Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

Abstract

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.

Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells

“…Altogether, the results obtained with the vitronectin receptor-specific antibody show that this receptor works as a receptor for enteroviruses and HPEV-1 in primary human islets. Similar results have been reported in established cell lines [19,20,21,70,71].…”

“…We studied the receptor specificity in primary human islets of a clinical strain, EV-9/DM, which had been isolated from a diabetic patient. This virus strain was more destructive in human islets than the corresponding prototype strains, as has been shown in comparative studies of different EV-9 strains and their tropism for pancreatic cells [21]. In the case of EV-9/DM the vitronectin receptor was responsible for beta-cell-specific destruction and virus replication in islets.…”

“…Results obtained by several different methods from one complete experiment are shown. The blocking effect of the anti-receptor antibody was also evident in other islet cell preparations as shown by double-immunofluorescence staining with insulin and virus-specific antisera and live/dead-cell stainings least in an established cell line of green monkey kidney [21]. However, replication of this virus in human islets, as well as virus-induced cellular death, was prevented by the polyclonal antibody to integrin αvβ3 (Fig.…”

“…Three serotypes of poliovirus use poliovirus receptor (PVR), a member of the immunoglobulin superfamily [17] and members of human enterovirus C use intercellular adhesion molecule-1 [18]. Integrin αvβ3, also known as the vitronectin receptor, works as a receptor for coxsackievirus A 9 (CAV-9) [19] and other arginineglycine-aspartic acid motifs (RGD) containing enteroviruses [20,21], whereas another integrin, α2β1, works as a receptor for EV-1 [22,23]. In addition, a complement regulatory protein, i.e.…”

Complete Nucleotide Sequence of a Coxsackievirus B4 Strain that Establishes Infection in ICR Mice Pancreas and Induces Glucose Intolerance

“…Recent studies have demonstrated the association of the virulence with the genetics of the virus strain (Yin et al, 2002b;Paananen et al, 2003;Al-Hello et al, 2005;Dotta et al, 2007). Based on the sequence comparison of the diabetogenic E2 strain of CVB4 and the nondiabetogenic JVB strains, it was revealed that some amino acid changes in the capsid and noncapsid proteins of the E2 strain might be responsible for determination of its diabetogenicity (Kang et al, 1994).…”