Genomic typing of BK virus in clinical specimens by direct sequencing of polymerase chain reaction products.

Two hundred and twelve urine specimens, from several clinical groups, were examined for BK virus (BKV) using the polymerase chain reaction (PCR) to detect the VP1 region of BKV DNA. Positive results were obtained on 14 specimens from 44 post-transplant patients (31.8%), 10 specimens from 39 pregnant women (25.6%), and 5 specimens from 100 children (5%) but not on any specimens from 29 laboratory staff. Twenty-eight of the amplified BKV genomes, 19 from urine specimens, eight from culture fluid of inoculated tissue, and also one from a throat washing were directly sequenced from single-stranded templates immobilized via a biotinylated primer; it was possible to assign all to one of the four subtypes of BKV which had previously been identified on the basis of variation in nucleotide sequence of the VP1 region. Serological subgroup classification correlated with the genomic subtyping results in 21 of the isolates. The distribution of the BKV subtypes and the clinical status of the infected individuals are discussed.

Detection of JC virus DNA in the cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy.

JC virus (JCV) DNA was detected by polymerase chain reaction (PCR) amplification in the cerebrospinal fluid (CSF) of 10 of 13 patients with a diagnosis of progressive multifocal leukoencephalopathy (PML) previously confirmed by histological and virological techniques. It was not found in 42 CSF samples from 41 patients who did not have PML. Four sets of primers were used to amplify polyomavirus DNA. One was from the region of the genome coding the T antigen, a conserved region common to JC and BK viruses. A second set nested to these was used as a confirmatory test in a secondary PCR. The remaining two were from the region of the genome coding VP1, one specific for JCV and the other for BKV. CSF did not inhibit the PCR and preliminary DNA extraction was not considered necessary.

BK virus antigenic variants: sequence analysis within the capsid VP1 epitope.

DNA sequences for the VP1 gene which codes for the major capsid protein of BK virus (BKV) and may be responsible for antigenic variability were determined for seven BKV isolates. The observed sequence differences and those previously reported correlate with the typing of isolates into four groups by haemagglutination inhibition. Amino acid coding alterations were found to be clustered within residues 61 to 83. Each antigenic group was found to have a characteristic amino acid sequence between residues 61 and 83. Several clones originating from a single isolate, although differing slightly in restriction enzyme digestion patterns, were found to be identical in this region. The VP1 sequences of three of the four groups were analysed by hydropathy plots and two hydrophilic areas of high antigenicity were identified. One of these corresponds to residues 61 to 83 and it is postulated that this region is the epitope responsible for serotypic differences between BK isolates.

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.

Detection of human papillomavirus DNA by PCR in semen from patients with and without penile warts.

OBJECTIVES: To determine the prevalence of urethral HPV infection, as indicated by the presence of HPV DNA in semen, in males with and without penile warts. DESIGN: Prevalence study of HPV types 6/11 and 16 DNA using PCR and Southern blot hybridisation analysis of semen. SETTING: Department of Genitourinary Medicine, Blundell Street Clinic, Leeds General Infirmary and the Assisted Conception Unit (ACU) Kings' College, London. SUBJECTS: Patients attending the Genitourinary Clinic for treatment of sexually transmitted diseases including penile warts and males attending Kings' ACU for investigations of infertility. MAIN OUTCOME MEASURES: HPV DNA detected by polymerase chain reaction (PCR) and/or Southern blot hybridisation in semen. RESULTS: HPV DNA was detected by PCR in 23 of 27 (85%) specimens from patients attending the GUM clinic for treatment of genital warts and in one of two specimens from patients attending the clinic for other conditions. By Southern blot, nine (33%) of the 29 specimens from GUM clinic patients were HPV DNA-positive. HPV DNA was detected by PCR in 43 of 104 (41%) of specimens from males attending the ACU, whilst 70 of these tested by Southern blot hybridisation were all negative for HPV DNA. CONCLUSIONS: The data suggest that urethral HPV infections, as indicated by the presence of HPV DNA in semen, are prevalent in males with and without genital warts.

Human papillomavirus type 16 infection of the cervix: a comparison of differing DNA detection modes and the use of monoclonal antibodies against the major capsid protein.

