Human coronavirus NL63: a clinically important virus?

Respiratory tract infection is a leading cause of morbidity and mortality worldwide, especially among young children. Human coronaviruses (HCoVs) have only recently been shown to cause both lower and upper respiratory tract infections. To date, five coronaviruses (HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and HCoV HKU-1) that infect humans have been identified, four of which (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU-1) circulate continuously in the human population. Human coronavirus NL63 (HCoV-NL63) was first isolated from the aspirate from a 7-month-old baby in early 2004. Infection with HCoV-NL63 has since been shown to be a common worldwide occurrence and has been associated with many clinical symptoms and diagnoses, including severe lower respiratory tract infection, croup and bronchiolitis. HCoV-NL63 causes disease in children, the elderly and the immunocompromised, and has been detected in 1.0–9.3% of respiratory tract infections in children. In this article, the current knowledge of human coronavirus HCoV-NL63, with special reference to the clinical features, prevalence and seasonal incidence, and coinfection with other respiratory viruses, will be discussed.


Future Microbiology
Acute respiratory tract infections (ARTIs) are among the most common causes of disease in humans [1]. The majority of ARTIs are caused by viruses with rhinovirus, respiratory syncy tial virus, influenza virus, enterovirus, human metapneumovirus and parainfluenza virus con sidered the major pathogens [1][2][3]. Those most at risk of severe complications from these viral infections include young children, the elderly or persons with compromised cardiac, pulmonary or immune systems [2,4]. The high burden of disease caused by respiratory viruses in young children has led to the development of diag nostic tests and vaccines for the treatment of these infections [5].
Until recently, it was commonly accepted that the known human coronaviruses (HCoVs), with the exception of severe acute respiratory syn drome Cov (SARSCoV), mainly cause mild upper respiratory tract infections (URTIs) [6]. For this reason, the circulation of HCoVs was not monitored and no attempt to develop vac cines or drugs against these viruses was made [7]. CoVs are ssRNA viruses that infect humans and animals. In animals, CoVs cause a wide spectrum of diseases, including respiratory, enteric, hepatic and neurological diseases, with symptoms rang ing from mild to severe [8,9]. HCoVs causing URTIs were first isolated from patients in the 1960s [8], with HCoV229E and HCoVOC43 the best characterized. Then, following the outbreak of SARS in China in 2003, three additional human coronaviruses were identi fied -SARSCoV [10-12], HCoVNL63 [13,14] and HCoVHKU1 [15]. Of the five known HCoVs, four (HCoV229E, HCoVOC43, HCoVNL63 and HCoVHKU1) are circulating continuously in the human population.
This article aims to summarize the current knowledge of HCoVNL63 with reference to the clinical features, prevalence and seasonal incidence, and coinfection with other respira tory viruses. Finally, perspectives for future developments in the field are discussed.
In a study testing the serum samples of 139 children, HCoVNL63 seroconversion occurred before the age of 3.5 years, with 75% of children in the age group 2.5-3.5 years found to be sero positive [34]. In another report, the seropreva lence of HCoVNL63 in 6-12monthold chil dren was 28.6-40.0% [35]. Maternally acquired antibodies were present in newborns and usu ally decreased within the first 4-5 months of Respiratory tract infection is a leading cause of morbidity and mortality worldwide, especially among young children. Human coronaviruses (HCoVs) have only recently been shown to cause both lower and upper respiratory tract infections. To date, five coronaviruses (HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and HCoV HKU-1) that infect humans have been identified, four of which (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU-1) circulate continuously in the human population. Human coronavirus NL63 (HCoV-NL63) was first isolated from the aspirate from a 7-month-old baby in early 2004. Infection with HCoV-NL63 has since been shown to be a common worldwide occurrence and has been associated with many clinical symptoms and diagnoses, including severe lower respiratory tract infection, croup and bronchiolitis. HCoV-NL63 causes disease in children, the elderly and the immunocompromised, and has been detected in 1.0-9.3% of respiratory tract infections in children. In this article, the current knowledge of human coronavirus HCoV-NL63, with special reference to the clinical features, prevalence and seasonal incidence, and coinfection with other respiratory viruses, will be discussed.  [34,35]. Three studies have calculated the incidence of HCoVNL63 infections [5,19,36]. The design of these studies varied, however, and differences in the calculated incidences were reported. The comprehensive study by van der Hoek and colleagues [5] calculated the overall annual incidence in outpatients as seven per 1000 children per year, with the hospitaliza tion rate estimated at 22 per 100,000 children; in contrast, a hospitalization rate of 224 per 100,000 (children younger than 6 years old) as previously reported [19].
At present, the accuracy of HCoV detection is hampered by three difficulties. First, the suitability of the clinical samples examined: a recent study has demonstrated that there are differences between respiratory samples col lected by nose/throat swabs, and those col lected by nasopharyngeal aspiration, specifi cally regarding their usefulness in detecting and identifying respiratory pathogens [37]. The second problem is that diagnostic tests for HCoVs are not frequently used in the routine testing for respiratory viruses, which probably results in the percentage of HCoVs infections being greatly underestimated [38]. Lastly, over the years several molecular methods of variable sensitivity were used to determine the incidence of the virus [39][40][41]. Unless sensitive and specific nucleic acid amplification tests are used for the detection of CoVs (including those infections with low viral load) as part of a respiratory virus surveillance strategy, the CoVs will always be underdiagnosed [42].
Even though the majority of initial reports identified winter as the peak season for HCoVNL63 infections, other studies reported seasonal variations for HCoVNL63 infections, including spring and autumn for China [19], and summer for Taiwan [43]. Also, although a study from Thailand, with its tropical climate, showed yeartoyear variation in prevalence of HCoVNL63 infections, the virus was detected throughout the year [44]. A recent comprehen sive 2year populationbased study, using data from different countries, on children under 3 years of age with lower respiratory tract infec tion (LRTI) shows that HCoVNL63 infections peak in winter months. This study also shows large yeartoyear differences in the frequency of HCoVNL63 infections, with results indicating an interepidemic period of 2 years [5].

