ABSTRACT
BACKGROUND: Varicella zoster virus (VZV) vasculopathy produces stroke secondary to viral infection of cerebral arteries. Not all patients have rash before cerebral ischemia or stroke. Furthermore, other vasculitides produce similar clinical features and comparable imaging, angiographic, and CSF abnormalities. METHODS: We review our 23 published cases and 7 unpublished cases of VZV vasculopathy. All CSFs were tested for VZV DNA by PCR and anti-VZV IgG antibody and were positive for either or both. RESULTS: Among 30 patients, rash occurred in 19 (63%), CSF pleocytosis in 20 (67%), and imaging abnormalities in 29 (97%). Angiography in 23 patients revealed abnormalities in 16 (70%). Large and small arteries were involved in 15 (50%), small arteries in 11 (37%), and large arteries in only 4 (13%) of 30 patients. Average time from rash to neurologic symptoms and signs was 4.1 months, and from neurologic symptoms and signs to CSF virologic analysis was 4.2 months. CSF of 9 (30%) patients contained VZV DNA while 28 (93%) had anti-VZV IgG antibody in CSF; in each of these patients, reduced serum/CSF ratio of VZV IgG confirmed intrathecal synthesis. CONCLUSIONS: Rash or CSF pleocytosis is not required to diagnose varicella zoster virus (VZV) vasculopathy, whereas MRI/CT abnormalities are seen in almost all patients. Most patients had mixed large and small artery involvement. Detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). Determination of optimal antiviral treatment and benefit of concurrent steroid therapy awaits studies with larger case numbers.
Subject(s)
Cerebrovascular Disorders/cerebrospinal fluid , Cerebrovascular Disorders/virology , Herpesvirus 3, Human , Cerebrovascular Disorders/diagnosis , Cerebrovascular Disorders/etiology , Chickenpox/cerebrospinal fluid , Chickenpox/complications , Chickenpox/virology , Exanthema/cerebrospinal fluid , Exanthema/diagnosis , Exanthema/virology , Herpes Zoster/cerebrospinal fluid , Herpes Zoster/complications , Herpes Zoster/virology , Humans , Magnetic Resonance Imaging/methodsABSTRACT
BACKGROUND: Factors that may obscure the diagnosis of varicella zoster virus (VZV) vasculopathy include the absence of rash before TIAs or stroke as well as similar clinical features and imaging, angiographic, and CSF abnormalities to those of other vasculopathies. Diagnosis relies on virologic confirmation that detects VZV DNA, anti-VZV IgG antibody, or both in the CSF. METHODS: We reviewed our current 14 cases of patients diagnosed with VZV vasculopathy based on combined clinical, imaging, angiographic, or CSF abnormalities. All CSFs must have been tested for VZV DNA by PCR and for anti-VZV IgG antibody by enzyme immunoassay and found to be positive for either or both. Of the 14 subjects, 8 had a history of recent zoster, whereas 6 had no history of zoster rash before developing vasculopathy. RESULTS: All 14 subjects (100%) had anti-VZV IgG antibody in their CSF, whereas only 4 (28%) had VZV DNA. The detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). CONCLUSIONS: In varicella zoster virus (VZV) vasculopathy, the diagnostic value of detecting anti-VZV IgG antibody in CSF is greater than that of detecting VZV DNA. Although a positive PCR for VZV DNA in CSF is helpful, a negative PCR does not exclude the diagnosis of VZV vasculopathy. Only when the CSF is negative for both VZV DNA and anti-VZV IgG antibody can the diagnosis of VZV vasculopathy be excluded.
