Echovirus type 11 (E11) is the most prevalent subtype belonging to the family Picornaviridae, genus Enterovirus. In most cases, echovirus infections follow a benign, self-limiting course, manifesting either asymptomatically or with fever. However, the impact on newborns, particularly premature infants, can often lead to severe complications such as myocarditis, liver failure, coagulation disorders with reduced platelets, and even death (Chuang and Huang, 2019; Zhang et al., 2021). In recent years, sporadic outbreaks of E11 infection have been reported in Europe (Grapin et al., 2023; Piralla et al., 2023), and prior to the prevalence of the coronavirus disease 2019, Asia also witnessed widespread outbreaks (Patel et al., 1985; Ho et al., 2020; Chen et al., 2021; Wang et al., 2023b). Reports of serious consequences in premature infants due to this virus have been on the rise. According to World Health Organization data, the proportion of severe infections in newborns has increased from 6.2% in 2016 to 55% in 2022. Clinical manifestations may include coagulation disorders and liver damage, with critically ill patients developing acute fulminant liver failure. However, the reasons for the severe liver damage caused by E11 in premature infants remain unclear, primarily due to the lack of comparative clinical histopathology.

This study described findings on a pair of premature male twins who suffered from liver damage and succumbed to E11 infection. It offered a comprehensive analysis of the pathological and immunological characteristic differences in liver tissues between infants with E11 infection and control group. By combining clinical biochemical indicators, histopathology, and cytokine immunology, we elucidated the abnormal expression of interferon α (IFNα) and interferon-inducible protein-10 (IP10) in severe cases of E11 infection and discussed potential molecular mechanisms. The aim was to offer new insights into the early diagnosis and treatment of severe liver damage caused by E11 infection in premature infants.

E11 is notably associated with sepsis and acute fulminant hepatitis in newborns, often leading to neonatal deaths (Bubba et al., 2017; Zhang et al., 2021). Severe cases have received increased attention from researchers. Reports from different countries in recent years have documented cases of severe liver failure and even death in newborns infected with E11 (Grapin et al., 2023; Piralla et al., 2023), with premature birth and male sex identified as potential susceptibility factors, but the specific pathogenic mechanisms remain unclear. One study reported nine cases, with eight being male premature twins (Grapin et al., 2023). The cases presented in this study also concern male premature twins infected around the age of 1 week, rapidly progressing to acute fulminant hepatitis, similar to cases reported in Italy and France (Grapin et al., 2023; Piralla et al., 2023). Based on clinical manifestations, this study delved deeper into the pathogenesis from the perspectives of histopathology and cytokine immunology.

This study was novel in comparing the histopathological changes in different tissues following E11 infection with normal controls. Viral quantification studies were also conducted on various tissue samples from patients. Unlike other enteroviruses such as Enterovirus A71 (EV71) (Huang et al., 2020; Moshiri et al., 2023) and Coxsackievirus B3 (CVB3) (Massilamany et al., 2014; Garmaroudi et al., 2015), severe E11 infection prominently manifests as notable liver damage. The histopathological findings from liver autopsy samples of the twins were consistent with the pathological changes of liver failure. Immunofluorescence staining revealed a significantly higher number of virus-positive cells in the liver compared with the intestine and lungs. This confirmed, for the first time in human samples, that the liver was a crucial target organ for E11-induced sepsis. A previous study by Wells et al. reported a similar phenomenon in mouse model of echovirus infection (Wells et al., 2022), but severe E11 infection occurred only when the IFNAR gene was knocked out. However, in this study, a comparison of IFNAR expression in liver tissue between the case and control groups revealed that the expression in Kupffer cells of the case group was significantly higher than that in the control group, with no statistical difference in hepatocytes. This result suggested that, although the key point leading to severe infection in animal experiments was the knockout of IFNAR, the key regulatory mechanism in severe cases might be associated with abnormalities in other key points of the IFNα signaling pathway.

IFNα originating from extraepithelial sources is crucial for constraining enterovirus replication in the human intestine and is secreted by most types of cells (Vilcek and Oliveira, 1994). They can initiate signal transduction through autocrine or paracrine mechanisms, forming ternary receptor complexes with their respective receptors to induce IFN-mediated immune responses (Schneider et al., 2014; Saxena et al., 2017).Our team previously validated significantly lower levels of IFNα in the peripheral-blood serum of patients with severe liver injury infected with E11 compared with mild cases (Wang et al., 2023b). Animal experiments have demonstrated that, without knocking out the IFNAR gene, E11 infection remains localized to the intestine. However, after the gene is knocked out, blocking the normal function of IFNα allows E11 infection to spread systemically (Wells et al., 2022). The decreased levels of IFNα in patients with severe liver injury infected with E11 might contribute to the virus spreading and causing liver failure in severe cases. The expression of upstream transcription factors of IFNα in the liver tissue of these twins was examined to further explore the specific regulatory mechanisms. The expression level of IRF5 in the liver tissue was observed to be significantly lower than that in the control group. A weak positive correlation between IFNα and IRF5 was observed in Kupffer cells within the liver. Numerous studies have demonstrated that IRF3, IRF5, and IRF7 are involved in the activation and secretion of Type-I IFNs after viral invasion, playing a synergistic role in this process (Barnes et al., 2001; Barnes et al., 2004; Paun et al., 2008; Jefferies, 2019; Nie et al., 2019). Upon activation of Toll-like receptors (TLRs) 7, 8, or 9 by exogenous nucleic acids, a supramolecular organizing center is formed, connecting endosomal TLRs with enzymes and ubiquitin-modified proteins. The formation of this complex in the early endosome leads to the activation of NF-κB and the transcription of pro-inflammatory cytokines. Simultaneously, IRF5 is recruited to lysosomes and undergoes phosphorylation. Subsequently, IRF5 translocates to the cell nucleus, initiating the transcription of Type-I IFN genes (Elkon and Briggs, 2020). IRF5 may directly impact the secretion of IFNα and also influence the synergistic action among various regulatory factors, affecting the body’s antiviral capabilities. Therefore, IRF5 may be one of the therapeutic targets for treating acute fulminant liver failure caused by E11 in newborns. Additionally, studies have reported that viral proteins selectively act on a specific target within the IFN signaling pathway, causing damage to the IFN signal transduction pathway (Rasti et al., 2019; Ji et al., 2023). Further studies are needed to confirm whether the downregulation of IRF5 expression in severe E11 infection is due to a specific viral component.

