Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

Human herpesvirus 6 is closely related to human cytomegalovirus.

A sequence of 21,858 base pairs from the genome of human herpesvirus 6 (HHV-6) strain U1102 is presented. The sequence has a mean composition of 41% G + C, and the observed frequency of CpG dinucleotides is close to that predicted from this mononucleotide composition. The sequence contains 17 complete open reading frames (ORFs) and part of another at the 5' end of the sequence. The predicted protein products of two of these ORFs have no recognizable homologs in the genomes of other sequenced human herpesviruses (i.e., Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], herpes simplex virus [HSV], and varicella-zoster virus [VZV]). However, the products of nine other ORFs are clearly homologous to a set of genes that is conserved in all other sequenced herpesviruses, including homologs of the alkaline exonuclease, the phosphotransferase, the spliced ORF, and the major capsid protein genes. Measurements of similarity between these homologous sequences showed that HHV-6 is clearly most closely related to HCMV. The degree of relatedness between HHV-6 and HCMV was commensurate with that observed in comparisons between HSV and VZV or EBV and herpesvirus saimiri and significantly greater than its relatedness to EBV, HSV, or VZV. In addition, the gene for the major capsid protein and its 5' neighbor are reoriented with respect to the spliced ORFs in the genomes of both HHV-6 and HCMV relative to the organization observed in EBV, HSV, and VZV. Three ORFs in HHV-6 have recognizable homologs only in the genome of HCMV. Despite differences in gross composition and size, we conclude that the genomes of HHV-6 and HCMV are closely related.     

Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays

Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays. Five patients with nonherpetic vesiculobullous disorders were included as negative controls. Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative. Five controls were all negative for HSV or VZV. Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays. VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays. HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis. For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory.     

Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.     

Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates.

The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.     

Herpesvirus DNA in Peripheral Blood Mononuclear Cells of Some Patients with Meniere's Disease

Herpes simplex virus‐1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV‐1,2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV‐1 DNA or VZV DNA was detected in PBMCs of 50 age‐ and sex‐matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV‐1 or VZV may be associated with the development of some cases of Meniere's disease.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.     

Engineering large viral DNA genomes using the CRISPR‐Cas9 system

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus‐infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time‐consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat‐Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene‐ablated HSV but also gene knock‐in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein–Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.     

Varicella-zoster virus (VZV) ORF17 protein induces RNA cleavage and is critical for replication of VZV at 37 degrees C but not 33 degrees C

Varicella-zoster virus (VZV) open reading frame 17 (ORF17) is homologous to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). HSV vhs induces degradation of mRNA and rapid shutoff of host protein synthesis. An antibody to ORF17 protein detected a 46-kDa protein in VZV-infected cells. While HSV vhs is located in virions, VZV ORF17 protein was not detectable in virions. ORF17 protein induced RNA cleavage, but to a substantially lesser extent than HSV-1 vhs. Expression of ORF17 protein did not inhibit expression from a beta-galactosidase reporter plasmid, while HSV type 1 vhs abolished reporter expression. Two VZV ORF17 deletion mutants were constructed to examine the role of ORF17 in virus replication. While the ORF17 VZV mutants grew to peak titers that were similar to those of the parental virus at 33 degrees C, the ORF17 mutants grew to 20- to 35-fold-lower titers than parental virus at 37 degrees C. ORF62 protein was distributed in a different pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37 degrees C compared to 33 degrees C. Inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus. The importance of ORF17 protein for viral replication at 37 degrees C but not at 33 degrees C suggests that this protein may facilitate the growth of virus in certain tissues in vivo.     

Varicella-zoster virus (VZV)

Varicella-zoster virus (VZV) causes varicella in primary infection and zoster after reactivation from latency. Both herpes simplex virus (HSV) and VZV are classified into the same alpha-herpesvirus subfamily. Although most VZV genes have their HSV homologs, VZV has many unique biological characteristics. In this review, we summarized recent studies on 1) animal models for VZV infection and outcomes from studies using the models, including 2) viral dissemination processes from respiratory mucosa, T cells, to skin, 3) cellular receptors for VZV entry, 4) functions of viral genes required uniquely for in vivo growth and for establishment of latency, 5) host immune responses and viral immune evasion mechanisms, and 6) varicella vaccine and anti-VZV drugs.     

The detection of the herpes simplex virus 1 and 2 antigens in clinical specimens by using monoclonal antibodies

The possibility of using monoclonal antibodies (McAb), obtained earlier, for the detection of herpes simplex virus (HSV) in clinical specimens taken from sick and infected persons was studied. The examination of 90 persons revealed that the mixture of McAb 4A and 2C could effectively detect the presence of HSV antigen in the indirect immunofluorescence assay (IFA) directly in cells contained in cytological preparations (smears, scrapes, impressions) obtained from different organs of patients. The search of optimum combinations of McAb for the detection of HSV antigens by the method of the solid-phase enzyme immunoassay (EIA) was carried out. This study, made on purified HSV used as an experimental model, revealed that the maximum sensitivity could be achieved with the use of two McAb (4f6 and 7c4) out of three McAb (4f6, 7c4 and 3d10). The approbation of both variants of EIA on clinical specimens taken from 99 patients (blood clots, seminal fluid, scrapes of cervical canal cells, peripheral blood lymphocytes) showed that the addition of McAb 3d10 made it possible to detect 8 more positive specimens. 754 specimens from 337 patients were studied with the use of McAb-based EIA, and in 204 of these patients (61%) HSV antigen was detected. The results obtained with the use of our McAb were compared with the data obtained with certified commercial test systems. The coincidence of the EIA data with those obtained with the use of the Murex Wellcozyme HSV test system (UK) was registered in 75% of cases (in 15 out of 20 cases). The coincidence of the IFA data with those obtained with the use of the Sanofi test system (France) was observed in all 19 cases (100%).     

Susceptibility of varicella-zoster virus to thymidine analogues

Ten strains of varicella-zoster virus (VZV) were tested for susceptibility to 17 nucleoside analogues by a plaque reduction assay using human embryonic lung fibroblast cells. The compounds employed were 5-substituted arabinosyluracils and 2'-deoxyuridines, 2'-fluoro-arabinosylpyrimidines (F-araPyr) and acyclovir. In terms of the 50% plaque reduction dose (PD50), 4- to 40-fold difference were found between the 10 strains of VZV in susceptibilities to each compound. VZV was highly susceptible to 5-halogenovinyl-arabinosyluracils (XV-araUs); the PD50 values of these compounds were less than 0.001 micrograms/ml. VZV was much more susceptible than herpes simplex virus (HSV) type 1 to XV-araUs, but less susceptible than either HSV type 1 or type 2 to 5-ethyl-2'-deoxyuridine, 5-ethyl-arabinosyluracil and acyclovir.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.