Subject(s)
Antibodies, Viral/cerebrospinal fluid , Chickenpox/complications , Herpesvirus 3, Human/immunology , Immunoglobulin G/cerebrospinal fluid , Vasculitis, Central Nervous System/cerebrospinal fluid , Vasculitis, Central Nervous System/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Cerebral Arteries/immunology , Cerebral Arteries/pathology , Cerebral Arteries/physiopathology , DNA, Viral/analysis , DNA, Viral/genetics , Female , Herpesvirus 3, Human/genetics , Humans , Male , Middle Aged , Predictive Value of Tests , Vasculitis, Central Nervous System/diagnosisABSTRACT
BACKGROUND: Encephalitis is a complex syndrome, and its etiology is often not identified. The California Encephalitis Project was initiated in 1998 to identify the causes and further describe the clinical and epidemiologic characteristics of encephalitis. METHODS: A standardized report form was used to collect demographic and clinical data. Serum, cerebrospinal fluid, and respiratory specimens were obtained prospectively and were tested for the presence of herpesviruses, arboviruses, enteroviruses, measles, respiratory viruses, Chlamydia species, and Mycoplasma pneumoniae. The association between an identified infection and encephalitis was defined using predetermined, organism-specific criteria for confirmed, probable, or possible causes. RESULTS: From 1998 through 2005, a total of 1570 patients were enrolled. Given the large number of patients, subgroups of patients with similar clinical characteristics and laboratory findings were identified. Ten clinical profiles were described. A confirmed or probable etiologic agent was identified for 16% of cases of encephalitis: 69% of these agents were viral; 20%, bacterial; 7%, prion; 3%, parasitic; and 1%, fungal. An additional 13% of cases had a possible etiology identified. Many of the agents classified as possible causes are suspected but have not yet been definitively demonstrated to cause encephalitis; these agents include M. pneumoniae (n=96), influenza virus (n=22), adenovirus (n=14), Chlamydia species (n=10), and human metapneumovirus (n=4). A noninfectious etiology was identified for 8% of cases, and no etiology was found for 63% of cases. CONCLUSIONS: Although the etiology of encephalitis remains unknown in most cases, the recognition of discrete clinical profiles among patients with encephalitis should help focus our efforts toward understanding the etiology, pathogenesis, course, and management of this complex syndrome.
Subject(s)
Encephalitis/physiopathology , Research Design/standards , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Encephalitis/microbiology , Encephalitis/virology , Female , Humans , Infant , Male , Middle Aged , Syndrome , Viruses/isolation & purificationSubject(s)
Breast Neoplasms/pathology , Carcinoma/secondary , Ganglia, Spinal/pathology , Herpes Zoster/pathology , Meningeal Neoplasms/secondary , Meningitis, Aseptic/complications , Adult , Antibodies, Viral/cerebrospinal fluid , Antiviral Agents/administration & dosage , Breast Neoplasms/complications , Carcinoma/complications , Chronic Disease , Fatal Outcome , Female , Herpes Zoster/complications , Herpes Zoster/drug therapy , Herpesvirus 3, Human/immunology , Humans , Hydronephrosis/complications , Magnetic Resonance Imaging , Meningeal Neoplasms/complications , Meningitis, Aseptic/pathology , Meningitis, Aseptic/physiopathology , Psoas Muscles/pathology , Renal Insufficiency/etiology , Spinal Nerve Roots/pathologyABSTRACT
BACKGROUND: As part of assessing the possibility of transfusion transmission of human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated herpesvirus), HHV-8 seroprevalence was estimated among US blood donors, the performance of HHV-8 serologic tests was compared, and the presence of HHV-8 DNA was tested for in donated blood. STUDY DESIGN AND METHODS: Replicate panels of 1040 plasma specimens prepared from 1000 US blood donors (collected in 1994 and 1995) and 21 Kaposi's sarcoma patients were tested for antibodies to HHV-8 in six laboratories. HHV-8 PCR was performed on blood samples from 138 donors, including all 33 who tested seropositive in at least two laboratories and 22 who tested positive in at least one. RESULTS: The estimated HHV-8 seroprevalence among US blood donors was 3.5 percent (95% CI, 1.2%-9.8%) by a conditional dependence latent-class model, 3.0 percent (95% CI, 2.0%-4.6%) by a conditional independence latent-class model, and 3.3 percent (95% CI, 2.3%-4.6%) by use of a consensus-derived gold standard (specimens positive in two or more laboratories); the conditional dependence model best fit the data. In this model, laboratory specificities ranged from 96.6 to 100 percent. Sensitivities ranged widely, but with overlapping 95 percent CIs. HHV-8 DNA was detected in blood from none of 138 donors evaluated. CONCLUSIONS: Medical and behavioral screening does not eliminate HHV-8-seropositive persons from the US blood donor pool, but no viral DNA was found in donor blood. Further studies of much larger numbers of seropositive individuals will be required to more completely assess the rate of viremia and possibility of HHV-8 transfusion transmission. Current data do not indicate a need to screen US blood donors for HHV-8.
