ABSTRACT
The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are translocated directly to ND10, like L2 expressed alone. Interestingly, accumulation of wild-type L1 is observed only after L2-induced release of the ND10-associated protein Sp100. Third, nuclear translocation of L2 but not of L1 was blocked by the proteasome inhibitor MG132. Our data suggest that L1 and L2 interaction occurs after L2-induced reorganization of ND10 subnuclear domains.
Subject(s)
Capsid Proteins , Capsid/chemistry , Oncogene Proteins, Viral/chemistry , Papillomaviridae/chemistry , Active Transport, Cell Nucleus , Animals , COS Cells , Capsid/analysis , Female , Humans , Oncogene Proteins, Viral/analysis , Virus AssemblyABSTRACT
To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.
Subject(s)
Capsid Proteins , JC Virus/chemistry , Leukoencephalopathy, Progressive Multifocal/pathology , Leukoencephalopathy, Progressive Multifocal/virology , Viral Proteins/analysis , Adult , Antigens, Viral, Tumor/analysis , Antigens, Viral, Tumor/immunology , Astrocytes/virology , Capsid/analysis , Capsid/immunology , Female , Humans , Male , Middle Aged , Neuroblastoma , Oligodendroglia/virology , Tumor Cells, Cultured , Viral Proteins/immunology , Viral Regulatory and Accessory Proteins , Virion/chemistryABSTRACT
The complete nucleotide sequence of an attenuated Pepper mild mottle virus (PMMoV C-1421) RNA genome has been determined. There were two differences from the type isolate in Japan (PMMoV-J). The mutations were located in the middle of the 126-kDa protein (126 K) gene; one mutation influenced amino acid substitution at 649th Val to Ala (V649A), and the other was silent. The analyses using the reverse genetic system of PMMoV-J revealed that symptom attenuation on pepper related to V649A. Accumulations of 126 K and coat protein (CP) in V649A mutant-infected pepper were lower than those of PMMoV-J in immunoblotting. These results suggest that V649A substitution in 126 K affects the accumulation of 126 K leading to a limitation of CP accumulation.
Subject(s)
Capsicum/virology , Genome, Viral , Plant Diseases/virology , RNA-Dependent RNA Polymerase/genetics , Tobamovirus/genetics , Amino Acid Substitution , Capsicum/economics , Capsid/analysis , Capsid/biosynthesis , Immunoblotting , Japan , Molecular Sequence Data , Mutation , Plant Diseases/economics , RNA-Dependent RNA Polymerase/analysis , RNA-Dependent RNA Polymerase/biosynthesis , Tobamovirus/enzymology , Tobamovirus/pathogenicity , Valine/geneticsABSTRACT
The epizootic hematopoietic necrosis virus (EHNV) is a strain of the Iridovirus genus, which includes viruses seriously affecting native and aquacultured fish and amphibians. Despite its growing importance as a threat to fish farming, very little information is available on the biochemical and immunological nature of this virus. To identify and characterize the main antigenic determinants of EHNV, a panel of murine monoclonal antibodies was produced upon parenteral inoculation with live virus. A total of 124 primary hybridoma cultures from two fusions was found to produce antibodies reacting with EHNV by ELISA, but no neutralizing monoclonal antibody was detected. Twenty hybridoma cultures were randomly chosen for further study, and the antibodies secreted were analyzed by Western blotting, radioimmunoprecipitation, and immunostaining of infected cells. Only three MAbs immunoprecipitated the 50-kDa EHNV major capsid protein (MCP) from infected cell lysates, but they did not stain this protein in Western blotting. Eight and five further MAbs recognized peptides of approximately 15 and 18 kDa, respectively. Four antibodies could not be mapped into any viral protein, although they specifically immunostained virus-infected cells and reacted with purified EHNV virions by ELISA. These latter MAbs and the three antibodies directed at the MCP are likely to recognize conformation-dependent epitopes on the virus capsid proteins.
