A novel varicella-zoster virus gE mutation discovered in two Swedish isolates.

BACKGROUND: Two VZV glycoprotein E (gE D150N) mutant strains were collected in North America in 1995 and in 1999. We now report a novel VZV gE mutant virus discovered in Europe in two VZV strains collected in Stockholm, Sweden, in 1990 and 1999. OBJECTIVES: To characterize the two isolates identified among a total of 634 VZV isolates collected over a 15-year period at the Karolinska University Hospital. STUDY DESIGN: VZV genes were amplified by polymerase chain reaction and their sequences were compared to the genomic sequence of VZV-Dumas prototype strain. RESULTS: A mutation within the gE gene in an epitope recognized by the 3B3 monoclonal antibody was identified in both isolates. A different residue was changed (R152S) compared to the North American strains (D150N). CONCLUSIONS: The occurrence of a VZV gE mutant virus is unusual, but probably a recurring event in Europe and North America. It is unknown whether widespread varicella vaccination programs will alter the rate at which these mutant viruses are isolated.     

Lamivudine and Famciclovir resistant hepatitis B virus associated with fatal hepatic failure.

BACKGROUND: Lamivudine (LMV) is the only nucleoside analogue approved for the treatment of chronic hepatitis B (CHB). LMV, as with other nucleoside analogues including Famciclovir (FCV), suppresses the replication of hepatitis B virus (HBV) by targeting the viral polymerase. However, prolonged antiviral therapy results in the emergence of drug resistance HBV which can contribute to virological breakthroughs and recurrent hepatitis flares. OBJECTIVES: A 38-year-old hepatitis B e antigen (HBeAg) positive Chinese female infected with genotype B HBV commenced treatment with FCV and LMV combination therapy but was later maintained on LMV monotherapy. The patient remained HBeAg positive throughout treatment. Virological breakthrough occurred with the emergence of drug resistant HBV. This coincided with worsening liver function and the patient died of subacute fulminant hepatitis. This study evaluated the virological factors that contributed to the clinical decline of the patient. STUDY DESIGN: Biochemical analysis and full-length HBV genomic sequencing were performed on serial serum samples collected from the patient before and during antiviral therapy. RESULTS: Virological analysis revealed that the pre-treatment dominant HBV quasispecies in the patient had a number of non-consensus genotype B mutations which were located in the basal core promoter (BCP), polymerase, X, core and S genes. Subsequent to the instigation of antiviral therapy, the dominant drug resistant HBV which caused virological breakthrough and was associated with hepatic failure displayed a series of unique mutations particularly in the BCP (A1762T and G1764A) and in the polymerase (rtL180M, rtM204V, rtA222T and rtL336V), core (cP5T, cS26A, cV85I and cP135A), surface (sI195M and sM213I) and X (xK95Q, xN118T, xK130M and xV131I) proteins. CONCLUSIONS: Monitoring for the accumulation of unique mutations within the genome of drug resistant HBV mutants isolated during long term antiviral therapy appears warranted in the clinical management of patients with CHB.     

Serine protein kinase associated with varicella-zoster virus ORF 47.

Varicella-zoster virus (VZV) ORF 47 lies in the unique long region of the VZV genome. Sequence homology studies have demonstrated that gene 47 possessed conserved protein kinase motifs. In this study, we investigated the properties of the ORF 47 product. First, a rabbit antiserum was raised against a protein generated from the fusion of the most antigenic ORF 47 domain with Escherichia coli beta-galactosidase. The high-titer antiserum reacted specifically with ORF 47 polypeptides translated in vitro. When incubated with VZV-infected cell lysate, the antiserum immunoprecipitated a phosphoprotein of M(r) 54,000, a size comparable with the predicted molecular mass. The precipitated viral protein was phosphorylated in a protein kinase assay; subsequent phosphoamino acid analysis indicated that the phosphotransferase associated with the ORF 47 protein was a serine protein kinase. Synthesis of the ORF 47 product in VZV-infected cell culture increased in the first and second days and plateaued after the third day of infection. The protein kinase activity associated with VZV ORF 47 had several distinctive biochemical properties: (i) its phosphotransferase activity was enhanced more by manganese than by magnesium, (ii) it utilized both ATP and GTP as donors of phosphate, and (iii) it phosphorylated both acidic and basic substrates. In summary, this report lends support to the computer homology data which predicted that VZV ORF 47 would encode a serine protein kinase.     

