Biomedical subjects

B Forghani

Publications and source records attributed to B Forghani.

At least 37 records · Page 2Linked to original sources

PubMed1996

Improved detection of varicella zoster infection with a spin amplification shell vial technique and blind passage.

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.

Journal Article↗

PubMed1994

Neutralization epitope of the varicella-zoster virus gH:gL glycoprotein complex.

Varicella-zoster virus (VZV) glycoprotein gpIII is the homolog of herpes simplex virus gH. Through the use of panels of monoclonal antibodies, VZV gpIII is known to possess a complement-independent neutralization epitope which is conformational in nature. Monoclonal antibody to this same epitope, when added postinfection, inhibits both syncytia formation and egress of virus. The nature of the neutralization epitope was investigated to determine whether its formation was dependent on gpIII alone or required a second VZV glycoprotein. To this end, VZV ORF 37 (gH) and VZV ORF 60 (gL homolog) were cloned into a vaccinia virus-pTM1 expression system. Analyses of the transfected products demonstrated that gpIII alone was not fully glycosylated nor was it transported to the cell surface. When both ORF 37 and ORF 60 were cotransfected, the gpIII product was transported to the cell surface, where it formed a neutralization epitope recognized by a previously characterized monoclonal antibody reagent. In summary, the VZV homologs of the herpes simplex virus gH:gL complex included a M(r) 118,000 product (gpIII or gH) and a M(r) 20,000 product (ORF 60 or gL).

Antibodies, Monoclonal↗

PubMed1993

Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system.

The unique short region of the varicella-zoster virus (VZV) genome contains two open reading frames which encode glycoproteins designated gpI and gpIV (herpes simplex virus homologs gE and gI, respectively). Like its herpesviral counterpart gE, the VZV gpI gene product functions as a cell surface receptor (V. Litwin, W. Jackson, and C. Grose, J. Virol. 66:3643-3651, 1992). To evaluate the biosynthesis of the two VZV glycoproteins and further explore their relationship to one another, the two glycoprotein genes were individually cloned into a pTM1 vector under control of the T7 promoter. Transfection of the cloned gpI or gpIV construct into HeLa cells previously infected with vaccinia recombinant virus expressing bacteriophage T7 polymerase resulted in a much higher level expression of each VZV glycoprotein than previously achieved. Synthesis of both gpI and gpIV included intermediary partially glycosylated forms and mature N- and O-linked final product. Transfections in the presence of 32Pi demonstrated that the mature forms of both gpI and gpIV were phosphorylated, while similar experiments with [35S]sulfate showed that only the mature gpI was sulfated. When gpI and gpIV were coexpressed in the same cell, the two glycoproteins were complexed to each other, as both proteins could be immunoprecipitated by antibodies against either gpI or gpIV. Coprecipitation did not occur as a result of a shared epitope, because gpI expressed alone was not precipitated by antibody to gpIV, and gpIV expressed alone was not precipitated by antibody to gpI. Pulse-chase analysis demonstrated that the gpI-gpIV association occurred early in processing; furthermore, this complex formation interfered with posttranslational modifications and thereby reduced the M(r)s of the mature forms of both gpI and gpIV. Similarly, the molecular masses of the cotransfected gene products corresponded with those of the infected cell glycoproteins, a result which suggested that authentic gpI and gpIV were ordinarily found within a complex. Thus, the adjacent open reading frames 67 and 68 code for two glycoproteins which in turn form a distinctive sulfated and phosphorylated cell surface complex with receptor properties.

Antibodies, Monoclonal↗

PubMed1993

Quantitation of latent varicella-zoster virus DNA in human trigeminal ganglia by polymerase chain reaction.

Competitive polymerase chain reaction was used to quantitate latent varicella-zoster virus (VZV) DNA in human trigeminal ganglia. Ganglionic DNA from five subjects was amplified with oligonucleotide primers specific for VZV gene 28. Two of the samples were also analyzed with primers specific for VZV gene 62. Our results indicated that there are 6 to 31 copies of the VZV genome in every 100,000 ganglionic cells.

Adult↗

PubMed1992

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

Blotting, Southern↗

PubMed1992

Comparison of six commercial human T-cell lymphotropic virus type I (HTLV-I) enzyme immunoassay kits for detection of antibody to HTLV-I and -II.

Three licensed and three research human T-cell lymphotropic virus type I (HTLV-I) enzyme immunoassay (EIA) kits were evaluated with a panel of 213 serum or plasma samples which were previously tested by the indirect immunofluorescence method for HTLV-I and -II antibodies. The three research kits were found to be more sensitive and specific than the three licensed kits.

Evaluation Studies as Topic↗

PubMed1991

Detection of the human immunodeficiency virus genome with a biotinylated DNA probe generated by polymerase chain reaction.

A non-radiolabelled DNA probe was developed for detection of the human immunodeficiency virus type 1 (HIV-1) genome using the polymerase chain reaction (PCR) technology. Primers amplifying a 395 base pair segment of a portion of the polymerase region of the HIV-1 genome were used both to amplify sample target DNA and to generate a biotinylated DNA probe used in Southern blot hybridization. This probe performed as well as one produced by nick translation using biotinylated nucleotides or an enzyme labelled oligonucleotide probe.

