New human rhinovirus species and their significance in asthma exacerbation and airway remodeling

Introduction

Asthma is the most common chronic disease of childhood, affecting 10-15% of all children [1]. Several different stimuli including allergens, tobacco smoke, certain drugs, and viral or bacterial infections are known to exacerbate asthma symptoms. Among these triggers, viruses are frequent inducers of asthma exacerbations with human rhinoviruses (HRV) as the most common in children and adults [2-11]. Moreover, HRV are associated with a significant burden of lower respiratory disease [2, 12-15] and this may contribute to the development of asthma during infancy [11, 16].

HRV history

Human rhinoviruses (HRV) are members of the Picornaviridae family (‘pico’ = small, ‘rna’ = ribonucleic acid genome) that were first identified in culture in 1956 [17, 18]. More than 100 serotypes of HRV have been identified to date [17, 19-21]. HRV are the most common cause of the common cold in both adults and children. Although once thought to cause only the common cold, it is now known that rhinoviruses are associated with lower respiratory illness [4, 5, 11-15, 23-25]. In addition, HRV is an important contributor to the etiology of bronchiolitis, pneumonia, and otitis in infants and school-aged children [27-29]. During recent years, in studies using the more sensitive reverse transcriptase-polymerase chain reaction (RT-PCR), HRV have been associated with a significant burden of disease in infants and young children [26 14]. In one United States population-based surveillance study of children <5 years of age who were hospitalized with respiratory symptoms or fever, rhinovirus was detected in nasal/throat swabs by RT-PCR in 26%, representing 5 hospitalizations per 1000 children. HRV-associated hospitalization was more frequent in children <6 months of age than in those 6-23 months or 24-59 months of age (17 versus 6 versus 2 hospitalizations per 1000, respectively) [14]. Rhinoviruses are best identified by RT-PCR performed on nasal secretions from infected individuals. However, not all rhinovirus infections are associated with symptoms. HRV has been detected by RT-PCR in an average of 15% of asymptomatic individuals [22].

HRV are distributed worldwide with no predictable pattern of infection by serotype. Multiple types may be present in a community or in a person at one time; and HRV strains belonging to the same genetic cluster may be isolated during consecutive epidemic seasons, suggesting persistence in a community over an extended period. In temperate climates the incidence of HRV infection peaks in fall, with another peak in spring, but HRV infections occur year-round [30]. Rhinoviruses are the major infectious trigger for asthma among young children, and studies have described a sharp increase in asthma exacerbations in this age group when school opens each fall (often referred to as the “September asthma epidemic”) [31]. Peak HRV incidence in the tropics occurs during the rainy season from June to October.

HRV and asthma

HRV are the most common trigger of asthma exacerbations in children and adults [2-11]. In one study, population-based rates of HRV in children <5 years of age hospitalized with respiratory symptoms or fever were more frequent in children with a history of wheezing or asthma than in those without such a history (25 versus 3 hospitalizations per 1000 children). More than 80% of pediatric asthma is diagnosed before five years of age [32]. Bronchiolitis, a lower respiratory infection in infants presenting with wheezing, rales, and respiratory distress, has typically been associated with respiratory syncytial virus (RSV) infection [15-16], but recent studies have identified HRV as another important cause of bronchiolitis [15, 17-19]. A study from Greece found that the presence of rhinovirus in bronchiolitis increased the risk for developing severe disease almost five-fold [27]. Bronchiolitis caused by RSV has been linked to the subsequent development of asthma [12-14]. HRV-related wheezing in infancy also has been hypothesized to play a causal role in the development of childhood asthma [11, 16]. One study demonstrated that infants of atopic families who wheeze with HRV in the first three years of life have an increased likelihood of developing subsequent childhood asthma at age six (OR = 25.6). In this study, 90% of children who wheezed with HRV in the third year of life had asthma at six years of age [11]. Another study implicated HRV as a causative agent for asthma based on bronchiolitis occurring during the HRV season and subsequent diagnosis of childhood asthma, but did not include viral testing [33]. The association of HRV with bronchiolitis [7, 12, 14] and asthma exacerbations suggests that HRV may be associated with the inception of asthma.

Ongoing studies are investigating the role of the host response in HRV-related illness and comparing it to the effects of viral pathogenicity on asthma exacerbations. Both allergen exposure and elevated IgE levels predispose patients with asthma to more severe respiratory symptoms in response to HRV infection. Studies suggest that abnormalities in the host cellular response to viral infection that result in impaired apoptosis and increased viral replication may be responsible for the severe and prolonged symptoms typical of asthmatic individuals. These mechanisms have yet to be elucidated.