Subject(s)
Blood Donors/statistics & numerical data , Herpesviridae Infections/blood , Herpesviridae Infections/epidemiology , Herpesvirus 8, Human/isolation & purification , Antibodies, Viral/blood , Blood Banks/standards , Herpesviridae Infections/transmission , Herpesvirus 8, Human/immunology , Humans , Reference Standards , Sensitivity and Specificity , Seroepidemiologic Studies , United States/epidemiologyABSTRACT
After lumbar-distribution zoster, an HTLV-1-seropositive woman developed chronic radicular sacral-distribution pain (zoster sine herpete), cervical-distribution zoster paresis and thoracic-distribution myelopathy. Detection of anti-varicella zoster virus (VZV) IgM and VZV IgG antibody in cerebrospinal fluid (CSF), with reduced serum/CSF ratios of anti-VZV IgG compared to normal serum/CSF ratios for albumin and total IgG, proved that VZV caused the protracted neurological complications. Diagnosis by antibody testing led to aggressive antiviral treatment and a favorable outcome.
Subject(s)
Herpes Zoster/complications , Herpesvirus 3, Human , Paresis/etiology , Spinal Cord Diseases/etiology , Zoster Sine Herpete/etiology , Chronic Disease , Female , HTLV-I Antibodies/blood , HTLV-I Antibodies/cerebrospinal fluid , HTLV-I Antibodies/metabolism , Herpes Zoster/blood , Herpes Zoster/cerebrospinal fluid , Herpes Zoster/virology , Humans , Middle Aged , Paresis/blood , Paresis/cerebrospinal fluid , Paresis/virology , Spinal Cord Diseases/blood , Spinal Cord Diseases/cerebrospinal fluid , Spinal Cord Diseases/virology , Time Factors , Zoster Sine Herpete/blood , Zoster Sine Herpete/cerebrospinal fluid , Zoster Sine Herpete/virologyABSTRACT
Data obtained in the third National Health and Nutrition Examination Survey (NHANES III), conducted during 1988-1994, were analyzed to determine the epidemiology of rubella seropositivity in the United States, including risk factors for low rubella seropositivity. Serological samples obtained from NHANES III study participants > or =6 years of age were tested for rubella IgG antibodies. "Rubella seropositivity" was defined as serum rubella IgG antibody level > or =10 IU by enzyme immunoassay. Overall, rubella seropositivity rates in the United States were 92% in persons aged 6-11 years, 83% in persons aged 12-19 years, 85% in persons aged 20-29 years, 89% in persons aged 30-39 years, and >or =93% in persons aged > or =40 years. The lowest rate (78%) of any United States birth cohort of the 20th century occurred among persons born from 1970-1974. Eliminating rubella and chronic rubella syndrome in the United States will require international efforts, including vaccination of preschool- and school-age children and all susceptible young adults.
Subject(s)
Antibodies, Viral/blood , Rubella virus/immunology , Rubella/epidemiology , Adolescent , Adult , Age Distribution , Child , Female , Health Surveys , Humans , Immunoenzyme Techniques/methods , Immunoglobulin G/blood , Male , Middle Aged , Seroepidemiologic Studies , United States/epidemiologyABSTRACT
Immunizing infants against measles at the youngest age possible has the potential to reduce morbidity and mortality. The ability of infants at 6, 9, or 12 months to respond to measles and mumps vaccines was evaluated by measuring T cell proliferation, interferon-gamma production, and neutralizing antibody titers before and after vaccination. Infants in all age groups had equivalent cellular immune responses to measles or mumps viruses, with or without passive antibodies when immunized. In contrast, 6-month-old infants without passive antibodies had low geometric mean titers of antibody to measles or mumps viruses and low seroconversion rates. Geometric mean titers of antibody to measles virus increased if infants were revaccinated at 12 months. Six-month-old infants had limited humoral responses to paramyxovirus vaccines, whereas cellular immunity was equivalent to that of older infants. T cell responses can be established by immunization with these live attenuated virus vaccines during the first year, despite the presence of passive antibodies.