Bacterial Proteins↗

PubMed1991

A human monoclonal antibody against varicella-zoster virus glycoprotein III.

Hybridomas producing human monoclonal antibodies (HMAbs) against varicella-zoster virus (VZV) were generated by fusing murine myeloma cells with human lymphocytes immunized in vitro. An assay system was developed to select anti-glycoprotein (gp)III HMAbs from the pool of anti-VZV HMAbs. A murine anti-gpIII MAb, 4B7, did not react with a VZV-infected cell homogenate, but did react with a VZV-infected cell monolayer, whereas anti-gpI and anti-gpII MAbs reacted with both antigens. Hybridomas were screened to obtain HMAbs having a reaction profile similar to that of 4B7 and one such clone, V3, stably produces human IgG1 (kappa). HMAb V3 immunoprecipitated a VZV antigen of 115K to 120K, which was not immunoabsorbed by an anti-gpII HMAb, implying that V3 recognizes gpIII. V3 neutralized VZV independently of complement, unlike anti-gpI and anti-gpII HMAbs. All five strains of VZV tested were completely neutralized by V3, and the dose of V3 required to reduce the number of virus plaques by 50% ranged from 0.027 to 0.15 micrograms/ml. V3 was also able to inhibit the spread of virus infection from infected to uninfected cells, whereas anti-gpI and anti-gpII HMAbs could not. In addition, V3 mediated antibody-dependent cellular cytotoxicity but not complement-dependent cytotoxicity of VZV-infected cells. The results suggest that an anti-gpIII HMAb may provide a new means of passive immunoprophylaxis and also help to identify an antigenic epitope appropriate for a subunit vaccine.

Animals↗

PubMed1991

Advantages of a human immunodeficiency virus type 1 (HIV-1) persistently infected HeLa T4+ cell line for HIV-1 indirect immunofluorescence serology.

A HeLa T4+ cell line persistently infected with human immunodeficiency virus type 1 (HIV-1) was used in an indirect immunofluorescent antibody assay (IFA) system to explore its potential suitability as an alternative source of viral antigen for confirmatory IFA in HIV serology. In a study of 121 serum samples chosen because they were reactive on repeat examination by enzyme immunoassay but nonspecific by IFA by using HIV-1-infected H9 cells (H9 IFA) or gave discrepant results by enzyme immunoassay and H9 IFA, the specificity and sensitivity of the HeLa T4+ IFA were comparable to those of Western blot (immunoblot), and identification of the true positive samples among these discrepant or nonspecific samples by HeLa T4+ IFA was approximately twice that by H9 IFA. The primary advantages of using the HeLa cell line rather than lymphoid cell lines in IFA are that cells can be grown as a monolayer and that the individual cells are much larger. The cell membrane, cytoplasm, and nucleus are easily discernible; this allows specific and nonspecific staining to be distinguished. At least eight different nonspecific nuclear and cytoplasmic staining patterns were identified in this study by using T4+ cells.

Evaluation Studies as Topic↗

PubMed1991

Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.

We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly.

Biotin↗

PubMed1991

Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization.

A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy.

DNA Probes↗

PubMed1990

Serologic specificity of antibodies to herpes simplex virus glycoprotein B present in human sera. Analysis by transient expression of glycoprotein B-derivatives.

Glycoprotein B (gB) is an essential glycoprotein of herpes simplex virus (HSV) and a major target for cellular and humoral immune response in the infected host. In the present study, we have analyzed the pattern of reactivity of a panel of 23 HSV-seropositive patient sera using as test antigens gB derivatives made in COS cells in a transient expression assay. Our results show that nearly all the sera tested, reacted with wild-type gB or tgB (772) (that lacks 102 amino acids cytoplasmic domain). However, when tgB (477 amino acids) or gBdl (an inframe deletion between amino acids 477-772) were used as test antigens only 12 out of 23 sera tested positive. Further analysis using competition assays revealed that these sera can be classified into at least two groups: (i) that contain gB-reactive antibodies reactive to intact gB or tgB (772); (ii) that contain antibodies that recognize all forms of gB-derivatives tested. The results presented here underscore the potential limitations in using certain truncated forms of gB as antigens for subunit vaccine or in the serodiagnosis of HSV infection.

Antibodies, Viral↗

PubMed1990

Varicella-zoster virus infection of human astrocytes, Schwann cells, and neurons.

