| Abstract |
Respiratory syncytial virus is the most common pathogen of lower respiratory tract in
infancy and early childhood worldwide. Human RSV primarily infects epithelium of the
nasopharynx and it usually presents with symptoms from the upper respiratory tract,
however in about 40% of cases it can be followed by clinical signs of bronchiolitis,
including tachypnea, hyperinflation, recession, respiratory wheezing and crackles.
The primary aim of our study was to record the incidence of RSV bronchiolitis during the
virus epidemic season in Cretan children less than 24 months of age. During the one year
recruitment period (September 2004-August 2005) 153 children were hospitalized for
bronchiolitis. Eighty eight cases (mean age 5.2±0.5 months; male/female ratio, 1.1:1)
proved RSV-associated and 82 met the enrolment criteria. This year’s epidemic was
identified in mid February to early May and accounted for 75 percent of hospitalized cases
RSV is a negative sense RNA virus. It is a member of the genus Pneumovirus of the family
Paramyxoviridae. The virally encoded surface transmembrane glucoproteins F (fusion) and
G (attachment) constitute the main antigenic factors encoding host’s immune response.
Although, currently, our knowledge of the pathogenesis of RSV in humans is incomplete,
constituting a field of intense inquiring activity, bibliographic data support the crucial role
of the host’s immune response in the pathogenesis and clinical manifestations of RSV
infection.
It is already known that the immune response to the RSV infection is characterized by mass
inflammatory filtering with neutrophils, monocytes, eosinophils and natural killer cells in
the respiratory epithelium. Responsible for the mobilisation, accumulation and filtering of
inflammatory cells (mainly neutrophils) in the inflammation area are considered
proinflammatory mediators (cytokines-chemokines), which are highly expressed from the
infected respiratory epithelial cells and the macrophages.
Recent studies in humans and animal models associate the wide range of RSV disease
severity with this cytokine expression pattern.
Chemokines consist a family of low molecular weight proinflammatory proteins
(cytokines), which act as chemoattractant molecules in cells that express specific receptors
playing regulatory role in the process of inflammation. Chemokines coordinate the immune
response by directing leukocyte trafficking and activation in the sight of inflammation.
They consist of a continuously expanding family of approximately 50 chemoattractant
proteins and 20 receptors which are classified in four sub-families. Chemokines are
involved in almost every inflammatory process and play an important role in the
pathogenesis of a large number of inflammatory and immuno-modulatory diseases such as
bronchiolitis. A wide variety of chemokines has been recognized in the blood and
nasopharyngeal secretions of patients with RSV infection, such as RANTES (CCL 5), MCP
1 (CCL 2), IL 8, and the recently discovered Fractalkine (CX3CL or FKN).
Fractalkine (FKN) is the only known member of CX3C chemokine family. It has the unique
ability to exist in two forms; membrane anchored or shed soluble glycoprotein and acts both
as a chemoattractant and an adhesion molecule for CX3CR1+ leukocytes (monocytes, T
lymphocytes and NK cells). The receptor of fractalkine (CX3CR1) is a seven
transmembrane domain G protein-coupled receptor, and fractalkine binds to it with high
affinity.
The interaction FKN/CX3CR1 activates pathogenetic mechanisms that lead to the
migration and adhesion of immune cells. FΚΝ has been involved in the pathogenesis of
RSV infection in a distinctive and viral specific way. Studies in animal models
demonstrated that in a non-glycosylated, central conserved region, G protein contains a
CX3C chemokine attachment motif at amino-acid positions 182-186. Due to that structural
similarity G protein has the ability to interact with the CX3C chemokine receptor. This
interaction appears to have at least two important roles in the pathogenesis of RSV
infection. Firstly, G glycoprotein through binding to CX3CR1 facilitates infection.
Secondly, this interaction seems capable of modifying the host’s immune response via
inhibition of activation and immigration of CX3CR1+ leukocytes leading to a less efficient
antiviral immune response.
The interaction of G glycoprotein with CX3CR1 receptor expressed in neurons and
microglia lead also to the release of neuronic products, such as factor P, which has been
connected with alteration of respiratory frequency (apnoea) and also with mucus
overproduction, edema and spasm of smooth muscular fibres, inflammatory activities
responsible for many clinical manifestations of severe RSV disease.