IFNα and IP10 are two key factors that modulate the immune reaction of type 1 T helper cells (Th1 cells). IP10, a member of the CXC chemokine subfamily, acts as a chemoattractant for activated T cells (Chen et al., 2013). It increases the migration of Th1 cells. IFNα prompts the proliferation of cytotoxic CD8⁺ T cells and the release of IFNγ (Hervas-Stubbs et al., 2010). The dysfunction of excreting IFNα in premature infants cannot stimulate the anti-E11 reaction. More IP10 was produced to recruit additional lymphocytes as feedback to the low level of IFNα (Brownell and Polyak, 2013; Dai et al., 2020; Yu et al., 2022; Zhang et al., 2022). The elevated expression of IP10 in the liver of the twins in this study aligned with previous findings associating IP10 levels with hepatic injury. In vitro experiments have demonstrated that E11 can induce IP10 expression (Drummond et al., 2017), a phenomenon not observed with other enteroviruses such as EV71 and CVB3. The elevated expression of IP10 appears to be a characteristic feature of E11 among enteroviruses. Animal studies have further confirmed that E11 induces the secretion and aggregation of IP10 in the liver, leading to severe hepatic damage (Wells et al., 2021). Following invasion of liver cells, E11 triggers significant IP10 secretion, attracting macrophages and natural killer cells to the liver. This leads to acute inflammatory responses and cytotoxic immune reactions, ultimately resulting in extensive hepatocyte necrosis. Therefore, inhibiting IP10 may serve as one of the therapeutic targets for treating E11-induced liver injury. Animal experiments have revealed that, during sepsis, myeloid-related protein 8/14 (MRP8/14) induces sustained IP10 production through the IFN-β-JAK1/TYK2-STAT1-IRF7 pathway (Wang et al., 2023a). Our findings were consistent with the results of these animal experiments, suggesting that IRF7 might also be one of the targets implicated in severe E11 infection leading to liver injury.

E11, similar to other enteroviruses, primarily spreads through the fecal–oral route, using the gastrointestinal tract as its main entry point (Morosky et al., 2019). Virus-positive cells were detected in the intestinal epithelium of the twins examined in this study, exhibiting a focal rather than diffuse distribution. This pattern might be linked to the involvement of various cell types in the viral invasion of the intestinal epithelium. Previous studies have indicated E11 replication in intestinal epithelial cells and enteroendocrine cells, but not in goblet cells (Drummond et al., 2017). Our observations also revealed intestinal damage in severe cases, characterized by the disappearance of intestinal villi structure and the presence of reduced and disorganized intestinal epithelial cells. This finding contrasted with the results of animal experiments where virus replication was confined to intestinal epithelial cells, and no damage to the intestinal epithelium was observed (Wells et al., 2022).

This study was the initial investigation into immunological characteristics using patient tissue samples, validating findings from animal experiments and proposing potential mechanisms for developing severe illness. However, its limitations included the absence of liver tissue samples from patients with mild E11 infection for comparative analysis, as the control cases involved non-E11-infected individuals. Additionally, this study provided only a preliminary exploration of upstream factors affecting IFNα and IP10, necessitating further clinical data and animal experiments to reveal the regulatory roles of IRF5, IRF7, and IFNAR in severe E11 infection in neonates.

In summary, this study was novel in exploring severe E11 infection in premature male twins at the tissue level. The findings, coupled with multicolor immunofluorescence, might offer initial insights into the potential mechanisms and prospective targets for preventing and treating severe preterm E11 infection.

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by the Institutional Review Board of Guangzhou Women and Children’s Medical Centre (GWCMC-(2020)63201). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

WL: Data curation, Investigation, Writing – original draft, Writing – review & editing. LW: Formal analysis, Investigation, Software, Writing – original draft. ZC: Project administration, Writing – review & editing. ML: Methodology, Writing – review & editing. YZ: Investigation, Writing – review & editing. YW: Investigation, Writing – review & editing. BH: Data curation, Investigation, Writing – original draft, Writing – review & editing, Formal analysis, Project administration, Software. PW: Data curation, Investigation, Writing – original draft, Writing – review & editing, Methodology, Supervision.