**4. Interaction between respiratory viruses and bacteria**

In a 25-year retrospective review from the Danish CF clinic, the first isolation of *P. aeruginosa* was most likely between October and March [16] coinciding with the peak of the *RSV* season. However, there are a number of other possible viral agents that would broadly fit the winter season, most notably *influenza, rhinovirus* and *metapneumovirus;* therefore, these findings must be interpreted with caution.

An increase in immunoglobulin A (IgA) antibodies to the O-antigen of *P. aeruginosa* is noted in 62% of viral infections [60]. This suggests a possible 'microbial synergism' between bacterial infections and infections with respiratory viruses in CF.

The first bacterial isolation of a given organism in CF has also been shown to often follow a viral infection. In the 17-month prospective study reported by Collinson et al. [26], 5 of the 6 first isolations of *P. aeruginosa* were made during the symptomatic phase of an upper respira‐ tory tract infection or three weeks thereafter. In contrast, only one of the 6 initial infections with *P. aeruginosa* was identified during the asymptomatic period. Similarly, *H. influenzae* was recovered for the first time from 3 children within 3 weeks of an upper respiratory tract infection and the one new *S. aureus* infection was identified immediately following a viral infection.

Armstrong and colleagues have reported that 50% of CF respiratory exacerbations requiring hospitalisation are associated with isolation of a respiratory virus [21]. In their prospective study of repeated BAL in infants over a 5-year period, a respiratory virus was identified in 52% of infants hospitalised for a respiratory exacerbation, most commonly *RSV*; 11 of the 31 hospitalised infants (35%) acquired *P. aeruginosa* in the subsequent 12-60 month follow-up, compared to 3 of 49 (6%) non-hospitalised infants (Relative risk 5.8). This indicates that RSV infection was identified immediately following a viral infection.

mation to public health authorities such that public health policies can be adjusted

**4.** Utilisation of real-time multiplex amplification technique allows multiple viruses being

More recently, Virochip has been shown to be a pan-virus microarray platform that is capable of detection of known as well as novel viruses in a single assay simultaneously [59]. Probes chosen for Virochip can identify nodes in the viral taxonomy at the family, genus and species levels. As the Virochip probes are updated regularly, the extent of probes that can be covered are ever increasing, up to 36,000. It has a diagnostic sensitivity comparable to PCR for detecting respiratory genomes at levels as low as 100 genome copies. At the present time, Virochip is very much a research tool, and several issues must be addressed before it can be used as a routine test for virus detection in the clinical setting, including cost, diagnostic accuracy, repeatability, and sensitivity/specificity for virus detection. In addition, the clinical implication of novel viruses in the human respiratory tract is not yet defined. Therefore, the accurate interpretation of Virochip in the clinical setting remains a formidable task. For example, where specimens are polymicrobial or viral material are present at low levels, clinical and epidemio‐

accordingly, e.g. the outbreak of SARS and influenza H5N1 virus.

logical information might be required to draw clinically meaningful conclusions.

In a 25-year retrospective review from the Danish CF clinic, the first isolation of *P. aeruginosa* was most likely between October and March [16] coinciding with the peak of the *RSV* season. However, there are a number of other possible viral agents that would broadly fit the winter season, most notably *influenza, rhinovirus* and *metapneumovirus;* therefore, these findings must

An increase in immunoglobulin A (IgA) antibodies to the O-antigen of *P. aeruginosa* is noted in 62% of viral infections [60]. This suggests a possible 'microbial synergism' between bacterial

The first bacterial isolation of a given organism in CF has also been shown to often follow a viral infection. In the 17-month prospective study reported by Collinson et al. [26], 5 of the 6 first isolations of *P. aeruginosa* were made during the symptomatic phase of an upper respira‐ tory tract infection or three weeks thereafter. In contrast, only one of the 6 initial infections with *P. aeruginosa* was identified during the asymptomatic period. Similarly, *H. influenzae* was recovered for the first time from 3 children within 3 weeks of an upper respiratory tract infection and the one new *S. aureus* infection was identified immediately following a viral

Armstrong and colleagues have reported that 50% of CF respiratory exacerbations requiring hospitalisation are associated with isolation of a respiratory virus [21]. In their prospective study of repeated BAL in infants over a 5-year period, a respiratory virus was identified in 52% of infants hospitalised for a respiratory exacerbation, most commonly *RSV*; 11 of the 31

**4. Interaction between respiratory viruses and bacteria**

infections and infections with respiratory viruses in CF.

be interpreted with caution.