Subject(s)
Antigens, Viral/analysis , Capsid Proteins , Capsid/analysis , Iridovirus/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Antigens, Viral/immunology , Blotting, Western , Capsid/immunology , Fluorescent Antibody Technique , Mice , Microscopy, Immunoelectron , Peptides/analysis , Radioimmunoprecipitation Assay , Tumor Cells, CulturedABSTRACT
AIMS: To investigate the involvement of enterovirus infection in chronic, rheumatic heart disease. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded, surgical samples of valve tissue were examined for the presence of enteroviral RNA and virus capsid protein VP1 by in situ hybridization and immunostaining. Of 53 cases, 33 were patients with chronic rheumatic heart disease and 20 had Marfan's syndrome or degenerative valve disease. Enterovirus RNA was detected in 8 (24.2%) of 33 patients with chronic rheumatic heart disease by in situ hybridization using strand-specific oligonucleotide probes, complementary to conserved sequences in enterovirus genomic (positive strand) RNA. The replication template (negative strand) RNA also was found in seven of these eight cases. The viral capsid protein VP1 was detected in 16 (48.5%) of 33 patients with chronic rheumatic heart disease by immunohistochemistry and correlated with viral RNA detection. Virus was localized generally to valvular tissue. Neither viral RNA nor capsid protein VP1 were found in valvular tissue from any of the 20 comparison cases. CONCLUSIONS: This is the first demonstration of detection and localization of both enterovirus RNA and capsid protein in chronic rheumatic heart disease. The presence of negative strand RNA and VP1 indicates enteroviral RNA replication and protein synthesis and suggests an aetiological role of enterovirus in the pathogenesis of chronic rheumatic heart disease.
Subject(s)
Enterovirus Infections/complications , Heart Valves/virology , Rheumatic Heart Disease/virology , Adult , Aged , Capsid/analysis , Chronic Disease , Female , Humans , Immunohistochemistry , In Situ Hybridization , Male , Middle Aged , RNA, Viral/analysis , Virus ReplicationABSTRACT
Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains beta-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some beta-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.
Subject(s)
Antiviral Agents/pharmacology , Brefeldin A/pharmacology , Caveolae/virology , Endoplasmic Reticulum/virology , Simian virus 40/metabolism , Animals , Biological Transport/drug effects , Capsid/analysis , Capsid/metabolism , Caveolae/metabolism , Coatomer Protein/analysis , Coatomer Protein/metabolism , Endocytosis , Endoplasmic Reticulum/metabolism , Virus ReplicationABSTRACT
The whitefly, Bemisia tabaci biotype B, has been shown to cause pathogenesis-related (PR) proteins to accumulate in plants as a result of direct feeding, but their specific role in plant defensive systems is unclear. Our objective was to compare accumulation of tomato PR proteins (beta-1,3-glucanase, chitinase, peroxidase, P2 and P4) in response to whitefly, with or without tomato mottle virus (ToMoV) infection. Tomato PR protein response was measured over time in plants divided into three treatments: uninfected controls (with or without whiteflies) and plants infested with viruliferous (ToMoV) whiteflies. Five- to six-leaf plants were infested with approximately 5 adult whitefly per leaf. Plants were sampled prior to whitefly infestation and at 14, 28, 42, and 56 days. By 56 days, plants infested with viruliferous whiteflies had significantly more eggs (2.5-fold) and nymphs (4.5-fold) than plants with nonviruliferous whiteflies. A significant increase in the enzymatic activity of all measured PR proteins, as compared to control plants, was only seen in viruliferous whitefly-infested plants. No significant difference was observed in enzyme activities between the uninfected control plants either with or without whiteflies. The greatest differences for all PR proteins assayed were observed 42 days after treatment initiation. Protein blot analyses showed that the differences in PR protein activities among the treatments were due to changes in specific enzyme levels within the plant and were associated with concomitant increases in levels of P2 and P4 PR proteins. Under our experimental conditions, it is clear that PR protein response is much more intense when it is attacked by whiteflies carrying ToMoV than by whitefly alone.
