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Human rhinoviruses
PAEDIATRIC RESPIRATORY REVIEWS (2003) 4, 91–98 doi: 10.1016/S1526–0542(03)00030-7
MINI-SYMPOSIUM: RESPIRATORY VIRUSES – PART I
Human rhinoviruses Carita Savolainen*, Soile Blomqvist and Tapani Hovi National Public Health Institute (KTL), Department of Microbiology, Enterovirus Laboratory, Mannerheimintie 166, FIN-00300 Helsinki, Finland KEYWORDS human rhinoviruses; upper respiratory tract infections; classification; laboratory diagnosis; epidemiology; molecular epidemiology
Summary Human rhinoviruses are the most important causative agents of upper respiratory infections and are also implicated in more severe clinical entities. Although often present, very little is known about human rhinoviruses. Molecular methods have been used in the classification of this large group of viruses into two separate clades. In addition, one known serotype was found to be a member of enterovirus group D. Laboratory diagnosis of human rhinovirus infection is based on reverse transcription polymerase chain reaction methods or the more tedious virus culture but a rapid ‘‘bedside’’ method is unavailable. Anti-rhinoviral therapy has been under extensive study over the past few decades but symptomatic treatment of the common cold is still the only useful approach in clinical use. More data on circulating human rhinovirus strains would facilitate both detection and treatment of these common pathogens. ß 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Rhinoviruses are known to be the most frequent causative agents of mild upper respiratory tract infections, or common colds.1 Their impact on health and disease is often ignored because they are considered to cause the selflimiting common cold alone and because specific diagnosis has been tedious and expensive. However, in addition to upper respiratory illnesses, rhinoviruses are also associated with more severe diseases such as acute otitis media in children2 and sinusitis in adults.3 Rhinoviruses can also infect the lower respiratory tract and therefore account for lower respiratory tract symptoms such as pneumonia,4 wheezing in children5 and exacerbations of asthma and chronic obstructive pulmonary disease (COPD) in adults.6 The changing picture of the overall disease burden associated with rhinovirus infections7 is partly based on the introduction, from the late 1980s onwards, of rapid and sensitive nucleic-acid-based [reverse transcription polymerase chain reaction (RT-PCR)] methods for rhinovirus detection. We still know nothing about potential differences in pathogenicity between different rhinovirus sero*
Correspondence to: C. Savolainen. Tel: þ358-9-4744-8884; Fax: þ358-9-4744-8355; E-mail: [email protected] 1526–0542/03/$ – see front matter
types. Systematic genetic characterisation of all rhinovirus serotypes, carried out recently,8 promises rapid identification of individual strains thus enabling molecular epidemiological studies.
GENERAL CHARACTERISTICS Human rhinoviruses (HRVs) constitute a genus in the large family of Picornaviridae together with e.g. entero-, hepato-, kobu- and parechoviruses that also cause infections in humans.9 Traditionally, HRVs are stated to differ from their closest relatives, the enteroviruses, by their inability to withstand acidic conditions.10 However, there is evidence that this may not be the case with all serotypes and strains of HRV (Blomqvist et al., unpublished data). HRVs have a relatively low optimum temperature for growth, 33 8C, considered to reflect an evolutionary adaptation to the environment of the nasopharyngeal region. However, at least some HRVs also multiply at higher temperatures.11 HRVs (Fig. 1) share morphological similarities with other picornaviruses.9 They are small particles with a diameter of 25–30 nm and possess a non-enveloped icosahedral capsid consisting of 60 copies of each of the four capsid proteins (VP1–4). The viral genome is a single-stranded, messengersense RNA of approximately 7.2 kb in size with a single ß 2003 Elsevier Science Ltd. All rights reserved.
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comprises 91 HRV serotypes exploiting the intracellular adhesion molecule 1 (ICAM-1, CD54), an immunoglobulin-like molecule , for binding to the cell surface. In the minor receptor group, there are 10 HRV serotypes using the low-density lipoprotein receptor (LDLR). HRV87 alone is using an unidentified sialoprotein as a receptor for attachment to cell.12 (2) The anti-viral drug sensitivity pattern can be used to divide HRV serotypes into two groups.13
Figure 1 Human rhinovirus 14, radially depth cued with antigenic sites highlighted, as solved by X-ray crystallography adopted from http://virology.net/Big_Virology/BVRNApicorna.html by JeanYves Sgro, 1994.
open-reading frame (Fig. 2). Typical of picornaviruses, the HRV genome begins with a short peptide (VPg) covalently coupled to the 50 -non-coding region (50 -NCR) which is followed by the capsid-coding region (P1). The P2 and P3 regions encode for the non-structural proteins that include two viral proteases (2A and 3C) and the RNA-dependent RNA polymerase. These regions are followed by a short 30 non-coding region and a poly-A tail. The replication of HRVs takes place in the cytoplasm of the host cell. After entering the cell, the genome is first translated and then copied to a ‘‘negative strand’’, an RNA molecule with a complementary nucleotide sequence. The latter is subsequently used as a template for synthesising a large number of copies of the viral genome, which are partly used as messenger RNAs for viral protein synthesis and partly incorporated as genomes into the progeny virus particles.