Subject(s)
Measles Vaccine/administration & dosage , Measles/prevention & control , Morbillivirus/immunology , Mumps Vaccine/administration & dosage , Mumps/prevention & control , Rubulavirus/immunology , Vaccination , Adult , Age Factors , Antibodies, Viral/blood , Cohort Studies , Humans , Infant , Interferon-gamma/blood , Measles-Mumps-Rubella Vaccine/administration & dosage , T-Lymphocytes/immunologyABSTRACT
The authors report three patients with acute, chronic, and recurrent neuropathy associated with varicella zoster virus (VZV) infection but without zoster rash. CSF of all three patients contained VZV immunoglobulin G antibody, but not herpes simplex virus. In two patients, serum/CSF ratios of VZV immunoglobulin G were reduced compared to normal ratios for immunoglobulin G and albumin, and one patient also had VZV immunoglobulin M in CSF. All three patients received antiviral therapy and improved. The diagnosis of nervous system infection by VZV may be confirmed by the presence of antibody to VZV in CSF even without detectable VZV DNA.
Subject(s)
Central Nervous System Diseases/pathology , Central Nervous System/pathology , Herpes Zoster/pathology , Acute Disease , Adult , Aged , Aged, 80 and over , Central Nervous System Diseases/physiopathology , Female , Herpes Zoster/physiopathology , Humans , Magnetic Resonance Imaging , Male , RecurrenceABSTRACT
Varicella-zoster virus (VZV) glycoprotein E [gE] contains 623 amino acid residues. Fifty percent of the gE gene, codons 39 to 344 that encompasses two epitope coding regions e1 and c1, was sequenced and analyzed for variation among the 30 VZV isolates. A total of eleven isolates showed variance when compared with Dumas VZV strain sequence through base substitutions, with two isolates showing an amino acid change of tryptophan to arginine outside the coding regions of the epitopes e1 and c1 that are recognized by monoclonal antibodies 4F9 and c1, respectively. The results suggest that these epitopes were stable in the various VZV isolates. Thus, VZV glycoproteins with conserved epitopes are suitable candidates for both primary and booster vaccines.
Subject(s)
Antigens, Viral/genetics , Herpesvirus 3, Human/genetics , Herpesvirus 3, Human/immunology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Adult , Aged , Amino Acid Substitution , Base Sequence , Chickenpox Vaccine/genetics , Chickenpox Vaccine/immunology , Child , Child, Preschool , DNA Primers/genetics , DNA, Viral/genetics , Epitopes/genetics , Female , Herpesvirus 3, Human/isolation & purification , Humans , Male , Middle Aged , Polymorphism, Genetic , Sequence Analysis, DNAABSTRACT
Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first description in 1994. However, its three-dimensional (3D) structure remained heretofore undetermined largely due to difficulties in viral purification. We have used log-phase cultures of body cavity-based lymphoma 1 cells induced with 12-O-tetradecanoylphorbol-13-acetate to obtain HHV-8 capsids for electron cryomicroscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical members of alpha- and betaherpesviruses, respectively. The inner radius of the HHV-8 capsid is identical to that of the HSV-1 capsid but is smaller than that of the HCMV capsid, which is consistent with the relative sizes of the genomes they enclose. While the HHV-8 capsid exhibits many structural similarities to the HSV-1 capsid, our reconstruction shows two major differences: its hexons lack the "horn-shaped" VP26 densities bound to the HSV-1 hexon subunits, and the HHV-8 triplexes appear smaller and less elongated than those of HSV-1. These differences are in excellent agreement with our sequence comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus capsid structure complements previous structural studies on alpha- and betaherpesviruses in providing an account of structural similarities and differences among capsids representing all human herpesvirus subfamilies.