Neutralizing antibody responses to varicella-zoster virus.

Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.     

Human serum antibodies to varicella-zoster virus thymidine kinase.

The conditions required for the production of varicella-zoster virus (VSV)-induced deoxythymidine kinase (dTk) have been studied. Extracts from Vero cells harvested 62 h after VZV infection were found to contain VZV-induced dTk activity, with a minimal contribution from the cellular dTk activity. VZV dTK was shown to have a broad substrate specificity phosphorylating both deoxythymidine, deoxycytidine, and iododeoxyuridine. Deoxythymidine triphosphate inhibition studies revealed an intermediate deoxythymidine triphosphate sensitivity when compared with that of the cellular cytosolar enzyme and the deoxythymidine triphosphate-insensitive herpes simplex virus dTk. An assay for VZV dTk-blocking antibodies was developed, with [125I]iododeoxyuridine as a substrate in the presence of a deoxythymidine triphosphate concentration which selectively blocked the dTK of host cell origin. A total of 79 serum samples were studied; these included serum pairs from patients with varicella or herpes zoster and single sera from immune and nonimmune adults. VZV dTk blocking antibodies were detected exclusively in sera from patients with herpes zoster. All serum pairs showing VZV dTK seroconversion also showed a parallel conversion of complement fixation titers. The VZV dTk antibodies were found to be of the immunoglobulin G class. The immunological specificity of VZV dTK was investigated, and no cross-reactivity with herpes simplex virus type 1 or 2 dTk was found.     

A novel deletion mutant of hepatitis B virus surface antigen.

HBsAg is the most important serological marker for acute or chronic hepatitis B. Nevertheless, there are reports of HBsAg-negative virus carriers, either with anti-HBc as the only marker for hepatitis B virus (HBV) infection or even positive for anti-HBs and anti-HBc. We report isolates from a patient, in which a deletion in the HBs-gene was associated with persisting viremia in the presence of anti-HBs. The 62-year-old female, infected most likely by her husband, had detectable markers of chronic active hepatitis B, such as HBsAg, HBeAg, and anti-HBc-IgM, for 2 years. The patient then seroconverted to anti-HBs, although HBeAg and anti-HBc-IgM remained detectable. At this time, semiquantitative polymerase chain reaction showed about 10(4) viral genomes per milliliter of serum. Direct sequencing of the amplified products revealed a major population of DNA molecules with a deletion of nucleotide 31 of the HBs-gene, which up to now has not been described. This deletion led to a frame-shift and introduced a stop-codon after 21 amino acids of the sHBsAg. We suspect that this deletion, and the resulting HBsAg lacking the major epitopes recognized by specific antibodies, could favor ongoing viral replication, despite the presence of anti-HBs. However, because the reading frame of the polymerase was also severely damaged by this deletion, it is assumed that a minor population of intact genomes was present to help in the formation of virus particles.     

Novel varicella-zoster virus glycoprotein E gene mutations associated with genotypes A and D

Here, we describe the association of certain varicella-zoster virus (VZV) genotypes with unique glycoprotein E (gE) gene mutations. Within 45 analyzed VZV wild-type strains of genotypes A and D, five novel gE mutations were discovered. A statistically significant (P < 0.0001) association of certain gE mutations with VZV genotype D was found.     

Comparison of two methods for detecting varicella-zoster virus antibody with varicella-zoster virus cell-mediated immunity.

We evaluated an enzyme-linked immunoassay (EIA; BioWhittaker) and a latex agglutination (LA; Becton Dickinson) for varicella-zoster virus (VZV) antibody determination, using cell-mediated immunity (CMI) as a "gold standard." VZV EIA had a sensitivity, specificity, positive predictive value, and negative predictive value of 87, 91, 87, and 91%, respectively, compared with CMI. Correlation was excellent except when the varicella index was 0.9 to 1.2. We defined sera with varicella indices of 0.9 to 1.2 as indeterminate. LA had a sensitivity, specificity, positive predictive value, and negative predictive value of 96, 91, 97, and 90%, respectively, compared with EIA. LA reactivity only at a 1:2 dilution did not correlate with CMI, but sera reactive at dilutions of > or = 1:8 indirectly did. We defined indeterminate sera as those reactive at 1:2 and nonreactive at 1:8. EIA and LA were equivalent for determining VZV immune status, and both methods required modified criteria of interpretation to increase their specificity.     