An immuno-peroxidase technique using monoclonal antibodies against the major capsid protein L1 of HPV-16 was compared with dot-blot hybridisation of cervical scrapes and in situ hybridisation of cervical biopsy specimens for HPV-16 DNA. In a series of 20 patients all techniques were specific for HPV-16 infection. Ten patients were positive by dot-blot hybridisation and half of those were positive by in situ hybridisation. Only one of HPV-16 DNA positive cases showed L1 protein expression, apparently shortly after the onset of clinical infection. Whether major capsid protein expression reflects episodes of viral replication deserves further study.

Human papillomavirus types in anogenital warts of children.

Tissue from anogenital warts of 25 children, 10 of whom were suspected of being victims of sexual abuse, was investigated by dot blot and Southern blot techniques for human papillomavirus (HPV) types. HPV DNA was detected in 22 children, two of whom had double infections. The genital HPV types 6 and/or 11 were detected in 20 children, and in three children other HPV types were found. One had HPV 18 (as well as 11); in a second child a possible skin type, HPV 2, was detected; and the third child was infected with an unidentified type. In three cases genital wart material was available from one of the parents, and in all three the HPV type was the same as that of the child. For nine other children one or both parents were reported to have genital warts. The source of infection appeared to be the adult genital tract, but sexual contact might not be the only means of transmission.

Serological typing scheme for BK-like isolates of human polyomavirus.

Human polyomavirus BK-like isolates were subjected to restriction endonuclease analysis with the enzymes EcoRI and Hind III. End-point dilution was used to obtain homogeneous virus pools for DNA analysis and to remove JC virus from a mixed stock. The results of Hind III digestion suggested that two subgroups could be distinguished. Several BK-like isolates were purified and rabbit antisera raised. The isolates were compared with each other and with BK and JC viruses by haemagglutination-inhibition (HI) and by neutralisation. JC virus was serologically distinct, but all the other isolates showed some cross-reactivity. Two subgroups were again evident: GS and PG were with prototype BK in subgroup 1, and MG and IV were in subgroup 2. Two isolates, AS and SB, reacted with isolates of both subgroups 1 and 2 but were distinct from one another: their genome was similar to subgroup 1 isolates. Typing by HI or by neutralisation may form a basis for grouping BK-like polyomavirus isolates.

Human papillomavirus type 31 infection of the uterine cervix in England.

A total of 452 uterine cervical scrapes, from two centres in England, has been examined by dot blot hybridisation for the presence of human papilloma virus (HPV) types 6, 11, 16, 18 and 31. HPV DNA was found in 36.5% samples. Twenty-nine of the women were infected with more than one type. Of those with detectable HPV, 14.5% had HPV type 31 infections. This type appears to cause significant infection of the genital tract in England.

Progressive multifocal leucoencephalopathy and viral antibody titres.

Progressive multifocal leucoencephalopathy (PML) is caused by a papovavirus but serum antibody titres are generally considered unhelpful in clinical diagnosis because antibodies to the commonest causal agent (JC virus) are frequently found in normal adults. There is little published information about CSF titres but usually they have not been useful. Two cases of PML, confirmed by autopsy, are described where CSF antibody to JC virus was measured. In one case the JC antibody titre was significantly higher in the CSF than the serum and we suggest that this finding is diagnostically useful. In this case there was a transient stabilization of the disease following treatment with cytarabine with a change in antibody titres suggestive of reduced viral replication in the central nervous system and a host response to the infection. In the other case, which was untreated, rising serum antibody levels indicated active infection with a host response.

Use of a molecular probe for detecting JCV DNA directly in human brain material.

A hybridot assay using a radiolabelled JC virus probe has been used to detect the presence of JCV DNA in brain biopsy and postmortem brain samples from patients with neurological disease and possible progressive multifocal leucoencephalopathy. Sixty-nine brain samples from 45 patients were examined. Eleven samples from eight patients had detectable JCV DNA sequences. In seven of the eight patients this result was confirmed by electron microscopy and/or virus isolation or immunofluorescence.

Detection of human polyomavirus DNA in urine specimens by hybridot assay.

