Enteroviruses are important causes of acute clinical disease, and there is growing evidence that they may be involved in chronic conditions, including T1D (18
). T1D is characterized by autoimmunity leading to destruction of the islet cells (20
). The mechanism of enterovirus involvement is not currently understood, although both molecular mimicry and direct beta-cell damage leading to sequestration of proteins and presentation to autoreactive T cells have been suggested (17
). If there is an association, it is important to determine if an individual enterovirus serotype or groups of serotypes are involved or whether enteroviruses capable of triggering autoimmunity share specific molecular or biological features. This has important implications for future vaccines and other control strategies. To address this issue, a number of viruses putatively associated with diabetes need to be studied, but few such studies have been reported (21
). Here, we present the complete nucleotide sequence of an E3 strain isolated from a fecal sample taken 2.5 months before a patient developed a range of diabetes-associated autoantibodies, which reflect the initiation of the beta-cell-damaging autoimmune process. This is the first time a virus has been isolated from a patient close to the time of appearance of autoantibodies; thus, the strain represents an invaluable resource for studying potential molecular determinants associated with autoantibody induction and T1D.
E3 has been comparatively little studied, and in addition to the reference strain, there are very limited sequence data available for only 11 isolates. Thus, the present study greatly extends the molecular information available on this serotype. It is clear that E3 is a typical HEV-B member in terms of sequence (Fig. ). It also conforms to the pattern seen in several recent reports of uncoupling of capsid region evolution from that of the 5′ UTR and nonstructural region through frequent recombination (9
). E3-Morrisey is closely related to the E6 prototype, Charles, in the nonstructural region and much less so to PicoBank/DM1/E3 in this region (Fig. ), suggesting recombination since the divergence of the lineages giving rise to E3-Morrisey and PicoBank/DM1/E3. The recombination point between the two E3 lineages seems to be close to the P1/P2 boundary, as seen in a number of other enteroviruses (27
), as the lines representing the identity between E3-Morrisey and E6-Charles, and between E3-Morrisey and PicoBank/DM1/E3, cross over around position 3600 (Fig. ). In nucleotide sequence identity terms (Fig. ), the closest sequenced relative of PicoBank/DM1/E3 in the nonstructural region is a recent (1988) Russian isolate of E11 (Kar/87), supporting the suggestion that recombination may allow rapid exchange of nonstructural lineages between serotypes (27
). In this case, it is likely that a recombination event has occurred in the 3CD region, as the PicoBank/DM1/E3/E11-Kar/87 identity increases sharply there (data not shown).
Previous molecular and serology studies have tended to implicate CBVs in diabetes, and the identification of PicoBank/DM1/E3 as an E3 isolate extends the range of enterovirus serotypes that may be involved in diabetes. A recent report describes the molecular and biological characterization of an E9 strain, E9-DM, shown to be diabetogenic, while patients infected during an E16 outbreak in Cuba were shown to develop autoantibodies characteristic of T1D (5
). Thus, there is growing evidence that echoviruses could be an environmental factor in T1D. As yet, no other echoviruses have been identified in the DIPP study, but this may be a reflection of the fact that few viruses have been successfully isolated at the time of autoantibody seroconversion.
The PicoBank/DM1/E3 sequence was examined for previously suggested correlates of diabetogenicity. In common with most other HEV-B members, it has the 2C and VP1 sequences previously implicated in autoimmunity and so could have a similar effect (4
). More specific determinants of strains associated with diabetes have also been reported. A study of partial enterovirus 5′-UTR sequences detected in the blood of children presenting with T1D indicated that they were related to CBV3 and CBV4 and clustered together (10
). However, neither PicoBank/DM1/E3 nor the diabetogenic E9 isolate E9-DM clusters with these sequences (Fig. ), and the same is also true when comparisons are made between sequences (1
) from adult patients (data not shown). In addition, specific nucleotide differences between the sequences from blood and the CBV3 and CBV4 prototype strains are not seen in either PicoBank/DM1/E3 and E3-Morrisey comparisons or comparisons between E9-DM and the E9 prototype, Barty. It should be noted that the blood samples were geographically and temporally close in origin and that a limited region of the genome was analyzed. It may well be that the clustering seen is due to these factors rather than being a common feature of viruses linked to T1D. The whole-genome sequencing approach adopted here and in the E9 study (37
) promises to be more useful in determining such common features.
Comparisons with E3-Morrisey do not reveal any clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. There is also no obvious link with work done on encephalomyocarditis virus, a member of the genus Cardiovirus
of the family Picornaviridae
, where the critical difference between diabetogenic and nondiabetogenic strains was mapped precisely to VP1 position 167 (3
), as PicoBank/DM1/E3 shows no significant differences from E3-Morrisey and the recent European isolates in the corresponding region of VP1. However, when the capsid differences are positioned on a three-dimensional structure predicted using CAV9, another HEV-B member, as a model (16
), several differences cluster around the threefold axis (data not shown). These include a difference immediately at the threefold axis, in a location analogous to one that brings about altered cell tropism in CAV9 (Ç. H. Williams and G. Stanway, unpublished data), and a difference at this position could influence the tropism of PicoBank/DM1/E3.
The patient from whom PicoBank/DM1/E3 was isolated has remained positive for IA-2A and ICA for 6 years, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. This virus strain, isolated immediately prior to the appearance of autoantibodies, is particularly interesting, as it may be involved in the initiation of the beta-cell-damaging process. This is the first time that such a strain has been isolated, and further biological studies should give important information. Mouse models and human islets have been widely used in previous studies and are logical approaches for future work, although both have limitations. It is not clear how well animal models reflect the diabetogenicity of enteroviruses in humans, as there are significant differences in pathology (2
). In addition, there is no established mouse model for echovirus 3. The use of human islet cells is complicated by the observation that all enteroviruses seem to infect these cells, causing a wide range of alterations (apoptosis, necrosis, or changes in insulin secretion without clear damage), and which of these correlate with diabetogenicity is not clear (7
). Thus, a range of models and additional enterovirus strains isolated during prospective studies and at the time of onset are required to further address the role of enteroviruses in T1D. The identification of consistent molecular features in such strains would be additional strong evidence of a role for enteroviruses in T1D and would provide a basis for strategies, such as vaccination, aimed at preventing the disease.