HRV SUB-GROUPS AND GENETIC CLUSTERS Traditionally, virus isolates have been classified by dividing them into distinct antigenic entities, usually designated serotypes. For HRVs, more than 100 separate serotypes have been officially characterised. HRVs can also be grouped by other phenotypic properties: (1) According to receptor usage, HRVs can be divided into three groups. The major receptor group
Currently, virus classification is primarily based on phylogenetic relationships, i.e. on assessment of similarities and differences of genomic nucleotide and/or deduced amino acid sequences (Fig. 3). For the closely related enteroviruses, nucleotide and amino acid sequences in the capsidcoding region have been shown to correlate with the established serotype identity, and those of the VP1 protein are suitable for genetic typing of previously uncharacterised isolates.14,15 Partial genomic sequences in the VP4/VP2 region of the prototype strains of 102 HRV serotypes were determined recently (Fig. 4).8 All but one serotype were found to cluster in the two previously known major clades or species, HRV A and HRV B.16 HRV 87 was the only exception and this was closer to the human enterovirus species D (HEV D) than any of the HRVs (Fig. 3)8. This finding prompted further research on HRV 87 and led to the conclusion that together with enterovirus 68, HRV 87 belongs to a single serotype within the species HEV D, which thus presents both enterovirus and rhinovirus features.17 Based on these observations and the no-longer valid segregation of enteroviruses and rhinoviruses as acid stabile and labile, respectively, the taxonomical grouping of picornaviruses is being revised. In future, enteroviruses and rhinoviruses are likely to be grouped together as a single genus with the tentative name Enterhinovirus (unofficial proposal discussed within the Picornavirus Study Group of the International Committee for the Taxonomy of Viruses).
EPIDEMIOLOGY OF RHINOVIRUS INFECTIONS HRV infections are transmitted by the respiratory–salivary route. Both airborne and contact-mediated transmission has been documented but their relative roles in everyday life are not known.18 It is also possible that differences exist
Figure 2 Picornavirus genome and gene organisation. Codes for individual proteins in the boxes. Numbers from 1000–7000 indicate approximate locations of corresponding nucleotides.
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Figure 3 A dendrogram showing genetic relationships of different picornavirus groups based on nucleotide sequences in the partial VP1 (369 nt). Note that this region is not homologous among all genera. The GenBank accession numbers of the sequences are: CVA16 NC_001612; EV-71 U22521; CV-B1 M16560; CV-B2 AF081485; CV-B5 X67706; E-1 AF081314; E-30 AF162711; CV-A21 D00538; EV-70 D00820; HRV-87 AY062281; PV-1 V01150; HRV-1B D00239; HRV-2 X02316; HRV-89 A10937; HRV-14 K02121; FMDV-A X00429; HHAV M14707; HPeV-1 L02971; Ljungan NC_003976; ERBV-1 X96871; AiV AB040749; PTV-1 AJ011380.
between different rhinoviruses in this regard. HRV infections are common all over the world and children in particular suffer from HRV infections frequently. In a recent cohort study on children followed from 2 months of age to 2 years, more than 90% of the children had experienced at least one HRV infection by 2 years of age.19 In a serological study, it was shown that the number of distinct serotype specificities of neutralising antibodies increases with followup time until about 35 years of age, and then plateaus reflecting the cumulative exposure to new serotypes with increasing age.20 In temperate climates, rhinovirus infections may occur throughout the year but the occurrence typically shows two peak seasons, April–May and September.21
Molecular epidemiology Like other RNA viruses, independent HRV strains are likely to show wide genetic variation due to an error-prone RNA-dependent RNA polymerase catalysing the synthesis of viral RNA.22 Furthermore, during synthesis of the negative strands, strand switching leading to recombination occurs frequently in RNA viruses, extending the range of genetic variants.23 Molecular methods, especially partial genome sequencing and comparison of genetic information, have been helpful tools in monitoring the progress of the global Poliomyelitis Eradication Initiative.24,25 Phylogenetic analysis
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Figure 4 Dendrograms describing intragroupic evolutionary relationships among human rhinovirus A (left, only part of the prototype strains) and B (right) in the VP4/VP2 region (420 nt).
of viral genome sequences has also elucidated the epidemiology of many other important viruses. Molecular epidemiology is giving answers to two main classes of questions: where does the virus come from and how is it spread in a human population? For HRVs, molecular epidemiological research has only just begun and many questions remain open. Molecular epidemiological methods have been utilised in genetic characterisation of HRV clinical isolates collected from a cohort of young children followed for acute respiratory infections.26 The genetic diversity between different strains was shown to be striking, suggesting simultaneous circulation of several different serotypes in a single suburb during one epidemic season. Also, strains of a given genetic cluster were sometimes isolated during consecutive epidemic seasons, suggesting either persistence or re-appearance of the epidemic strains.