152 Cystic Fibrosis in the Light of New Research

infection.

quantified even if the copy number of the viral target is low.

Respiratory viruses can disrupt the airway epithelium and precipitate bacterial adherence. *Influenza A* infection has been shown to cause epithelial shedding to basement membrane with submucosal oedema and neutrophil infiltrate [61], while both influenza and adenovirus have a cytopathic effect on cultured nasal epithelium leading to destruction of the cell monolayer [62]. This epithelial damage results in an increase in the permeability of the mucosal layer [63, 64] and possibly facilitating bacterial adherence. Bacteria can also utilise viral glycoproteins and other virus-induced receptors on host cell membrane as bacterial receptors in order to adhere to virus-infected cells [65, 66].

Kim et al. [67] found that invariant natural killer T cells induce a type of macrophage activation driving the secretion of interleukin-13 leading to the production of globlet cell metaplasia and airway hyperactivity following infection with Sendai virus. The term 'invariant' stems from the fact that all invariant natural killer T cells in humans and mice use a unique T cell receptor that is essential for interaction with CD1d. CD1d molecules present lipid antigens to T lymphocytes rather than peptide antigens as in the case of major histocompatibility complex (MHC) class I and II molecules. Historically, MHC class II dependent CD4 and T lymphocytes, through their response to stimulation by environmental allergens, are keys to the pathogenesis of human asthma. The findings by the authors lead to the notion of the use of anti-interleu‐ kin-13 therapy as a potential therapy in patients.

Viral infections might predispose to secondary bacterial infections by impairing mucociliary function and triggering host inflammatory receptors [68, 69]. This phenomenon has been demonstrated both in vivo and in vitro [70, 71]. Avadhanula et al. [72] showed that different respiratory viruses use different mechanisms to enhance the adherence of bacteria to respira‐ tory epithelial cells. In particular, *RSV* and *PIV type 3* up-regulate intercellular adhesion molecule-1 (ICAM-1), carcinoembryonic adhesion molecule 1 (CEACAM1) and platelet activating factor receptor (PAFr) but not mucin on the surfaces of A549, BEAS-2B and NHBE but not SAE cell lines. Much of the increased bacterial adhesion following *RSV* infection could be blocked by antibodies directed against these receptors. A549 and BEAS-2B are transformed cell lines derived from type II alveolar and normal bronchial cells, respectively. NHBE and SAE cells are primary epithelial cells obtained from bronchi and distal bronchial tree and are likely to include a heterogeneous population of cells.

Mechanisms independent of the expression of conventional receptors for bacteria, such as binding to viral proteins, could be responsible for enhanced adhesion [73]. Immunofluores‐ cence microscopy demonstrates that bacteria binding to *RSV*-infected A549 cells adhere not only to these cells expressing viral antigens but also to uninfected epithelial cells. These data suggest that the ability to augment bacterial adhesion may result from a factor served by infected cells that exert a paracrine effect on adjacent epithelium. Cytokines or other inflam‐ matory molecules are potential good candidates for such a mediator.

*Rhinovirus* has been shown to potentiate bacterial infections by inhibiting the secretion of TNF alpha and interleukin-8 by macrophages in vitro following co-infection with gram negative bacterial products, lipopolysaccharide (LPS), and gram positive bacterial products, lipotei‐ choic acid (LTA) [74]. This rhinovirus-dependent impairment of the macrophage immune response was not mediated by autocrine production of the anti-inflammatory cytokines interleukin-10 and PGE2, or by down-regulation of the cell surface receptor for LTA and LPS. In addition, the authors also show that rhinovirus inhibit the phagocytosis of bacterial products by macrophages. These findings support the notion that *rhinovirus* exposure resulted in a reduced ability to innate and adaptive immune responses against bacterial products, hence promoting the occurrence of bacterial and viral co-infections.

