**2. Viral respiratory infections in CF**

caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) [3]. The most common mutation is caused by deletion of phenylalanine at position 508 (Delta F508) of the CFTR on chromosome 7, which accounts for approximately 70% of CF cases. The primary function of CFTR in many tissues is to regulate and participate in the transport of chloride ions across epithelial cell membranes. To date, more than 1,900 mutations have

CF is a multisystem disease as CFTR is expressed in different organs [4]; however, the lungs are the predominant organs that bear the brunt of the disease [5]. Recurrent pulmonary infections may start at very early stages in the lives of patients with CF. It has been hypothesised that low airway surface liquid volume and impaired mucociliary clearance are responsible for the pathogenesis of lung infections. These in turn lead to impaired bacterial clearance from respiratory epithelial cells [6]. Pulmonary infections remain the greatest cause of poor life quality, morbidity and mortality in CF that eventually lead to

Apart from chronic lung disease with recurrent exacerbations, exocrine pancreatic insuffi‐ ciency is also a feature that leads to malabsorption and subsequently growth retardation and maturation. Endocrine pancreatic insufficiency is another feature of CF with the manifestation

The median survival from CF has taken great strides over the past 40 years as a consequence of the introduction of specialist centre care, nutritional optimisation, prevention and aggres‐ sive management of pulmonary exacerbations [8]. In the United Kingdom (UK) CF population in 2012, the median survival was reported as 43.5 years, compared to 38.8 years for the population in 2008 as per the UK CF Registry [9]. It has been postulated that the continuing improvement in survival of CF patients in successive cohorts means that the previous prediction of patients with CF living beyond a median age of 50 years is not impossible. The recent introduction of Ivacaftor to the management of CF patients with G551D CFTR mutations

Historically, bacteria have been the predominant cause for respiratory exacerbations. The presences of some organisms including *Staphylococcus aureus*, *Pseudomonas aeruginosa* and *Burkhoderia cepacia* in the airways have been shown to lead to clinical deterioration [11-13] and may subsequently lead to morbidity and mortality. Pulmonary exacerbations are associated with acquisition of new organisms and increased concentration of airway flora [14]. The new acquisitions of *P. aeruginosa* in CF have been demonstrated to occur in the winter months coinciding with the peak of respiratory viral infections [15, 16]. In the event of a pulmonary exacerbation the absence of pyrexia, raised inflammatory markers and systemic response, pathogens other than bacteria can be the potential cause. Respiratory viruses have been implicated by a number of studies in the last 30 years as potentiators for CF exacerbations [17-29]. *Influenza* is a substantial health threat; it is associated with approximately 36,000 deaths and 220,000 hospitalisations in the USA on an annual basis [30]. The recent emergence of novel *influenza virus (H1N1)* further heightened the awareness of influenza-like illness. CF Pulmo‐ nary exacerbation rates have also been shown to be significantly increased during the winter months and are highly associated with the influenza season [31]. Respiratory viruses that are

of diabetes. Obstructive azoospermia in male CF patients leads to male infertility.

been described in this gene.

144 Cystic Fibrosis in the Light of New Research

premature death in this condition [7].

may further enhance the overall survival [10].

Early studies looking at respiratory viruses in CF relied on repeated serological testing, either alone [20] or in combination with viral cultures for viral detection [21-25]. These methods are relatively insensitive and more recent studies have utilised molecular-based methodologies [18, 26-28, 33-36]. All these studies produced different results in terms of prevalence of respiratory viruses in CF. The differences can be due to different methodologies, different sampling methods; the differences can also be accountable by different populations studied as the prognosis for CF has improved with each successive birth cohort.

Wang et al. [25] described the relationship between respiratory viral infections and deteriora‐ tion in clinical status in CF almost 30 years ago. In this 2- year prospective study [25], viruses were identified through serology and nasal lavage in 49 patients with CF (mean age 13.7 years) on a quarterly basis and at the onset of exacerbations. Although the CF patients had more respiratory illnesses than sibling controls (3.7 versus 1.7/year), there were no differences in virus identification rates (1.7/year). The rate of proven virus infection was significantly correlated with the decline in lung functions, nutritional status, radiology score, and frequency and duration of hospitalisation.

