RSV infection remains a major cause of LRTIs and hospital visits during infancy and childhood globally. Preterm infants are much more vulnerable to RSV attack and RSV related LRTIs can be much more severe and even fatal for preterm infants (19). However, the specific mechanism of RSV infection is poorly understood. Therapeutic options on RSV infection are limited to symptomatic treatments and supportive care and show suboptimal efficacy (20). Recent advances of preventive strategy of immunoprophylactic agent administration (monoclonal antibodies of RSV) shed a light on the prevention of RSV infection (21). Hence, identifying the candidate genes susceptible to RSV infection could be conducive to early intervention, further diagnose and treatment strategies.
In terms of previous studies, RSV infection among neonates has a strongly genetic background and a few independent microarray platforms have performed the DGEs analysis comparing RSV infected patients with healthy people (22). However, attributing to the heterogeneity between different platforms, the results yielded from one single independent platform may not be reliable. Thus, it is reasonable and meaningful to screen and identify the candidate genes from multiple microarray datasets and platforms. In the present study, to minimize the false positive rates generated from multiple microarray platforms, three datasets were normalized and merged as one expression matrix. The study batch effect was adjusted and the DEGs were calculated by meta-analysis. Totally, 4028 DEGs were identified and among which, 131 most significant DEGs were selected. To further select the hub nodes and understand the functions and pathways of the 131 most significant DEGs, the PPI network was constructed and KEGG and GO enrichments were also performed subsequently. Accordingly, thirteen DEGs were screened out as hub genes and were sorted to be involved in response to virus, defense response to virus, regulation of viral genome replication and regulation of viral life cycle. Besides, IFI27 showed the highest EPC score of 12.309 and was confirmed to be the most significant expressed either in bioinformatical analysis or in clinical sample verification.
IFI27 is a member of the interferon alpha inducible proteins that may participate in the pathogenesis of various viral infections (23). IFI27 was confirmed to be involved in a series of biological pathways: innate immune, interferon gamma signaling, RNA polymerase II activating transcription factor binding and lamin binding (24). Tang et al (25) showed that IFI27 alone provides equivalent diagnostic capability comparable to that of multi-gene biomarkers in differentiating between influenza and bacterial infections. In addition, IFI27 also plays a proinflammatory role by inducing the nuclear export of an anti-inflammatory nuclear receptor, NR4A1 (26). Consistently, our pathway enrichment analysis showed that IFI27 was involved in the immune system and response to virus.
As an interferon-α inducible protein, IFI27 was confirmed to be up-regulated in PBMCs of systemic lupus erythematosus patients (27)and in the lungs after influenza A infection in mice, mainly due to the infiltration of macrophages and lymphocytes(28). It is also up-regulated in inflammatory psoriatic skin and in some epithelial cancers, such as ovarian cancer, and its expression is associated with patient survival(29). IFI27 is a novel modulator of innate immune response and it regulates anti-inflammatory nuclear receptors. Experiments in IFI27-deficient mice indicated that a lack of IFI27 prolongs survival in experimental sepsis and endotoxemia(30). These observations suggest that IFI27 is involved in the regulation of inflammatory events in PBMCs. It is confirmed that immunological reaction gets involved in viral infectious diseases and such immunological reaction is not only limited in connective tissue diseases and cancers. Therefore, as a highly immune-connected gene, we can rationally deduce that IFI27 may also has a strong correlation with severe RSV disease. A latest research published in 2020 conducted by Min Zhu et al revealed that IFI27 may be served as a potential indicator for severe enterovirus-71 caused hand foot and mouth disease(31).
Altogether, IFI27 may be associated with RSV progression by multiple functions in activating immune system, regulation of viral genome and proinflammation. Thus, IFI27 may be served as a gene signature for diagnosis and therapeutic target of RSV infection. In further clinical verification, it was demonstrated that infants with relatively high IFI27 expression suffered from more incidence of severe cases, more requirements of invasive ventilation, more frequent hospitalization as well as longer hospital stay. Therefore, IFI27 may also get involved in the progression of RSV infection and may serve to predict the severity of RSV infection. By now, many researches about IFI27 focus on the connective tissue diseases and tumors whereas the studies regarding the correlation between IFI27 expression and viral infectious diseases are inadequate, therefore, it is meaningful and worthful to pay more attention on this topic.
Another four hub genes selected by bioinformatical analysis were IFITM1, IFI44, IFI44L and LY6E, respectively. Interferon induced transmembrane protein 1 (IFITM1) is a member of interferon-induced transmembrane proteins and can be activated by various viruses. IFITM1 inhibits the entry of viruses to the host cell cytoplasm, permits endocytosis and prevents subsequent viral fusion and release of viral contents into the cytosol (32-34). Interferon induced protein 44 (IFI44) and interferon induced protein 44 like (IFI44L), an important paralog of IFI44, are associated with the formation of microtubular structures as well as exhibiting a low antiviral activity against hepatitis C virus (33, 35, 36). Lymphocyte antigen 6 family member E (LY6E) may participate in T-cell development, metabolism of proteins and ectoderm differentiation (37-39). However, their significance in RSV infection is ambiguous since the clinical samples in our center showed no significant difference between groups.
Several limitations about our present study should be considered. Firstly, we did not compare RSV infected patients with patients whose respiratory tract infection were caused by non-RSV viruses. Actually, as a member of the interferon alpha inducible proteins, IFI27 may participate in the pathogenesis of various viral infections not solely limited in RSV infection. Even so, during infancy, RSV still remains the biggest threaten to preterm infants and early recognition of the status of IFI27 expression could provide certain values in clinical decision-making and disease prevention. Secondly, the clinical samples (n=72) were not adequate enough, which may lead to considerable bias of the result. We hope we could expand our sample size in the oncoming research.
In summary, our study may provide a novel understanding of the mechanism of RSV infection. Several key genes were screened out and were further verified by clinical samples. IFI27 was confirmed to be the most meaningful one that highly up-regulated in RSV infected infants. IFI27 may have the potential for screening preterm infants who are susceptible to RSV infection and for predicting the severity of RSV infection. In-depth researches of those genes identified in present study need to be further explored.