The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expecto‐ rated. *Influenza* viral infection has been shown to precipitate the loss of cilial beat, and shedding of the columnar epithelial cells generally within 48 hours of infection [75]. Pittet et al. [76] showed that a prior *influenza* infection of tracheal cells in vivo does not increase the initial number of *pneumococci* found during the first hour of infection, but it does significantly reduce mucociliary velocity, and thereby reduces *pneumococcal* clearance during the first 2 hours after *pneumococcal* infection at both 3 and 6 days after an *influenza* infection. The defects in *pneumo‐ coccal* clearance were greatest at 6 days after *influenza* infection. Changes to the tracheal epithelium induced by *influenz*a virus may increase susceptibility to a secondary *S. pneumo‐ niae* infection by increasing *pneumococcal* adherence to the tracheal epithelium and/or decreas‐ ing the clearance of *S. pneumoniae* via the mucociliary escalator of the trachea, and thus increasing the risk of secondary bacterial infection.

De Vrankrijker et al. [77] showed that mice that were co-infected with *RSV* and *P. aeruginosa* had a 2,000 times higher colony-forming units (CFU) count of *P*. *aeruginosa* in the lung homogenates compared to mice that were infected with *P. aeruginosa* alone. Co-infected mice also had more severe lung function changes. These results suggest that *RSV* can facilitate the initiation of acute *P. aeruginosa* infection.

Another study also showed that *H. influenzae* and *S. pneumoniae* bind to both free *RSV* virions and epithelial cells transfected with cell-membrane-bound G protein, but not to secreted G protein. Pre-incubation with specific anti-G antibody significantly reduces bacterial adhesion to G protein-transfected cells [78].

Stark et al. [79] showed that mice that were exposed to *RSV* had significantly decreased *S. pneumonia*, *S. aureus* or *P. aeruginosa* clearance 1 to 7 days after *RSV* exposure. Mice that were exposed to both *RSV* and bacteria had a higher production of neutrophil-induced peroxide but less production of myeloperoxidase compared to mice that were exposed to *S. pneumoniae* alone. This suggests that functional changes in the recruited neutrophils may contribute to the decreased bacterial clearance.

More recently, Chattoraj et al. [15] demonstrated that acute infection of primary CF airway epithelial cells with rhinovirus liberates planktonic bacteria from biofilm. Superinfection with *rhinovirus* stimulates robust chemokine responses from CF airway epithelial cells that were pre-treated with mucoid *P. aeruginosa*. The authors also showed that these chemokine re‐ sponses lead to a liberation of bacteria from mucoid *P. aeruginosa* biofilm and transmigration of planktonic bacteria from the apical to the basolateral surface of mucociliary-differentiated CF airway epithelial cells. Planktonic bacteria, which are more pro-inflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations and subsequent prolonged intravenous antibiotic use and hospitalisation.

*Rhinovirus* has been shown to potentiate bacterial infections by inhibiting the secretion of TNF alpha and interleukin-8 by macrophages in vitro following co-infection with gram negative bacterial products, lipopolysaccharide (LPS), and gram positive bacterial products, lipotei‐ choic acid (LTA) [74]. This rhinovirus-dependent impairment of the macrophage immune response was not mediated by autocrine production of the anti-inflammatory cytokines interleukin-10 and PGE2, or by down-regulation of the cell surface receptor for LTA and LPS. In addition, the authors also show that rhinovirus inhibit the phagocytosis of bacterial products by macrophages. These findings support the notion that *rhinovirus* exposure resulted in a reduced ability to innate and adaptive immune responses against bacterial products, hence

The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expecto‐ rated. *Influenza* viral infection has been shown to precipitate the loss of cilial beat, and shedding of the columnar epithelial cells generally within 48 hours of infection [75]. Pittet et al. [76] showed that a prior *influenza* infection of tracheal cells in vivo does not increase the initial number of *pneumococci* found during the first hour of infection, but it does significantly reduce mucociliary velocity, and thereby reduces *pneumococcal* clearance during the first 2 hours after *pneumococcal* infection at both 3 and 6 days after an *influenza* infection. The defects in *pneumo‐ coccal* clearance were greatest at 6 days after *influenza* infection. Changes to the tracheal epithelium induced by *influenz*a virus may increase susceptibility to a secondary *S. pneumo‐ niae* infection by increasing *pneumococcal* adherence to the tracheal epithelium and/or decreas‐ ing the clearance of *S. pneumoniae* via the mucociliary escalator of the trachea, and thus