More recent studies suggest no difference in the frequency of either upper respiratory tract illness (URTI) episodes [22] or proven respiratory viral infections [24] between children with CF and healthy controls, but children with CF have significantly more episodes of lower airway symptoms than controls [22, 24]. Ramsey et al. [24] prospectively compared the incidence and effect of viral infections on pulmonary function and clinical scores in 15 school-age patients with CF aged between 5 and 21 years and their healthy siblings. Over a 2-year period, samples were taken at regular two monthly intervals and during acute respiratory illnesses (ARI) for pharyngeal culture and serology for respiratory viruses. There was a total of 68 ARI episodes that occurred in the patients with CF and in 19 episodes there was an associated virus identified. A total of 49 infective agents were identified either during ARIs or at routine testing in the patients with CF; 14 were identified on viral isolation (*rhinoviru*s on 11 occasions), whilst 35 were isolated on seroconversion (*PIV* on 12, *RSV* on 9 and *M. pneumoniae* on 6 occasions). At the time of an ARI, the virus isolation and seroconversion rates were 8.8% and 19.1%, respectively, in children with CF compared to 15.0% and 15.0%, respectively, for the healthy siblings. In contrast, the rates of virus isolation and seroconversion at routine 2 monthly visits were 5.6% and 16.2%, respectively, for children with CF and 7.7% and 20.2%, respectively, for the healthy siblings. There was no significant difference in the rate of viral infections between the patients with CF and their sibling controls, as measured either by culture or serology. The rate of viral infections was higher in younger children (both CF and controls); however, the rate of decline in pulmonary function and severity score was both lower in the younger children with CF and the ones with more viral infections. The authors concluded that there were no significant adverse effects with viral infections in CF.

Likewise, Hiatt [22] assessed respiratory viral infections over three winters in 22 infants less than 2 years of age with CF (30 patient seasons), and 27 age-matched controls (28 patient seasons). The average number of acute respiratory illness per winter was the same in the control and CF groups (5.0 vs. 5.0). However, only 4 of the 28 control infants had lower respiratory tract symptoms in association with the respiratory tract illness, compared with 13 out of the 30 infants with CF (Odd ratio – 4.6; 95% confidence interval 1.3 and 16.5; p-value <0.05); 7 of the infants with CF cultured *RSV*, of whom 3 required hospitalisation. In contrast, none of the controls required hospitalisation. Pulmonary function measured by rapid chest compression technique was significantly reduced in the infants with CF after the winter months and was associated with two interactions; *RSV* infection with lower respiratory tract infection and male sex with lower respiratory tract infection.

From previous reports, two viral agents appear to have the greatest effect on respiratory status in CF, namely *RSV and influenza*, possibly because the uses of viral culture and serology have underestimated the effects of rhinovirus. In younger children, *RSV* is a major pathogen resulting in an increased rate of hospitalisation. Abman et al. [37] prospectively followed up 48 children with CF diagnosed through newborn screening and documented the effect of *RSV* infection. Eighteen of the infants were admitted into hospital a total of 30 times over a mean follow-up of 28 months (range 5-59]. In 7 of these infants *RSV* was isolated, and their clinical course was severe with 3 requiring mechanical ventilation and 5 necessitating chronic oxygen therapy. Over the next 2 years, these infants had significantly more frequent respiratory symptoms and lower Brasfield chest radiograph [38] scores than *non-RSV-*infected counter‐ parts.