NATURAL COURSE OF RHINOVIRUS INFECTION In the respiratory tract, HRVs mainly replicate in the ciliary epithelial cells of the nasal mucosa and, to a lesser extent, in the oral cavity and throat.27 It has been suggested that the epithelial cells in the lower respiratory tract are less susceptible to HRV infection than those in the nose.28 However, HRV-infected lower respiratory tract cells have been
found in bronchoalveolar lavage samples.29 The commonly held lay view that exposure to cold and subjective ‘‘chilling’’ would have an aetiological role in the common cold could not be proven in experimental infections.30 In contrast, there is evidence that significant psychosocial stress is likely to increase the occurrence and severity of the HRV infection’s clinical symptoms.31 The amount of HRV needed to infect a human being (human infecting dose, HID) is surprisingly small as measured in plaque-forming units (PFU). Figures as low as 0.032 PFUs have been reported for HID50.32 In the common cold, the usual consequence of HRV infection, typical symptoms such as coryza and cough can persist for 14 days but the virus may still be recovered in the excreta 3 weeks after the onset of infection. The virus is not considered to enter the blood circulation regularly and viraemia has been documented only exceptionally in immunosuppressed individuals.33 An immune response with circulating neutralising antibodies can be shown in most documented infections34 but other host factors, such as type I interferon, may have a major role in limiting the primary infection.2 Apart from serum, neutralising antibodies can be demonstrated in the mucosal excreta as well.35 As in other viral infections, antibody concentrations in blood and in the excreta decay with time and, in the case of HRV, lifelong immunity to a given serotype is not necessarily acquired.36
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CLINICAL ENTITIES ASSOCIATED WITH HRV INFECTIONS HRVs are responsible for most episodes of the common cold in all age groups. The common cold caused by rhinoviruses cannot be distinguished clinically from that caused by other viruses. The general features and complications of the common cold have been reviewed recently.2 After 1–4 days incubation, the symptoms usually peak 3–7 days after onset of infection. Sore throat, coryza and cough are typical symptoms. Fever, headache and malaise may also occur. The recovery time varies greatly and complete healing may take 2 weeks. Acute otitis media (AOM) is the most common complication of rhinovirus infection in children. More than 40% of documented nasopharyngeal rhinovirus infections were associated with AOM in small children in a recent prospective study.19,21 The true figure may be even greater as subsequent studies have demonstrated rhinovirus RNA in middle ear fluid in some cases where the concomitantly collected nasopharyngeal specimen tested negative (NoksoKoivisto et al., unpublished results). While an overt AOM evidently also involves pathogenic bacteria, the role of respiratory viral infection in the overall pathogenetic process of the disease is being given more and more attention. Placebo-controlled double-blind studies have revealed that no added benefit is derived from the common practice of prescribing antibiotic treatment in AOM.37 Together with the closely related enteroviruses, rhinoviruses are associated with more than half of all episodes of AOM in small children (Nokso-Koivisto et al., unpublished results). Apart from AOM, rhinoviruses appear to contribute to the aetiology of sinusitis, bronchitis, bronchiolitis and even pneumonia in children. Furthermore, pneumonia and other lower respiratory tract events in elderly people are often preceeded and perhaps precipitated by rhinovirus infections.38 An increasing amount of evidence is linking rhinovirus infections to exacerbations of asthma and COPD.39 Replication of rhinovirus in the bronchial epithelium is considered to result in the release of cytokines and kinins, which contribute to the bronchoconstrictory reactions typical of the attacks.2
LABORATORY DIAGNOSIS Virus isolation The detection of rhinoviruses has been based traditionally on virus isolation in cell culture followed by acid-sensitivity testing to differentiate rhinoviruses from enteroviruses.10 Efficient growth of rhinoviruses occurs mainly in human tissues. A variety of such tissues are available and favourable results can be achieved at least in human embryonic lung fibroblast cell lines WI-38 and MRC-5, and the HeLa-I line of HeLa cells overexpressing ICAM-1.40 The sensitivities of different cell lines vary considerably with regard to distinct rhinovirus serotypes, so the maximal isolation assay
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requires simultaneous use of a combination of cell lines. The other special growth requirements of the fastidious rhinoviruses include a lowered incubation temperature (þ33–34 8C), a relatively low pH of the culture medium and slow rotation of culture tubes.10 The isolation process is tedious and laborious and is not suitable for rapid diagnosis of rhinovirus infections.
RT-PCR methods RT-PCR was introduced to rhinovirus detection in 1988.41 Subsequently, about 20 different rhinoviral RT-PCR applications have been described (reviewed by Santti et al.42 and Steininger et al.43). These have been used extensively in several laboratories and have proven to be sensitive and convenient methods for both diagnostic and epidemiological studies of rhinoviral infections. Most of the rhinovirus RT-PCR assays take advantage of short highly conserved stretches in the 50 non-coding region as the binding sites for oligonucleotide primers. These assays are shown to be sensitive but not specific, with the same primer sites also being conserved in enteroviruses. The differentiation of rhinoviruses from enteroviruses needs additional steps, for example, restriction fragment length polymorphism,44 hybridisation with rhinovirus-specific probes,45–48 sequencing of PCR amplicons49 or (semi)nested-PCR with rhinovirus-specific primers.43,50 The differentiation of rhinoviruses and enteroviruses by the size of the RT-PCR amplicon is also possible, when the RT-PCR is performed from 50 NCR through VP2.51 However, the sensitivity of this application is probably lowered by the mismatches in the VP2 primer binding site but yet it is brilliant in, for example, replacing the acid-resistance test of clinical isolates.52 Although the greater sensitivity of RTPCR in the detection of rhinovirus infections compared with virus isolation is well established (e.g. Blomqvist et al.47, Hyypia¨ et al.53), there are still major problems in the optimisation of the diagnostic rhinovirus RT-PCR mainly due to the limited sequence information of rhinoviruses. Only five of >100 rhinovirus serotypes have been completely sequenced, and the primers and probes used today are based on these prototype viruses isolated decades ago and their homology to enteroviruses. Mismatches in primer binding are likely to occur and this is likely to affect both sensitivity and specificity of the RT-PCR. More sequence data of circulating viruses would greatly facilitate the primer design of specific diagnostic RT-PCR assays for rhinoviruses. Sequencing of the 50 NCR of 39 rhinoviruses, nine prototype strains and 30 Dutch isolates,50 confirmed the existence of six well-conserved regions in the 50 NCR, which have been previously adopted in other studies, suitable for primer design. However, the clinical isolates analysed in that study were collected during two limited seasons in a given region, and all the isolates were first passaged in cell culture, thus they probably represented only a small fraction of the large genus of rhinoviruses.