De Vrankrijker et al. [77] showed that mice that were co-infected with *RSV* and *P. aeruginosa* had a 2,000 times higher colony-forming units (CFU) count of *P*. *aeruginosa* in the lung homogenates compared to mice that were infected with *P. aeruginosa* alone. Co-infected mice also had more severe lung function changes. These results suggest that *RSV* can facilitate the

Another study also showed that *H. influenzae* and *S. pneumoniae* bind to both free *RSV* virions and epithelial cells transfected with cell-membrane-bound G protein, but not to secreted G protein. Pre-incubation with specific anti-G antibody significantly reduces bacterial adhesion

Stark et al. [79] showed that mice that were exposed to *RSV* had significantly decreased *S. pneumonia*, *S. aureus* or *P. aeruginosa* clearance 1 to 7 days after *RSV* exposure. Mice that were exposed to both *RSV* and bacteria had a higher production of neutrophil-induced peroxide but less production of myeloperoxidase compared to mice that were exposed to *S. pneumoniae* alone. This suggests that functional changes in the recruited neutrophils may contribute to the

promoting the occurrence of bacterial and viral co-infections.

154 Cystic Fibrosis in the Light of New Research

increasing the risk of secondary bacterial infection.

initiation of acute *P. aeruginosa* infection.

to G protein-transfected cells [78].

decreased bacterial clearance.

Contrary to the above reports, Chin et al. [32] performed a prospective study over a 2-year period on 35 adult CF patients. *P. aeruginosa* sputum density was analysed during stable, exacerbation and post-exacerbation assessments. PCR was used to detect respiratory viruses during exacerbations. The sputum density of *P. aeruginosa* in patients with or without a viral infection was compared using quantitative culture or by PCR. Twenty-two patients experi‐ enced 30 exacerbations during the study period; 50% were associated with a viral infection. There was no change in sputum density of *P. aeruginosa* from the stable to exacerbation state. Virus-associated exacerbations did not result in significant increases in *P. aeruginosa* sputum density compared to non-viral exacerbations.

Contrary to the above findings, Asner et al. [44] found the mean total bacterial density in sputum samples in virus-positive patients being two logs lower than that found in virusnegative patients (p=0.299). However, this could be explained by the fact that the median age of the virus-positive group was significantly lower than the virus-negative group. Viruspositive and virus-negative patients had similar IL-8, neutrophil percentage and neutrophil elastase levels.

Similarly, Kieninger et al. [80] performed a comprehensive investigation of the inflammatory response of CF airway epithelial cells on virus infection. Strong cytokine production was found in all cells studied, with the magnitude and type of inflammation differing depending on cell type and virus used. There was no exaggerated inflammatory response in CF, either during cytokine production or at the transcriptional level. Instead, there was a trend towards lower cytokine production in CF airway epithelial cells after virus infection, which was associated with increased cell death. The lower inflammatory response in CF can also be explained by additional pathophysiological mechanisms, such as interactions between anti-viral and proinflammatory pathways, which are likely to be involved [81]. It could also be speculated that because of chronic activation of pro-inflammatory pathways, CF airway epithelial cells are not able to respond sufficiently to further stimuli, such as virus infections. This might, in turn, lead to a lack of recruitment of effector immune cells resulting in longer duration and more severe respiratory symptoms.

TLRs are key mediators of type I interferon (IFN) during viral infections by recognizing various viral components. TLR7 and TLR9 have become apparent as universally important in inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells [82]. New intracellular viral pattern recognition receptors leading to type I IFN production have been identified. CFTR mutations have been shown to affect the epithelial induction of type I IFN expression by airway cells in response to *P. aeruginosa* infection [83]. This is achieved by abolishing this signalling pathway, an important component of the innate immune system that protects mucosal surfaces. Based on available evidence, chronic colonisation of *P. aeruginosa* in CF airways can be hypothesised to increase the predisposition of viral infections; however, more in-depth studies are required to elucidate this hypothesis.

Taken together, these findings suggest conflicting data regarding the inflammatory response of the CF airway epithelium on virus infection and to some extent the symbiotic relationship between viruses and bacteria. Nonetheless, respiratory viruses may lead to epithelial disrup‐ tion, increase neutrophil influx, inhibition of macrophage phagocytosis, destruction of mucociliary escalator, down-regulation of cilia beat, liberation of pro-inflammatory planktonic *P. aeruginosa* from biofilm and increased neutrophil-induced peroxide release, indirectly facilitating bacterial infection of the airway.