Respiratory clinical findings
Scientific and clinical evidence show that HCoVNL63 infects both the upper and lower respiratory tract [36], causing symptoms simi lar to those associated with HCoV229E and HCoVOC43. Commonly, patients diagnosed with HCoVNL63 infections of the upper respi ratory tract present with mild symptoms, such as fever, cough, sore throat and rhinitis [18]. It is Table 1. Incidence of HCoV-NL63 in clinical samples.

Country
Method used for detection Incidence (%; sample size) Ref.
future science group Human coronavirus NL63: a clinically important virus? Special Report important to remember though, that the reports of symptoms in young children, who represent the majority of cases, are based mainly on paren tal observations, which often do not include pos sible subjective signs and symptoms. In addi tion, most studies are case reports of patients who have been hospitalized for acute respiratory tract infections (and other milder symptoms are ignored in favor of treating the more severe ones) and studies are often limited by small sample sizes or short periods of assessment [36]. For these reasons the involvement of HCoVNL63 in many other diseases could be overlooked. Recent studies have reported an associa tion between HCoVNL63 infection and more severe LRTIs [21,30,45]. LRTIs are the leading cause of morbidity in children younger than 5 years of age worldwide [46,47]. A recent study by Dominguez and colleagues reports that even though HCoVNL63 and HCoVOC43 were equally prevalent during their 1year study period, HCoVNL63 was more often associated with more severe LRTIs and subsequent hospitalization [21]. One of the most worrying clinical diagnoses of HCoVNL63 infection is bronchiolitis, an inflam mation of the membranes lining the bronchioles [22,24,25,30,45,48,49], and although a study of chil dren hospitalized with fever and acute respiratory symptoms in China did not report an association of HCoVNL63 with bronchiolitis [19], it still believed to be one of the presenting symptoms.
Several research groups have linked HCoVNL63 infections to croup [5,17,19,28,30,43,[50][51][52]. Croup children present with pharangitis, sore throat and hoarseness of voice, and infected children are considered for hospitalization. The chance of developing croup is 6.6times higher in HCoV NL63infected children than in HCoVNL63 negative children [30]. Another study reports that HCoVNL63, when compared with other respiratory viruses, is the virus secondmost com monly associated with young children (median age 13 months) hospitalized with croup [17]. In fact, HCoVNL63 appears to be more frequently associated with croup than HCoV229E and HCoVOC43 [7]. Previously, the etiological agent of croup was generally assumed to be one of the more wellknown respiratory viruses, such as the parainfluenza viruses, but it is now clear that HCoVNL63 also plays a major role in this disease [9].
It is important to remember that since HCoVs (including HCoVNL63) are also frequently detected in asymptomatic individuals [44,53,54], the studies that lack of specimens from healthy indi viduals limit the inference of a disease association.
A recent study, however, reported that for all four circulating HCoVs combined, the detection fre quency in samples from patients with URTIs and LRTIs exceeds the proportion seen with samples taken from patients with no respiratory symptoms; this provides epidemiological evidence for the role of HCoVs in the etiology of respiratory disease [55]. Furthermore, since it has previously been shown that HCoV can be detected in clinical samples 14 days after illness [56], the possibility that some of the control patients in the earlier studies may have been shedding HCoV following an earlier symptomatic infection cannot be excluded.

