Enterovirus RNA was detected in seven of 356 whole-blood RNA samples, with five strongly enterovirus-positive and two weakly enterovirus-positive samples each taken from a different child.
To characterise enterovirus-associated changes in whole-blood transcriptome, we studied longitudinal gene expression profiles of these enterovirus-positive children by dividing all probe sets child-specifically into ten clusters (ESM Fig. 1; see also Dataset 2 published on https://www.btk.fi/1234-2/). For each child, the clusters with the highest and lowest centroid value at the time of enterovirus positivity were selected as the enterovirus-associated signals (Fig. 1a–g).
For four strongly enterovirus-positive children, the overlaps between peaking and dropping clusters were higher (average overlaps of 46% and 37%, respectively) than those between the other children (average overlaps of 8%) (Fig. 1h, i). In total, 593 probe sets mapping to 339 distinct genes were detected in the peaking clusters of all four children. This set was defined as the enterovirus-induced signature. However, approximately 20% of the probe sets in each of these peaking clusters were child-specific, indicating the presence of individual differences in enterovirus responses. The other three children had lower overlaps with each other and with all other children.
Genes involved in antiviral immune responses and especially interferon signalling were significantly enriched in both the peaking clusters of the four strongly enterovirus-positive children and the enterovirus-induced signature (Fig. 2a; see also Dataset 2 published on https://www.btk.fi/1234-2/). Also, child-specific expression profiles of two interferon signalling genes, STAT2 and MX1, showed clear peaks in the four children at enterovirus positivity (Fig. 2b–h). Furthermore, 74% of the signature genes were regulated by interferons based on the Interferome database. The B cell receptor signalling pathway was among the most significantly enriched pathways in the peaking clusters of the other three children, potentially reflecting the activation of adaptive immune responses. Based on the dropping clusters, enterovirus infection was also associated with downregulation of genes involved in mRNA processing, transcription or translation in six of the seven children (see Dataset 3 published on https://www.btk.fi/1234-2/).
Enterovirus RNA is detectable in blood for only a few days during the acute phase of infection. To estimate the timing of infection relative to sample collection, we compared our peaking and dropping clusters with whole-blood transcriptional changes during acute and recovery phases after influenza virus infection, as reported by Zhai et al [23] (see Dataset 2 published on https://www.btk.fi/1234-2/). Of the 25 top genes upregulated during the acute phase of influenza infection [23], 23 overlapped our enterovirus-induced signature. Also, all seven natural killer (NK) cell activation signature genes associated with the acute phase of influenza infection [23] peaked in more than one of the enterovirus-positive children. Overlaps with the top up- and downregulated genes specific for the recovery phase after influenza virus infection [23] were low for all the children.
We also compared our results with enterovirus-induced responses in human PBMCs infected in vitro with three different enteroviruses [14]. The genes upregulated in the in vitro infections were enriched with those associated with the defence response to virus and the type I interferon signalling pathway, similarly to the genes in our enterovirus-induced signature. Of the genes upregulated by any of the enteroviruses, 70% were present in at least one of the peaking clusters. Overall, 77 genes present in the enterovirus-induced signature were upregulated in all three in vitro infections (ESM Fig. 2a). Of these genes, 73 were interferon-regulated based on the Interferome database. Although only approximately 50% of the genes downregulated in the in vitro infections were present in any of the dropping clusters of enterovirus-positive children, genes associated with translation were enriched in both datasets.
As enteroviruses are known to infect pancreatic islets [5] and have been found in pancreases of individuals with type 1 diabetes more often than in non-diabetic control groups [24, 25], we also compared our results with enterovirus-induced responses in human pancreatic islets infected in vitro with enteroviruses [10, 26]. Approximately half of the enterovirus-induced genes in human pancreatic islets [10, 26] were also present in our enterovirus-induced blood transcriptomic signature in children at risk for developing type 1 diabetes (ESM Fig. 2b), while the overlaps with the genes present in the peaking clusters of the other three type 1 diabetes risk children were low (ESM Fig. 2b). In total, there were 64 enterovirus-induced genes common to the in vitro infected pancreatic islets [10, 26] and our enterovirus-induced blood transcriptomic signature, all of which were associated with antiviral interferon responses, including IFIH1, IRF7, MX1, STAT1 and STAT2.
Finally, we tested whether the 339 enterovirus-induced signature genes or the 77 genes also upregulated in all three in vitro infections of human PBMCs could differentiate between the enterovirus-positive and enterovirus-negative blood samples in the full transcriptomics dataset by Kallionpää et al [15]. The four enterovirus-positive samples with clear signs of interferon response activation were clearly separated from all other samples using either of the gene sets, while the other three enterovirus-positive samples were not separated from the enterovirus-negative samples (Fig. 3a, ESM Fig. 3a). To ensure that the observed separation was not due to the use of the same four enterovirus-positive children in defining the signature, a similar analysis was performed using the genes upregulated in the in vitro infected PBMCs by any of the three enteroviruses. Here also, the four strongly enterovirus-positive samples were clearly separated from the enterovirus-negative samples (ESM Fig. 3b).
Upregulation of interferon-regulated genes during enterovirus infection is one of the conspicuous features in this study, and activation of interferon signalling has been observed in the blood of children who have developed diabetes-related autoantibodies or clinical type 1 diabetes before the first detection of autoantibodies [15, 27]. However, the expression of the 339 enterovirus-induced signature genes (ESM Figs 3a, c and 4) or the 77 genes also upregulated with in vitro enterovirus infection (Fig. 3a, b) did not show marked differences between autoantibody-negative and autoantibody-positive children before or after seroconversion based on the longitudinal blood transcriptomics data from children at risk for type 1 diabetes reported by Kallionpää et al [15] or Ferreira et al [27]. Moreover, fewer than 50% of the genes upregulated in children who have developed diabetes-related autoantibodies or clinical type 1 diabetes in the two aforementioned studies [15, 27] overlapped with the enterovirus-induced signature (Fig. 3c, ESM Fig. 3c).