HRVC--a new rhinovirus species

In the past, some HRV strains were difficult to detect by conventional methods because they do not grow well in culture. In fact, recent studies suggest that there are many rhinoviruses that are not cultivatable but have significant correlation with clinical illness. Traditional methods of virus typing using immune antisera have identified about 100 serotypes, classified into HRVA and HRVB species based on genetic sequence similarity [17, 19-21]. More recent PCR-based studies have discovered a novel group, HRVC (also called HRVA2, HRVNY, HRVQPM, and HRVX) [34-42]. HRVC has been proposed as a new HRV species within the genus Enterovirus by the International Committee on Taxonomy of Viruses (http://talk.ictvonline.org/media/p/1035.aspx). HRVC contains a distinct subgroup of strains. Since the discovery of this new species of HRV, other related viruses have been identified from different countries from Africa, Asia, Australia, Europe, and North America [34-42]. In 2009, Palmenburg et al. published complete genome sequences of all 99 previously known HRV serotypes and numerous novel strains, and sequence analysis demonstrates that the HRVC group comprises a genetically distinct species [43]. It also identified a potential new group, HRVD (or a subgroup of HRVA) [43]. Figure 1 illustrates the genetic diversity of rhinoviruses detected in one study of hospitalized children. The novel group HRVC contributed to a substantial burden of HRV-related illness.

Figure 1.

Phylogenetic tree depicting relationships between known HRV serotypes and novel HRV. Previously known HRV serotypes are designated by ‘HRV’. Novel sequences identified in this study are designated by ‘RV’ and a black circle. The numbers in parentheses after the label of these sequences indicates how many additional specimens contained each virus. Novel sequences identified in recent studies are designated by ‘HRV’ and a grey circle.

The identification of a novel group of HRV, and the finding that a significant proportion of HRV-related illness is attributable to these new HRVs, raises the question of why these viruses were not identified previously. One reason appears to be the extreme difficulty in recovering viable virus. Several groups have been unable thus far to recover these viruses in culture, despite numerous classical and non-classical approaches [15,17, 2].

It is likely that HRVC has been circulating for some time, and is not a newly emergent virus. An extensive search of HRV sequences published in GenBank prior to 2006 was unable to identify any that belonged to the HRVC group (Miller et al., unpublished data). One group reported detection of a genetically similar virus in a specimen taken in New York in 2004 [13] and another showed that these viruses have been circulating since at least 2002 [2]. Our group has detected HRVC in respiratory specimens that were collected 20 years ago (Miller et al. unpublished data).

HRVC frequency and symptomatology

During recent years, RT-PCR based studies of HRV have shown that they are associated with a substantial portion of upper and lower respiratory illness in outpatient and hospitalized adults and children, [7,24-33] with evidence that HRVC accounts for a large proportion of this HRV-associated illness. HRVC has been detected in patients with symptoms including acute upper or lower respiratory illness, wheezing, bronchiolitis, asthma exacerbations, COPD exacerbations, pneumonia, LRI, cold and flu-like illness, fever, cough, rhinitis, nasal congestion, bronchitis, retractions, crackle, bronchopneumonia, dyspnea, otitis media, gastroesophageal reflux disease, pericarditis, poor appetite, and apparent life-threatening events [55]. In published studies of adults and children with respiratory symptoms that were tested for HRVC, the proportion of HRV attributable to HRVC ranged from 8 to 81% [2, 35, 37, 42, 44-54]. Overall frequency of HRVC in published studies of adults and children with respiratory symptoms ranges from 2 to 20% [2, 35, 37, 42, 44-54]. (See Table 1.) Like HRVA and HRVB, HRVC has also been detected in asymptomatic individuals [52].

Table 1.

Summary of HRVC rates in studies of adults and children with acute respiratory illness, by inpatient versus outpatient status.

	HRV+/Total Specimens (%)	HRVC/HRV Total (%)	Rates of HRVC
Adults
Inpatient	32%	17%	5%
Outpatient	45%	14%	6%
Children
Inpatient	16-52%	14-73%	4-20%
Outpatient	37%	28%	6%
Mixed	13-22%	8-81%	2-10%
Adults and Children, Inpatient and Outpatient	18%	39%	7%
Overall	13-52%	14-81%	2-20%*

^*Overall prevalence of HRVC in published studies of adults and children with respiratory symptoms ranges from 2 to 20%2, 30, 32, 37, 39-49.

HRVC seasonality

Among the studies that have tested for HRVC to date, the data on seasonality of HRVC is not conclusive. While many studies have detected HRVC as the predominant HRV species in the fall, suggesting it may play a role in the so-called September asthma epidemic [21-23], other studies in different regions and years have found otherwise. Han et al. reported HRVC to be more common in the spring and HRVA in the fall in South Korea in 2006, but co-circulation occurred during both seasons [56]. Consistent with our studies, Lau et al. found HRVA and HRVC to alternate as the most common HRV species at different times during the peak seasons [57]. Alternate disease activity by species and seasons suggests possible viral interference or serological cross-protection between HRVA and HRVC [57.