Subject(s)
Capsid/chemistry , Herpesvirus 8, Human/chemistry , Herpesvirus 1, Human/chemistry , Humans , Open Reading Frames , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: The genome of human herpesvirus 8 (HHV-8) contains at least 84 open reading frames, including the highly immunogenic K8.1. Other studies have determined that K8.1 gene generates at least two spliced transcripts in the HHV-8 infected BCBL-1 cells, termed as glycoprotein (gp)K8.1A and gpK8.1B. OBJECTIVE: To analyze the expression, post-translational modification and localization of HHV-8 gpK8.1 by monoclonal antibody (mAb). STUDY DESIGN: Mabs to HHV-8 produced by conventional hybridization and several clones identified. A mAb was used by various immunological assays to analyze HHV-8 K8.1 proteins in BCBL-1- and Sf9 insect cells. RESULTS: MAb clone 19B4 identified a 0.75-kb insert from the lambdaZAP cDNA expression library of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced BCBL-1 cells. Sequence analysis revealed that the cDNA insert corresponds to the published spliced ORF K8.1 mRNA of HHV-8. By immunofluorescence assay, the mAb stained the cell membrane, cytoplasm and perinuclear region of TPA induced BCBL-1 cells and showed no cross-reactivity with other herpesviruses. By immunoblotting assay, mAb 19B4 reacted with two species polypeptides giving a diffuse band with rMW from 42 to 64 kDa (gpK8.1A) and two closely migrating polypeptides of rMW 35/37 kDa (gpK8.1B). Both species were labeled by [14C]glucosamine, indicating that they are glycosylated and only gpK8.1A was detected in the virions. Expression of the full length K8.1 derived from cDNA in baculovirus system confirmed that these two glycoproteins are encoded by K8.1 gene. Enzymatic deglycosylation with endoF/peptide-N-glycosidase F, led to the reduction of rMW of both polypeptides whereas deglycosylation with O-glycosidase led only the reduction of rMW of K8.1A. CONCLUSION: The mAb 19B4 reacts specifically with BCBL-1 and Sf9 cells infected with recombinant baculovirus containing HHV-8 K8.1 gene. In several assays the mAb reacts with gpK8.1A and gpK8.1B. Only the mature spliced gpK8.1A is incorporated into virion. Enzymatic deglacosylation determined that gpK8.1A is N- and O-glycosylated, whereas gpK8.1B may lack O-glycosylation.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Cell Membrane/virology , Cell Nucleus/virology , Cytoplasm/virology , Glycoproteins/metabolism , Herpesvirus 8, Human/metabolism , Protein Processing, Post-Translational , Alternative Splicing , Animals , Baculoviridae/genetics , Cell Line , Glycoproteins/genetics , Glycoproteins/immunology , Glycoside Hydrolases/chemistry , Glycosylation , Herpesvirus 8, Human/drug effects , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/immunology , Humans , Mice , Microscopy, Fluorescence , Molecular Weight , Open Reading Frames , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , RNA, Viral/analysis , Spodoptera/virology , Tetradecanoylphorbol Acetate/pharmacology , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism , Virion/metabolismABSTRACT
Agreement between assays for the detection of human herpesvirus 8 (HHV-8) antibodies has been limited. In part, this disagreement has been because assay calibration (i.e., differentiating positive from negative results) has not been done in a standardized fashion with reference to a wide spectrum of HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) persons. To describe the performance of an assay for HHV-8 antibodies more accurately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodies to define two groups: a group of 135 HHV-8-infected individuals (true positives), including Kaposi's sarcoma patients and those asymptomatically infected, and a group of 234 individuals with a high likelihood of being HHV-8 uninfected (true negatives). A new enzyme immunoassay (EIA), using lysed HHV-8 virion as the antigen target, was then developed. With the above true positives and true negatives as references, the sensitivity and specificity of the EIA associated with different cutoff values were determined. At the cutoff that maximized both sensitivity and specificity, sensitivity was 94% and specificity was 93%. When the EIA was used to test a separate validation group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were seropositive, versus 6% seropositivity in a group of children, women, and heterosexual men. It is proposed that the EIA has utility for large-scale use in a number of settings and that the calibration method described can be used for other assays, both to more accurately describe the performance of these assays and to permit more-valid interassay comparison.
Subject(s)
Antibodies, Viral/blood , Herpesviridae Infections/diagnosis , Herpesviridae Infections/epidemiology , Herpesvirus 8, Human/immunology , Immunoenzyme Techniques/methods , Child , Female , Fluorescent Antibody Technique , Herpesviridae Infections/virology , Humans , Male , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The epitope stability of a varicella-zoster virus (VZV) glycoprotein E (gE) was analyzed with monoclonal antibodies (mAbs) in cells infected with different passages of various VZV strains and isolates. The gE-specific mAbs recognized same antigenic sites (epitopes) in VZV isolates with various passage history. All VZV strains and virus-isolates reacted with an anti-gE monoclonal antibody by immunoprecipitation, or indirect fluorescent antibody staining test. Sera from VZV seropositive individuals reacted with a truncated VZV gE glycoprotein, designated TgpI-511. Also, human mononuclear cells (MNCs) stimulated with TgpI-511 glycoprotein were shown to produce VZV-specific antibodies in vitro. The results demonstrated the stability of these gE epitopes tested in this study in TgpI-511 and among the VZV-isolates obtained from different passages. These results also suggest that VZV glycoproteins as well as live attenuated or killed varicella vaccines containing these epitopes could be used as therapeutic booster vaccines in adults and the elderly to prevent zoster.
