This study on human respiratory viral infections including Flu A, RSV and RV was carried out to determine gene expression patterns of host-based peripheral blood specific to the adaptive immune responses to these viral infections. For this purpose, 192 genes related to adaptive immunity were evaluated in PBMCs extracted from respiratory viral infected patients. In order to simplify the characterization of analysis, genes were categorized into three groups including genes associated with activation, proliferation and differentiation of T cells, genes associated with activation, proliferation and differentiation of B cells, and genes associated with antigen presentation. Results provide evidence that gene expression of peripheral blood can potentially be considered as biomarkers for specific infectious pathogens and may function as efficient diagnostic tools for triaging treatment decisions for respiratory infection. Triage and treatment decisions can be facilitated by distinction between infectious causes of illness. Although traditional culture and PCR based diagnostics are effective in determining aetiology, these techniques have some limitations especially the high false negative rate when the test is performed during the window period or late when immune response had cleared the virus in circulation.¹⁷ Classification of infectious pathogens according to host immune responses has potential to increase our diagnostic abilities and to provide additional understanding into the infection pathobiology. Woods et al¹⁸ has shown that, in H1N1 and H1N3 infections, the peripheral blood gene expression signature can distinguish between these two strains with good degree of accuracy. Additionally, their study demonstrated that genomic signatures were able to distinguish upper respiratory viral infection from pneumonia due to Streptococcus pneumoniae.¹⁹

There are different types of T cells that are distinguishable based on their cell surface markers and functional capabilities. These T cells that play a regulatory role in adaptive immunity, whether cellular or humoral immune responses, are referred to as T helper (Th) cells and typically express CD4+ molecules on their surface. Activation of virus specific CD4+ Th cells, recognize virus-derived MHC class II-associated peptides on antigen presenting cells. Viral epitopes associated with MHC class I molecules activate the naïve CD8+ T cells in the draining lymph nodes, which subsequently differentiates into cytotoxic T lymphocytes (CTLs).²⁰ Different gene sets encompass many areas of the T-cell receptor together with the associated signal transduction genes and this also include genes encoding transcriptional regulators and cell adhesion molecules.^21,22

In this study, we found that some genes are common among different respiratory viral infections; whilst some genes were differentially elevated in different respiratory viral infections. We found that genes related to cell adhesion and costimulatory molecules including CD40, CD40L, CD2, CD86, CD38, CD28 were the common genes upregulated in respiratory viral infected. It has been shown that costimulatory signals enable amplification of the T-cell receptor (TCR)-induced signal with a limited number of MHC–peptide complexes. With CD28 costimulation, the number of TCR molecules decreases and these require to be engaged on a single T cell to stimulate activation and phosphorylation (Noble, 2000). Additionally, other genes that were upregulated include TNFRS4, IL12RB1, PRF1, CXCRS in all samples of Flu A, RSV and RV. These genes tightly regulate the threshold, magnitude and duration of T cell responses against respiratory viral infections.^26,6

In another analysis, we found genes including CD4, CD8, TAP1/2, HLA-DMA, HLA-A/B/C, HLA-DRA were significantly increased in samples of Flu A and RSV. The protein encoded by TAP1/2 genes is associated with assembly of the class I molecules through transfer of cytosolic peptides to the endoplasmic reticulum into the membrane-bound compartment.²⁴ HLAs including A, B and C, present peptides from inside the cell. HLA-DMA and HLA-DR (HLA class II alpha chain paralogues) is a heterodimer consisting of an alpha (DMA) and a beta chain (DMB), both anchored in the membrane and found in intracellular vesicles. The DM molecule helps to release the CLIP molecule from the peptide binding site. Genes critical in B cell activation, function and signalling such as CD69, CD38, LRBA and CD27 as were also upregulated in samples of RSV and Flu A.^25,26

On the other hand, genes such as VAV1, CCL4, KLRB1, KLRD1, LCP1, AN32B, FCER2, TCL1A and SH2D1A were upregulated in samples of rhinovirus only. Among these genes, VAV1, KLRB1, KLRD1, AN32B and SH2D1A were found to be involved in NK cell activation. VAV1 was initially identified as a proto-oncogene,⁴ and later was showed to played a central role in NK cell-mediated cytotoxicity⁸. The both KLRB1 and KLRD1 are two classes of killer lectin-line receptor, while SH2D1A is an adaptor protein that involve in the activation of NK cell.⁵ While KLRB1 (also known as CD161) is an early differentiation marker for NK cells and act as an activation receptor;²⁷ KLRB1 (also known as NKG2A) is an inhibitory receptor that interrupt the activation of NK cells,^28,29 suggesting that perhaps this is the viral strategy to evade from NK cell surveillance. Intriguingly, FCER2 gene that encoded for Fc epsilon receptor 2 was also found to be upregulated among rhinovirus-infected patients. Given that Fcε is the receptor for IgE, this is at least in part explained why rhinovirus has often associated with allergic responses such as asthma among infected children.^30,31,9 In general, gene expression analysis revealed significant alterations of different genes among Flu A, RSV, and RV samples respectively. Respiratory viral infection stimulates alterations in gene expression of human peripheral blood cells that SH2D1A and TCL1A genes were upregulated only in samples of rhinovirus. Additionally, different kinds of genes such as PSMB7, CD4, CD8A, HLA-DMA, HLA-DRA and CD69 were upregulated in samples of Flu A and RSV while they were not significant in sample of RV compared to healthy individuals.