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PREVENTION AND TREATMENT Early attempts to develop vaccines against rhinovirus were promising in principle but only provided some protection against the homotypic strains. Further observations on the decay of immunity and recognition of the vast number of rhinovirus serotypes led to the conclusion that a vaccine against rhinovirus infection may be unrealisable.54 However, considerable efforts have been made to develop antiviral therapy against rhinovirus infection.2 A wide variety of compounds have shown promising activity in cell culture but none of them has made the way to common practice so far. These include preparations of type I interferon, a natural anti-viral cytokine, which was found to be active in in-vivo prophylaxis but failed to show any efficacy when administered after onset of symptoms.55,56 A problem in the development of anti-viral chemotherapy to rhinovirus infections is the lack of suitable animal models, which means that one has to jump from the cell-culture observations directly to human trials as regards the efficacy of the planned treatment. Three separate lines of anti-viral research are especially promising at the moment. The longest history can be presented for compounds that slip into the so-called lipid pocket, a hydrophobic molecular cave under the receptor canyon in the viral capsid (Fig. 1) and block the onset of infection by stabilising the capsid structure.57 Several of these compounds are also active against the related enteroviruses. The chemical structure of compounds acting in this way is surprisingly variable but they all share the property of having the capacity to form stabilising physicochemical bonds with the delineating amino acid residues. This will prevent conformational changes of the capsid structure that would be needed for uncoating and release of the genomic RNA at the onset of infection. Some of the compounds, when bound to the pocket, may also interfere with virus–receptor interaction. A frustrating series of clinical trials with a number of different promising compounds failed because of pharmacokinetic problems; in particular, an effective concentration of the active compound in the respiratory excreta could not be obtained by any tested mode of administration.57 The most recent candidate, pleconaril, was an exception as satisfactory concentrations were obtained and controlled trials revealed promising clinical efficacy against picornaviral respiratory infections. While regulatory approval was not granted for the oral formulation of the drug due to the induction of CYP 3A activity and associated concern with potential drug interactions (M. McKinlay, personal communication), the clinical results show that it is possible to reduce the duration and severity of rhinovirus infections with a targeted anti-viral agent. An intranasal formulation of the drug is currently under investigation. In the long term, interference with specific virus–hostcell receptor interactions is another potential intervention
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in anti-viral chemotherapy. Blocking of the receptors, none of which exist primarily for the viruses but for some physiological function, might in theory result in harmful effects, especially if the desired anti-viral therapy were administered systemically. The development of recombinant DNA technology and protein tailoring raises the possibility of interfering with virus–receptor interactions the other way around. Most rhinoviruses exploit intercellular adherence molecule 1 (ICAM-1) as the receptor molecule for binding to the host cell.58 A truncated derivative of this molecule, given the name tremacamra, was shown to have prophylactic activity in an experimental setting.59 Further studies are needed to confirm the clinical value, if any, of this approach of anti-rhinovirus therapy. The third line of anti-viral research is following the success in the development of anti-HIV chemotherapy: development of inhibitors of the viral proteases. Most viruses, including the rhinoviruses, encode for one or more proteases that are needed in the maturation of viral proteins. Inhibitors of HIV proteases are an important part of today’s successful multi-drug treatment regimen against HIV infections and AIDS60 and the success has facilitated testing the same concept for other virus infections as well.61 Drugs designed according to the X-ray crystallographic analysis of the HRV 3C-protease molecules have given promising results in the first clinical trials. Again, further studies and more experience in controlled trials are needed before attempts can be made to use these drugs in everyday practice. One has to remember that availability of safe and effective anti-rhinovirus drugs would not solve the everyday problems in clinical practice as almost half of all cases of the common cold are caused by other groups of viruses. Therefore, development of rapid diagnostic tests would also be necessary for optimal use of the putative anti-virals. In practice, symptomatic treatment of the common cold is still a useful approach, although there are not many treatment protocols for which the efficacy and tolerability have been documented in placebo-controlled trials (reviewed by Pitka¨ ranta and Hayden2).
FUTURE RESEARCH PROSPECTS HRV infections have been considered only a minor nuisance compared with many other viral diseases. However, it is well documented that they can be triggers of or associated with more serious infections, such as COPD and asthma. The development of anti-rhinoviral drug treatment is therefore very important and still possible. Molecular epidemiological research is producing new information on the relatedness of circulating HRV strains. These data are essential in developing tools for rapid diagnosis for HRV infections. With a reliable ‘‘bedside’’ test, correctly directed treatment of HRV-related diseases might become possible in future. Furthermore, this would decrease use of unnecessary antibiotic treatment.
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PRACTICE POINTS With specific anti-rhinoviral drugs unavailable, treatment of HRV infections is symptomatic. Intranasal or orally administered decongestants are used to relieve nasal stuffiness.62 Non-steroidal anti-inflammatory drugs reduce soreness of the throat and fever.63 The effect of other medications is poor or inconclusive. Prevention of HRV infections by hygienic methods, e.g. increased hand washing, could be one means. However, HRV infections are also transmitted aerogenically and therefore practically unavoidable to a large extent.
