Maria Lönnrot

Enterovirus RNA in serum is a risk factor for beta‐cell autoimmunity and clinical type 1 diabetes: A prospective study

Recent prospective studies have documented serologically an increased frequency of enterovirus infections in prediabetic children, indicating that these infections may initiate and accelerate the beta‐cell damaging process several years before the clinical manifestation of type 1 diabetes. The aim of the present study was to establish whether these serological findings would be supported by the detection of enterovirus RNA in a unique prospective series of sera collected from prediabetic children 0–10 years before the manifestation of clinical type 1 diabetes. Reverse transcription followed by polymerase chain reaction employing highly conserved primers among enteroviruses were used to amplify enteroviral sequences. Viral RNA was found in 22% (11/49) of follow‐up samples from prediabetic children but in only 2% (2/105) of those from controls (OR 14.9, P < 0.001). Persisting RNA positivity was not observed in any of these children. The presence of enterovirus RNA was associated with concomitant increases in the levels of autoantibodies against islet cells (OR 21.7, P < 0.01) and glutamic acid decarboxylase (OR 15.4, P < 0.05), but not in the levels of antibodies against insulin or the tyrosine phosphatase‐like IA‐2 protein. In contrast to the prediabetic children, those with newly diagnosed type 1 diabetes were negative for enterovirus RNA. The results thus complement previous serological data, suggesting that enterovirus infections are an important risk factor underlying type 1 diabetes and associated with the induction of beta‐cell autoimmunity even years before symptoms appear. J. Med. Virol. 61:214–220, 2000.

Diagnosis of enterovirus and rhinovirus infections by RT-PCR and time-resolved fluorometry with lanthanide chelate labeled probes.

Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time‐consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription‐polymerase chain reaction (RT‐PCR) have been introduced. An RT‐PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid‐phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus‐ or rhinovirus‐specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time‐resolved fluorometry. The sensitivity and specificity of the RT‐PCR‐hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT‐PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT‐PCR‐hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections. J. Med. Virol. 59:378–384, 1999© 1999 Wiley‐Liss, Inc.

Enterovirus antibody levels during the first two years of life in prediabetic autoantibody-positive children

Aims/hypothesis. We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study. Methods. Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected. Results. IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype. Conclusions/interpretation. Our data show that children who seroconverted for diabetes-associated autoantibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes. [Diabetologia (2001) 44: 818–823]

Enterovirus infections as a risk factor for type I diabetes: virus analyses in a dietary intervention trial.

This study evaluated the possible role of enterovirus infections in the pathogenesis of type I (insulin‐dependent) diabetes in a prospective dietary intervention trial. Children participated in the second pilot of the Trial to Reduce IDDM in Genetically at Risk (TRIGR) project. They were randomized into two groups receiving either a casein hydrolysed formula (Nutramigen®) or a regular formula, whenever breast milk was not available over the first 6–8 months of life. Altogether 19 children who turned positive for autoantibodies associated with type I diabetes by 2 years of age and 84 matched control children were analysed for enterovirus antibodies and enterovirus RNA in serum. Enterovirus infections were common during the first 2 years of life and more frequent among boys than girls (P = 0·02). Autoantibody‐positive children had more enterovirus infections than autoantibody‐negative children before the appearance of autoantibodies (0·83 versus 0·29 infection per child, P = 0·01). The average levels of IgG antibodies to echovirus antigen were also higher in autoantibody‐positive than in autoantibody‐negative children (P = 0·0009). No difference was found in the frequency of enterovirus infections between children receiving the casein hydrolysed formula or regular formula. These results suggest that enterovirus infections are associated with the induction of β‐cell autoimmunity in young children with increased genetic susceptibility to type I diabetes.

Antibody cross‐reactivity induced by the homologous regions in glutamic acid decarboxylase (GAD65) and 2C protein of coxsackievirus B4

GAD65 contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4‐2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross‐reactivity between CBV4‐2C and GAD65 was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross‐reactivity between 2C, GAD65 and GAD67 derived peptides in ELISA. There was substantial cross‐reactivity between 2C and GAD65 peptides, but not between 2C and GAD67 peptides. The most cross‐reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide‐specific sera to the GAD65 protein was analysed in immunoprecipitation, two sera were found to be cross‐reactive (anti‐FIEWLKVKILPEVKEK and anti‐WLKVKILPEVKEKHEF). One of these (anti‐WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)‐infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin‐dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD65 antibody‐negative non‐diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD65, and that peptide immunization induces antibodies which cross‐react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent‐onset IDDM and in control subjects.

Enterovirus infections and enterovirus specific T-cell responses in infancy

The development of enterovirus specific T‐cell and antibody responses were examined in a cohort of 60 healthy infants at the ages of 3, 6, 9, and 12 months. By the age of 6 months, 68% of the infants had developed T‐cell responses against enterovirus antigens by lymphocyte proliferation test, whereas only 30% had serological evidence of an enterovirus infection. By this age, only 7% of the infants had adenovirus specific T‐cell responses and 3% had serologically verified adenovirus infection. Enterovirus specific T‐cell responses correlated with the lack of enterovirus antibodies in cord blood and the number of sibs reflecting protection by maternal antibodies and the rate of exposures, respectively. T‐cell responses cross‐reacted between different enterovirus serotypes. The results show that enterovirus infections occur frequently in infancy and induce T‐cell immunity. Cellular immunity may be a more sensitive indicator of neonatal enterovirus infections than antibodies. J. Med. Virol. 54:226–232, 1998.

