Abbas Vafai

Latent Varicella–Zoster Viral DNA in Human Trigeminal and Thoracic Ganglia

BACKGROUNDnSome human herpesviruses become latent in dorsal-root ganglia. Primary infection with the varicella-zoster virus causes chickenpox, followed by latency, and subsequent reactivation leading to shingles (zoster), but the frequency and distribution of latent virus have not been established.nnnMETHODSnUsing the polymerase chain reaction, we performed postmortem examinations of trigeminal and thoracic ganglia of 23 subjects 33 to 88 years old who had not recently had chickenpox or shingles to identify the presence of latent varicella-zoster viral DNA. Oligonucleotide primers representing the origin of replication of the varicella-zoster virus and varicella-zoster virus gene 29 were used for amplification.nnnRESULTSnAmong the 22 subjects seropositive for the antibody to the virus, both the viral origin-of-replication and gene-29 sequences were detected in 13 of 15 subjects (87 percent) in whom trigeminal ganglia were examined and in 9 of 17 (53 percent) in whom thoracic ganglia were examined. Viral DNA was not detected in brain or mononuclear cells from the seropositive subjects. None of three thoracic ganglia from the one seronegative subject contained varicella-zoster viral DNA.nnnCONCLUSIONSnThese findings indicate that after primary infection with varicella-zoster virus (varicella), the virus becomes latent in many ganglia--more often in the trigeminal ganglia than in any thoracic ganglion--and that more than one region of the viral genome is present during latency.

Varicella-zoster virus infection of human mononuclear cells

Varicella-zoster virus (VZV) DNA was detected in mononuclear cells (MNC) of 7 humans with acute zoster 1-23 days after the onset of skin lesions. To further study the interaction of VZV with human MNC, cells obtained from seropositive normal donors were infected with VZV and analyzed for the presence of viral DNA and proteins. VZV-DNA was detected in T, B, and OKM 1 (monocyte-macrophage) positive cells, and virus-specific proteins were demonstrated by indirect immunofluorescence and immunoprecipitation. Hybridization studies revealed that VZV-DNA did not replicate in human MNC.

Persistence of varicella-zoster virus DNA in blood mononuclear cells of patients with varicella or zoster

Varicella-zoster virus (VZV) DNA was detectable by in-situ hybridization in blood mononuclear cells (MNCs) of patients with varicella or zoster for 2–56 days after the onset of a rash. VZV DNA was present in many MNCs from one acute varicella patient 2 days after the onset of the rash and was rarely found in MNCs during acute zoster, convalescent zoster, and convalescent varicella. The morphology of MNCs containing VZV was heterogenous, although most viral-DNA-containing MNCs were large monocytoid cells. Serial examination of blood MNCs from one adult with varicella revealed VZV DNA up until 8 weeks, but not 16 weeks, after the appearance of the rash; parallel studies in four zoster patients showed VZV DNA up until 3 weeks, but not later than 7 weeks after the appearance of the rash. These results indicate that MNCs become infected with VZV during the primary encounter with VZV (varicella) and during reactivation (zoster) and that infection continues for weeks after the onset of the skin rash. Furthermore, the detection of VZV DNA in blood MNCs of uncomplicated zoster patients coincides with the period during which these patients experience pain.

Detection of Antibodies to Varicella-Zoster Virus Proteins in Sera from the Elderly

Sera from 40 elderly individuals ranging in age from 60 to 94 years were tested for the presence of antibodies to varicella-zoster virus (VZV)-specific proteins. Sodium dodecylsulfate polyacrylamide gel electrophoresis analysis of lysates of VZV-infected BSC-1 cells labeled with either [35S]methionine or [3H]mannose and immunoprecipitated with human sera revealed the variable presence of VZV-specific antibodies to four VZV glycoproteins (gpI, gpII, gpIII, and gpIV), and three nonglycosylated proteins (155, 140, 32 kilodaltons, kDa). The predominant antibody response in the sera from the elderly was to VZV gpII and the 155-kDa species. In addition, some sera from elderly individuals without an identifiable history of varicella or zoster contained antibodies to VZV proteins, suggesting a possible subclinical infection in these patients. Finally a history of zoster in the elderly was significantly correlated (p = 0.02) with the presence of antibody to gpIV.

Affinity-purified varicella-zoster virus glycoprotein gp1/gp3 stimulates the production of neutralizing antibody

Varicella-zoster virus glycoprotein gp1/gp3 was purified by affinity chromatography using anti-gp1/gp3 monoclonal antibody 19.1 linked to CNBr-activated Sepharose CL-4B. Rabbits immunized with purified glycoprotein gp1/gp3 developed mono-specific neutralizing antibody.

Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein

Human sera from varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) seropositive individuals contain antibody to a 155-kilodalton (155 kDa) viral protein. In this study, we show that monoclonal antibodies (mAb10.1 and mAb1A1.4) prepared against VZV and HSV-1 proteins, respectively, reacted with nuclear antigens and recognized a 155 kDa protein in the infected cells. Immunoprecipitation of whole virions and viral nucleocapsids with these mAbs showed that the 155 kDa protein is located in VZV and HSV-1 nucleocapsids. In addition, immunofluorescence and cross-reaction experiments revealed the antigenic cross-reactivity between the VZV and HSV-1 155 kDa nucleocapsid proteins. To map the coding region of the VZV 155 kDa protein, a truncated DNA fragment from the predicted open reading frame 40 was cloned into an in vitro transcription vector (pGEM). The RNA transcribed from the inserted DNA was translated in vitro and immunoprecipitated with mAb10.1. The reactivity of the in vitro translation products with mAb10.1 indicated that the 155 kDa nucleocapsid protein is encoded by VZV gene 40. These findings demonstrated that the VZV 155 kDa nucleocapsid protein encoded by gene 40 induces humoral response which cross-reacts with both VZV and HSV.

Existence of similar antigenic-sites on varicella-zoster virus gpI and gpIV

We previously identified the antibody-binding site of a monoclonal antibody (mAb 79.0) on varicella-zoster virus (VZV) glycoprotein I (gpI) and showed that this monoclonal antibody binds to both VZV gpI and gpIV (Vafai et al., J. Virol. 62, 2544, 1988). In this study, a synthetic peptide comprising the mAb 79.0 binding site (designated el) was prepared and anti-peptide antibodies (RAnti-el) were raised in rabbit. RAnti-el recognized the primary translation products encoded by VZV genes 67 (gpIV) and 68 (gpI). To further localize the binding site of RAnti-el on VZV gpIV, the gpIV gene cloned in pGEM transcription vector was cleaved at different locations to generate four truncated DNA fragments. RNA was transcribed from each truncated gpIV fragment, translated in vitro and immunoprecipitated with RAnti-el. The results indicated that RAnti-el binds an antigenic determinant within the first 153 amino acid residues on the primary translation product of VZV gpIV. In addition, RAnti-el recognized the high-mannose intermediate but not the mature from of gpI in the infected cells or the translation products of gpIV glycosylated in vitro in the presence of canine microsomal membrane. These results: (a) confirmed the existence of a shared antigenic determinant on both VZV gpI and gpIV; and (b) indicated that the addition of terminal sugar modification may influence the conformation of gpI and gpIV with respect to the antigenic determinant recognized by RAnti-el.

Polypeptides encoded by varicella-zoster virus unique short sequences.

The SalI-I and K DNA fragments which lie within the unique short sequences (Us) and contain a portion of the inverted repeat sequences (IRs/TRs) of varicella-zoster virus (VZV) DNA were cloned in an in vitro transcription vector system (pGEM-2). RNA was transcribed from both strands, translated in vitro and analyzed by SDS-PAGE. The results showed that SalI-I and K each codes for three primary translation products. Polypeptides with Mrs of 19,000 (19K), 47K, 93K/90K are encoded by SalI-I and polypeptides of 12K, 19K, and 50K are encoded by SalI-K. These results are consistent with the predicted genetic expression of the VZV SalI-I and SalI-K DNA fragments.

Induction of antibody against in vitro translation products encoded by varicella-zoster virus glycoprotein genes

Antibodies were raised in rabbit against the in vitro translation products encoded by the varicella-zoster virus (VZV) glycoprotein genes gpI and gpIV. The antisera neutralized VZV infectivity and specifically identified two late VZV glycoproteins, gpI and gpIV, in VZV-infected cells and in the envelope of VZ virions. Pulse-chase experiments revealed a 55K precursor protein to gpIV (60K) and a 82K precursor protein to gpI (95K). Immunoprecipitation of 32P-labeled VZV-infected cells showed that the precursor-products of gpI are phosphorylated. These results demonstrate that translation products synthesized in vitro can be used to produce antibodies that recognize native viral proteins and therefore facilitate the identification and analysis of viral gene products in the infected cells.

Expression of varicella-zoster virus glycoprotein I in cells infected with a vaccinia virus recombinant

BSC-1 cells infected with a vaccinia virus recombinant containing the coding sequences for varicella-zoster virus (VZV) glycoprotein I (gpI) were analyzed by indirect immunofluorescence and immunoprecipitation for the expression and processing of gpI. The processing of gpI in cells infected with recombinant virus was the same as that observed during VZV infection. Immunofluorescence revealed localization of gpI to the membranes of recombinant virus-infected cells.