When viruses infect the cells of the respiratory tract, the host reacts by activating parallel signalling pathways that then leads to transcription of cytokine genes essential for activation of the adaptive immune responses.³¹ Programmed cell death or apoptosis is another strategy of the cells to eliminate viral infection¹¹. Viruses developed several strategies to evade the immune defence mechanism against infection. On the other hand, viruses profit from apoptosis as dissemination of virus progeny can occur via induction of the death of the infected cells.³² Therefore, our experiment was also designed to describe alterations in the expression level of some important cytokines and apoptosis proteins in patients with clinical syndromes of respiratory infection.

Significant increase in the levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12, IL-15, TNF-a, and IFN-γ in all resultant groups showed that infection of studied respiratory viruses resulted in a cascade of signalling events leading to the expression of pro-inflammatory, type 1 T helper (Th1) and type 2 T helper (Th2) cytokines which may be of importance for viral clearance. It has been investigated that the presence of many respiratory viruses including RSV²⁹, RV¹⁴, and influenza²⁷ with single stranded RNA cause to activate the TLR3 or TLR8. Moreover, double-stranded RNA and CpG DNA can be recognized by TLR7 and TLR9, respectively¹⁰. TLR7 and TLR9 use MyD88 as an essential adaptor for the activation of NF-B and IRF-7 which leads to the production of proinflammatory cytokines³³ and type I IFNs¹⁵. The RIG-I and MDA5 (melanoma differentiation-associated gene 5) induces an antiviral response and this has been seen with RSV where it stimulates replication-mediated activation of the IFN-β promoter via RIG-I signalling²². Small RNA viruses such as rhinoviruses can be detected by MDA5 and it is very important in transcription of IFN- β, IFN- λ and proinflammatory cytokines²⁶. Additionally, negative-sense ssRNA viruses, such as influenza virus and RSV are detectable by RIG-I and play a critical role in response to infection with these viruses.²⁶ The important role of NLRP3 has also been indicated and here signalling vis this molecule results in activation of pro-inflammatory cytokines such as IL-1β and IL-18.^13,25,34

In this study, among 14 cytokines analysed the significant increase in the levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12, IL-15, TNF-a, and IFN-γ were observed in samples of Flu A, H1N1, RV + H1N1, FluB + H1N1, RV, RSV, RV + RSV + EV with single-stranded RNA, and HAdV with double-stranded DNA. This result corresponds with a previous study showing the recognition of single-stranded RNA viruses by TLR3/8 and double-stranded DNA by TLR 7/9 which resulted in activation of type I IFN and inflammatory cytokines.³⁵ Type I IFN production is one of the key cytokines that directs T cell responses toward Th1-type and cell-mediated immunity.³⁶ In the present study, respiratory viral infection due to FluB+H1N1was associated with significantly higher concentrations of IL-1β, TNF-a, IL-8, IFN-γ when compared with other viruses. It has been documented that influenza virus elicited higher levels of INF-γ, IL-4, and IL-2 compared to human metapneumovirus and RSV in infants.²⁸ Studies on influenza virus and RSV have shown that the differential induction shown is due to the involvement of plasmacytoid dendritic cells (pDCs) at a lower level in the RSV-infected animal.²¹ However, in this study rhino virus infection showed the lowest expression level of IL-2, IL-4 IL-6, IL-8, TNF-a, IFN-γ and GS-CSF as compared to other viruses.

Interestingly, our results showed significant decrease in level of IL-10 in samples of Flu A, Flu H1N1, FluA + H1N1, RV + H1N1, FluB + H1N1, RV + RSV + EV and HAdV. IL-10’s directly affects T and NK cell function via monocyte-macrophages which prevents expression of MHC class II and costimulatory molecule B7-1/B7-2 on these cells, thus restricting the production of some cytokines such as IL-6, IL-18, TNF-a, IL-1a and β and IL-12. IL-10 also controls the CD4 T cell activity and inhibits proliferation and production of IL-2, IFN-γ, IL-4, IL-5 and TNF-a. Thus IL-10 controls innate and adaptive Th1 and Th2 reactions and this occurs via limiting activation and differentiation of T cells in the lymph nodes apart from suppressing pro-inflammatory responses in tissues.^23,24 Downregulation of IL-10 in our results and upregulation of IL-2, IL-4, IL-12, IFN-γ and TNF-a suggested that innate and adaptive Th1 and Th2 responses has been activated against studied respiratory viruses. Additionally, higher expression of GM-CSF was observed in all groups especially in patients with Flu B and H1N1. It has been investigated that GM-CSF is an essential factor to provide the protection against infection with some respiratory viruses such as influenza and RSV.