In older children and adults with CF, *influenza* seems to have the greatest effect. Pribble et al. [23] assessed acute pulmonary exacerbation isolates from 54 patients with CF. Over the year of the study, 80 exacerbations were identified, of which 21 episodes were associated with an identified viral agent (*influenza A* – 5 episodes; *influenza B* – 4 episodes; *RSV* – 3 episodes) with most agents identified on serology. Compared to other respiratory viruses, infection with *influenza* was associated with a more significant drop in pulmonary function (FEV1 declined by 26% compared with 6%). There were also a higher proportion of patients with a greater than 20% drop in FEV1 within the *influenza* infected cohort. A retrospective study in older patients with chronic *P. aeruginosa* infection reported an acute deterioration in clinical status in association with *influenza A* virus infection, which was confirmed by serology [39].

symptoms than controls [22, 24]. Ramsey et al. [24] prospectively compared the incidence and effect of viral infections on pulmonary function and clinical scores in 15 school-age patients with CF aged between 5 and 21 years and their healthy siblings. Over a 2-year period, samples were taken at regular two monthly intervals and during acute respiratory illnesses (ARI) for pharyngeal culture and serology for respiratory viruses. There was a total of 68 ARI episodes that occurred in the patients with CF and in 19 episodes there was an associated virus identified. A total of 49 infective agents were identified either during ARIs or at routine testing in the patients with CF; 14 were identified on viral isolation (*rhinoviru*s on 11 occasions), whilst 35 were isolated on seroconversion (*PIV* on 12, *RSV* on 9 and *M. pneumoniae* on 6 occasions). At the time of an ARI, the virus isolation and seroconversion rates were 8.8% and 19.1%, respectively, in children with CF compared to 15.0% and 15.0%, respectively, for the healthy siblings. In contrast, the rates of virus isolation and seroconversion at routine 2 monthly visits were 5.6% and 16.2%, respectively, for children with CF and 7.7% and 20.2%, respectively, for the healthy siblings. There was no significant difference in the rate of viral infections between the patients with CF and their sibling controls, as measured either by culture or serology. The rate of viral infections was higher in younger children (both CF and controls); however, the rate of decline in pulmonary function and severity score was both lower in the younger children with CF and the ones with more viral infections. The authors concluded that there

Likewise, Hiatt [22] assessed respiratory viral infections over three winters in 22 infants less than 2 years of age with CF (30 patient seasons), and 27 age-matched controls (28 patient seasons). The average number of acute respiratory illness per winter was the same in the control and CF groups (5.0 vs. 5.0). However, only 4 of the 28 control infants had lower respiratory tract symptoms in association with the respiratory tract illness, compared with 13 out of the 30 infants with CF (Odd ratio – 4.6; 95% confidence interval 1.3 and 16.5; p-value <0.05); 7 of the infants with CF cultured *RSV*, of whom 3 required hospitalisation. In contrast, none of the controls required hospitalisation. Pulmonary function measured by rapid chest compression technique was significantly reduced in the infants with CF after the winter months and was associated with two interactions; *RSV* infection with lower respiratory tract

From previous reports, two viral agents appear to have the greatest effect on respiratory status in CF, namely *RSV and influenza*, possibly because the uses of viral culture and serology have underestimated the effects of rhinovirus. In younger children, *RSV* is a major pathogen resulting in an increased rate of hospitalisation. Abman et al. [37] prospectively followed up 48 children with CF diagnosed through newborn screening and documented the effect of *RSV* infection. Eighteen of the infants were admitted into hospital a total of 30 times over a mean follow-up of 28 months (range 5-59]. In 7 of these infants *RSV* was isolated, and their clinical course was severe with 3 requiring mechanical ventilation and 5 necessitating chronic oxygen therapy. Over the next 2 years, these infants had significantly more frequent respiratory symptoms and lower Brasfield chest radiograph [38] scores than *non-RSV-*infected counter‐

were no significant adverse effects with viral infections in CF.

146 Cystic Fibrosis in the Light of New Research

infection and male sex with lower respiratory tract infection.

parts.