Human fetal cell cultures enriched for astrocytes, Schwann cells, or dorsal root ganglia neurons were infected with cell-free varicella-zoster virus (VZV), and the course of these infections was compared with that in fetal fibroblasts. Virus replication was detected in each neural cell type as early as 10-16 hr postinfection. Permissiveness of each cell type was confirmed by electron microscopy. However, the kinetics of virus spread varied between the neural cell types. Moreover, the accumulation, progression, and localization of VZV putative immediate early (IE), early (E), and late proteins was neural cell-type specific. VZV replication was slower in Schwann cells and neurons than in astrocytes. In Schwann cells and neurons VZV E proteins could be detected before IE proteins, a reversal of the order of accumulation noted with astrocytes and fibroblasts. There was also relatively more VZV IE protein in the perinuclear cytoplasm of Schwann cells and neurons, suggesting a delay in the transport of this antigen to its nuclear site. The permissiveness of non-neuronal cell types suggests that they could play a role in the pathogenesis of herpes zoster.

Antigens, Viral↗

PubMed1990

Monoclonal antibody to immediate early protein encoded by varicella-zoster virus gene 62.

Monoclonal antibodies (mAbs) were prepared against varicella-zoster virus (VZV)-infected cell proteins, and 10 mAbs which reacted with nuclear antigens were selected. These mAbs recognized a major 175-180 kDa and three minor VZV-specific phosphoprotein species. Immunofluorescence staining of VZV-infected cells showed that the 175-180 kDa protein was synthesized within 6 h after infection. The synthesis of this protein was inhibited by cycloheximide (CH); however, reversal of CH treatment and addition of actinomycin D (ActD) resulted in the synthesis of the 175-180 kDa protein. To determine whether the 175-180 kDa protein seen in the infected cells is encoded by VZV immediate early (IE) gene 62, the predicted open reading frames of VZV genes 61 and 62 were cloned into pGEM transcription vectors. RNA was transcribed from each gene, translated in vitro and immunoprecipitated with a mAb which recognizes a major 175-180 kDa and three minor proteins. The reactivity of the in vitro translation products encoded by gene 62 with this mAb suggested that the 175-180 kDa protein is encoded by VZV IE gene 62.

Animals↗

PubMed1990

Epitopes functional in neutralization of varicella-zoster virus.

By competition neutralization assay using monoclonal antibodies (MAbs) to varicella-zoster virus (VZV) glycoproteins (gps), we attempted to determine the topographical relationship of epitopes which are functional in VZV neutralization. MAbs against gpI interfered moderately to strongly with neutralization of MAbs against gpIII, and one antigenic domain with two distinct epitopes was identified on gpIII. Competition neutralization assays performed with MAbs to gpI revealed at least three distinct antigenic domains: the first contained two complement-dependent neutralizing epitopes; the second contained five complement-dependent neutralizing, overlapping epitopes and one nonneutralizing, nonoverlapping epitope; and the third contained one complement-enhanced neutralizing epitope. Competition neutralization assays performed with MAbs to gpIV showed one antigenic domain with two distinct epitopes which competed with nonneutralizing gpI MAbs. gpII did not interfere with neutralization of gpI, gpIII, or gpIV. Our data suggest that neutralizing and nonneutralizing MAbs can interfere with the action of viral neutralization either by inhibition or by enhancement. This report describes the epitope mapping of VZV gps by a functional biological assay.

Antibodies, Monoclonal↗

PubMed1986

New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products.

The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted.

Genes, Viral↗

PubMed1985

Rapid detection of herpes simplex virus DNA in human brain tissue by in situ hybridization.

A commercial system for detection of herpes simplex virus (HSV) DNA by in situ hybridization gave positive results on 16 of 17 stored human brain specimens that were positive for HSV on initial testing by virus isolation and immunofluorescence staining, and the hybridization system gave negative results on 13 brain specimens that showed no evidence of HSV by isolation or immunofluorescence staining.

Animals↗

PubMed1984

Antibody class capture assays for varicella-zoster virus.

Pooled monoclonal antibodies to varicella-zoster virus (VZV) were used as "detector" antibodies in a four-phase enzyme immunofluorescence assay for determination of immunoglobulin M (IgM), IgA, and IgG antibodies to VZV. Polyclonal antisera specific for heavy chains of human IgM, IgA, and IgG were employed as "capture" antibodies on the solid phase. The antibody class capture assay (ACCA) for VZV IgM antibody detected high titers of virus-specific IgM in all patients with varicella and in 5 of 10 zoster patients. VZV IgM antibody was not detected in patients with primary herpes simplex virus infections or in other individuals without active VZV infection, with one exception, a patient with encephalitis who had other serological findings compatible with a reactivated VZV infection. VZV-specific IgA and IgG antibody titers demonstrable by ACCA were compared with those measured by solid-phase indirect enzyme immunofluorescence assay (EIFA). VZV IgA antibody titers detected in patients with varicella and zoster were variable and could not be considered to be reliable markers of active VZV infection. IgA antibody titers detected by ACCA tended to be higher than those demonstrated by solid-phase indirect EIFA in varicella and zoster patients. VZV IgG antibody titers detected by ACCA in patients with varicella, and to a lesser extent in zoster patients, were as high as or higher than those demonstrated by solid-phase indirect EIFA. However, ACCA was totally insensitive in detecting VZV IgG antibody in individuals with past infections with VZV and would not be a suitable approach for determination of immunity status to VZV.

Antibodies, Monoclonal↗