Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Viral infections in acute and chronic respiratory diseases: genetic diversity of viruses and interferon defence mechanisms

Introduction Respiratory diseases account for a large proportion of all acute morbidities, as well as mortalities, worldwide, among which viral respiratory tract infections are the leading cause (appropriate 80%). Respiratory viruses can also trigger 60%-85% of acute exacerbation in chronic lung diseases. Notably, viruses and bacteria often occupy the same niches and innate immunity pathways can exert positive as well as negative effects during the respiratory tract infections. In the light of these consideration, my Life Science PhD project planned (i) to determine the prevalence of respiratory viruses and (ii) to characterize virus-bacteria-host interactions and their impact on the innate antiviral response in the respiratory tract of patients suffering from bronchiolitis, an acute respiratory syndrome, and cystic fibrosis (CF), a persistent respiratory disorder. Methods Detection of respiratory viruses [respiratory syncytial virus (RSV), rhinovirus (HRV), influenza virus, metapneumovirus (hMPV), bocavirus (hBoV), human coronaviruses (hCoVs) -229E, -OC43, -NL63, -HKU1 and SARS-CoV-2] was performed through reverse transcriptase (RT)-PCR based tests or Real-Time PCR reactions on respiratory samples from bronchiolitis infants (n=431, nasopharyngeal aspirates) and cystic fibrosis patients (n=265, oropharyngeal aspirate and sputum samples). Nasal bacterial community was examined in infants hospitalized for bronchiolitis, positive for RSV infection (RSV-A, n=30 and RSV-B, n=17) and negative for respiratory viruses (n=28) via next-generation sequencing (NGS). Transcript levels of interferon (IFN)-related genes [IFN-III receptors (IFNLR1 and IL10RB) and IFN stimulated genes (ISGs: ISG56 and MxA)] in infants infected with RSV-NA1 (n=46), RSV-ON1 (n=47) or RSV-BA (n=28) and of Toll-like receptors (TLRs: TLR2, TLR3, TLR4, TLR8 and TLR9) in the respiratory samples of HRV positive (n=55) and HRV negative (n=55) CF patients were analyzed using quantitative RT/Real-Time PCR. Ex-vivo induction of ISG15-mRNA was carried out in 2x10^6 cells from oropharyngeal swabs of CF patients treated with IFNβ (5ng/mL) for 24 hours in 48 well plates. Statistical analyses were performed with SPSS v.17.0. Results Molecular epidemiology analysis of respiratory viruses indicated that RSV (ON1 strain), and HRV were the most frequently detected viruses, in infants with bronchiolitis and in all-age cystic fibrosis patients, respectively. Analysis of the nasal microbiota of bronchiolitis infants revealed that RSV-positive group, as well as the RSV-A group, had a lower biodiversity (including beneficial species: probiotic or anti-inflammatory species), a specific microbial profile with a relative abundance of pathobiont species S. pneumonia (p=0.0004) and a general loss of connectivity among microbes with respect to the RSV-negative one. As far as the evaluation of innate immunity response in infants infected with different RSV strains or in HRV infected CF patients was concerned, our results indicated that the gene expression levels of IFNLR1 and IL10RB positively correlated to each other (p=0.037, r=0.230) and did not differ in infants infected with RSV strains, NA1, ON1 and BA. The ISGs levels also correlated between them (p<0.0001, r=0.630) but differed among RSV groups. In particular, MxA and ISG56 mRNA levels were higher in NA1-infected infants than in those with ON1 and BA (p<0.05); interestingly, a moderate negative correlation existed between RSV load and both ISGs values in ON1-infected infants (MxA: p=0.010, r=-0.378; ISG56: p=0.035, r=-0.316). Furthermore, CF patients infected with HRV A, B or C species exhibited higher levels of TLR2, TLR4 and TLR8 as compared to HRV negative patients (p<0.05). These TLRs correlate with high level of HRV load (TLR2: p=0.003, r=0.39; TLR4: p=0.03, r=0.29; TLR8: p=0.001, r=0.42). Also, HRV positive patients co-colonized by S. aureus or P. aeruginosa showed enhanced amounts of TLR2 and TLR2/4-mRNAs expression, respectively (p<0.05 for all genes). In the case of presence of both bacteria TLR8 and TLR9-mRNAs levels were elevated in positive HRV patients. Moreover, ex vivo IFN-β stimulation of nasopharyngeal cells collected from CF patients, induced a lower increase of ISG15 levels (3-fold) compared to that detected in respiratory cells from healthy donors (18-fold increase, p<0.05). Conclusions Results obtained during my PhD project in Life Science underlined a key role played by RSV and HRV in bronchiolitis and cystic fibrosis, respectively. On one hand, RSV infection in bronchiolitis infants (and specifically that caused by RSV-A strain) emphasized a significant perturbation in the nasal microbiota structure, defining a state of dysbiosis. RSV strains could also differentially regulate IFN pathways; the reduced ISGs levels elicited during infections with RSV-A ON1 (and BA) might cause a weaker control of RSV replication and or an inadequate antiviral immune response. On the other hands, HRV infection in CF resulted in a profound dysregulation of the antiviral defence, as indicated by the alterations observed in the expression of TLRs as well as by the reduced responsiveness of the respiratory cells to ex vivo IFN-I stimulation. In addition, HRV can promote bacteria colonization resulting in detrimental effects in CF individuals.