Virus mutants (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus have been previously reported by this laboratory to differ from the wild-type virus (NDV(o)) in several physical and biological properties. It has now been determined that, in addition to these differences, the NDV(pi) mutants are also spontaneously selected temperature-sensitive mutants. The temperature sensitivity of 10 NDV(pi) clones was confirmed by temperature inhibition, plaquing efficiency, and single-cycle yield experiments. The cut-off temperature, at which more than 90% of virus replication is inhibited was between 41 and 42 C. All 10 NDV(pi) clones were also found to be defective in virus-specific ribonucleic acid (RNA) synthesis in infected chick embryo cells at 42 C and are tentatively classified as RNA(-). The possible relationships of the temperature sensitivity, the other NDV(pi) properties, and the maintenance of the persistently infected state are discussed.