Over a 1-year period, Smyth et al. [27] prospectively investigated 108 patients with CF (mean age of 7.9 years) using a combination of viral immunofluorescence, culture and seroconversion to identify respiratory viruses. With the exception of *rhinovirus*, a semi-nested reverse tran‐ scriptase PCR technique was used. During the study, 76 subjects had 157 respiratory exacer‐ bations (1.5 episodes/patient/year) and a viral agent was identified in 44 episodes, 25 of which were *rhinovirus* and an equal distribution of other viruses was identified almost always on seroconversion. Rhinovirus identification was associated with significantly more days of intravenous antibiotics, whereas, those children in whom a non-rhinovirus was identified had a significantly greater decrease in FEV1 over the year of the study. When all viruses were considered as a whole, patients had significantly greater decline in Shwachman score [40] and days of intravenous antibiotics use.

Collinson et al. [26] followed 48 children with CF over a 15-month period using viral cultures for viral detection, with the exception of *picornaviruses* where polymerase chain reaction (PCR) was used; 38 children completed the study and there were 147 symptomatic upper respiratory tract infections (URTIs), 2.7 episodes/child/year, with samples available for 119 episodes. *Picornaviruses* were identified in 51 (43%) of these episodes, of which 21 (18%) were *rhinovi‐ ruses*. In those children old enough to perform spirometry, there was significant reduction in both FVC and FEV1 in association with URTIs, with little difference in severity of reduction whether a picornavirus was identified or not. Maximal mean drop in FEV1 was 16.5%, at 1-4 days after onset of symptoms, but a deficit of 10.3% persisted at 21-24 days. Those with more URTIs appeared to have greater change in total Shwachman score [40] and Chrispin-Norman score [41] over the study. Six children isolated a *P.aeruginosa* for the first time during the study, 5 at the time of a URTI and only 1 was asymptomatic at the time of first isolation. However, the data from this study have to be handled with care as the term 'URTI' does not necessarily imply a positive viral isolation.

Punch et al. [42] used a multiplex reverse transcriptase PCR (RT-PCR) assay combined with an enzyme-linked amplicon hybridization assay (ELAHA) for the identification of seven common respiratory viruses in the sputum of 38 CF patients; 53 sputum samples were collected over 2 seasons and 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for *influenza B*, 3 for *parainfluenza type 1*, 3 for *influenza A* and 2 for *RSV*). There were no statistical associations between virus status and demographics, clinical variables or isolation rates for *P. aeruginosa, S. aureus or A. fumigatus*.

Olesen and colleagues [28] obtained sputum and laryngeal aspirates from children with CF over a 12-month period in outpatient clinics. They achieved a viral detection rate of 16%, with *rhinovirus* being the most prevalent virus. FEV1 was significantly reduced during viral infection (-12.5%, p=0.048), with the exception of *rhinovirus* infection. The authors were not able to demonstrate a positive correlation between respiratory viruses and bacterial infections in their studied population as the type or frequency of bacterial infection during or after viral infections were not altered. They also concluded that clinical viral symptoms had a very poor predictive value (0.39) for a positive viral test.

Our group in 2004 [36] utilised 'real-time' multiplex Nucleic Acid Sequenced Based Amplifi‐ cation to examine the role of respiratory viruses in CF children. Over an 18-month period, a viral detection rate of 46% was achieved during reported episodes of respiratory illness. The results compared favourably with previous studies and it may be that earlier studies relied heavily on repeated serological testing, either alone [20] or in combination with viral isolation [21-25]. The viral detection rate was 18.3% from routine nasal samples. However, this was comparable to the seroconversion rate of 12.3% as reported by Wang et al. [25]. Ramsey and colleagues [24] also achieved a similar seroconversion rate of 16.2% from asymptomatic samples. These results suggest that a laboratory method with a higher sensitivity for viral detection does not increase the detection rate in asymptomatic samples, implying that false positives are not necessarily more common than less sensitive diagnostic methods. *Influenza A and B* viruses were the major viruses in causing respiratory exacerbations in CF and both viruses are more commonly detected during pulmonary exacerbations; 22 of 88 [23%) viruses found in this study were *influenza viruses (A & B)*. The result is consistent with majority of the previous studies which showed that *influenza* virus represented between 12% and 27% of all viruses detected. However, the findings are in contrast with other studies where rhinovirus is the major virus in CF exacerbations [26, 28, 43]. The *influenza* vaccine uptake rate during the study period was up to 70% [36]. It is possible that the detection rate for *influenza* virus could have been higher had the vaccine coverage not been this high.