Antibody to varicella-zoster virus after passive immunization against chickenpox.

Antibody titers to varicella-zoster virus were measured in varicella-susceptible immunocompromised children 48 h after they received either one of two lots of zoster immune globulin (ZIG) or a selected lot of immune serum globulin (ISG). Globulin was given to modify varicella in these children after exposure to varicella or zoster. Indirect immunofluorescence antibody titers (FAMA) of children after receipt of globulin ranged from less than 1:2 to 1:32. Geometric mean FAMA titers were highest after 1.2 ml of ISG per kg (FAMA titer 1:128) and 0.16 ml of ZIG lot A per kg (FAMA titer 1:1,024). Selected batches of ISG titering 1:128 or greater by FAMA, at a dose of 1.2 ml/kg, may be used to attempt to modify varicella in susceptible high-risk individuals when ZIG is not available.     

Specificity of skin test with varicella-zoster virus antigen in varicella-zoster and herpes simplex virus infections.

Specificity of the skin test with varicella-zoster virus (VZV) antigen was examined in guinea pigs infected with herpes simplex virus (HSV) type 1 or VZV and in children with a history of HSV infection who developed varicella. Infected guinea pigs responded positively only to homologous virus. No cross-reaction between HSV and VZV was detected in the skin test, as well as in the neutralization test in infected guinea pigs, suggesting that the VZV skin test is specific for immunity to VZV infection. Twelve children were infected with HSV during an HSV epidemic and subsequently developed varicella in institutional settings. During the 2.5-month period between the HSV and VZV infections, the immune status of the children to VZV was negative both in the skin test and in the antibody test, although antibody to HSV was detected by an immune adherence hemagglutination test. After VZV infection, all responded positively both in the skin test and in the antibody test (immune adherence hemagglutination test) to VZV. These results suggest that the VZV skin test is specific for immunity to VZV infection, not cross-reactive to HSV infection in humans. This specificity will be of value in screening susceptibility or immunity to VZV, irrespective of prior HSV infection.     

Improved yields of cell-free varicella-zoster virus.

Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and harvesting of cultures and have led to the production of cell-free virus preparations with infectivity titers of greater than or equal to 10(6) plaque-forming units per ml. The highest yields of cell-free virus were obtained by (i) sonic treatment of the cellular phase of cultures inoculated with trypsin-dispersed infected cells at ratios of 1 infected cell to 6 to 10 uninfected cells in the monolayer and (ii) harvesting cells after 24 to 36 h of incubation at 36 degrees C. At this time the cultures showed minimal viral cytopathic effect. Spread of infectivity occurred much more rapidly in cultures inoculated with whole infected cells than in those infected with cell-free virus. Complement-fixing antigens with improved titers of greater than or equal to 1:128 were prepared from varicella-zoster virus-infected cell cultures in the same manner as cell-free virus, but harvested after 3 to 4 days of incubation when the cultures showed an advanced cytopathic effect.     

Enzyme-linked immunosorbent assay for detection of antibody to varicella-zoster virus.

Primary varicella-zoster virus (VZV) infection is a serious illness in immunocompromised individuals, and a rapid, sensitive, and reliable assay to identify high-risk VZV-susceptible patients would be clinically useful. An enzyme-linked immunosorbent assay (ELISA) for antibody to VZV was compared with the fluorescent antibody-to-membrane antigen (FAMA) assay and found to be similar in both sensitivity and specificity. The antibody titers determined by both assays were also similar. The absence of antibody detected by ELISA correlated with susceptibility to VZV infection. Because it is simple to perform and has equivalent sensitivity to FAMA, ELISA should be useful for VZV antibody testing in diagnostic and research laboratories.     

Use of genomic analysis of varicella-zoster virus to investigate suspected varicella-zoster transmission within a renal unit.

BACKGROUND: The source of hospital-acquired chickenpox infection may be presumed from a known exposure, but has not been previously proven using genomic analysis. OBJECTIVE: Investigation of suspected VZV transmission was done using single nucleotide polymorphism genomic analysis. STUDY DESIGN: Comparison was made of viral isolates from two patients with chickenpox on the same ward who were not known to have had direct contact. RESULTS: An identical genotype in the variable R1 region of the VZV was isolated from the two patients. CONCLUSION: Inapparent hospital-acquired transmission was the most likely route of infection.     

