Wendy A. Knowles

Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.

Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. Methods: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. Results: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. Conclusion: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

Prevalence of long-term BK and JC excretion in HIV-infected adults and lack of correlation with serological markers

The natural history of polyomavirus infection, and sensitivity of diagnostic assays remain unclear. A stratified group of 94 human immunodeficiency virus (HIV)‐infected patients was studied for both virological and serological markers of active infection with both JC virus and BK virus. JC DNA was detected in the urine of 18 of 81 (22%) patients and BK DNA in 30 (37%) patients. Whilst patients with a low CD4 cell count (P = .009), CD4/CD8 ratio (P = .031) and β2M concentration (P = .042) were significantly more likely to be excreting BK, JC excretion did not correlate with any of the immunological markers measured. Furthermore, when all the immunological factors were taken into account, there was no association between either BK or JC excretion and age of the patient (P = .149 for BK, P = 0.891 for JC). BK IgM antibody was detected in only 3 of 30 (10%) BK excretors. JC IgM was detected in 5 of 18 (27.7%) JC excretors but also in 11 of 63 (17.5%) patients without demonstrable JC excretion. Therefore IgM was a very poor indicator of viruria. One year follow‐up on a subset of patients showed that both DNA detection in urine and IgM antibody remain stable over many months despite falling CD4 cell counts, and would indicate that events leading to enhanced viral production probably occur early after HIV infection. Replication of JC virus in the brain leading to the onset of progressive multifocal leukoencephalopathy (PML) could not be predicted using any of the markers studied. J. Med. Virol. 59:474–479, 1999.

The JC virus antibody response in serum and cerebrospinal fluid in progressive multifocal leucoencephalopathy

BACKGROUND A clinical diagnosis of progressive multifocal leucoencephalopathy (PML) can be confirmed by histological or virological examination of brain material. Whilst a less invasive method is provided by the detection of JC DNA in cerebrospinal fluid (CSF), very few studies have been done to assess the value of JC virus (JCV) serology in PML diagnosis. OBJECTIVES To study the JCV antibody response in the serum and CSF of PML patients. STUDY DESIGN A retrospective study was done using haemagglutination inhibition (HI), M-antibody capture radioimmunoassay (MACRIA) and JC-specific oligoclonal IgG banding on one or more sera and/or CSFs from 28 confirmed PML patients. Seventy-one serum and CSF samples were tested from patients with memory loss or dementia as a control group. RESULTS Twenty-seven PML patients (96%) had detectable JCV HI antibody in the serum, with titres ranging from 1 : 10 to > 1 : 20480, compared to 48 (68%) of the controls (P = <0.005). JCV IgM antibody was detected in the serum of 12/22 (55%) PML patients. JCV HI antibody was detected in the CSF in 12 of 18 (67%) PML patients, antibody index measurements being used to control for a possible breakdown of the blood-brain barrier. Intrathecal JCV antibody was not found in any control patient. Locally produced JCV-specific IgG bands were detected in the CSF of 7 PML patients tested, confirming the intrathecal origin and specificity of the HI antibody. CONCLUSIONS The presence of intrathecal JCV antibody indicates active central nervous system infection with JC virus, and provides a useful diagnostic test for PML, with a sensitivity of 67% and a specificity of 100%. The absence of serum JCV antibody nearly always excludes a diagnosis of PML, but the titre of antibody, IgG or IgM, correlates with the underlying condition rather than the development of neurological symptoms.

In-vitro cultivation of human cytomegalovirus in thyroid epithelial cells.

SummarySeveral strains of human cytomegalovirus including recent isolates, were grown in epithelial cells derived from thyroid tissue. All the strains tested grew in these cultures without pre-treatment of the cells, and no difference in cytopathic effect was detected between strains of genital and non-genital origin. 4 strains of CMV were isolated directly from urine in thyroid cells; however the failure to isolate 6 further strains in these cultures from other specimens may indicate that a higher multiplicity of infection is required to infect epithelial than fibroblast cells.

The genomic sequence of a contemporary wild-type mumps virus strain

Vaccination has the potential to eradicate mumps, and 82 countries now include a live attenuated mumps vaccine as part of their childhood vaccination programme. Although, monotypic, genetic variants of mumps virus (MuV) have been described based on comparison of the SH gene sequences, and at least seven genotypes have been identified. We now report the entire sequence of a recently isolated wild type MuV strain, Glouc1/UK96 (Glouc1) by direct sequencing of the cDNA obtained from cell culture fluid. The genome of this recent isolate was 15384 nucleotides in length. There were 579 nucleotide differences (3.8%) and 71 amino acid differences (1.5%) between Glouc1, a genotype G strain and Ur-AM9, a genotype B strain. Other MuV strains with available sequences were also compared with this pathological strain. The sequence of the contemporary strain reported here provides a picture of the variability of MuV over its entire genome (GenBank accession no. AF280799).