Recently, two single nucleotide polymorphisms of the CX3CR1 gene have been identified
to be associated with decreased receptor expression and reduced receptor’s affinity for its
ligand. The well established pivotal role of G-glycoprotein-CX3C receptor interaction in
the pathogenesis of RSV infection, along with the functional consequences of the genetic
variability of the receptor, led us to the hypothesis that the V249I and T280M
polymorphisms of the CX3CR1 receptor gene could also affect G glycoprotein CX3CCX3CR1
pathway, influencing the RSV disease’s clinical manifestations and outcome. To
test this hypothesis, our study explored the differences in frequency of T280M and V249I
variations between 82 children hospitalized for RSV bronchiolitis in a year period and one
hundred twenty sex matched healthy adults, without history of severe lower respiratory tract
infections who formed the control group.
Genotyping revealed predominance of the 280M containing genotypes (MM or TM) in
cases than in controls (odds ratio, 2.0; 95% confidence interval, 1.1-3.9; p=0.025),
The 280M allele carriers were significantly more common among case subjects compared
with controls (OR, 2.0; 95% CI, 1.1-3.9; p=0.025). The calculated odds ratio (OR) suggests
that a 280M allele carrier (M/M or T/M) is twice as likely to be hospitalized for severe
RSV-induced bronchiolitis compared with a wild-type homozygote (T/T).
We furthermore demonstrated that our results retained their significance when the studied
population was limited to the most severe cases assessed by objective clinical criteria,
indicating a strong association between the 280M allele and susceptibility to severe RSV-
induced bronchiolitis. These results provide additional evidence to support the role of
CX3CR1 in RSV infection. They are also consistent with previous studies suggesting that
the T280M polymorphism causes functional defects of the CX3CR1 protein.
As we mentioned before, severe RSV infection is characterized by increased chemokine
activity. In order to further explore the association of RSV infection and chemokine
activity, we evaluated the CC chemokine, RANTES, through a similar population genetic
perspective. Several studies have already linked increased RANTES expression with severe
RSV disease. Three single nucleotide polymorphisms, -28C/G, -403G/A and In1.1T/C in
the RANTES gene, have been correlated with the gene’s transcriptional activity. In the
present study, we explored the possible correlation of the genetic variability of the
RANTES gene with the clinical manifestation of RSV disease.
We studied the frequency of these three polymorphisms in 106 children hospitalized for
RSV bronchiolitis, in a 2-year period and in one hundred and twenty sex-matched healthy
adults, without a history of severe lower respiratory tract infections.
No association was established between -28C/G polymorphism and RSV-induced
bronchiolitis, mainly due to its extreme rarity in the studied population. No statistically
significant differences were observed in cases and controls regarding genotype and allele
frequencies of each of the In.1.1T/C and -403G/A polymorphisms. Nevertheless, the -
28C/C-403G/AIn1.1T/T combined genotype was significantly more common in cases than
controls.
Our results indicate an association between a common RANTES genotype with severe RSV
infection. This observation supports the previously reported results indicating RANTES as
an important mediator of RSV infection.
In summary, we concluded that the interaction of CX3C pattern of G glycoprotein with the
CX3CR1-M280 variation of the FKN receptor leads to dysfunctional biochemical pathways
with final conclusion the facilitation of RSV infection and the manifestation of severe RSV
bronchiolitis. Our findings also indicate an association between a common genotype with
severe RSV infection, confirming the crucial mediatory role of chemokine RANTES in the
pathogenesis of the disease. The results of our study contribute in the better comprehension
of the protective mechanisms connected with RSV infection. The comprehension of these
mechanisms is essential for the development of safe and effective vaccines and therapies for
the more sufficient confrontation of RSV disease.
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Μελέτη της επίπτωσης της λοίμωξης από αναπνευστικό συγκυτιακό ιό (RSV) στα βρέφη, συσχετισμός της βαρύτητας των κλινικών εκδηλώσεων με γενετικούς πολυμορφισμούς του υποδοχέα CX3CR1