Asner et al. [44] performed an observational cross-sectional study of CF children from a large paediatric referral centre investigating the association between respiratory viruses and pulmonary exacerbations by taking mid-turbinate swabs, sputum and throat swab samples that were tested by a direct immunofluorescent antibody assay and a multiplex PCR panel. Forty-three patients were recruited into the study. Pulmonary function tests, quality of life and severity scores were recorded. Sputum cell counts, bacterial density and cytokines were measured. Twenty-six (60.5%) subjects were tested positive for at least one respiratory virus by any diagnostic method applied to any sample type. Of the 26 virus positive subjects, 17 (65.4%) were positive for one virus and the remaining 9 (34.6%) were positive for two or more viruses. *Coxsackie/echovirus* was the most commonly identified pathogen (29.4%) amongst the 17 subjects that were positive for one virus. Virus-positive patients were younger (p=0.047) and more likely to be male (p=0.029). They were also more likely to present with fever (p=0.019), have higher CF clinical severity (p=0.041) and lower quality of life scores (p=0.022). However, virus-positive and negative patients had similar IL-8, neutrophil percentage, elastase levels and 26 additional cytokines levels between both groups. The authors reported a higher rate of viral detection with mid-turbinate swabs than with sputum samples. The study was primarily conducted in the outpatient setting and subjects may have milder form of exacerbations which may in turn explain the lack of inflammatory response in virus-positive subjects. There was also a significant median age difference between the virus-positive and negative groups (6.9 vs. 13 years, p=0.047), with respiratory viral infection being more common in younger children presumably related to the delay maturation of the immune system.

Olesen and colleagues [28] obtained sputum and laryngeal aspirates from children with CF over a 12-month period in outpatient clinics. They achieved a viral detection rate of 16%, with *rhinovirus* being the most prevalent virus. FEV1 was significantly reduced during viral infection (-12.5%, p=0.048), with the exception of *rhinovirus* infection. The authors were not able to demonstrate a positive correlation between respiratory viruses and bacterial infections in their studied population as the type or frequency of bacterial infection during or after viral infections were not altered. They also concluded that clinical viral symptoms had a very poor predictive

Our group in 2004 [36] utilised 'real-time' multiplex Nucleic Acid Sequenced Based Amplifi‐ cation to examine the role of respiratory viruses in CF children. Over an 18-month period, a viral detection rate of 46% was achieved during reported episodes of respiratory illness. The results compared favourably with previous studies and it may be that earlier studies relied heavily on repeated serological testing, either alone [20] or in combination with viral isolation [21-25]. The viral detection rate was 18.3% from routine nasal samples. However, this was comparable to the seroconversion rate of 12.3% as reported by Wang et al. [25]. Ramsey and colleagues [24] also achieved a similar seroconversion rate of 16.2% from asymptomatic samples. These results suggest that a laboratory method with a higher sensitivity for viral detection does not increase the detection rate in asymptomatic samples, implying that false positives are not necessarily more common than less sensitive diagnostic methods. *Influenza A and B* viruses were the major viruses in causing respiratory exacerbations in CF and both viruses are more commonly detected during pulmonary exacerbations; 22 of 88 [23%) viruses found in this study were *influenza viruses (A & B)*. The result is consistent with majority of the previous studies which showed that *influenza* virus represented between 12% and 27% of all viruses detected. However, the findings are in contrast with other studies where rhinovirus is the major virus in CF exacerbations [26, 28, 43]. The *influenza* vaccine uptake rate during the study period was up to 70% [36]. It is possible that the detection rate for *influenza* virus could

