Trial of a supplemental dose of four poliovirus vaccines.

BACKGROUND: The immunogenicity of oral poliovirus vaccine (OPV), particularly the type 3 component, is lower in infants in most developing countries than in infants in industrialized countries. We conducted a multicenter trial in Oman to evaluate the response to a supplemental dose of four poliovirus vaccine formulations. METHODS: At nine months of age, infants were randomly assigned to receive inactivated-poliovirus vaccine (IPV), administered subcutaneously; trivalent OPV manufactured in the United States or in Europe; or monovalent type 3 OPV. Serum samples were collected at enrollment and 7 and 30 days later. All of the infants had previously received five doses of OPV. RESULTS: We enrolled 1025 infants; 785 (76.6 percent) met all the study requirements. At enrollment, 96.8 percent of the infants were seropositive for poliovirus type 1, 98.0 percent for type 2, and 88.0 percent for type 3. At 30 days there were no significant increases in type 3 seroprevalence or in the median antibody titer in the groups of infants who received OPV. Among the recipients of IPV, type 3 seroprevalence increased from 87.8 percent at enrollment to 97.1 percent at 30 days (P<0.001), and the median antibody titer increased from 1:228 to 1:1448 or higher (P<0.001). The rapid initial increase in the antibody titer suggests a secondary immune response. CONCLUSIONS: A supplemental dose of IPV has excellent immunogenicity and leads to increases in the titer of antibodies against type 3 poliovirus, whereas supplemental doses of the oral vaccines do not have these effects.

A study of enterovirus isolations in Glasgow from 1977 to 1997.

The number and range of enteroviruses isolated in the Regional Virus Laboratory, Glasgow during 1977-1997 was determined. Over this period, 3,039 enterovirus isolations were reported. The echoviruses represented 67% of isolations with echovirus 4 (due to an outbreak in 1990), echovirus 30 and echovirus 11 being the most frequently isolated types. The pattern of prevalence of non-polio enteroviruses had changed from the previous 20-year period with echovirus types isolated more often (77% vs. 55.4%) and coxsackieviruses isolated less often (23% vs. 44.6%). The polymerase chain reaction (PCR) introduced into the routine diagnostic service in 1996 increased the detection of enteroviruses from cerebrospinal fluid samples compared with traditional cell culture methods. Finally, the 5' nontranslated region (NTR, bases 63-475) and the VP4/VP2 region (bases 581-1199) of selected echovirus 30 and coxsackie B3 isolates were sequenced. These represented endemic and epidemic types respectively and were shown to be closely related within their type, but different from the published sequences. The current echovirus 30 strains differed from 1966 isolates by 16-20% in both the 5' NTR and VP4/VP2 regions. The coxsackie B3 isolates, predominant in 1997 after 5 years of absence, were also dissimilar from previously isolated strains, causing a small outbreak.

Enterovirus variants in the serum of children at the onset of Type 1 diabetes mellitus.

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.

Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin's disease.

DNA from malignant cells is present in the serum/plasma of cancer patients and DNA from this source is amenable to analysis by polymerase chain reaction (PCR). In the present study, we evaluated whether Epstein-Barr virus (EBV) DNA is present in the serum of patients with EBV-associated Hodgkin's disease (HD). Using conventional PCR, EBV DNA was detected in serum from 30/33 patients with EBV-associated HD but in only 6/26 patients with non-EBV-associated disease (p < 0.001). Samples from healthy individuals were negative and only 5/12 infectious mononucleosis samples were positive. Real-time quantitative PCR was subsequently employed to determine the concentration of EBV DNA present in serum; among positive samples the level ranged from 1 to 705 copies per 125 microliter of serum. Post-treatment samples from 5/14 cases with EBV-associated HD contained detectable EBV DNA; analysis of this small group of cases suggests that positivity in post-treatment samples correlates with risk factors indicative of a poor prognosis. Overall, our results are consistent with the notion that DNA from Reed-Sternberg cells is present in the serum of HD patients, and further suggest that serum EBV should be evaluated as a prognostic marker. Int. J. Cancer (Pred. Oncol.) 84:442-448, 1999.

