A simplified immunofluorescence technique for antibody to varicella-zoster membrane antigen (FAMA)

Indirect fluorescent antibody to varicella-zoster membrane antigens (FAMA) was measured by a new technique. The procedure gives rapid, sensitive and accurate results and is suitable for use in diagnosis or screening of susceptibility to varicella-zoster virus (VZV) infection. The test procedure was simplified by using Terasaki tissue culture plates for the reaction and for direct observation by fluorescence microscopy. Preparations of VZV-infected Vero cells stored in liquid nitrogen could be used as antigen in this FAMA-test.     

A quantitative immunofluorescence test for detection of serum antibody to Sendai virus in mice

A simple, semi-automated immunofluorescence assay, (TRACK XI), was developed for the detection and quantitation of circulating antibodies to Sendai virus in mice. The assay was validated by selecting Sendai virus-free and naturally infected mice from six different colonies and testing each serum in both ELISA and quantitative immunofluorescence (QIF) assays. The QIF test utilizes Sendai viral proteins immobilized within a dried colloid gel and permits serum antibody quantitation in 30 minutes. Using a dedicated fluorometer, antibody titers in test sera are calculated automatically from a three-point best fit calibration line. The QIF test gave 92.9% agreement with the ELISA and proved to be a reproducible, accurate and convenient assay for the quantitative measurement of serum antibody to Sendai virus in mice.     

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.     

An immunofluorescence test for detection of serum antibody to rodent coronaviruses

An indirect immunofluorescence test, using cultured cells infected with two strains of mouse hepatitis virus, was developed for detection of antibody to rodent coronaviruses. The immunofluorescence test detected serum antibody to mouse hepatitis virus and rat sialodacryoadenitis virus earlier than the neutralization test. In the case of mouse hepatitis virus, the results of the immunofluorescence test closely paralleled those obtained with a commercially available enzyme-linked immunosorbent assay.     

Recombinant monoclonal antibody recognizes a unique epitope on varicella-zoster virus immediate-early 63 protein

We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.     

Accuracy of the endpoint assay for virus titration

The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.     

Structure of varicella-zoster virus DNA

Varicella-zoster virus (VZV) DNA was prepared from nucleocapsids and from enveloped virions of a laboratory strain (Ellen) and directly from the vesicle fluids of patients with zoster infections. VZV Ellen nucleocapsid DNA was cleaved with 11 different restriction endonucleases and electrophoresed in agarose gels. The restriction profiles of the nucleocapsid DNA were identical to those of the DNA recovered from purified virions, but differed from those of another VZV strain (KM). In vitro-labeled VZV K.M. DNA purified directly from vesicle fluid yielded a distinct restriction pattern which appeared to be unchanged after several tissue culture passages of the isolate from that fluid. Restriction endonuclease analysis (EcoRI or BglII) of VZV DNA revealed the presence of four cleavage fragments with a molar ratio of approximately 0.5. No individual fragments with molar ratios of 0.25 were noted. This observation suggests that the VZV genome may contain one invertible segment. Comparison of the electrophoretic migrations of VZV DNA fragments relative to those of DNAs of known size permitted calculation of the VZV genome size to be 72 X 10(6) to 80 X 10(6) daltons. These results were confirmed by electron microscopy which demonstrated a genome size of about 76 X 10(6) daltons for passaged and unpassaged VZV DNA. Electron microscopy also revealed that some of the DNA molecules recovered from nucleocapsids or directly from vesicle fluids were superhelical circles.     

Radioimmunoassay for antibodies in varicella-zoster virus serology.

The method of RIA for antibodies was employed with success in VZ virus serology. The method is suitable for testing VZ antibodies in the course of varicella or herpes zoster disease as well as for determining anamnestic titres. Its advantages are stability of antigen, objective reading of results and applicability to testing large serum sets.     

A single serum dilution method for the quantitation of neutralizing antibodies to varicella-zoster virus

A one-point serum dilution method for determination of neutralizing antibody in human sera to varicella-zoster (V-Z) virus instead of the serial serum dilution method was investigated. Focus counting was performed under a microscope on day 5 to 6 after inoculation of V-Z virus into 6-well plastic trays in which human embryonic lung cells were grown. A table was constructed to estimate the ND50 titers by the per cent reduction of the focus count from the control at only one dilution of test sera. The estimated ND50 values agreed well with those determined by the serial serum dilution method. Test sera showed a slight nonspecific reactivity at low serum dilutions, but reliable results could usually be obtained at a serum dilution of 1:8 or more. This method, which saves materials and labor, was applied to the quantification of neutralizing antibody against V-Z virus in human sera with satisfactory accuracy and reproducibility.     

Effect of HLA on the cellular immune response to varicella-zoster virus

The effect of HLA on varicella-zoster virus (VZV)-specific lymphocyte transformation (LTF) was studied in 100 normal immune adults and 64 children who were immunized with live attenuated varicella vaccine. In the normal adults, a statistically significant association was observed between low responsiveness and the presence of A2 (p less than 0.025), and also between high responsiveness and the presence of Aw24 (p less than 0.05). A similar but clearer association, i.e. low responsiveness with A2 (p less than 0.005) and high responsiveness with Aw24 (p less than 0.025), was observed in the vaccinated children. In these children, Aw31 was also found to be related to low responsiveness (p less than 0.05). These results suggest that the VZV-specific cellular immune response is in some way influenced by HLA.     

Analysis of dose-dependent antibody titration curves

Several types of dose-response titration curves were considered. It was demonstrated that the use of the so-called coordinates of dilution suggested earlier by us allows one to analyze the titration curves, obtained either by ELISA, or by agglutination. Theoretical curves, obtained by the developed theory are very similar to those obtained in experiments. It was shown, that the analysis of the titration curves could give important information concerning antibody-blocking factors in titration sera or other samples of studied antibodies.     

A qualitative analysis of a model for the transmission of varicella-zoster virus

Varicella-zoster virus (VZV) is a herpesvirus which is the known agent for causing varicella (chickenpox) in its initial manifestation and zoster (shingles) in a reactivated state. The standard SEIR compartmental model is modified to include the cycle of shingles acquisition, recovery, and possible reacquisition. The basic reproduction number R(0) shows the influence of the zoster cycle and how shingles can be important in maintaining VZV in populations. The model has the typical threshold behavior in the sense that when R(0)1, the virus persists over time and so chickenpox and shingles remain endemic.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Requirement of varicella-zoster virus immediate-early 4 protein for viral replication

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZV open reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.     

Characterization of varicella-zoster virus enhancement by the pesticide carbaryl

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.