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The discovery of prion proteins and the diseases which are associated with them still present scientists and clinicians with a number of problems. There are clearly risks with the use of living cells and materials of animal origin to produce therapeutic compounds with respect to the transmission of prion protein. However the medical benefit many of these compounds has to be weighed against this. It is clear a number of groups are continuing to unravel the highly complex relationships of prion biology and pathology and it is only when this is clearly established that the community can decide on these issues. Until this time the scientific community must rely on the best research available and provide guidance from this.

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The present study details the design and demonstrates function for a series of reagents and methods to allow the detection of exposure to antigens specific for Porcine endogenous retrovirus (PERV). The detection of PERV is carried out by the means of a variety of immunological screening methods including, indirect immunofluorescence, Western blotting and enzyme linked immunosorbent assay (ELISA) for the detection of antibodies in serum specific for PERV gag and env antigens. Alternatively, PERV-specific antisera for gag and env can be used to detect viral antigen in serum or other samples. PERV env peptides with potential specificity for the known PERV types are also described. Antisera against the peptides can be used to detect PERV antigens directly or to characterise viral type. Using electron microscopy coupled with labelled PERV-gag-specific antisera it was possible to visualise PERV virions.

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AIMS: This study was designed to assess further the possible links between enterovirus infection and Type 1 diabetes mellitus (DM). METHODS: Sera from 110 children in the age range 0-15 years was obtained shortly after the diagnosis of Type 1 DM, in paediatric centres throughout the UK. They were tested for the presence of enteroviral sequences by the polymerase chain reaction (PCR) of the 5' nontranslated region (5' NTR). One hundred and eighty-two controls tested were matched for age, geographical location and time of year. RESULTS: A significantly greater number of diabetic children (27% vs. 4.9%, P <0.005) had evidence of enteroviral RNA sequences. Proportionally, more younger children were enterovirus PCR positive, thus eight out of 20 children aged < or =2 years were enterovirus PCR positive. Sequence analysis showed that there was considerable variation in the sequences detected, although all appeared to be of the coxsackie/echovirus type. CONCLUSION: This study re-emphasizes that a link exists between enteroviral infection and the onset of Type 1 DM, particularly at a very early age, and suggests that these viruses are aetiologically important in diabetes in a significant proportion of children.

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We have sought evidence of enteroviral persistence in humans. Eight individuals with chronic fatigue syndrome (CFS) were positive for enteroviral sequences, detected by PCR in two serum samples taken at least 5 months apart. The nucleotide sequence of the 5' non-translated region (bases 174-423) was determined for each amplicon. Four individuals had virtually identical nucleotide sequences ( > 97%) in both samples. The sequence pairs also each had a unique shared pattern indicating that the virus had persisted. In one individual (HO), it was clear that there had been infection with two different enteroviruses. In the remaining three individuals, the lack of unique shared features suggested that re-infection had occurred, rather than persistence. With the exception of HO, the sequences fell into a subgroup that is related to the Coxsackie B-like viruses.

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We used a nested polymerase chain reaction (nPCR) to seek evidence for enteroviruses in clinical samples from patients with symptoms of aseptic meningitis. When compared with conventional virus isolation methods on a total of 366 samples collected during 1994-1995, an increase in positivity from 6% to 27% was shown. The results indicate that nPCR would be a valuable aid to the laboratory diagnosis of enteroviral infections as it can detect those enteroviruses that cannot be identified by current isolation methods.

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Coxsackie B enteroviruses have been implicated repeatedly as agents associated with chronic fatigue syndrome (CFS). The objective of this study was to compare the serological evidence for the presence of Coxsackie B virus neutralising antibody, with the polymerase chain reaction (PCR) detecting a portion of the 5' nontranslated region (NTR) of the enterovirus genome. Serum samples from 100 chronic fatigue patients and from 100 healthy comparison patients were used in this study. In the CFS study group, 42% patients were positive for enteroviral sequences by PCR, compared to only 9% of the comparison group. Using the neutralisation assay, 34% of study patients were positive, compared to 41% of comparison patients. In the study group, 66/100 patient results correlated, i.e., they were either positive/positive or negative/negative for both tests. Of those that did not correlate, the majority were PCR-positive/Coxsackie B antibody-negative (21/34). In the comparison group, 58/100 patient results correlated. Of those that did not, the majority were PCR-negative/Coxsackie B antibody-positive (37/42). The Coxsackie B antibody neutralisation assay was not able to differentiate the CFS study group from the healthy comparison group, and thus the clinical relevance of this assay may be questioned. The PCR assay did differentiate the two groups with significantly more CFS patients having evidence of enterovirus than the comparison group.

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Infection with Coxsackie B viruses has been linked to insulin-dependent diabetes mellitus. Nine of 14 serum samples (64%) taken from children at the onset of diabetes were positive for enterovirus RNA by PCR. All of the children were under age six, and five were under age three. By contrast, enterovirus sequences were detected in only two of 45 serum samples from appropriate comparison children (4%). Sequences from six of the positive patients showed strong homology with Coxsackie B3 and B4 viruses, and there were some common patterns among the sequences from infected diabetic children. This is evidence for a role for enteroviruses in childhood diabetes.

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This study used phylogenetic analysis based on a region of the 5' non-translated region (5'NTR) of a variety of enteroviral sequences to compare sequences associated with chronic fatigue syndrome (CFS) and those from enteroviruses causing acute infections. Direct sequencing of PCR products was used to obtain the nucleic acid sequences from CFS patients. The inferred phylogenetic tree identified three groupings, one correlating with the diagnosis of CFS. The analysis identified a close relationship between the chronic fatigue enteroviral sequences, and showed that 19/20 were distinct from previously described enteroviruses. These results suggest there is persistence of enterovirus infection in some CFS patients and indicate the presence of distinct novel enterovirus sequences.

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The serum of 88 chronic fatigue patients was screened for enteroviral specific sequences by polymerase chain reaction (PCR) assay. The PCR method used was "nested" PCR targetting the 5' nontranslated region of the enteroviral genome which yielded a final fragment length of 264 base pairs. Samples were obtained from patients during 1990-1991. In addition, buffy coat specimens and stool specimens were examined in some patients. Samples from two cohorts of comparison individuals were also obtained. The comparison groups were firstly, acutely ill individuals with symptoms consistent with a presumed enteroviral infection (matched by age, sex, and date of receipt of specimen) and secondly, healthy individuals (matched by age and date of receipt of specimen). Enteroviral specific sequences were detected in 36 of 88 serum samples from chronic fatigue patients, 22 of 82 acutely ill individuals, and 3 of 126 healthy individuals. The enteroviral PCR positivity did not correlate with any one particular feature of chronic fatigue nor did it reflect any history of illness at onset of fatigue, duration of fatigue, or age of patient. These results provide new evidence for the presence of enteroviral specific sequences in serum, buffy coat, and stool samples in many patients with chronic fatigue. This may reflect a persistent enterovirus infection in a proportion of chronic fatigue patients.

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