Subject(s)
Epitopes/immunology , Herpesvirus 3, Human/immunology , Viral Envelope Proteins/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Cell Line , Chickenpox/immunology , Chickenpox/virology , Child , Child, Preschool , Female , Fluorescent Antibody Technique, Indirect , Herpes Zoster/immunology , Herpes Zoster/virology , Herpesvirus 3, Human/growth & development , Humans , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Radioimmunoprecipitation AssayABSTRACT
The most abundantly expressed latent transcripts encoded by the Kaposi's sarcoma (KS)-associated herpesvirus derive from the genomic region surrounding open reading frame (ORF) K12 (kaposin A). Here we show that these transcripts, initially described as limited to ORF K12 itself, more frequently encompass upstream sequences spanning two sets of 23-nucleotide GC-rich direct repeats (DRs) (DR1 and DR2). Although the DRs lack AUG codons and were previously presumed to be noncoding, a monoclonal antibody raised to infected cells detected multiple polypeptides encoded by this region. These proteins are expressed during latency and upon induction of lytic viral replication in both primary effusion lymphoma (PEL) cell lines and KS tumors. Biochemical and genetic analyses reveal that these proteins are derived from variant translational initiation at CUG codons. The predominant translation product in the PEL cell line BCBL-1 derives from the 5'-most CUG codon in the transcript, resulting in a protein (termed kaposin B) which is encoded largely by the repeats themselves and which does not include K12 sequences. Other non-AUG codons in alternate reading frames are also used at lower efficiency, including one that initiates translation of a DR-K12 fusion protein (kaposin C) that is predicted to sort to a different subcellular locale than kaposin B. Thus, the products of the K12 region, which is the most abundantly transcribed region in latency, are surprisingly complex and may encompass multiple biological functions.
Subject(s)
Herpesvirus 8, Human/genetics , Protein Biosynthesis , Viral Proteins/genetics , 3T3 Cells , 5' Untranslated Regions , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , DNA, Viral , Gene Expression , Humans , Mice , Molecular Sequence Data , RNA, Messenger , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Human herpesvirus 8 (HHV-8) infection is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. In this study, we used monoclonal antibodies (MAbs) directed against HHV-8 lytic cycle-associated proteins encoded by open reading frame (ORF) 59 (nuclear PF-8 protein) and ORF K8.1 (viral envelope glycoprotein K8.1 [gpK8.1]) to investigate HHV-8 lytic infection in single cells. Lytically infected cells were labeled with MAbs, stained with fluorescently conjugated secondary Abs, and analyzed by flow cytometry. A 3-day stimulation of HHV-8-positive PEL cell lines (BCBL-1 and BC-3) with 12-O-tetradecanoylphorbol-13-acetate (30 nM) or n-butyric acid (0.3 mM) maximized the expression of lytic-phase viral proteins and minimized cell toxicity. The absolute number of expressing cells was inducer and cell line dependent. Expression of PF-8 occurred earlier and more frequently (in up to 20% of cells) than did expression of gpK8.1. A subset of PF-8 positive cells (25%) co-expressed gpK8.1, representing the majority of gpK8.1 expressing cells. Acyclovir, foscarnet, cidofovir, and PMEA reduced the number of cells expressing gpK8.1, but not the number expressing the nonstructural early lytic gene product PF-8. By contrast, alpha interferon (IFN-alpha) and IFN-beta reduced expression of both PF-8 and gpK8.1, implying an overall inhibitory effect on viral gene transcription or translation. In summary, we have characterized and quantified HHV-8 lytic infection in single cells by dual measurement of early- and late-lytic-cycle HHV-8 protein expression. This technique should prove useful for screening of possible antiherpesvirus agents and for detailed phenotypic characterization of HHV-8-infected cells in vitro and in patients with HHV-8-associated diseases.