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transcription-polymerase chain reaction. J Clin Microbiol 1993; 31: 2544–2546. Monto AS, Bryan ER, Ohmit S. Rhinovirus infections in Tecumseh, Michigan: Frequency of illness and number of serotypes. J Infect Dis 1987; 156: 43–49. Hyypia¨ T, Puhakka T, Ruuskanen O, Ma¨ kela¨ M, Arola A, Arstila P. Molecular diagnosis of human rhinovirus infections: comparison with virus isolation. J Clin Microbiol 1998; 36: 2081–2083. Hayden FG, Albrecht JK, Kaiser DL, Gwaltney JM Jr. Prevention of natural colds by contact prophylaxis with intranasal alpha 2interferon. N Engl J Med 1986; 314: 71–75. Hayden FG, Kaiser DL, Albrecht JK. Intranasal recombinant alfa-2B interferon treatment of naturally occurring common colds. Antimicrob Agents Chemother 1988; 32: 224–230. Arruda E, Hayden FG. Clinical studies of antiviral agents for picornaviral infections. In: Jeffries DJ, DeClercq E (Eds) Antiviral Chemotherapy. Chichester: John Wiley 1995; pp 321–355. Colonno RJ, Callahan PL, Long WJ. Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. J Virol 1986; 57: 7–12. Turner RB, Wecker MT, Pohl G et al. Efficacy of soluble ICAM-1 (sICAM) for prevention of rhinovirus infection and illness. Abstr Proc 37th Intersc Conf Antimicr Ag Chemother (ICAAC), Toronto, Sept 28–Oct 1, 1997. Abstr H-85. Hammer SM, Squires KE, Hughes MD et al. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic meter or less. N Engl J Med 1997; 337: 725–733. Webber SE, Tikhe J, Worland ST et al. Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease. J Med Chem 1996; 39: 5072–5082. Sperber SJ, Sorrentino JV, Riker DK, Hayden FG. Evaluation of an alpha agonist alone in combination with a nonsteroidal antiinflammatory agent in the treatment of experimental rhinovirus colds. Bull NY Acad Med 1989; 65: 146–160. Sperber SJ, Hendley JO, Hayden FG et al. Effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial. Ann Intern Med 1992; 117: 37–42.
MINI-SYMPOSIUM: RESPIRATORY VIRUSES – PART I
Human rhinoviruses Carita Savolainen*, Soile Blomqvist and Tapani Hovi National Public Health Institute (KTL), Department of Microbiology, Enterovirus Laboratory, Mannerheimintie 166, FIN-00300 Helsinki, Finland KEYWORDS human rhinoviruses; upper respiratory tract infections; classification; laboratory diagnosis; epidemiology; molecular epidemiology
Summary Human rhinoviruses are the most important causative agents of upper respiratory infections and are also implicated in more severe clinical entities. Although often present, very little is known about human rhinoviruses. Molecular methods have been used in the classification of this large group of viruses into two separate clades. In addition, one known serotype was found to be a member of enterovirus group D. Laboratory diagnosis of human rhinovirus infection is based on reverse transcription polymerase chain reaction methods or the more tedious virus culture but a rapid ‘‘bedside’’ method is unavailable. Anti-rhinoviral therapy has been under extensive study over the past few decades but symptomatic treatment of the common cold is still the only useful approach in clinical use. More data on circulating human rhinovirus strains would facilitate both detection and treatment of these common pathogens. ß 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Rhinoviruses are known to be the most frequent causative agents of mild upper respiratory tract infections, or common colds.1 Their impact on health and disease is often ignored because they are considered to cause the selflimiting common cold alone and because specific diagnosis has been tedious and expensive. However, in addition to upper respiratory illnesses, rhinoviruses are also associated with more severe diseases such as acute otitis media in children2 and sinusitis in adults.3 Rhinoviruses can also infect the lower respiratory tract and therefore account for lower respiratory tract symptoms such as pneumonia,4 wheezing in children5 and exacerbations of asthma and chronic obstructive pulmonary disease (COPD) in adults.6 The changing picture of the overall disease burden associated with rhinovirus infections7 is partly based on the introduction, from the late 1980s onwards, of rapid and sensitive nucleic-acid-based [reverse transcription polymerase chain reaction (RT-PCR)] methods for rhinovirus detection. We still know nothing about potential differences in pathogenicity between different rhinovirus sero*
Correspondence to: C. Savolainen. Tel: þ358-9-4744-8884; Fax: þ358-9-4744-8355; E-mail: [email protected] 1526–0542/03/$ – see front matter
types. Systematic genetic characterisation of all rhinovirus serotypes, carried out recently,8 promises rapid identification of individual strains thus enabling molecular epidemiological studies.
GENERAL CHARACTERISTICS Human rhinoviruses (HRVs) constitute a genus in the large family of Picornaviridae together with e.g. entero-, hepato-, kobu- and parechoviruses that also cause infections in humans.9 Traditionally, HRVs are stated to differ from their closest relatives, the enteroviruses, by their inability to withstand acidic conditions.10 However, there is evidence that this may not be the case with all serotypes and strains of HRV (Blomqvist et al., unpublished data). HRVs have a relatively low optimum temperature for growth, 33 8C, considered to reflect an evolutionary adaptation to the environment of the nasopharyngeal region. However, at least some HRVs also multiply at higher temperatures.11 HRVs (Fig. 1) share morphological similarities with other picornaviruses.9 They are small particles with a diameter of 25–30 nm and possess a non-enveloped icosahedral capsid consisting of 60 copies of each of the four capsid proteins (VP1–4). The viral genome is a single-stranded, messengersense RNA of approximately 7.2 kb in size with a single ß 2003 Elsevier Science Ltd. All rights reserved.
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comprises 91 HRV serotypes exploiting the intracellular adhesion molecule 1 (ICAM-1, CD54), an immunoglobulin-like molecule , for binding to the cell surface. In the minor receptor group, there are 10 HRV serotypes using the low-density lipoprotein receptor (LDLR). HRV87 alone is using an unidentified sialoprotein as a receptor for attachment to cell.12 (2) The anti-viral drug sensitivity pattern can be used to divide HRV serotypes into two groups.13
Figure 1 Human rhinovirus 14, radially depth cued with antigenic sites highlighted, as solved by X-ray crystallography adopted from http://virology.net/Big_Virology/BVRNApicorna.html by JeanYves Sgro, 1994.
open-reading frame (Fig. 2). Typical of picornaviruses, the HRV genome begins with a short peptide (VPg) covalently coupled to the 50 -non-coding region (50 -NCR) which is followed by the capsid-coding region (P1). The P2 and P3 regions encode for the non-structural proteins that include two viral proteases (2A and 3C) and the RNA-dependent RNA polymerase. These regions are followed by a short 30 non-coding region and a poly-A tail. The replication of HRVs takes place in the cytoplasm of the host cell. After entering the cell, the genome is first translated and then copied to a ‘‘negative strand’’, an RNA molecule with a complementary nucleotide sequence. The latter is subsequently used as a template for synthesising a large number of copies of the viral genome, which are partly used as messenger RNAs for viral protein synthesis and partly incorporated as genomes into the progeny virus particles.