Simultaneous onset of type 1 diabetes mellitus in identical infant twins with enterovirus infection.

This report describes classical Type 1 insulin deficient diabetes mellitus (DM) arising in twins aged 14 months, both of whom had evidence of enterovirus infection. The diagnosis of Type 1 DM was made in the second twin within 12 days of the first. Enterovirus infection was detected in each twin at diagnosis by polymerase chain reaction (PCR). Both twins were negative for enterovirus by PCR 5 months following diagnosis, although both were then positive for islet cell antibodies. Sequencing of the amplicons produced by PCR suggested that the viruses from each twin were not the same but that they were both variants related to echovirus 6.

Consequences of live poliovirus vaccine administration in chronic fatigue syndrome.

The effect of live oral polio virus vaccination on chronic fatigue syndrome (CFS) patients was examined in a double-blind study. CFS patients were allocated randomly to placebo (N = 7) or vaccine (N = 7) conditions. All controls subjects received the vaccine (9). Vaccine administration was not associated with clinical exacerbation of CFS. However, objective responses to the vaccine revealed differences between patients and controls: increased poliovirus isolation, earlier peak proliferative responses, lower T-cell subsets on certain days post vaccination and a trend for reduced gamma-interferon in the CFS-vaccine group. Polio vaccination was not found to be clinically contraindicated in CFS patients, however, there was evidence of altered immune reactivity and virus clearance.

Evidence for enteroviral persistence in humans.

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.

Enteroviral polymerase chain reaction in the investigation of aseptic meningitis.

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.

Comparison of coxsackie B neutralisation and enteroviral PCR in chronic fatigue patients.

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.

Coxsackie B virus infection and onset of childhood diabetes.

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.

Phylogenetic analysis of short enteroviral sequences from patients with chronic fatigue syndrome.

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.

Detection of enterovirus-specific RNA in serum: the relationship to chronic fatigue.

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.

Sequences specific for enterovirus detected in spinal cord from patients with motor neurone disease.

OBJECTIVE: To investigate the association of enteroviruses with motor neurone disease, also known as amyotrophic lateral sclerosis. DESIGN: Analysis by enterovirus polymerase chain reaction of wax embedded material from spinal cords taken at necropsy from subjects with motor neurone disease and from age and sex matched controls. SETTING: Specimens were collected in the west of Scotland and in London between 1982 and 1992. RESULTS: Sequences specific for a non-poliovirus type enterovirus were detected in spinal cord tissue from subjects with motor neurone disease. Amplification of a 414 base RNA target sequence in the conserved enterovirus 5' untranslated region from wax embedded tissue sections was successful in tissue from eight of 11 cases of sporadic motor neurone disease, one of two cases of familial motor neurone disease, and the one case of poliomyelitis, but not in the six matched controls or one case of antecedent poliomyelitis. In addition, sequences were detected in spinal cords from one monkey infected with wild type poliovirus and one monkey infected with polio vaccine. Comparison of sequences from cases of motor neurone disease with sequences of corresponding regions of the 5' untranslated regions of known picornaviruses showed them to be tightly grouped within the enterovirus genus closely related to coxsackievirus type B but not to polioviruses. Sequences derived from different parts of the spinal cord of the same subjects were identical, but sequences differed between individual subjects. CONCLUSIONS: Conserved enteroviral sequences closely related to coxsackie B virus sequences were detectable in spinal cords from subjects with sporadic motor neurone disease and from one subject with possible familial motor neurone disease.

Simple technique for detecting RNA viruses by PCR in single sections of wax embedded tissue.

