The present study shows that CVB infections may contribute to the initiation of islet autoimmunity characterised by IAA as the first appearing autoantibody. This particular group of children are known as one of the phenotypes of islet autoimmunity in man . This recently discovered phenotype of type 1 diabetes differs from the other typical disease phenotype characterised by initiation of autoimmunity against GAD [5, 6]. The fact that the initiation of islet autoimmunity in these two phenotypes is associated with distinct HLA-DQ alleles indicates that they might have different triggers for the disease process leading to overt type 1 diabetes and different pathogenetic mechanisms [5, 6]. Our study suggests that specific CVB infections may contribute to the activation of the ‘insulin-driven’ pathogenetic pathway. This finding motivates further exploration of the pathogenic mechanisms underlying human type 1 diabetes related to CVB infections.
The current study supports the previous observations that enterovirus infections (including CVB) may increase the risk of islet autoimmunity and type 1 diabetes [7, 8]. In addition, the results imply that certain CVB infections might trigger the insulin-driven autoimmune process. The observation that no risk associations were seen between CVB infections and the GADA-driven pathway and that the association appears to be specific to CVB1 argues against the effect of unknown confounding factors. Additionally, to minimise any sampling bias and the effect of possible confounders, case and control children were individually matched for HLA-DQB1 genes, sex, age, sampling dates and the region of birth. The findings from the present study are also in line with those recently published by The Environmental Determinants of Diabetes in the Young (TEDDY) study . In the TEDDY study parent-reported recent respiratory infections, particularly common cold, influenza-like illness, sinusitis and laryngitis/tracheitis were associated with the subsequent risk of islet autoimmunity. The causative agents could not be identified since laboratory testing was not carried out. However, it is known that these symptoms are commonly caused by enterovirus infections.
CVB shares an important biological feature, which may explain their tropism to beta cells; they are the only EVs that use the Coxsackie and adenovirus receptor (CAR) to enter the cell. CAR is expressed more strongly on the surface of beta cells than other pancreatic cells  being selectively used by CVBs to gain entry into the beta cells hence implying that they may be responsible for the initiation of the process leading to type 1 diabetes. This also indicates that all CVB types might potentially cause beta cell damage. This feature could then be modulated by other viral characteristics and/or beta cell sensitivity to these viruses. In vitro studies have suggested that EVs infect mainly beta cells in cultured human islets [15, 16], and that CAR expression is upregulated in islets of individuals with type 1 diabetes compared with controls . In fact, in vivo tropism of CVB to human pancreatic islets has also been documented in post mortem examination of children with a fatal CVB infection [18, 19]. Enterovirus capsid proteins have been detected in beta cells from individuals with type 1 diabetes as well, while other islet cells have been mostly virus negative [20,21,22]. A large proportion of individuals affected by type 1 diabetes were reported to carry enterovirus in a small number of pancreatic beta cells, supporting the hypothesis of a chronic/persistent viral infection being present in these individuals . A chronic/persistent enterovirus infection(s), with a slower rate of virus propagation, has already been demonstrated in cell culture and mice models [23,24,25] and it has been shown that a deletion at the 5′UTR end of the viral genome could contribute to virus persistence in selected human tissues [26, 27]. The presence of the 5′ end deletion was also reported from a fatal case of fulminant enterovirus myocarditis in Japan . Therefore, we postulate that such a persistent/chronic infection of the beta cells might be one of the mechanisms initiating the autoimmune process.
The underlying mechanisms by which certain CVBs could contribute to the autoimmune response against insulin but not GAD are yet to be identified. One possible explanation, though, would be the tropism of CVBs to beta cells where a direct infection could induce autoimmunity against the most abundant and exclusively expressed protein in these cells, namely insulin. CVB infection could potentially induce post-translational modifications of the insulin molecule rendering it highly immunogenic. Interestingly, a recent publication showed that an incomplete response to CVB is associated with insulin autoimmunity but not with GAD autoimmunity in young children . Collectively, these studies suggest that further work is required to address the possible role of EVs in type 1 diabetes particularly focusing on the IAA phenotype of the disease.