HRV SUB-GROUPS AND GENETIC CLUSTERS Traditionally, virus isolates have been classified by dividing them into distinct antigenic entities, usually designated serotypes. For HRVs, more than 100 separate serotypes have been officially characterised. HRVs can also be grouped by other phenotypic properties: (1) According to receptor usage, HRVs can be divided into three groups. The major receptor group
Currently, virus classification is primarily based on phylogenetic relationships, i.e. on assessment of similarities and differences of genomic nucleotide and/or deduced amino acid sequences (Fig. 3). For the closely related enteroviruses, nucleotide and amino acid sequences in the capsidcoding region have been shown to correlate with the established serotype identity, and those of the VP1 protein are suitable for genetic typing of previously uncharacterised isolates.14,15 Partial genomic sequences in the VP4/VP2 region of the prototype strains of 102 HRV serotypes were determined recently (Fig. 4).8 All but one serotype were found to cluster in the two previously known major clades or species, HRV A and HRV B.16 HRV 87 was the only exception and this was closer to the human enterovirus species D (HEV D) than any of the HRVs (Fig. 3)8. This finding prompted further research on HRV 87 and led to the conclusion that together with enterovirus 68, HRV 87 belongs to a single serotype within the species HEV D, which thus presents both enterovirus and rhinovirus features.17 Based on these observations and the no-longer valid segregation of enteroviruses and rhinoviruses as acid stabile and labile, respectively, the taxonomical grouping of picornaviruses is being revised. In future, enteroviruses and rhinoviruses are likely to be grouped together as a single genus with the tentative name Enterhinovirus (unofficial proposal discussed within the Picornavirus Study Group of the International Committee for the Taxonomy of Viruses).
EPIDEMIOLOGY OF RHINOVIRUS INFECTIONS HRV infections are transmitted by the respiratory–salivary route. Both airborne and contact-mediated transmission has been documented but their relative roles in everyday life are not known.18 It is also possible that differences exist
Figure 2 Picornavirus genome and gene organisation. Codes for individual proteins in the boxes. Numbers from 1000–7000 indicate approximate locations of corresponding nucleotides.
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Figure 3 A dendrogram showing genetic relationships of different picornavirus groups based on nucleotide sequences in the partial VP1 (369 nt). Note that this region is not homologous among all genera. The GenBank accession numbers of the sequences are: CVA16 NC_001612; EV-71 U22521; CV-B1 M16560; CV-B2 AF081485; CV-B5 X67706; E-1 AF081314; E-30 AF162711; CV-A21 D00538; EV-70 D00820; HRV-87 AY062281; PV-1 V01150; HRV-1B D00239; HRV-2 X02316; HRV-89 A10937; HRV-14 K02121; FMDV-A X00429; HHAV M14707; HPeV-1 L02971; Ljungan NC_003976; ERBV-1 X96871; AiV AB040749; PTV-1 AJ011380.
between different rhinoviruses in this regard. HRV infections are common all over the world and children in particular suffer from HRV infections frequently. In a recent cohort study on children followed from 2 months of age to 2 years, more than 90% of the children had experienced at least one HRV infection by 2 years of age.19 In a serological study, it was shown that the number of distinct serotype specificities of neutralising antibodies increases with followup time until about 35 years of age, and then plateaus reflecting the cumulative exposure to new serotypes with increasing age.20 In temperate climates, rhinovirus infections may occur throughout the year but the occurrence typically shows two peak seasons, April–May and September.21
Molecular epidemiology Like other RNA viruses, independent HRV strains are likely to show wide genetic variation due to an error-prone RNA-dependent RNA polymerase catalysing the synthesis of viral RNA.22 Furthermore, during synthesis of the negative strands, strand switching leading to recombination occurs frequently in RNA viruses, extending the range of genetic variants.23 Molecular methods, especially partial genome sequencing and comparison of genetic information, have been helpful tools in monitoring the progress of the global Poliomyelitis Eradication Initiative.24,25 Phylogenetic analysis
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Figure 4 Dendrograms describing intragroupic evolutionary relationships among human rhinovirus A (left, only part of the prototype strains) and B (right) in the VP4/VP2 region (420 nt).
of viral genome sequences has also elucidated the epidemiology of many other important viruses. Molecular epidemiology is giving answers to two main classes of questions: where does the virus come from and how is it spread in a human population? For HRVs, molecular epidemiological research has only just begun and many questions remain open. Molecular epidemiological methods have been utilised in genetic characterisation of HRV clinical isolates collected from a cohort of young children followed for acute respiratory infections.26 The genetic diversity between different strains was shown to be striking, suggesting simultaneous circulation of several different serotypes in a single suburb during one epidemic season. Also, strains of a given genetic cluster were sometimes isolated during consecutive epidemic seasons, suggesting either persistence or re-appearance of the epidemic strains.