The detection of specific RNA species in wax-embedded tissue sections using the polymerase chain reaction (PCR) means that gene expression can be studied and RNA viruses detected in stored histological tissue samples. This technique potentially allows the distribution of gene expression and viral replication to be studied in finely subdivided tissues. A technique is presented that has been used successfully to detect short RNA target sequences (130-420 bases) from proto-oncogene Abelson, human enteroviruses, and the sheep retrovirus Maedi-Visna virus using RNA PCR in single wax sections (20-30 microns). Various tissues were used which had not been deliberately prepared for this purpose. In a simple procedure hot xylene dewaxing is followed by acid phenol extraction of RNA and RNA PCR.

Surveillance of Mycoplasma pneumoniae infections in Scotland 1986-1991.

Mycoplasma pneumoniae is the second most common cause of community-acquired pneumonia. Infection is found worldwide and epidemics are said to occur in 4-yearly cycles. In Scotland this pattern has been noted since 1982 and, in common with England and Wales as well probably as other parts of Europe, there is a current epidemic which began in the autumn of 1990. The disease has been noted predominantly in children and young adults, with lower respiratory tract infection as the most common manifestation. At present, diagnosis is based on a serological response and various tests are available for detecting both primary infection and reinfection. In view of the present epidemic, initial treatment of respiratory-tract infection, especially in children and young adults, should include adequate cover against Mycoplasma pneumoniae.

Distribution of HIV and acute hepatitis B infection among drug injectors in Glasgow.

This study compared the distributions of known human immunodeficiency virus (HIV) and acute hepatitis B virus (HBV) infection among drug injectors in Glasgow over a 3.5 year period. Data were obtained from all relevant laboratory request forms submitted to Glasgow's 3 virology laboratories during the period 1 January 1986 to 30 June 1989. The overall prevalence of HIV among those tested was 3.7% (66/1786). There were 125 cases of acute HBV infection. The male:female ratios for HIV and acute HBV were 1:1 and 2:1, respectively. Thirty-four per cent of persons with HIV were aged under 21 years compared with 53% with acute HBV. Three out of 10 areas of the city accounted for 92% of HIV infection but only 66% of acute HBV infection. HIV infection was not detected among drug injectors in 4 areas of the city but at least 2 cases of acute HBV infection were recorded in all 10 areas. The geographical and age distribution of acute HBV infection in Glasgow suggests that the potential for future spread of HIV among drug injectors remains considerable.

Immune responsiveness in chronic fatigue syndrome.

We have endeavoured to find immunological indications of chronic virus infection in patients with chronic fatigue syndrome (myalgic encephalomyelitis) and to investigate immune responsiveness to viruses in such patients in comparison with normal subjects and patients with muscular dystrophy. Levels of circulating IgM immune complexes were elevated (above the 95% normal control range) in 10 (17%) of 58 patients with chronic fatigue syndrome, which was not significantly different from the normal controls or from dystrophy controls (by Mann Whitney U test). Levels of IgG complexes were only increased in 10% of patients. Lymphocyte proliferation in response to concanavalin A (Con A), assessed by increase in 3H-thymidine incorporation, did not differ between 14 patients and 18 normal subjects. The proliferative response to Coxsackie B virus antigen did not differ between chronic fatigue patients and normal subjects when expressed either as an increase in counts or as a stimulation index. Adjustment of the counts in relation to the proliferation response to Con A, as an indication of the overall proliferative response of the cell preparation, did not reveal any hidden difference. IgM antibodies to Coxsackie B viruses were not found in any of 20 patients and in 1 of 20 dystrophy controls. Significant levels of neutralizing antibodies to Coxsackie B viruses 1-5 were found in 6 out of 19 (32%) patients compared with 4 out of 17 (24%) dystrophy controls, which does not differ from currently expected normal incidence. Antibody titres to other respiratory viruses were also not notably different between the patient and control groups. In conclusion we can find no evidence for a definable viral aetiology for the chronic fatigue syndrome, neither in terms of a persistent infection nor an altered ability to respond to virus.

Enteroviral RNA sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome.