Quantitation of antibodies to varicella-zoster virus by immune adherence hemagglutination.

Immune adherence hemagglutination was compared with the complement fixation test as a means of measuring antibodies to varicella-zoster virus. Analysis of acute- and convalescent-phase sera from patients infected with varicella-zoster or with herpes simplex virus showed the immune adherence hemagglutination test to be more sensitive than the complement fixation test, and greater cross-reactivity between the two viruses appeared to be associated with the increased sensitivity. The two assay methods were used to measure antibodies to varicella-zoster virus in 265 sera obtained from patients of different ages as well as sera from 26 patients with leukemia. There were 35 cases where antibodies were detected by immune adherence hemagglutination but not by complement fixation, whereas in five cases the converse was found. Our findings support the contention that immune adherence hemagglutination is the method of choice for detecting antibodies to varicella-zoster virus.     

A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis

BACKGROUND. A nosocomial outbreak of fulminant hepatitis B occurred in five patients in Haifa, Israel. Previous investigations identified the suspected source as a carrier of hepatitis B surface antigen who was positive for antibodies to hepatitis B e antigen and had chronic liver disease. We examined the strain of hepatitis B virus (HBV) that caused this epidemic, in order to identify specific mutations in the precore or core region. METHODS. The presence of HBV was identified by polymerase-chain-reaction amplification of viral DNA in serum from the source patient, the five patients with fulminant hepatitis B, and five controls with acute, self-limited hepatitis B. The amplified viral HBV DNA samples were then cloned and sequenced. RESULTS. Sequence analysis of viral DNA established that the same HBV mutant with two mutations in the precore region was present in the source patient and the five patients with fulminant hepatic failure. This HBV mutant had significant sequence divergence from other known HBV subtypes in the X, precore, and core regions. Cloned HBV DNA derived from a hospitalized patient who had subclinical hepatitis B at the same time as the outbreak and from four other control subjects with acute, self-limited hepatitis B all contained the wild-type sequence in the precore region. CONCLUSIONS. In the outbreak we studied, a mutant hepatitis B viral strain was transmitted from a common source to five patients who subsequently died of fulminant hepatitis B infection. Naturally occurring viral mutations hepatitis B infection. Naturally occurring viral mutations in the HBV genome may predispose the infected host to more severe liver injury.     

戊型肝炎病毒新潜在中和性表位的发现

目的 探讨戊型肝炎病毒(HEV)衣壳蛋白是否存在除主要免疫优势中和表位aa459～606以外的其他中和表位.方法 通过对几株单克隆抗体及其表位的性质进行分析,对比位于主要免疫优势表位区aM59～606和位于该表位N端序列aa394～458区域的数个表位的中和活性.结果 发现位于aa423～437的表位对应的单抗具有潜在中和活性,不同于已知的HEV中和性表位(aa459～606),该表位是一个线性非免疫优势表位.结论 HEV ORF2 aa423～437为新的潜在的线性非免疫优势中和表位,该发现丰富了对HEV衣壳结构的认识,为HEV预防与治疗提供了新的针对靶点.     

Antibody avidity following varicella-zoster virus infections.

The avidity of IgG antibodies following varicella-zoster virus (VZV) infections was investigated using urea treatment of antigen-bound serum antibody by indirect radioimmunoassay (RIA) and immunoblotting techniques. Sequential sera from 16 patients with varicella and 17 patients with zoster were tested, as well as sera from 80 seropositive individuals without a recent history of VZV disease. Both types of assay showed that low-avidity antibodies predominate early after primary infection, but that antibody avidity increases markedly during convalescence. Using RIA, all sera taken up to 12 weeks after the onset of varicella showed greater than 50% reduction in antibody titre after treatment with 8 M urea but thereafter the proportion of urea resistant antibody increased with time. In contrast, after recurrent infection, high avidity antibodies were found to predominate at all times. Only 6 of 47 sera tested from zoster cases showed greater than 30% reduction after urea treatment and all these were taken within 2 weeks after onset of rash. Immunoblotting also showed that the highly immunogenic p32/p36 nucleoproteins appear to induce predominantly low avidity antibodies, even after recurrent VZV infection. The results of this study indicate that treatment with 8 M urea in RIA for IgG antibodies may be a simple and reliable method for distinguishing primary and anamnestic antibody responses against VZV.