A hybridot assay method using a labelled BK virus probe has been used to detect the presence of human polyomavirus DNA in 81 urine specimens from 61 patients, most of whom were immuno-compromised to some degree. Twenty-eight urines from 23 patients had detectable DNA. The results have been compared to virus isolation and electron microscopy on the same specimens. In 90 per cent a comparable result was obtained by at least one of the other two tests and in the 73 specimens on which all 3 tests were performed there was 80 per cent agreement between all. In 6 cases the hybridot assay was more sensitive in detecting infection.

BK virus specific IgM responses in cord sera, young children and healthy adults detected by RIA.

An IgM capture solid-phase radioimmunoassay (MACRIA) for BK virus (BKV) specific IgM is described. This test was found to be more sensitive in detecting BKV specific IgM than both haemagglutination inhibition and immune electron microscopy with serum fractions from sucrose density gradients. The use of this specific assay allowed large numbers of sera to be examined with ease so that the distribution of BKV specific IgM in different populations could be studied more fully. BKV specific IgM was detected in 11/300 sera from London blood donors, in 24/114 sera from children aged between 2 and 11 years admitted to a paediatric unit and 14/79 sera taken from children aged between 2 and 5 years for the investigation of anti-streptolysin 0 titres. BKV specific IgM was not detected in 404 cord sera examined to investigate the transplacental transmission of BK virus.

Strain differences and some serological observations on several isolates of human polyomaviruses.

Six new strains of BK polyomavirus are described and compared by serological reactions and restriction endonuclease digest patterns of their DNA to BKV (prototype) and BKV (GS). One strain (PG) had a restriction endonuclease pattern similar to BKV (prototype) but with two EcoRI cleavage sites. Four strains were similar to BKV (GS) and one strain (SB) appeared to be a variant of BKV, having a smaller defective genome. Two mixed polyomavirus infections have been identified and a new human polyomavirus (ASV), distinct from BKV and JCV, has been described.

Occurrence of IgM antibodies against BK and JC polyomaviruses during pregnancy.

In a serological survey of 430 pregnant women 45 had high or rising titres of BK-virus haemagglutination-inhibiting antibodies. The presence of BK-virus-specific IgM was confirmed in 10 of these women. No BK-virus-specific IgM was detected in the samples of cord blood from the babies born to these women. The sera from 40 women known to be excreting inclusion-bearing cells during pregnancy were tested for the presence of BK-virus and JC-virus-specific IgM and IgG. The presence of BK-virus-specific IgM was confirmed in three cases and JC-virus-specific IgM in seven cases. Specific IgM persisted for several months in some pregnant women. No Bk-virus-specific IgM was detected in any of the samples of cord blood from the babies born to these women with evidence of polyomavirus infection. No JC-virus-specific IgM was detected in 36 out of 37 of the cord bloods; however, in one it is possible that minute amounts of JC-virus-specific IgM were present.

A prospective study of human polyomavirus infection in pregnancy.

Urine samples from 1,235 pregnant women were examined by light microscopy for cytologic evidence of virus infection. Smears of urine sediment from 40 women (3.2%) were observed to contain inclusion-bearing cells; polyomavirus infection was confirmed by virologic methods in 24 (60%). A polyomavirus was isolated from 12 women. Five isolates were identified as JC virus and one as BK virus. Another isolate designated AS virus appeared to be unique. Serologic studies on the 40 women were consistent with a high frequency of reactivation of JC virus, and virus excretion was related to gestation. The evidence suggests that selective excretion of JC virus may occur in pregnancy. Among 390 pregnant women without inclusion-bearing cells in their urine, 78 (20%) had a high or rising titer of serum antibody to JC or BK virus or both, a result suggesting virus reactivation, but virus excretion was not detected. In contrast to other reports, no evidence was found for transmission of BK virus to the fetus.

Occurrence of e antigen in acute hepatitis B.

The occurrence of e antigen in 44 patients with acute hepatitis B was studied in order to determine if e was related to the outcome of the illness. In 13.6% of cases e was detected, and in none of these did the carrier state develop. The HBsAg carrier state developed in 4.6% of the patients and in none of these was e antigen detected. Anti-e was detected in only three cases. Only in the very early acute phase of illness e was detected and the time serum samples were taken for the detection of e was considered to be very significant. The occurrence of e was not found to affect the duration of HBs antigenaemia in those patients who recovered completely.