While one study reported HRVC year-round without peaks in the spring and fall [52], most studies do suggest that HRV species vary by season, year, and geographic location. This variation underscores the importance of studies over multiple years and seasons to understand fully the geographical and seasonal rhinovirus epidemiology.

Asymptomatic infection with HRVC

It is clear that HRV infection occurs in ill individuals and experimental inoculation of patients with HRV induces respiratory symptoms; however, asymptomatic HRV infection with HRV has been documented. The rates of asymptomatic infection have been little studied. One study that included asymptomatic controls identified HRVC more often in sick than in well patients, with HRV detected in only 3 of 93 asymptomatic individuals [52]. There is some evidence that HRVC may interfere with infection by other viruses [58].

HRVC and asthma/wheezing

Certain viruses may pose a greater risk for severe disease and asthma. There is much data to support a relationship of HRVC to wheezing/asthma (Table 2), but whether this differs from the symptoms produced by HRVA or HRVB strains is uncertain. In 2007, four retrospective studies showed a possible association of HRVC with wheezing or bronchiolitis [35, 37, 40, 42]. In those studies, however, sample size was insufficient to detect statistically significant clinical differences between HRVC and other species. In 2009, a large, prospective population-based study of young hospitalized children in two U.S. cities over two years found several clinically significant differences among patients infected with HRVC compared to HRVA, the other predominant HRV species. Of note, children with HRVC were twice as likely to have a discharge diagnosis of asthma than those with HRVA, and children with HRVA were more likely to present with fever [2]. In a similar study published in 2009, in a population of children less than five years of age hospitalized with fever or acute respiratory illness in Amman, Jordan, HRVC was significantly more often associated with wheezing and supplemental oxygen use, as compared with HRVA [53]. An unpublished asthma case-control study of 400 school-aged children in Argentina found slightly more HRVC in asthmatics with URI who wheezed than in asthmatics with URI and no wheezing (p = 0.036) (Miller et al, unpublished data). One retrospective study investigated hospitalized children <11 years of age and found a trend for HRVC to be detected more in patients with underlying asthma (p = 0.09) [46]. HRVC was the most prevalent HRV infection in this Thai study population and was detected in 22% of 50 children aged 4-23 months with first wheezing episode. There were also two patients in this study who had been admitted with five different episodes of acute respiratory infection and subsequent recurrent wheezing associated with HRVA and HRVC [47].

Table 2.

Summary of HRVC in patients with wheezing, bronchiolitis, or asthma.

Study Type	Patient Age	Study Dates	Sample Size	Wheezing/Asthma Status	Different from HRVA/B (p<0.05)	Reference
Inpatient, Outpatient Retrospective	<80 yrs	1/03-12/03	1244	Wheeze, Persistent Cough		(39, 40)
Inpatient	<14 yrs	1/04-12/08	1555	Asthma, Recurrent Wheeze, Bronchiolitis	No	(61)
Inpatient Retrospective	<18 yrs	11/04-10/05	26	Wheeze		(37)
Inpatient, Outpatient	<18 yrs	12/04-7/05	44	Wheeze		(49)
Prospective Case-Control	≥2yrs	3/03-2/04	29	Asthma Exacerbation, Lower FEV	A and C in cases	(51)
Inpatient, Outpatient	<2 yrs	1/04-12/04	447 (93 controls)	Wheezing	No	(52)
Inpatient Retrospective	<11 yrs	2/06-2/07	289	Bronchiolitis, Wheezing, Asthma	Trend	(46, 47)
Inpatient Prospective	<5 yrs	10/01-9/03	1052	Asthma	Yes	(41)
Inpatient PICU	<18 yrs	02/07-10/07	43	Wheeze		(54)
Inpatient Retrospective	<14 yrs	1/06-12/06	470	Asthma exacerbation, Bronchiolitis		(56)
Inpatient Retrospective	<12 yrs	10/05-3/07	500	Asthma, Bronchiolitis		(60)
Inpatient Prospective	<5 yrs	1/07-3/07	728	Wheezing, Supplemental O2	Yes	(53)
Inpatient	2wks-5 yrs	03-06	97	Bronchiolitis	No	(42)
Inpatient	<18 years, adults	04/04-03/05	1200	Asthma, COPD, Febrile Wheeze	No	(57)
Outpatient	>18 yrs	Fall 01-12/04	83	Asthma		(35)

^*Other signs and symptoms associated with HRV-C in some studies but not included in this table include pneumonia, LRI, cold and flu-like illness, fever, cough, rhinitis, nasal congestion, bronchitis, retractions, crackle, bronchopneumonia, upper respiratory tract illness, dyspnea, otitis, gastroesophageal reflux disease, pericarditis, poor appetite, and apparent life-threatening events.