OBJECTIVE: To determine the presence of enteroviral sequences in muscle of patients with the postviral fatigue syndrome. DESIGN: Detection of sequences with the polymerase chain reaction in a well defined group of patients with the syndrome and controls over the same period. SETTING: Institute of Neurological Sciences, Glasgow. SUBJECTS: 60 consecutive patients admitted to the institute with the postviral fatigue syndrome who had undergone extensive investigation to exclude other conditions. 41 controls from the same catchment area without evidence of fatigue, all undergoing routine surgery. MAIN OUTCOME MEASURES: Routine investigations, serological screen for antibodies to a range of viruses, and presence of enteroviral RNA sequences in muscle biopsy specimens. RESULTS: 15 (25%) patients and 10 (24.4%) controls had important serological findings. 12 patients had neutralising antibody titres of greater than or equal to 256 to coxsackieviruses B1-5 (six positive for enteroviral RNA sequences, six negative); three were positive for Epstein-Barr virus specific IgM (two positive, one negative). Six controls had similar neutralising antibody titres to coxsackieviruses (all negative); one was positive for Epstein-Barr virus specific IgM (negative); and three had titres of complement fixing antibody greater than or equal to 256 to cytomegalovirus (all negative). Overall, significantly more patients than controls had enteroviral RNA sequences in muscle (32/60, 53% v 6/41, 15%; odds ratio 6.7, 95% confidence interval 2.4 to 18.2). This was not correlated with duration of disease, patient and age, or to raised titres of antibodies to coxsackieviruses B1-5. CONCLUSIONS: Persistent enteroviral infection of muscle may occur in some patients with postviral fatigue syndrome and may have an aetiological role.

Studies on the pathogenesis of neurological diseases associated with Varicella-Zoster virus.

Immunocytochemical techniques and in situ hybridization with three different Varicella-Zoster Virus (VZV)-specific RNA probes have been used to study the pathogenesis of VZV-associated neurological syndromes. Varicella-Zoster Virus antigens were not detected using the avidin-biotin peroxidase technique with a polyvalent anti-VZV antibody in any of the formalin-fixed tissue sections from eight cases of VZV-associated neurological disease (encephalitis, myelitis, ganglionitis); one case was immunosuppressed although inflammatory lesions were present. Intense labelling was detected within the inflammatory lesions in several representative VZV cases with a monoclonal antibody against Class II MHC antigens, whereas cases of Herpes Simplex Virus encephalitis and normal controls were not so labelled. Three VZV probes from open reading frames 62, 16 and 40, which show homology with the Herpes Simplex Virus immediate early 175 kd protein, the 65 kd DNA binding protein and the major capsid protein respectively, were used for in situ hybridization studies in these VZV tissues. Although the probes were able to detect VZV RNA in VZV-infected CV-1 and Flow 2002 cell cultures and formalin-fixed VZV skin biopsy sections, positive hybridization was not seen in any of the neurological cases studied. Thus neither VZV nucleic acid nor antigens were detected in any of the cases of VZV-associated neurological disease. It is proposed that the mechanism of neurological damage in the syndromes is immune-mediated, there being increased expression of Class II MHC antigens associated with persistent inflammation.

The JH2604 deletion variant of herpes simplex virus type 2 (HG52) fails to produce necrotizing encephalitis following intracranial inoculation of mice.

The pathological changes and distribution of virus antigen in mouse brains were studied following intracranial inoculation of 3 week old BALB/c mice with the herpes simplex virus (HSV) type 2 strain HG52 and its deletion variant JH2604. The variant JH2604 failed to produce necrotizing encephalitis compared to the parental HG52. The morphological changes induced in JH2604-infected brains consisted of localized perivascular cuffing by lymphocytes and infiltration by immune cells. Immunohistochemical studies using polyclonal anti-HSV serum showed that JH2604 antigens were localized at the site of inoculation with no evidence of neuronal involvement. Wild-type HSV-infected brains demonstrated a wide distribution of antigens both in neuronal and supporting cells. These data provide evidence that the non-neurovirulent phenotype of JH2604 is due to inability to replicate within neuronal cells of the central nervous system and pinpoints a precise role for the HG52 sequences contained within the 1488 bp subfragment of TRL/IRL deleted in JH2604.