Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR.
We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at -80 degrees C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs. (+info)
Varicella zoster virus infection associated with high-dose chemotherapy and autologous stem-cell rescue.
A retrospective evaluation of 215 consecutive recipients of high-dose chemotherapy (HDC) and autologous stem cell rescue (ASCR) was conducted to ascertain the incidence, temporal course, and outcome of varicella zoster virus (VZV) infection. Herpes zoster was identified in 40 individuals at a median of 69 days following ASCR. Six of these cases occurred at a median of 33 days prior to ASCR but following the initiation of high doses of stem cell mobilization chemotherapy. Twenty-five percent of patients demonstrated cutaneous or systemic dissemination and 32.5% required medical intervention for post-herpetic neuralgia. All except two individuals received antiviral chemotherapy. One patient with active VZV infection died of multiorgan failure 39 days after ASCR. Multivariate analysis of risk factors disclosed the significance of prophylactic acyclovir use in Herpes simplex virus seropositive individuals in reducing the risk of VZV infection. Moreover, the use of busulfan, thiotepa and carboplatin as the conditioning chemotherapy regimen was associated with an increased risk of subsequent VZV infection. The incidence of VZV reactivation after HDC and ASCR is similar to that observed following bone marrow transplantation but has an earlier onset. This may be related to an earlier induction of immunosuppression by stem cell mobilization chemotherapy administered prior to ASCR. We demonstrated a marked reduction in the proliferative and synthetic capacities of peripheral blood mononuclear cells obtained prior to and following stem cell mobilizing chemotherapy. Moreover, greater than 80% of VZV infections occurred within 6 months following ASCR and late cases were seldom observed compared to allogeneic and autologous bone marrow transplantation. The role of antiviral chemoprophylaxis during the period of maximum immunocompromise needs to be studied further in the HDC-ASCR setting. (+info)
Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth.
Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication. (+info)
Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay.
A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF. (+info)
AIDS related eye disease in Burundi, Africa.
AIMS: To determine the prevalence of ocular manifestations in AIDS patients hospitalised in Bujumbura, Burundi, according to their CD4+ lymphocyte count, serological status for CMV and VZV, and general health status. METHODS: Prospective study of 154 consecutive patients who underwent general and ophthalmological examinations, including dilated fundus examination. AIDS was diagnosed on the basis of Bangui criteria and HIV-1 seropositivity. CD4+ lymphocyte counts were determined by the Capcellia method. CMV and VZV antibodies were detected with ELISA methods. RESULTS: The mean age was 37 (SD 9) years and 65% of the patients were male. Active tuberculosis was the most frequent underlying disease (61%). Almost all the patients (99%) were seropositive for CMV and VZV. Among the 115 patients for whom CD4+ lymphocyte counts were available, 86 (75%) had more than 100 cells x 10(6)/l. Ocular involvement comprised 16 cases of microangiopathy, six of opalescence of the anterior chamber, five of retinal perivasculitis, two of zoster ophthalmicus, two of viral retinitis, and one of opalescence of the vitreous. CONCLUSION: In Africa, the prevalence of ocular involvement in HIV infection is far lower than in Europe and the United States, possibly because most African patients die before ocular opportunistic infections occur. (+info)
Transcript mapping and transregulatory behavior of varicella-zoster virus gene 21, a latency-associated gene.
Gene 21 is one of at least four genes transcribed during latent infection of varicella-zoster virus (VZV) in human ganglia. It may encode a nucleocapsid protein, but its function in lytic and latent infection is not clear. To characterize further the structure and the function of the gene 21 open reading frame (ORF 21), precise localization of its transcripts and their termini was determined by using Northern analysis, S1 nuclease or RNase protection, and primer extension assays. One abundant 3.5-kb transcript that spans ORF 21 was identified. A predominant transcription start site was defined at -78 nucleotide (nt) relative to the ORF 21 translation start codon ATG, and two potential TATA elements were identified at 26 and 83 nt upstream of the 5' end of gene 21 transcripts. Transcription was found to terminate 210 nt beyond the ORF 21 translation stop codon and immediately before the start codon of ORF 22. In transient expression assays, the ORF 21 showed no significant transregulatory activity on promoters of diverse kinetic classes. The ORF 21 promoter, however, was transactivated strongly by VZV infection or by ORF 62. (+info)
Latent Varicella-zoster virus in human dorsal root ganglia.
To understand further the molecular events underlying the process of Varicella-zoster virus (VZV) latency in human ganglionic tissues, in situ hybridisation (ISH) for VZV RNA and DNA, and PCR in situ amplification for VZV DNA were used in human dorsal root ganglia from 12 individuals (3 normal and 9 who had died with AIDS). The results showed that (a) two separate regions of the VZV genome, represented by genes 4 and 40, were detected in neurons in two normal and three AIDS ganglia, (b) evidence of transcription of VZV genes 4, 21, 29, and 63 was found in normal and AIDS cases, and (c) VZV DNA and RNA for the same gene (gene 29) was detected in neurons in serial tissue sections in three cases. Thus more than one region of the VZV genome is present in neurons during VZV ganglionic latency, and the presence of both a VZV gene and its corresponding RNA transcript can be shown to occur in the same localised region of DRG tissue. (+info)
Varicella-zoster virus-specific cellular immunity in subjects given acyclovir after household chickenpox exposure.
The time course of primary cell-mediated immune responses to varicella-zoster virus (VZV) among persons receiving acyclovir prophylaxis after exposure to chickenpox has not been well defined. Fifteen children who had household exposure to varicella received prophylactic acyclovir (40 mg/kg/day for 7-14 days after exposure) and were studied for development of both antibody and cell-mediated immunity (CMI) to VZV. Twelve developed antibodies and/or CMI; 10 had no symptoms and 2 manifested mild varicella. Two were already immune to varicella and had booster immune responses. One was not infected and subsequently developed full-blown varicella. Although acyclovir given after exposure to VZV is highly effective and does not appear to attenuate the immune response, it remains necessary to confirm whether, in the absence of clinical varicella, persons acquire specific immunity. (+info)