Other studies have reported HRVC in patients with wheezing, bronchiolitis, or asthma but either did not compare or did not find a significant difference between HRVC and HRVA or HRVB. McErlean et al. detected HRVC (then called HRVQPM) in 17/1244 (1.4%) of ill adult and pediatric patients. It was the sole virus detected in 62% of the patients, and those with HRVC infection alone were sicker than those with co-infections. Most patients with HRVC in this study were less than two years of age and 46% had lower respiratory illness, many with wheezing and bronchodilator use [40]. The same group later published an intensive chart review of 17 patients with HRVC and found 36% had bronchiolitis, 14% asthma, 7% COPD, and 21% persistent or hacking cough. Three subjects with HRVC did not have clinical data to assess. Of those with bronchiolitis or asthma, no other viruses were detected [39, 59].

Lau et al. studied 21 children with HRVC and found that wheezing or asthmatic exacerbations were common (16/21 cases, 76%), especially in those with a history of febrile wheeze or asthma [37]. Louie et al. studied 43 children admitted to the intensive care unit and detected HRV in 21:3 of the 10 sequenced had HRVC, many of the patients enrolled were noted to have wheezing [54]. Han et al. detected HRVC in 17 Korean children hospitalized with lower respiratory illness and the diagnoses were asthma exacerbation in eight patients, bronchiolitis in eight, and pneumonia in one [56]. Tan et al reported that 2/64 HRV-positive specimens tested had HRVC, both patients with asthma and bronchiolitis [60]. Another study determined HRVC to be associated with respiratory distress, hypoxia, and wheezing, but the numbers with HRVC were small [49].

Four studies have not detected a clinically significant difference between HRVC and HRVA or HRVB. In a retrospective study of virus-negative bronchiolitis, Renwick et al. found that the frequency of bronchiolitis with HRV A/B (11%) was comparable to that with HRV C (12%) [42]. Similarly, Piotrowska et al. studied 447 symptomatic children younger than two years of age and 93 asymptomatic controls and detected no associations between particular HRV genotypes and wheezing or asthma symptoms [52]. Khetsuriani et a.l performed a case-control study of asthmatics with acute exacerbation versus asthmatics without any respiratory symptoms, and found both HRVA and HRVC to be significantly associated with wheezing [51]. Calvo et al. detected HRV in 424/1555 (27%) hospitalized children. A subset was sequenced, and there was substantial wheezing in children with HRVA and HRVC, but the investigators did not detect a difference in wheezing between the species. Of note, 12% of healthy controls had HRV [61].

Several other studies have identified an association between HRVC and asthma or wheezing. Lau et al studied 1200 hospitalized adults and children with nasopharyngeal aspirate specimens negative for other viruses over a one-year period and found that of the 91 HRVC-positive specimens, 78 were detected in children, and 13 were detected in adults. Of note, in this study adults who had HRVC were more likely to present with lower respiratory illness than were children. Nonetheless, 33 (42%) of the 78 children with HRVC infection experienced asthma exacerbations or febrile wheeze. Sixty-two (68%) infections of adults or children occurred in patients with underlying diseases, with asthma or other chronic lung diseases being the most common. Wheezing episodes were more common in patients with HRVC (37%) and HRVA (20%) infection, compared with that in patients with HRVB (0%) infection [57].

Taken together, these data suggest that viruses in the HRVC group may be either more prone to stimulate asthma reactivity, or intrinsically more virulent. Further studies are required to determine whether these novel rhinoviruses have a greater propensity to cause the exacerbation of asthma and other respiratory illnesses. Moreover, while the role of HRV in the ontogeny or onset of asthma is a provocative area of research [16, 33], the question of whether HRVC may drive this relationship has not been studied. The discovery of an association between disease severity or asthma risk and particular groups or strains of HRV could help inform preventive and treatment measures.

Conclusions

It is well known that HRVs are a major trigger of asthma exacerbations in adults and children and are now under investigation for their potential involvement in the inception of asthma. It is also clear that the newly described HRVC species account for a large proportion of HRV-related illness, including asthma and wheezing exacerbations. HRVC is genetically diverse and appears to have seasonal variations. Whether HRVC or other as yet undiscovered divergent HRV species and strains, are consistently more pathogenic or ‘asthmagenic’ is uncertain, and further studies are warranted.

Should an association between HRVC or certain HRV strains and more severe disease and asthma development exist, focused vaccine development targeting these strains could offer benefit for asthmatic patients of all ages. While antigenic diversity complicates passive and active prophylactic interventions (i.e. antibodies or vaccines), further characterization of individual serotypes (and their neutralizing antigens) associated with severe disease could provide valuable information for developing potential preventive strategies in the future. In addition, the availability of rapid detection techniques for HRV could be implemented in the clinical setting to allow appropriate diagnosis and treatment of HRV-associated bronchiolitis and asthma.