Subject(s)
Geminiviridae/growth & development , Hemiptera/virology , Plant Diseases/virology , Plant Proteins/biosynthesis , Solanum lycopersicum/virology , Animals , Capsid/analysis , Capsid/biosynthesis , Enzyme-Linked Immunosorbent Assay , Female , Hemiptera/growth & development , Solanum lycopersicum/metabolism , Solanum lycopersicum/parasitology , Male , Plant Diseases/parasitology , Plant Proteins/analysisABSTRACT
OBJECTIVE: To study VP7 type of rotavirus from children with diarrhea in Chongqing area from 1998 to 2000. METHODS: Double-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. Nucleotide sequence analysis was used for VP7 typing. RESULTS: Among the 130 fecal specimens from pediatric patients with acute gastroenteritis during the epidemic season of 1998-1999, fifty specimens (38.46%) were identified as rotavirus-positive by RT-PCR. Of these, serotype G1 was found to be largely predominant and accounted for 88% (44/50), while serotype G3 accounted for 8% (4/50), coinfection of G1 and G3 accounted for 4% (2/50) respectively. While during the next epidemic season from 1999 to 2000, thirty-eight (33.93%) rotavirus positive specimens were detected from 112 stool samples by RT-PCR. Serotype G3 increased significantly-up to 78.95% (30/38) and became the most prevalent serotype in that season. It was found that only 13.16% (5/38) were serotype G1, 7.89% (3/38) were G1 and G3 mixture. The nucleotide sequence analysis results agreed with the RT-PCR typing assay. CONCLUSIONS: Serotype G1 and G3 were the prevalent VP7 types in Chongqing, China from 1998 to 2000. A shift from G1 to G3 was observed in the last two successive epidemic seasons of rotavirus epidemics.
Subject(s)
Antigens, Viral , Capsid Proteins , Capsid/analysis , Diarrhea/virology , Rotavirus Infections/virology , Amino Acid Sequence , Capsid/genetics , China/epidemiology , Diarrhea/epidemiology , Humans , Infant , Molecular Sequence Data , Polymerase Chain Reaction , Rotavirus Infections/epidemiology , Sequence Homology, Amino AcidABSTRACT
Epizootic ulcerative syndrome (EUS) has been infecting a wide range of fishes in the South and Southeast Asia for the last 2 decades. One reovirus-like agent (snakehead reovirus, SKRV), isolated from an EUS-infected snakehead fish and investigated in the present study, is the only reovirus so far isolated from an EUS-infected fish. SKRV was characterised by the presence of a double-stranded RNA genome with icosahedral symmetry and double capsid. The virus had an average size of 71 nm, a buoyant density of 1.36 g ml(-1) in CsCl and lacked a lipid-containing envelope. Apart from the above, the presence of a segmented genome and structural proteins falling into 3 specific size classes confirmed that the virus belongs to the family Reoviridae. SKRV differed from aquareoviruses by the lack of a cytopathic effect (CPE) with syncitium formation and in the segmentation pattern of RNA genome. The resistance to pH (3.0 to 9.0) and heat treatment and inability to multiply in mammalian cell lines and haemagglutinate human 'O' red blood cells (RBCs) differentiated SKRV from the rest of the similar genera in the family Reoviridae. Serological comparison indicated the antigenic distinctness of the isolate from selected American and European aquareoviruses. SKRV grew well in SSN-1 and SSN-3 cells at 25 to 30 degrees C but not in the most common Aquareovirus susceptible coldwater fish cell line--CHSE-214.
Subject(s)
Fish Diseases/virology , Perciformes , Reoviridae Infections/veterinary , Reoviridae/isolation & purification , Animals , Capsid/analysis , Cell Line , Cytopathogenic Effect, Viral , Microscopy, Electron/veterinary , RNA, Double-Stranded/analysis , RNA, Viral/analysis , Reoviridae/classification , Reoviridae/genetics , Reoviridae/ultrastructure , Reoviridae Infections/virology , Serologic Tests , Virus ReplicationABSTRACT
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.
Subject(s)
Infectious bursal disease virus/physiology , Viral Structural Proteins/physiology , Virus Assembly , Animals , Capsid/analysis , Capsid/physiology , Chick Embryo , Infectious bursal disease virus/immunology , Microscopy, Electron , Viral Structural Proteins/analysis , Viral Structural Proteins/chemistry , Viral Vaccines/immunologyABSTRACT
Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids ("empty" or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both "empty" vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.
Subject(s)
Capsid Proteins , Capsid/metabolism , Caveolae/physiology , Cell Nucleus/virology , Polyomavirus/physiology , Virion/physiology , beta-Cyclodextrins , Adsorption , Animals , Biological Transport , Capsid/analysis , Caveolin 1 , Caveolins/physiology , Cell Line , Cell Nucleus/metabolism , Cyclodextrins/pharmacology , Mice , Tubulin/analysisABSTRACT
During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.
