Heikki Hyöty

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.

Several different enterovirus serotypes can be associated with prediabetic autoimmune episodes and onset of overt IDDM

In a prospective multicentre study described previously on prediabetic events in siblings of index cases with insulin‐dependent diabetes mellitus, 31 children developed clinical diabetes during the observation period and 51 children seroconverted for islet cell antibodies or insulin autoantibodies. By using nonserotype specific EIA and RIA, it has shown recently that enterovirus infections in both groups were frequently associated with increases of islet cell antibody and/or insulin autoantibody titres. Serum specimens sequentially collected from 12 children during the prediabetic period were still available and were then tested for serotype‐specific neutralizing antibodies. Plaque‐neutralization assays were carried out for coxsackievirus A9, coxsackievirus B types 1 to 6, and echovirus types 1 and 11. An unequivocal monotypic increase in neutralizing antibodies was observed on seven occasions in six children, on one occasion with coxsackievirus A9, one with coxsackievirus B1, two with coxsackievirus B2, two with coxsackievirus B3, and one with coxsackievirus B5. In four patients, the infection was associated temporally with increases in the levels of islet cell antibodies, insulin autoantibodies and/or antibodies to glutamic acid decarboxylase, and in three other patients, it coincided with the clinical onset of insulin‐dependent diabetes mellitus. These results suggest that the association of enterovirus infections with insulin‐dependent diabetes mellitus is not restricted to serotype 4 of coxsackie B viruses suspected previously, but that several different serotypes might play a role in the pathogenesis of the disease. J. Med. Virol. 56:74–78, 1998.

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 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.

Interaction of genetic and environmental factors in the pathogenesis of insulin-dependent diabetes mellitus.

The current concept of the pathogenesis of insulin-dependent diabetes mellitus (IDDM) is based on the view that environmental factors, either alone or in combination, trigger in a genetically, susceptible individual an autoimmune process which leads to the destruction of the insulin-secreting beta cells. The identification of environmental risk factors for IDDM is of utmost importance with regard to possibilities for implementing preventive measures. Studies on the interaction between genetic and environmental factors may be complicated by the observations that genetic markers, e.g. HLA risk alleles, may vary from one population to another, and combinations of predisposing genes may differ between populations. In addition, the role of a given environmental trigger in initiating the autoimmune process may be variable in various individuals depending on the genetic set-up and other host-related characteristics. As prevention trials are already being initiated, and genetic screening is essential in the identification of individuals at increased risk of IDDM, it is crucial to learn more about the interaction of genetic and environmental factors in this disease.