Asner et al. [44] performed an observational cross-sectional study of CF children from a large paediatric referral centre investigating the association between respiratory viruses and pulmonary exacerbations by taking mid-turbinate swabs, sputum and throat swab samples that were tested by a direct immunofluorescent antibody assay and a multiplex PCR panel. Forty-three patients were recruited into the study. Pulmonary function tests, quality of life and severity scores were recorded. Sputum cell counts, bacterial density and cytokines were measured. Twenty-six (60.5%) subjects were tested positive for at least one respiratory virus by any diagnostic method applied to any sample type. Of the 26 virus positive subjects, 17 (65.4%) were positive for one virus and the remaining 9 (34.6%) were positive for two or more viruses. *Coxsackie/echovirus* was the most commonly identified pathogen (29.4%) amongst the 17 subjects that were positive for one virus. Virus-positive patients were younger (p=0.047) and more likely to be male (p=0.029). They were also more likely to present with fever (p=0.019), have higher CF clinical severity (p=0.041) and lower quality of life scores (p=0.022). However, virus-positive and negative patients had similar IL-8, neutrophil percentage, elastase levels and 26 additional cytokines levels between both groups. The authors reported a higher rate of

value (0.39) for a positive viral test.

148 Cystic Fibrosis in the Light of New Research

have been higher had the vaccine coverage not been this high.

A CF centre in Milan (Italy) led by Esposito and colleagues [43] showed that human *rhinovi‐ rus* was the most frequently isolated virus from CF patients <25 years of age during respiratory exacerbations and when subjects were clinically stable over a 1-year period. Molecular techniques were utilised to isolate viruses from nasopharyngeal samples. The authors demonstrated that human *rhinovirus* was more common among patients with pulmonary exacerbations than among clinically stable patients. The human *rhinovirus* viral load was however similar in subjects with or without acute respiratory exacerbations (p=0.46). There were no correlations between the associated clinical condition and viral load as well as between bacterial colonisation, colonising bacterial, and viral infections between the 2 groups. This finding is similar to de Almeida et al. [17] who did not show a difference in viral infection between the exacerbation and clinically stable groups. Therefore, this raises a possibility that isolation of *rhinovirus* from nasopharyngeal swabs does not always indicate that it is a cause of exacerbation as it may be explained by a coincidental upper airways infection, a carrier state, or prolonged shedding of a pathogen that caused a previous infection [45].

In 2009, a novel swine pandemic *influenza A virus (H1N1)* was identified. Nash et al. [46] showed that the symptoms of CF patients infected with H1N1 tend to be mild. There was no significant reduction in FEV1 % predicted, FVC % predicted and body mass index regardless of whether the patients were positive or negative for *H1N1*. Colombo et al. [47] performed a multi-centre survey showing that diagnostic testing did not identify clinical characteristics specifically associated with *H1N1* infections. Similarly, they did not show a significant decline in lung function associated with this infection. To date, the significance of *H1N1* infection in CF remains undefined.

In contrast, the data regarding respiratory viral infection in adults is sparse. An observational study conducted by Hoek et al. [48] over a 1-year period amongst adult CF patients yielded a viral isolation rate of 33% [8/24] utilising molecular techniques and conventional methods. Etherington and colleagues [18] from an adult CF centre published a retrospective case control study looking at the prevalence of respiratory viruses during exacerbations. Viral throat swabs were taken from all patients presenting with an acute pulmonary exacerbation requiring intravenous antibiotic treatment over a 12-month period. Viral isolation was performed by PCR. There were 432 pulmonary exacerbations in 180 adults. In total, there was a total positive viral isolation in 42 exacerbations indicating a prevalence of 9.7%. *Rhinovirus* was the com‐ monest isolated virus and was found on 29 occasions (69%). *Influenza A/H1N1* was isolated in seven patients (16.7%). They demonstrated a measurable impact of viral infections in CF as exacerbations associated with a positive viral PCR had a greater fall in lung function at presentation with higher levels of inflammatory markers. These patients also received more days of intravenous antibiotics, showed less response to treatment and had a shorter time to next pulmonary exacerbation compared to matched controls.

