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A fatal case of myocarditis in a neonate is described. The clinical features were evident at birth, and enteroviral RNA was detected in the blood of the baby on the day of birth and again 10 days later by a generic enterovirus nested reverse transcription-polymerase chain reaction (RT-PCR) assay. The enterovirus RNA was subsequently retested by a separate, newly developed nested RT-PCR assay yielding a PCR product within the VP1 coding region suitable for sequencing. Identical 239-base pair sequences were obtained from the RNA of the two blood samples and this sequence most closely resembled coxsackievirus B3 (94% identity). The baby's mother was pyrexial immediately postpartum and an early antenatal serum and a serum sample collected 10 days postpartum tested in parallel for enterovirus IgM antibody showed negative to strong-positive seroconversion. Infection of the mother was the likely primary event with in utero transfer of the virus to the fetus in the last few days of pregnancy. Neonatal blood is a valuable specimen for enterovirus diagnosis by RT-PCR. A newly developed nested RT-PCR assay was successful in typing the enterovirus from stored RNA extracted directly from the blood samples. Serology for enterovirus IgM antibody can be useful for convalescent diagnosis of enterovirus infection in the mother, especially with earlier serum for comparison., (Copyright 2003 Wiley-Liss, Inc.)

Primers were designed and tested for their ability to distinguish rhinoviruses from enteroviruses. A primer set derived from the 5'-UTR/VP coding region junction was able to amplify all the rhinovirus serotypes tested. Enteroviruses were either not amplified by these primer pairs or produced a band of larger size that could easily be discriminated from the rhinovirus-specific product. In contrast, primers embedded in the 5'-UTR region alone were able to amplify both rhinovirus and enterovirus RNA. It is shown that rhinoviruses could be specifically typed by sequencing the amplicon derived from the 5'-UTR set. The sequences of the 5'-UTR region of ten previously unsequenced rhinoviruses were derived. The sequences obtained cluster into two groups: 1B, 41, 15, 30, 63, 31, 56, and 44; and 17, 69, and 70. Amplicons from serotypes 17,69, and 70 also group by sequence with the equivalent region of HRV14 from the genetic database, while the others group with 2 and 89.

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.

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.

We present the electron microscopy observations on samples from 38 pregnancies that were investigated for B19 parvovirus infection. Thirty-four had resulted in fetal loss thought to be due to a virus infection and 22 of the 38 were positive for B19 parvovirus in one or more of the tissues. Twenty-one placentas and 75 fetal tissue samples were examined. Fresh samples were investigated by immune electron microscopy while formalin-fixed tissues were examined as thin sections and by negative staining of tissue extracts with direct electron microscopy. Electron microscopy was more sensitive on fresh than on fixed samples. The ultrastructural observations on thin sections of fixed tissues yielded new information locating B19 parvovirus particles in both nucleus and cytoplasm of infected fetal cells. The diagnostic results of the range of electron microscopy assays were compared with those of two hybridization methods. The fresh samples yielded comparable results from electron microscopy and hybridisation assays but on formalin-fixed materials hybridisation was more sensitive.

Evidence of B19 parvovirus infection was sought by in situ hybridisation with biotinylated probes in 65 tissue samples from 32 pregnancies (fetuses, products of conception and/or placentas). Twenty-seven samples were reactive and the results were confirmed by other methods for B19 virus detection in 22 cases. The other methods used were in situ hybridisation with 3H and 35S labelled probes; dot-blot hybridisation with biotin and 32P labelled probes; polymerase chain reaction assay; negative stain and thin section electron microscopy; and radioimmunoassay for B19 antigen. The five false positive results by in situ hybridisation with biotinylated probes were considered to be due to non-specific biotin capture and were more frequent with unfixed samples than with formalin fixed material. It was concluded that while biotinylated probes offered advantages over radioactive probes for detecting B19 DNA by in situ hybridisation, positive findings should be confirmed by other methods.

Human parvovirus B19 commonly infects children, causing erythema infectiosum (fifth disease). However, there is a significant adult population which has not been exposed to the virus and, consequently, does not have protective antibody. Recent reports have associated B19 infection during pregnancy with fetal death, although normal outcome of pregnancy is more common. To characterise further the role of B19 infection in fetal deaths, a series of laboratory investigations has been undertaken on tissues obtained at autopsy. These have demonstrated the presence of virion-sized DNA by Southern blotting, viral antigen by radioimmunoassay, and viral particles by electron microscopy, all from tissues of hydrops fetalis. These data confirm that the human parvovirus B19 can cross the placenta and replicate in fetal tissues.

Half of the genomic DNA of the human parvovirus (B19) was cloned in the plasmid pBR322. The cloned DNA was used as a molecular probe for the detection of parvovirus in serum by means of a dot hybridization test. In an assay of 26 samples, the dot hybridization test was found to be of comparable sensitivity and to be as rapid as radioimmunoassay for viral antigen detection; it is potentially useful as a diagnostic test.