Subject(s)
Capsid Proteins , Capsid/physiology , DNA, Viral/physiology , Virus Assembly , Amino Acid Sequence , Animals , Capsid/analysis , Capsid/chemistry , Chlorocebus aethiops , Microscopy, Electron , Molecular Sequence Data , Vero Cells , Viral ProteinsABSTRACT
The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase chain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000-40,000 copies in 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10(5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV-induced transverse myelitis with an increased number of EBV-genome copies in peripheral blood mononuclear cells and EBV-positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplantation, as well as for monitoring the response to therapy.
Subject(s)
DNA, Viral/analysis , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/isolation & purification , Acute Disease , Antigens, Viral/analysis , Antigens, Viral/genetics , Capsid/analysis , Capsid/genetics , DNA Primers , Epstein-Barr Virus Infections/cerebrospinal fluid , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/immunology , Herpesvirus 4, Human/genetics , Humans , Leukocytes, Mononuclear/virology , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral LoadABSTRACT
Cardamom mosaic virus, a possible member of the family Potyviridae has been associated with the mosaic disease (Katte disease) of small cardamom in India. A virus isolated from the symptomatic cardamom leaves was positive in ELISA only with antiserum to the Guatemalan isolate of cardamom mosaic virus and not with a number of other potyviruses. The size of the viral RNA (8.5 kb) and the molecular weight of the coat protein (CP) (38 kDa) were determined. A 1.8-kb product containing the partial nuclear inclusion body (NIb) gene, the entire coat protein gene and the 3' untranslated region (UTR) was amplified by reverse transcription (RT) and polymerase chain reaction (PCR), cloned and sequenced. The viral origin of the clone was confirmed by Northern hybridization with viral RNA. The experimentally determined N-terminal sequence of the CP matched with the deduced amino acid sequence of the CP gene. Sequence analysis of the clone suggests that the cardamom mosaic virus is a member of the Maclurvirus genus of the family Potyviridae.
Subject(s)
3' Untranslated Regions , Elettaria/virology , Genome, Viral , Potyvirus/genetics , RNA, Viral , Amino Acid Sequence , Base Sequence , Binding Sites , Capsid/analysis , Capsid/genetics , DNA-Directed RNA Polymerases , Electrophoresis, Polyacrylamide Gel/methods , Endopeptidases/metabolism , India , Molecular Sequence Data , Phylogeny , Potyvirus/classification , Potyvirus/isolation & purification , Sequence Analysis, RNA , Sequence Homology, Amino Acid , Sodium Dodecyl Sulfate , Viral Proteins/analysis , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.
Subject(s)
Capsid/analysis , DNA-Directed RNA Polymerases/analysis , Eimeria/virology , Virion/isolation & purification , Animals , Antigens, Viral/analysis , Chickens/parasitology , Eimeria/growth & development , Immunohistochemistry , Life Cycle Stages , Microscopy, Immunoelectron , Molecular Weight , Virion/immunology , Virion/ultrastructureABSTRACT
Detection and typing of human papillomavirus (HPV) infection may have a major impact in cervical-screening and follow-up. In this study various commercially available techniques for the detection of HPV were evaluated. HPV-status was determined in 86 samples of cervical cancer by PCR and direct sequencing, catalyzed signal amplified colorimetric DNA in situ hybridization (CSAC- ISH) (GenPoint system, DAKO), immunohistochemistry (IHC) and in 12 selected cases also by conventional, non-amplified ISH. Twenty-one samples of cervical intraepithelial neoplasias grade III (CIN III) were investigated by CSAC-ISH, conventional ISH and by IHC, in corresponding PAP smears HPV-detection and typing was performed by CSAC-ISH and Hybrid Capture test II (HC). In additional 20 PAP smears HPV typing was performed using HC and a novel immunocytochemical system for HPV detection and-typing. CSAC-ISH showed good correlation with PCR analysis in cervical cancers: In 87% of PCR positive cases, HPV infection was also detected by CSAC- ISH (66/76). HPV 16 was detected in 75% of PCR-positive cases (44/59), HPV 18 in 71% of PCR positive cases (5/7). CSAC-ISH detected HPV 31 in only 29% of PCR positive cases (2/7), and HPV 33 in 64% of PCR-positive cases (23/36). Nevertheless, CSAC-ISH- false negative cases for HPV 31 or 33 were nearly always combined infections with other HPV types, which were detectable by CSAC-ISH in most cases. CSAC-ISH revealed HPV infection in 20 of 21 HC-positive cervical smears, while in corresponding biopsies (CIN III) CSAC-ISH detected 100% of HPV infections. Conventional, non-amplified ISH showed significantly lower sensitivity compared with CSAC-ISH, and immunocyto- and -histochemistry were of very low sensitivity for detection of HPV. CSAC-ISH is an easy-to-handle method for detection and typing of cervical HPV infection, and shows sufficient sensitivity for clinical practice.