Nonrespiratory clinical findings
To date, one group has reported an association between HCoVNL63 and Kawasaki disease, a form of early childhood systemic vasculitis that presents as prolonged fever, polymorphic exan them, oropharyngeal erythema and bilateral con juctivitis [57]. In view of this paper, the editorial by McIntosh [8] made a compelling argument for a possible association between respiratory HCoVs and Kawasaki disease, motivating further study using broader epidemiological and nonepidemio logical criteria. All subsequent reports, however, have conclusively demonstrated that no statisti cal significant link between HCoVNL63 and Kawasaki disease exists [8, [58][59][60][61][62][63][64].
HCoVNL63 infections have previously been associated with gastrointestinal find ings [21,25,29,[65][66][67]. However, this is not unique to HCoVNL63 infections, as coronaviruslike particles [21], as well as SARSCoV and HCoV HKU1 RNA [68,69] have been previously detected in patient diarrheic samples. These manifestations appear to be a direct consequence of viral invasion of the intestinal mucosa [7]. In one study, 4 out of 878 stool samples from children with acute gas troenteritis tested positive for HCoVNL63, and these samples are usually positive for other gastro enteritis viruses as well [70]. Another study using stool samples collected from 479 patients, reported the absence of HCoVNL63 [71]. However, this study had several shortcomings, including a short study period, the absence of a control group with out gastrointestinal disease, and specimen selec tion that might be biased towards individuals with more severe disease. Also, the majority of patients included were adults (older than 18 years of age); this is a problem since it has previously been shown that young children are more sus ceptible to HCoVNL63 infections. Therefore, the findings of this study cannot exclude the role of HCoVNL63 in gastrointestinal disease [71]. Thus far, data show that HCoVNL63 may, future science group Special Report Fielding at most, have a minor etiological role in acute gastro enteritis in children, but since other viruses are frequently associated with gastroenteritis in HCoVNL63 infected individuals, the exact role of HCoVNL63 is not clear.

Coinfections with other respiratory viruses
Coinfections of HCoVNL63 and other respira tory viruses, including other HCoVs, influenza A virus, respiratory syncytial virus, parainfluenza virus and human metapneumovirus (hMPV), are common [19,23,27,30,31,43,44,72,73]. Interestingly, coinfected patients are more likely to be hospi talized, indicating the severity of this kind of superinfection [74]. In a study from Germany, respiratory syncytial virus A and HCoVNL63 was the most common coinfection identified in children less than 3 years of age. This is probably due to the high incidence of respiratory syncytial virus A in winter and the overlap in the seasonal ity of the viruses [30]. Coinfection in hospital ized children with HCoVNL63 and bocavirus is reported [75]. The viral load of HCoVNL63 is lower in coinfected patients than in patients infected with HCoVNL63 only [5,30]. The clini cal significance of these coinfections are not clear, but various plausible explanations for the lower HCoVNL63 viral load have previously been discussed: HCoVNL63 causes the initial infec tion that weakens the immune system enough for a second viral infection to gain a foothold. By the time this second infection shows symptoms, the HCoVNL63 infection might have already been brought under control by the host immune system; HCoVNL63 and the other virus may be in competition for the same cellular receptor or target cell in the respiratory organs; the acti vation of the innate immune response triggered by the second respiratory virus may cause inhibi tion of HCoVNL63; or prolonged persistence of HCoVNL63 at low levels of expression [30,74]. Even though these reports may reflect biological complexity or interaction, it is important to keep in mind that virtually all the published studies comprise solely of cohorts of children hospitalized for ARTI and thus are extremely biased.