Characterization of a new polyomavirus (Polyomavirus papionis-2) isolated from baboon kidney cell cultures

SummaryViruses with papovavirus morphology were seen in fluids from baboon kidney cell cultures on three separate occasions (isolates A, B, and C). The size of the virions, 47.9 nm, placed the virus in the polyomavirus genus. It grew well in baboon kidney and Vero cells and less well in human embryo lung (HEL) fibroblasts.The virus could not be identified as the previously described baboon polyomavirus, SA 12, or as any of the other known primate polyomaviruses BK, JC or SV 40, the non-primate viruses mouse polyoma, K, rabbit kidney vacuolating virus (RKV) or bovine polyomavirus (FRKV) by immunofluorescence, immune electron microscopy or hemagglutination inhibition (HI) tests. A rabbit antiserum to the new virus (isolate A) reacted only with the three isolates and not with the other primate polyomaviruses studied.Thirteen percent of 118 wild-caught baboons (Papio anubis) had HI antibody to the new polyomavirus and 21 percent were seropositive for SA 12; only two baboons had antibody to both viruses. These results suggest that in baboons there are two antigenically distinct polyomaviruses which circulate independently. The two viruses may also be distinguished by their hemagglutinating properties: SA 12 agglutinated erythrocytes from a wider range of species but only the newly recognized polyomavirus agglutinated baboon erythrocytes.We propose that the two baboon viruses, SA 12 and the new virus, should be namedPolyomavirus papionis-1 andPolyomavirus papionis-2 respectively.

Comparison of cell culture-grown JC virus (primary human fetal glial cells and the JCI cell line) and recombinant JCV VP1 as antigen for the detection of anti-JCV antibody by haemagglutination inhibition.

JC virus (JCV) is the causative agent of the demyelinating disease progressive multifocal leucoencephalopathy (PML), which can be diagnosed by detection in the cerebrospinal fluid (CSF) of both JCV DNA and intrathecally-produced anti-JCV antibody. However, the restricted in-vitro species and cell tropism shown by JCV has made antigen production difficult and limited serological investigations both in PML diagnosis and for JCV epidemiology. In this study antigen prepared as a crude cell lysate of JCV-infected primary human fetal glial (PHFG) cells was compared in a haemagglutination inhibition (HI) assay with antigen produced from the JCV carrier cell line, JCI, and yeast-expressed JCV VP1. Forty-two sera were tested with each antigen and there was a high level of correlation between the assays: 96.5% between the HI assays with PHFG and JCI antigens and 98.1% between the HI assays with PHFG and recombinant VP1 (rVP1) antigens. The JCI antigen gave HI titres 19% lower than the PHFG antigen (P=0.022). Titres with the rVP1 antigen were 2% higher than with the PHFG antigen (P=0.83). When serum/CSF pairs from 11 PML patients were tested, the antibody index calculated in each case confirmed the production of intrathecal anti-JCV antibody. Antibody testing for JCV is no longer reliant on PHFG cells and JCV serological tests should be available more widely.

Genetic and antigenic characterisation of the haemagglutinin protein of measles virus strains recently circulating in the UK

The complete nucleotide sequence of the H protein gene of seven measles virus (MV) strains, representing three MV genotypes circulating in the UK in recent years, was determined. Compared to the MV vaccine strain Moraten (Mor-v), the divergence of the coded H gene (aal-600) of the seven UK strains was between 1.8% and 2.8%. Representative isolates from each of the genotypes were tested by radio-immunoprecipitation using a panel of H protein-specific MAbs. Different patterns of MAb reactivity were shown between the three genotypes and between the wild-type strains and the vaccine strain. Plaque reduction neutralising antibody titres against strains UK350/94 (genotype I) and UK226/94 (genotype III) were measured in sera from 11 vaccinees. Vaccine derived antibody neutralised both strains and the GMTs were not significantly lower against the wild-type strains than against strain Mor-v.

An M-antibody capture radioimmunoassay (MACRIA) for detection of JC virus-specific IgM

A solid-phase M-antibody capture radioimmunoassay (MACRIA) for detecting JC-specific IgM is described. The assay is based on a JC-specific monoclonal antibody (17.7.6) and Nonidet P40-treated, glycine-extracted antigen. MACRIA is more sensitive for JC IgM detection than haemagglutination inhibition (HI) following serum fractionation on a sucrose density gradient, and can be applied to large numbers of sera. The specificity of the assay was confirmed by examining sera from several acute virus infections and also those containing rheumatoid factor. Sera collected from renal transplant recipients with known active JC virus infection were found to contain more than 5 units of JC IgM. In this group of patients JC IgM represents either primary or reactivated JC infection. JC IgM was detected by MACRIA in 15 of 100 unselected blood donors, indicating that JC IgM is frequently produced in healthy seropositive individuals. Thirteen of the 15 sera positive from blood donors contained only low levels of JC IgM (< 5 units), but the specificity of all these results was confirmed in a blocking assay. It is suggested that these low levels of JC IgM may occur in up to 28% of seropositive individuals and result from active JC antigenic stimulation in healthy immunocompetent adults.

An indirect immunofluorescence method for detection of infectious BK virus in urine

An indirect immunofluorescence (IF) method is described for the detection of infectious BK virus in urine within seven days in contrast to up to three months or longer using routine tissue culture. Virus is pelleted from the urine, inoculated onto pre-formed monolayers of human embryo lung (HEL) fibroblasts, and infected cells are detected in an indirect fluorescent antibody test using a human serum. The sensitivity of the IF method is 91%. Positive isolates may be confirmed as BK virus using specific rabbit antisera on the original inoculated cultures. Furthermore, a virus stock may be grown up by passage from the original culture fluids for further studies such as DNA analysis. As a broadly-reactive human serum is used for screening the cultures, other viruses which grow in HEL cells, such as CMV, may also be detected.