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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.

Two DNA clones were obtained from faecal specimens containing a parvovirus-like small round virus from a 1977 outbreak of gastroenteritis, and their nucleotide sequences were determined and found to be essentially identical with parts of the published sequence of serum parvovirus B19 and with a B19 isolate (JB) partially sequenced in this study. The clones corresponded mainly to genome regions coding for non-structural proteins, but also include a sequence of some 160 bp coding for structural proteins. Southern blotting experiments with a full-length B19 probe revealed a virion-sized 5 x 5 kbp DNA band in specimens from gastroenteritis cases in both 1977 and 1986. Thus the nucleotide sequence and hybridization results suggest that the virus seen in these studies is very similar to B19. Further work is necessary to clarify the antigenic relationship of these viruses.

Owing to the lack of a practical means of production of large quantities of virus in cell culture, present tests to screen for parvovirus antibody rely on the use of virus obtained from the sera of blood donors during the brief, intensely viraemic phase of acute infection (Cohen et al, 1983). Accordingly, antigen is scarce and testing for parvovirus antibody is confined to a few centres. Availability of recombinant antigen would make the test more widely available, and by circumventing the requirement for use of whole virus render the test safer (Cohen et al, 1988). Therefore, constructs of parvovirus DNA encompassing the region of the genome coding for the structural proteins in expression vectors with T7 or trc or tac promoters were made, and proteins produced by the resulting clones tested for their ability to react with B19 antibodies from human sera. Additionally, recombinant B19 DNA was used as a hybridization probe for virus in clinical specimens, and a sensitive polymerase chain reaction assay was investigated as a tool for parvovirus diagnosis.

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.

The buoyant density, nucleic acid, and proteins of the human serum parvovirus-like agent were investigated. Evidence is presented which suggests that the virus has genomic single-stranded DNA, and that complementary strands may be encapsidated in separate virions. Three proteins of 48 000, 68 000 and 80 000 mol. wt. were found to co-purify with viral antigen at a density of 1.43 g/ml on CsCl gradients. On the basis of these properties it is suggested that this virus is a parvovirus.

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.

A nonradioactive dot blot hybridization assay for human parvovirus B19 DNA was set up by using a biotin-labeled DNA probe and streptavidin-alkaline phosphatase conjugate. The assay was used to examine 4,895 specimens referred for B19 virus diagnosis during 1987. Of 48 specimens that gave positive reactions for B19 DNA, 41 were confirmed virus positive by electron microscopy (n = 36), radioimmunoassay (n = 26), or counterimmunoelectrophoresis (n = 20). In 7 samples which were not confirmed and in 11 samples giving weak reactions for B19 DNA, there was serological or epidemiological evidence of recent B19 infection. A further 70 specimens gave weak, apparently false-positive reactions. By electron microscopy, 13 of 16 were contaminated by bacteria, and plasmid DNA was demonstrated in one specimen. Of 55 specimens tested, 52 reacted with streptavidin-alkaline phosphatase conjugate alone. These were probable sources of nonspecificity in an otherwise practical and economic screening method for B19 virus.

The polymerase chain reaction (PCR) was used to detect parvovirus B19 DNA in a panel of sera from individuals recently infected with B19, as demonstrated by the presence of anti-B19 immunoglobulin M. Of 95 serum samples, 60 (63%) were found positive by PCR, whereas only 1 was also found positive by dot hybridization. In a control panel of 100 serum samples from individuals with other infections, only 1 serum sample was found positive by PCR, and this was also found positive by dot hybridization. This was probably just a fortuitous discovery of viremia. Placental tissues from women (n = 89) who had proven B19 infections in pregnancy but who gave birth to healthy infants at term were also tested. A total of 74 (83%) were found positive for B19 DNA by PCR. The high rate of detection by PCR probably represents "decay" of viral DNA after the peak of viremia and is not a clinically significant phenomenon.

DNA from human parvovirus B19 was prepared from infected serum and examined by electron microscopy. Double-stranded molecules were seen, often with characteristic 'fold-back' ends that were assumed to be due to the inverted terminal repeats of the genome DNA. This double-stranded DNA was mapped with 13 restriction enzymes. More than 40 isolates, including the virus from the original B19 serum, were compared. Although isolates could be grouped by this method, there was no correlation between a particular restriction endonuclease map and any of the several disease presentations of the virus.