One important aspect of our study is to report the existence of a possible interaction between different CVB types: CVB1-associated risk of islet autoimmunity was the highest in children who experienced mainly CVB1 infections (or CVB1/5) alone or prior to the other CVB infections. This indicates that additional infections by other CVBs may attenuate the risk association of CVB1 with islet autoimmunity. One possible explanation to this phenomenon could be immunological cross-protection between different CVB types, when earlier CVB infections could provide partial protection against subsequent CVB1 infection. In fact, an earlier study in mice has demonstrated that infection by CVB3 can attenuate later infection by CVB1  supporting this hypothesis. T cell cross-reactivity can be another possible mechanism explaining a cross-protection process [31,32,33,34,35]. An alternative to the cross-protection hypothesis could be the presence of an immunoregulatory effect in which autoimmune responses could be downregulated by earlier infections as reported before . In this case, CVB1 could possibly lack such an immunoregulatory property due to different intrinsic properties of various CVB group viruses in vivo. It is also possible that their potential diabetogenic effect could overcome their immunoregulatory effect.
The present observation that different CVB types can show contrasting associations with type 1 diabetes and possibly interact with each other, demonstrates the importance of the type of assays that are used to discriminate between the type of the enterovirus infections involved, i.e. virus neutralising assays which are highly specific for the serotype used in the assay. In fact, when all CVBs were analysed as a group, they showed no association with insulin-driven autoimmunity even though CVB1 was strongly associated with the appearance of IAA, thus demonstrating the importance of neutralising antibody assessments. In addition, the results demonstrate the value of prospective studies, which allows a time-ordered analysis to be conducted (e.g. IAA or GADA being the first appearing autoantibody and chronological order of different CVB infections). Therefore, the large population size and the prospective birth cohort design have clearly provided an important advantage, giving us an excellent opportunity to identify the association of CVB infections with type 1 diabetes.
It is also important to consider the limitations of the current study when interpreting the results. The study samples are representative of the Finnish population; therefore, it would be important to perform similar studies in other populations where the epidemiology of CVBs might be different. Previous studies have suggested that CVBs other than CVB1 (CVB4 in particular) could be diabetogenic [20, 37]. Hence, it is also important to keep in mind that the diabetogenic effect might not be the characteristic of a single virus serotype. In other words, other enterovirus types may also be associated with type 1 diabetes, depending on the circulation and accumulation of diabetogenic strains in different populations. In the current study CVB5 association with the risk of islet autoimmunity or type 1 diabetes was not statistically significant on its own but in combination with CVB1 there was some association. The lack of statistical significance for CVB5 could be due to its substantially lower frequency compared with CVB1. It is noteworthy that circulating strains of a single enterovirus serotype may also widely differ for their non-structural proteins, which may further affect the diabetogenicity of the viral strains . Therefore, it would be important to identify recombination events in circulating EVs in different populations. In line with this, we have previously shown that the CVB1 strains have different properties encountering innate immunity  and causing cell death in human islet cell cultures , which further emphasises the possible role of strain variations. Population dynamics of enterovirus infections and levels of maternal enterovirus antibodies, which protect the offspring against enteroviral infections, may also matter. For example, the prevalence of enterovirus infections and levels of maternal enterovirus antibodies are considerably lower in Finland than in most other countries. It has been proposed that this makes Finnish children particularly susceptible to the diabetogenic effect of these viruses . In fact, in the present study, 47.3–83.4% of the children lacked maternal CVB antibodies in cord blood, depending on the CVB serotype in question (data not shown).
Another limitation of the present study is that the identification of CVB types was based solely on neutralising antibody analyses. Therefore, further studies identifying enterovirus serotypes by direct sequencing from diabetic and prediabetic children should be conducted. However, such studies can easily be compromised by difficulties in identifying the individual enterovirus serotypes in sufficient numbers, leading to limited statistical power to address this question. For example, we have previously observed an excess of enterovirus RNA by RT-PCR in serial stool and blood samples collected prior to autoantibody seroconversion from prospectively followed children in the DIPP study [42, 43]. However, the identification of the serotype of these enteroviruses by sequencing the viral genome was successful only in a portion of samples where too few CVB viruses were identified to enable us to analyse their association with the initiation of islet autoimmunity. We also have to appreciate the general limitations of the case−control study design including the risk of selection bias. However, this study had a nested case−control design that minimises selection and recall bias compared with a conventional case−control study . Nonetheless, it is difficult to completely exclude a possible contribution of unknown confounding factors to the observed association between CVBs and islet autoimmunity. However, this was minimised using strict matching criteria for the selection of control children at an individual level.
In conclusion, the present study suggests that CVB infections could contribute to the development of type 1 diabetes via a specific pathway characterised by IAA as the first appearing islet autoantibody. An early infection by CVB1 or possibly by some other CVBs such as CVB5, might lead to the induction of an autoimmune response against insulin. However, the underlying mechanism(s) remain to be identified and further studies are needed to fully characterise this phenomenon. Large prospective studies conducted in different populations are also crucial tools to achieve this goal.