Future perspective
New data concerning HCoVNL63 and other HCoVs indicate that HCoVs may be more clini cally important in children and the immuno compromised than previously thought. Since vaccines are not currently available for these respiratory viruses, it is necessary to monitor epidemic patterns and investigate the spread of respiratory infections to efficiently identify, control and prevent epidemics. More compre hensive populationbased studies are required to determine the involvement of HCoVNL63 in other body systems. Also, the development of technologies to accurately identify HCoVNL63 infections will shed light on the true incidence of this virus in the human population.
Finally, a detailed manipulation of the HCoVNL63 genome to understand the role of the HCoVNL63 viral genes in pathogenesis and replication, and for the subsequent development of HCoVNL63 as a vaccine vector, is needed. This, however, is hampered by the poor growth of the virus in cell culture, as well as the lack of an appropriate animal model. The recent devel opment of the first fulllength infectious clone of HCoVNL63 allows for the systematic experi mental study -genes can be modified and/or deleted from the genome -of the functions of the various corresponding HCoVNL63 pro teins, which will lead to a better understanding of the role of the viral genes in infectivity and pathogenicity. This manipulation of the virus genome, in turn, provides a reverse genetics platform that can lead to the development of HCoVNL63based vector vaccines [76].

Conclusion
The detection of HCoVNL63 in samples col lected in 1981 [36] and 1988 [16] shows that the virus has been circulating and causing disease in the human population for a long time. However, the discovery of HCoVNL63 and other novel HCoVs does not necessarily represent a sudden increase in emerging infections by 'new' CoVs. Of the CoVs isolated from patients in the 1960s, at least four were shown to be serologically dis tinct [77][78][79]. Unfortunately, these clinical sam ples were lost before they could be characterized and it will never be known whether these 'old' HCoVs and the current 'new' HCoVs represent the same strains [80]. HCoVNL63 infections vary in frequency between years, but appear to peak during the winter months. HCoVNL63 causes LRTIs and URTIs in 1.0-9.3% of children, the elderly and the immunocompromised, with symptoms ranging from mild to severe. Although unlikely, the high prevalence of coinfections of HCoVNL63 and other respiratory viruses increases the chances of genetic recombination between these viruses in the host. In theory, these types of recombination events could enable highly pathogenic virus variants to arise [81]. Current data clearly show that HCoVNL63 is clinically more important that previously suspected.

Respiratory clinical findings
n HCoV-NL63 infects both the upper and lower respiratory tracts. n Infection with HCoV-NL63 is often associated with more severe lower respiratory tract infections and subsequent hospitalization. n Lower respiratory tract symptoms include croup and bronchiolitis.

Nonrespiratory clinical findings
n The majority of studies did not find an association between HCoV-NL63 infection and Kawasaki disease. n HCoV-NL63 has been detected in a small number of diarrhea stool samples.

Coinfections with other respiratory viruses
n Coinfections with other well known respiratory viruses are very common. n HCoV-NL63 viral load is higher in patients infected with HCoV-NL63 alone. n Patients with coinfections are more likely to be hospitalized.

Future perspective
n More comprehensive population-based studies are needed to determine the involvement of HCoV-NL63 in the infection of systems other than the respiratory tract.
n The development of the HCoV-NL63 infectious clone will greatly aid in understanding this virus. n An animal model is required for virus study and vaccine development.

Bibliography
Papers of special note have been highlighted as: n of interest nn of considerable interest with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.