Flight et al. [35] followed up 100 adult CF patients prospectively for 12 months. Sputum, nose swabs and throat swabs were collected every 2 months and at the onset of pulmonary exacerbation for virus detection. PCR assays for *adenovirus, influenza A&B, human metapneu‐ movirus, parainfluenza 1-3, respiratory syncytial virus* and *human rhinovirus* were performed on each sample. Symptom scores, spirometry and inflammatory markers were measured at each visit. Overall, virology results were available for 626 of 649 completed study visits. Of these, 191 (30.5%) were positive for a respiratory virus including 9 episodes of dual viral infection. Human *rhinovirus* accounted for 72.5% of viruses. Overall incidence of viral respiratory infection (VRI) was 1.66 (95% CI 1.39 to 1.92) cases/patient-year. VRI was associated with increased risk of pulmonary exacerbation (OR=2.19; 95% CI 1.56 to 3.08; p<0.001) and pre‐ scription of antibiotics (OR=2.26; 95% CI 1.63 to 3.13; p<0.001). Virus-positive visits were associated with higher respiratory symptom scores and greater C-reactive protein levels. Virus-positive exacerbations had a lower acute fall in FEV1 than virus-negative exacerbations (12.7% vs. 15.6%; p=0.040). The incidence of exacerbations, but not VRI, was associated with greater lung function decline over 12 months (-1.79% per pulmonary exacerbation/year; 95% CI -3.4 to -0.23; p=0.025).

Experimental data on the effects of viral infections in CF are limited. Toll-like receptors (TLRs) have recently been identified as key mediators of the innate response and they recognise pathogens through detection of conserved microbial structures that are absent from the host. Kurt-Jones et al. [49] found that *RSV* persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Haynes et al. [50] also demonstrated that TLR4-deficient mice when challenged with *RSV* exhibited impaired natural killer cell trafficking and impaired virus clearance compared to normal ones. Limited human studies have demonstrated the important role of TLRs in host response against many major groups of mammalian pathogens [51]. The relationship between TLR and respiratory virus including *RSV* in humans will require further studies before it can be established.

Some studies have suggested a higher viral replication when there is an impairment of the innate host defence in CF. *Influenza* titres were significantly increased in a mouse model which were chronically infected with *P. aeruginosa* compared to control model [52]. This in turn led to an increase in susceptibility to fatal streptococcus pneumonia infection. Increased virus replication was also found after *PIV* infection of CF human airway epithelial cells, compared to controls [53]. One of the possible causes of increased virus replication and of virus persis‐ tence might be a reduced production of respiratory nitric oxide (NO), which is a vital part of innate antiviral defence mechanism [54]. Increased production of NO protects against viral infections. In CF patients, expression of the NO producing enzyme NO synthase type 2 (NOS2) is considerably reduced.

Xu et al. [55] showed that CF cells that were infected with influenza A had less IFN-related antiviral gene induction at 24 h but more significant inflammatory cytokine gene induction at 1 h after infection. Therefore, the lesser antiviral and greater early inflammatory response may explain the severe respiratory illness of CF patients with viral infections. Sutanto and coworkers [56] showed that CF airway epithelial cells had a marked increase in IL-8 production, a reduction in apoptosis and an increased viral replication compared with airway epithelial cells from healthy children following exposure to human rhinovirus. This is despite the fact that CF and healthy airway epithelial cells have similar basal and stimulated expression of IL-8 in response to pro-inflammatory stimuli. The increment of IL-8, together with a reduction of apoptotic responses by CF cells to human rhinovirus, could contribute to augmented airway inflammation in the setting of recurrent viral infections early in life.

Azithromycin has previously been shown to offer anti-rhinoviral activity in bronchial epithe‐ lial cells and, during rhinovirus infection by increasing the production of interferon-stimulated genes [57]. However, the role of anti-viral properties of Azithromycin in CF is not clearly defined. Schögler et al. [58] showed that primary bronchial epithelial cells from CF children that were pre-treated with Azithromycin had a seven-fold reduction in rhinovirus replication without inducing cell death. Azithromycin also increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels when measured by real-time quantitative PCR. Therefore, it is likely that Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway.