NATURAL COURSE OF RHINOVIRUS INFECTION In the respiratory tract, HRVs mainly replicate in the ciliary epithelial cells of the nasal mucosa and, to a lesser extent, in the oral cavity and throat.27 It has been suggested that the epithelial cells in the lower respiratory tract are less susceptible to HRV infection than those in the nose.28 However, HRV-infected lower respiratory tract cells have been
found in bronchoalveolar lavage samples.29 The commonly held lay view that exposure to cold and subjective ‘‘chilling’’ would have an aetiological role in the common cold could not be proven in experimental infections.30 In contrast, there is evidence that significant psychosocial stress is likely to increase the occurrence and severity of the HRV infection’s clinical symptoms.31 The amount of HRV needed to infect a human being (human infecting dose, HID) is surprisingly small as measured in plaque-forming units (PFU). Figures as low as 0.032 PFUs have been reported for HID50.32 In the common cold, the usual consequence of HRV infection, typical symptoms such as coryza and cough can persist for 14 days but the virus may still be recovered in the excreta 3 weeks after the onset of infection. The virus is not considered to enter the blood circulation regularly and viraemia has been documented only exceptionally in immunosuppressed individuals.33 An immune response with circulating neutralising antibodies can be shown in most documented infections34 but other host factors, such as type I interferon, may have a major role in limiting the primary infection.2 Apart from serum, neutralising antibodies can be demonstrated in the mucosal excreta as well.35 As in other viral infections, antibody concentrations in blood and in the excreta decay with time and, in the case of HRV, lifelong immunity to a given serotype is not necessarily acquired.36
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CLINICAL ENTITIES ASSOCIATED WITH HRV INFECTIONS HRVs are responsible for most episodes of the common cold in all age groups. The common cold caused by rhinoviruses cannot be distinguished clinically from that caused by other viruses. The general features and complications of the common cold have been reviewed recently.2 After 1–4 days incubation, the symptoms usually peak 3–7 days after onset of infection. Sore throat, coryza and cough are typical symptoms. Fever, headache and malaise may also occur. The recovery time varies greatly and complete healing may take 2 weeks. Acute otitis media (AOM) is the most common complication of rhinovirus infection in children. More than 40% of documented nasopharyngeal rhinovirus infections were associated with AOM in small children in a recent prospective study.19,21 The true figure may be even greater as subsequent studies have demonstrated rhinovirus RNA in middle ear fluid in some cases where the concomitantly collected nasopharyngeal specimen tested negative (NoksoKoivisto et al., unpublished results). While an overt AOM evidently also involves pathogenic bacteria, the role of respiratory viral infection in the overall pathogenetic process of the disease is being given more and more attention. Placebo-controlled double-blind studies have revealed that no added benefit is derived from the common practice of prescribing antibiotic treatment in AOM.37 Together with the closely related enteroviruses, rhinoviruses are associated with more than half of all episodes of AOM in small children (Nokso-Koivisto et al., unpublished results). Apart from AOM, rhinoviruses appear to contribute to the aetiology of sinusitis, bronchitis, bronchiolitis and even pneumonia in children. Furthermore, pneumonia and other lower respiratory tract events in elderly people are often preceeded and perhaps precipitated by rhinovirus infections.38 An increasing amount of evidence is linking rhinovirus infections to exacerbations of asthma and COPD.39 Replication of rhinovirus in the bronchial epithelium is considered to result in the release of cytokines and kinins, which contribute to the bronchoconstrictory reactions typical of the attacks.2
LABORATORY DIAGNOSIS Virus isolation The detection of rhinoviruses has been based traditionally on virus isolation in cell culture followed by acid-sensitivity testing to differentiate rhinoviruses from enteroviruses.10 Efficient growth of rhinoviruses occurs mainly in human tissues. A variety of such tissues are available and favourable results can be achieved at least in human embryonic lung fibroblast cell lines WI-38 and MRC-5, and the HeLa-I line of HeLa cells overexpressing ICAM-1.40 The sensitivities of different cell lines vary considerably with regard to distinct rhinovirus serotypes, so the maximal isolation assay
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requires simultaneous use of a combination of cell lines. The other special growth requirements of the fastidious rhinoviruses include a lowered incubation temperature (þ33–34 8C), a relatively low pH of the culture medium and slow rotation of culture tubes.10 The isolation process is tedious and laborious and is not suitable for rapid diagnosis of rhinovirus infections.
RT-PCR methods RT-PCR was introduced to rhinovirus detection in 1988.41 Subsequently, about 20 different rhinoviral RT-PCR applications have been described (reviewed by Santti et al.42 and Steininger et al.43). These have been used extensively in several laboratories and have proven to be sensitive and convenient methods for both diagnostic and epidemiological studies of rhinoviral infections. Most of the rhinovirus RT-PCR assays take advantage of short highly conserved stretches in the 50 non-coding region as the binding sites for oligonucleotide primers. These assays are shown to be sensitive but not specific, with the same primer sites also being conserved in enteroviruses. The differentiation of rhinoviruses from enteroviruses needs additional steps, for example, restriction fragment length polymorphism,44 hybridisation with rhinovirus-specific probes,45–48 sequencing of PCR amplicons49 or (semi)nested-PCR with rhinovirus-specific primers.43,50 The differentiation of rhinoviruses and enteroviruses by the size of the RT-PCR amplicon is also possible, when the RT-PCR is performed from 50 NCR through VP2.51 However, the sensitivity of this application is probably lowered by the mismatches in the VP2 primer binding site but yet it is brilliant in, for example, replacing the acid-resistance test of clinical isolates.52 Although the greater sensitivity of RTPCR in the detection of rhinovirus infections compared with virus isolation is well established (e.g. Blomqvist et al.47, Hyypia¨ et al.53), there are still major problems in the optimisation of the diagnostic rhinovirus RT-PCR mainly due to the limited sequence information of rhinoviruses. Only five of >100 rhinovirus serotypes have been completely sequenced, and the primers and probes used today are based on these prototype viruses isolated decades ago and their homology to enteroviruses. Mismatches in primer binding are likely to occur and this is likely to affect both sensitivity and specificity of the RT-PCR. More sequence data of circulating viruses would greatly facilitate the primer design of specific diagnostic RT-PCR assays for rhinoviruses. Sequencing of the 50 NCR of 39 rhinoviruses, nine prototype strains and 30 Dutch isolates,50 confirmed the existence of six well-conserved regions in the 50 NCR, which have been previously adopted in other studies, suitable for primer design. However, the clinical isolates analysed in that study were collected during two limited seasons in a given region, and all the isolates were first passaged in cell culture, thus they probably represented only a small fraction of the large genus of rhinoviruses.