Subject(s)
Cervix Uteri/virology , In Situ Hybridization/methods , Papillomaviridae , Papillomavirus Infections/virology , Tumor Virus Infections/virology , Capsid/analysis , Cervix Uteri/pathology , DNA Probes, HPV , DNA, Viral/genetics , Female , Humans , Immunohistochemistry , Papanicolaou Test , Papillomaviridae/genetics , Papillomavirus Infections/pathology , Paraffin Embedding , Polymerase Chain Reaction , Tumor Virus Infections/pathology , Uterine Cervical Dysplasia/pathology , Uterine Cervical Dysplasia/virology , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology , Vaginal SmearsSubject(s)
Apoproteins/analysis , Capsid/analysis , Myoglobin/analysis , Peptide Hydrolases/metabolism , Proteins/metabolism , Ribonuclease, Pancreatic/analysis , Apoproteins/metabolism , Capsid/metabolism , Myoglobin/metabolism , Proteins/analysis , Ribonuclease, Pancreatic/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , Thermolysin/metabolismABSTRACT
Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.
Subject(s)
Capsid/analysis , Picornaviridae Infections/immunology , Picornaviridae/chemistry , Picornaviridae/immunology , Viral Proteins , 3C Viral Proteases , Animals , Antibodies, Viral/analysis , Antigens, Viral/immunology , Blotting, Western , Capsid/chemistry , Capsid/immunology , Cysteine Endopeptidases/analysis , Horses , Immune Sera/analysis , Molecular Weight , Neutralization Tests , Picornaviridae/radiation effects , Picornaviridae Infections/virology , Rabbits , Ultraviolet RaysABSTRACT
Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.
Subject(s)
Antigens, Viral , Capsid/analysis , Luminescent Proteins/analysis , Rotavirus/ultrastructure , Animals , Baculoviridae/physiology , Baculoviridae/ultrastructure , Capsid/genetics , Capsid Proteins , Cell Line , Cryoelectron Microscopy , Green Fluorescent Proteins , Image Processing, Computer-Assisted , Luminescent Proteins/genetics , Microscopy, Confocal , Recombinant Fusion Proteins/analysis , Spodoptera , TransfectionABSTRACT
There is substantial evidence that Kaposi sarcoma-associated herpesvirus (KSHV) plays an important role in the pathogenesis of all forms of Kaposi sarcoma (KS). It has been noted that KS commonly occurs in locations, such as the feet, where tissue may be poorly oxygenated. On the basis of this observation, the potential role of hypoxia in the reactivation of KSHV replication was explored by studying 2 KSHV-infected primary effusion lymphoma B-cell lines (BC-3 and BCBL-1) latently infected with KSHV. Acute and chronic exposure of these cells to hypoxia (1% O(2)) induced KSHV lytic replication, as indicated by an increase in intracellular lytic protein expression and detection of virus in cell supernatants by Western immunoblotting. In addition, hypoxia increased the levels of secreted viral interleukin-6. Moreover, hypoxia enhanced the lytic replication initiated by the viral inducer 12-O-tetradecanoylphorbol-13-acetate. Desferoxamine and cobalt chloride, 2 compounds that increase the intracellular levels of hypoxia-inducible factor 1, were also able to induce KSHV lytic replication. These studies suggest that hypoxia is an inducer of KSHV replication. This process may play an important role in the pathogenesis of KS.