Subject(s)
Antiviral Agents/pharmacology , Glycoproteins , Herpesvirus 8, Human/drug effects , Viral Proteins/biosynthesis , Acyclovir/pharmacology , Blotting, Western , Cell Line , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Foscarnet/pharmacology , Herpesvirus 8, Human/metabolism , Herpesvirus 8, Human/physiology , Herpesvirus 8, Human/ultrastructure , Humans , Interferon-alpha/pharmacology , Interferon-beta/pharmacology , Interferon-gamma/pharmacology , Microscopy, Electron , Time FactorsABSTRACT
BACKGROUND: Diagnosis of acute and past infection with parvovirus B19 is based on detection of IgM and IgG antibodies. OBJECTIVES: To evaluate two commercial recombinant antigen-based enzyme immunoassay (EIA) test kits for detection of IgM and IgG antibodies to parvovirus B19 and to compare the commercial EIAs to in-house EIA test procedures. STUDY DESIGN: A panel of 121 sera was used to compare the three IgM EIAs. The panel included 84 sera submitted for parvovirus B19 testing and 37 sera that were IgM positive for other viral pathogens. The same serum panel plus an additional 14 sera submitted for B19 testing was used to compare the three IgG EIAs. The commercial EIAs were performed according to manufacturers' instructions. Using the in-house EIA test procedures as the reference, sensitivity and specificity for each of the commercial EIAs was determined. RESULTS: The commercial B19 IgM EIAs showed agreements of 95.0 and 93.4% to the in-house IgM EIA. Compared to the in-house B19 IgM EIA, the commercial B19 IgM EIAs were 97.4 and 97.5% sensitive, respectively. Specificities were 93.5 and 91.4%, respectively. Sensitivities for the commercial IgG EIAs, compared to in-house IgG EIA, were 88.0 and 85.2%, respectively, and specificities were 94.1 and 98.0%. CONCLUSION: We found that the commercial parvovirus B19 IgM and IgG EIAs are comparable to standard in-house EIAs and are suitable for testing for B19 antibodies in human sera.
Subject(s)
Antibodies, Viral/blood , Immunoenzyme Techniques , Immunoglobulin G/blood , Immunoglobulin M/blood , Parvovirus B19, Human/immunology , Reagent Kits, Diagnostic , HumansSubject(s)
Molluscum contagiosum virus/growth & development , Skin Transplantation , Skin/virology , Adult , Animals , Child , Fetus/virology , Humans , Mice , Mice, Nude , Mice, SCID , Transplantation, HeterologousABSTRACT
The genome of varicella-zoster virus (VZV), encodes at least six glycoproteins and they elicit the formation of complement-independent, complement-dependent, and non-neutralizing antibody responses. We have used our library of MAbs to VZV glycoprotein E (gE) to determine the neutralizing epitopes of gE, and shown that gE has 3 distinct neutralizing domains. In this report we have used the baculovirus expression system to identify the antigenic domains of gE. We have generated 3 recombinant baculoviruses, expressing the full-length gE and two overlapping truncated forms (the amino-terminal and the carboxy-terminal) of gE. By immuno-fluorescence and immunoblotting we have explored the physical interactions of Mabs to gE on these constructs. Our panel of MAbs revealed 3 district antigenic domains on gE. All MAbs reacted with the full-length gE; MAbs with high titered complement-dependent neutralizing activities reacted with the N-terminal truncated gE; MAbs with low titered or non-neutralizing activities reacted with the C-terminal truncated gE; MAbs with complement-enhanced neutralizing activities reacted with both truncated constructs. However, although the antibody binding in immunofluorescence and immunoblotting was carried out under denatured conditions, whereas the neutralization is under non-denatured conditions, still the antigenic mapping was similar in both conditions.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Herpesvirus 3, Human/immunology , Viral Envelope Proteins/immunology , DNA, Recombinant , DNA, Viral , Epitope MappingABSTRACT
BACKGROUND: Among the methods available for the diagnosis of varicella zoster virus (VZV) infections, only tissue culture yields an isolate available for antiviral susceptibility testing or pathogenesis studies. However, conventional tube tissue culture (TC) has a low sensitivity. OBJECTIVE: To increase the recovery rate of VZV in tissue culture. STUDY DESIGN: Clinical specimens submitted for VZV isolation were processed by TC and rapid shell vial (SV) techniques followed by a blind passage (BP). For SV, two incubation times and two mAbs, directed against viral-early or immediate-early antigens, were compared. RESULTS: Isolation of VZV using the SV stained at 72 h postinoculation was more sensitive (88-96%) than TC (50-67%), or the SV technique at 48 h (66-70%). It was also more rapid than TC (9.6 days). The comparison of mAbs yielded similar results in SV. Blind passage of SV at 7 days postinoculation further increased detection. CONCLUSIONS: SV combined with BP is the method of choice for VZV isolation.