Activation of cytokines involves the comprehensive host immune responses against viral infection. One should not rule out the possibilities of having different level of cytokines in the host once the virus has been cleared. Therefore, future studies should consider having two time points (during and post infection) of serum cytokine assessment in order determine signature cytokines that are raised only in patients who are positive for RNA of the viruses.¹¹ The present study, also investigated programmed cell death in infected host cells. The effect of the different respiratory viruses on the receptors for the TNF related apoptosis inducing ligand (TRAILR) indicated significant upregulated levels of TRAIL-R1 and TRAIL-R2 in all virus detected samples. It has been reported that up-regulation of TRAIL-R1 and TRAIL-R2 leads to induction of apoptosis. However, over expressions of TRAIL-R3 and TRAIL-R4 inhibit the TRIAL. In this study, no significant upregulated or downregulated expression levels of TRAIL-R3 and TRAIL-R4 was observed in all samples showing the apoptosis may have activated through TRAILR receptors. TRAILR-3 prevents the formation of DISC (TRAIL-R2 associated death inducing signalling complex) while TRAIL-R4 binds to TRAIL-R2 DISC to prevent caspase recruitment within DISC and activating NF-kB to inhibit TRAIL induced apoptosis.

Various pro-apoptotic and anti-apoptotic proteins up-regulated in samples including Flu A, H1N1, RV + H1N1 + Flu A + H1N1, Flu B + H1N1, RV, RSV, RV + RSV + EV, HAdV and RV + H1N1. Pro-apoptotic bcl-2 markers such as BAX, BAD and BIM, CD40, CD40L, Caspase 3 and Caspase 8, P21 and p53 was most frequent among studied groups compared to healthy individuals. Pro-apoptotic markers of the bcl-2 family induce the caspase activation through the caspase release from the death antagonists and this prompts release of mitochondrial apoptogenic factors into the cytoplasm. Up-regulation of Caspase 3 and caspase 8 in virus detected samples imply that caspase 8- dependent activation of caspase 3 leads to induction of apoptosis. Significantly, up-regulated expression level of CD40 and CD40L is able to induce apoptosis through caspase-depended pathways or activation of nuclear factor-κB (NF-κB), AP-1 and CD95.

Of anti-apoptotic proteins, HSP27, sTNF-R1, sTNF-R2, IGF-II, were upregulated in some virus detected samples including Flu A, H1N1, Flu A + H1N1, Flu B+ H1N1, RV, RSV, RV + RSV + EV, HAdV and RV + H1N1. Upregulated of these anti-apoptotic proteins indicates that the viruses might modulate host cell apoptotic responses to supress host immune system. We also detected several down-regulations in anti- apoptotic proteins in virus detected samples. Among the down-regulated proteins, bcl-2 and bcl-w showed the highest fold-differences compared with controls in samples including Flu A, H1N1, RV + H1N1, Flu A + H1N1, Flu B+ H1N1, PIV3 + PIV4, HboV + HCoV-HKU1 + HCoV-229E, and HAdV. Anti-apoptotic bcl-2 markers prevent apoptosis in the mitochondria through inhibition of release of apoptogenic factors, such as cytochrome c or by deactivation of caspases which is through their proficiencies to sequester pro-caspases. The other down-regulated proteins were HSP60, HSP70, livin, Survivin, IGF-I, IGF-1sR, XIAP and cIAP-2. Heat shock proteins (HSPs) bind to apoptosis protease-activating factor 1 (Apaf-1) disrupting apoptosome formation. It has been shown that viruses are able to activate HSPs to reduce apoptosis, but later, control these proteins to induce apoptosis for effective viral dissemination. Survivin, Livin, XIAP and cIAP-2 are members of inhibitor of apoptosis proteins (IAP) that exert their anti-apoptotic activity through binding to caspase 3, 7 and 9 as the effector caspases in the signalling pathway of apoptosis. IGF-I and IGF-II can activate insulin-like growth factor type I (IGF-I) receptor (IGF-IR) to introduce several signal transduction pathways that facilitate apoptosis suppression. Downregulation of these anti-apoptotic proteins suggesting that immune response in studied individuals had a protective role in inhibiting the virus to complete its replication and to disseminate infectious progeny viruses., while down-regulation of some pro-apoptotic markers such as p21, p27, BAX, CD40 or IGFBPs shows that these respiratory viruses have the capacity to disable the host cell apoptotic mechanisms that may be obligatory for viral life cycle completion.