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PREVENTION AND TREATMENT Early attempts to develop vaccines against rhinovirus were promising in principle but only provided some protection against the homotypic strains. Further observations on the decay of immunity and recognition of the vast number of rhinovirus serotypes led to the conclusion that a vaccine against rhinovirus infection may be unrealisable.54 However, considerable efforts have been made to develop antiviral therapy against rhinovirus infection.2 A wide variety of compounds have shown promising activity in cell culture but none of them has made the way to common practice so far. These include preparations of type I interferon, a natural anti-viral cytokine, which was found to be active in in-vivo prophylaxis but failed to show any efficacy when administered after onset of symptoms.55,56 A problem in the development of anti-viral chemotherapy to rhinovirus infections is the lack of suitable animal models, which means that one has to jump from the cell-culture observations directly to human trials as regards the efficacy of the planned treatment. Three separate lines of anti-viral research are especially promising at the moment. The longest history can be presented for compounds that slip into the so-called lipid pocket, a hydrophobic molecular cave under the receptor canyon in the viral capsid (Fig. 1) and block the onset of infection by stabilising the capsid structure.57 Several of these compounds are also active against the related enteroviruses. The chemical structure of compounds acting in this way is surprisingly variable but they all share the property of having the capacity to form stabilising physicochemical bonds with the delineating amino acid residues. This will prevent conformational changes of the capsid structure that would be needed for uncoating and release of the genomic RNA at the onset of infection. Some of the compounds, when bound to the pocket, may also interfere with virus–receptor interaction. A frustrating series of clinical trials with a number of different promising compounds failed because of pharmacokinetic problems; in particular, an effective concentration of the active compound in the respiratory excreta could not be obtained by any tested mode of administration.57 The most recent candidate, pleconaril, was an exception as satisfactory concentrations were obtained and controlled trials revealed promising clinical efficacy against picornaviral respiratory infections. While regulatory approval was not granted for the oral formulation of the drug due to the induction of CYP 3A activity and associated concern with potential drug interactions (M. McKinlay, personal communication), the clinical results show that it is possible to reduce the duration and severity of rhinovirus infections with a targeted anti-viral agent. An intranasal formulation of the drug is currently under investigation. In the long term, interference with specific virus–hostcell receptor interactions is another potential intervention
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in anti-viral chemotherapy. Blocking of the receptors, none of which exist primarily for the viruses but for some physiological function, might in theory result in harmful effects, especially if the desired anti-viral therapy were administered systemically. The development of recombinant DNA technology and protein tailoring raises the possibility of interfering with virus–receptor interactions the other way around. Most rhinoviruses exploit intercellular adherence molecule 1 (ICAM-1) as the receptor molecule for binding to the host cell.58 A truncated derivative of this molecule, given the name tremacamra, was shown to have prophylactic activity in an experimental setting.59 Further studies are needed to confirm the clinical value, if any, of this approach of anti-rhinovirus therapy. The third line of anti-viral research is following the success in the development of anti-HIV chemotherapy: development of inhibitors of the viral proteases. Most viruses, including the rhinoviruses, encode for one or more proteases that are needed in the maturation of viral proteins. Inhibitors of HIV proteases are an important part of today’s successful multi-drug treatment regimen against HIV infections and AIDS60 and the success has facilitated testing the same concept for other virus infections as well.61 Drugs designed according to the X-ray crystallographic analysis of the HRV 3C-protease molecules have given promising results in the first clinical trials. Again, further studies and more experience in controlled trials are needed before attempts can be made to use these drugs in everyday practice. One has to remember that availability of safe and effective anti-rhinovirus drugs would not solve the everyday problems in clinical practice as almost half of all cases of the common cold are caused by other groups of viruses. Therefore, development of rapid diagnostic tests would also be necessary for optimal use of the putative anti-virals. In practice, symptomatic treatment of the common cold is still a useful approach, although there are not many treatment protocols for which the efficacy and tolerability have been documented in placebo-controlled trials (reviewed by Pitka¨ ranta and Hayden2).
FUTURE RESEARCH PROSPECTS HRV infections have been considered only a minor nuisance compared with many other viral diseases. However, it is well documented that they can be triggers of or associated with more serious infections, such as COPD and asthma. The development of anti-rhinoviral drug treatment is therefore very important and still possible. Molecular epidemiological research is producing new information on the relatedness of circulating HRV strains. These data are essential in developing tools for rapid diagnosis for HRV infections. With a reliable ‘‘bedside’’ test, correctly directed treatment of HRV-related diseases might become possible in future. Furthermore, this would decrease use of unnecessary antibiotic treatment.
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PRACTICE POINTS With specific anti-rhinoviral drugs unavailable, treatment of HRV infections is symptomatic. Intranasal or orally administered decongestants are used to relieve nasal stuffiness.62 Non-steroidal anti-inflammatory drugs reduce soreness of the throat and fever.63 The effect of other medications is poor or inconclusive. Prevention of HRV infections by hygienic methods, e.g. increased hand washing, could be one means. However, HRV infections are also transmitted aerogenically and therefore practically unavoidable to a large extent.
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