Enterovirus infections and insulin dependent diabetes mellitus : evidence for causality

BACKGROUND Insulin-dependent diabetes mellitus (IDDM) has a long subclinical period characterised by gradually progressing autoimmune damage of insulin producing beta-cells. Clinical IDDM is manifested when 90% of beta-cells have been destroyed. Several studies have indicated that enterovirus infections, coxsackievirus B (CVB) infections especially, are frequent at the manifestation of clinical IDDM suggesting that they can precipitate the symptoms of IDDM in individuals who already have an advanced beta-cell damage. Recently, the first prospective studies have been published suggesting that enterovirus infections can also initiate the process several years before clinical IDDM. This implies that enterovirus infections may have a crucial role in the pathogenesis of human IDDM. OBJECTIVE The recent findings have brought up the question whether the time has come when a causal association between enterovirus infections and IDDM could finally be confirmed. This review focuses on this question summarising the current knowledge and the prospects of future research. STUDY DESIGN Review of the recent progress in studies evaluating the role of enterovirus infections in human IDDM. CONCLUSIONS The currently available information supports the assumption that the role of enterovirus infections may be more important than previously estimated. Enterovirus infections are obviously associated with increased risk of IDDM, but whether this association reflects causal relationship remains to be confirmed in future studies. Prospective birth-cohort studies will be among the most important ones giving important data on the etiologic fraction of enterovirus infections, the properties of diabetogenic virus variants and the mechanisms of beta-cell damage.

Onset of type 1 diabetes mellitus in infancy after enterovirus infections

Enterovirus infections may initiate and accelerate the beta‐cell damaging process leading to Type 1 (insulin‐dependent) diabetes mellitus (Type 1 DM). Recent prospective studies have suggested that this can happen long before overt disease and even in utero. We describe an infant, followed regularly from birth, who progressed to clinical Type 1 DM at the age of 14 months. He had a strong enterovirus exposure exceptionally early in life; the first enterovirus infection occurred before the age of 3 months and the second between the age of 9 and 12 months. The first infection probably occurred at birth, when the child had symptoms of a respiratory infection. This infection was followed by the appearance of beta‐cell autoimmunity, and clinical Type 1 DM was diagnosed shortly after the second infection. The child had a low level of maternal enterovirus antibodies and short duration of breast‐feeding, both associated with increased risk for enterovirus infections during the fetal period and infancy. This case fits with the current hypothesis that enterovirus infections can induce the process resulting in Type 1 DM, especially when occuring early in life. Furthermore, this demonstrates the feasibility of the present study design, which is applicable also in large‐scale birth‐cohort studies.

Temporal Relationship between Human Parechovirus 1 Infection and Otitis Media in Young Children

BACKGROUND Human parechovirus (HPeV) 1 is a common virus that infects almost everyone during childhood. Because clinical symptoms are poorly documented, we evaluated the symptoms associated with HPeV1 infection in a cohort of children followed prospectively from birth at 3-month intervals. METHODS Symptoms such as fever, cough, those of the common cold, otitis media, and gastroenteritis were determined from hospital records and from questionnaires administered to the parents of 59 children during regular study visits. HPeV1 infections were diagnosed by measuring neutralizing antibodies in follow-up serum samples. Additionally, HPeV RNA was analyzed in middle ear fluid (MEF) and nasopharyngeal aspirate samples from 33 patients with otitis media by reverse-transcription polymerase chain reaction. RESULTS Otitis media showed a clear association with HPeV1 infection-it developed in 50% of the 3-month follow-up periods that yielded evidence for HPeV1 infection but in only 14% of the HPeV1-negative periods (odds ratio [OR], 6.14 [95% confidence interval {CI}, 2.75-13.77]). In children with recurring otitis media, MEF samples were positive for HPeV in 15% of episodes. Cough was also associated with HPeV1 infection, but this association was weaker (OR, 3.67 [95% CI, 1.66-8.09]). Other symptoms were not linked to HPeV1 infection. CONCLUSIONS HPeV1 infections are common in childhood and may cause otitis media and cough.

Comparison of enterovirus-specific cellular immunity in two populations of young children vaccinated with inactivated or live poliovirus vaccines

Enterovirus‐specific cellular immunity was studied in Estonian and in Finnish children at the age of 9 months. The aim was to evaluate the level of responsiveness in two neighbouring countries with different poliovirus immunization practices and striking differences in the incidence of insulin‐dependent diabetes mellitus (IDDM), a disease in which early enterovirus infections are an aetiological risk factor. The Estonian children immunized with live attenuated polio vaccine had stronger T cell responses to coxsackievirus B4 and poliovirus type 1 when compared with Finnish children immunized with inactivated polio vaccine (median stimulation indices 10.4 and 6.3 in Estonian children and 1.9 and 2.9 in Finnish children, respectively; P < 0.05). Lymphocytes stimulated by poliovirus type 1 antigen expressed interferon‐gamma (IFN‐γ) mRNAs, which strongly correlated with the level of proliferation responses. Lymphocytes of Estonian children had a tendency towards stronger expression of IFN‐γ upon poliovirus challenge when compared with Finnish children. The number of children who had experienced coxsackievirus B infections, as determined by the presence of neutralizing antibodies, did not differ between Estonian and Finnish children. The results show that Finnish children have weaker cellular immunity against enteroviruses at the age of 9 months compared with Estonian children at the same age. This is most probably due to the difference in polio vaccination schedules; in Estonia live poliovirus vaccine is used and given at earlier ages than the inactivated vaccines in Finland. This leads to stronger T cell immunity which cross‐reacts with other enterovirus serotypes. This may explain the lower incidence of IDDM in Estonia by providing effective protection against diabetogenic enterovirus strains in Estonian children.