Human rhinoviruses (HRVs) frequently cause mild upper respiratory tract infections and more severe disease manifestations such as bronchiolitis and asthma exacerbations. HRV is classified into three species within the genus Enterovirus of the family Picornaviridae. HRV species A and B contain 75 and 25 serotypes identified by cross-neutralization assays, although the use of such assays for routine HRV typing is hampered by the large number of serotypes, replacement of virus isolation by molecular methods in HRV diagnosis and the poor or absent replication of HRV species C in cell culture. To address these problems, we propose an alternative, genotypic classification of HRV-based genetic relatedness analogous to that used for enteroviruses. Nucleotide distances between 384 complete VP1 sequences of currently assigned HRV (sero)types identified divergence thresholds of 13, 12 and 13 % for species A, B and C, respectively, that divided inter- and intra-type comparisons. These were paralleled by 10, 9.5 and 10 % thresholds in the larger dataset of >3800 VP4 region sequences. Assignments based on VP1 sequences led to minor revisions of existing type designations (such as the reclassification of serotype pairs, e.g. A8/A95 and A29/A44, as single serotypes) and the designation of new HRV types A101–106, B101–103 and C34–C51. A protocol for assignment and numbering of new HRV types using VP1 sequences and the restriction of VP4 sequence comparisons to type identification and provisional type assignments is proposed. Genotypic assignment and identification of HRV types will be of considerable value in the future investigation of type-associated differences in disease outcomes, transmission and epidemiology.

Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13 % divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing .10 % divergence in VP4/ VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties. IntroductionHuman rhinoviruses (HRVs) are highly prevalent respiratory pathogens, most commonly associated with mild upper respiratory tract disease and exacerbations of preexisting respiratory disease such as asthma. They are also increasingly recognized as underlying more severe disease manifestations, such as bronchiolitis in young children and in the immunosuppressed. The increasing use of molecular methods for respiratory virus screening has contributed to this reappraisal of rhinoviruses, as has the recent discovery of an entirely novel rhinovirus group, refractory to previously used virus isolation methods but now known to be highly prevalent and widely circulating worldwide (Arden et al., 2006;Kaiser et al., 2006;Lamson et al., 2006;Kistler et al., 2007;Lau et al., 2007;Lee et al., 2007;McErlean et al., 2007;Renwick et al., 2007;Olenec et al., 2010).These newly characterized rhinoviruses have been proposed to belong to a novel species of rhinovirus (designated species C; HRV-C), recognizing their substantial sequence divergence from other classified species within the genus Enterovirus of picornaviruses (Carstens, 2010;Knowles, 2010) (Fig. 1a). Clinically and biologically, they share many attributes with the other designated HRV species, HRV-A and -B....

Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of neonatal sepsis-like disease and meningitis. The relative frequencies of specific EV and HPeV types were determined over a 5-year surveillance period using highly sensitive EV and HPeV PCR assays for screening 4,168 cerebrospinal fluid (CSF) specimens collected from hospitalized individuals between 2005 and 2010 in Edinburgh. Positive CSF samples were typed by sequencing of VP1. From the 201 EV and 31 HPeV positive (uncultured) CSF samples on screening, a high proportion of available samples could be directly typed (176/182, 97%). Highest frequencies of EV infections occurred in young adults (n = 43; 8.6%) although a remarkably high proportion of positive samples (n = 98; 46%) were obtained from young infants (<3 months). HPeV infections were seen exclusively in children under the age of 3 months (31/1,105; 2.8%), and confined to spring on even-numbered years (22% in March 2006, 25% in April 2008, and 22% in March 2010). In contrast, EV infections were distributed widely across the years. Twenty different EV serotypes were detected; E9, E6, and CAV9 being found most frequently, whereas all but one HPeVs were type 3. Over this period, HPeV3 was identified as the most prevalent picornavirus type in CNS-related infections with similarly high incidences of EV infection frequencies in very young children. The highly sensitive virus typing methods applied in this study will assist further EV and HPeV screening of sepsis and meningitis cases as well as in future molecular epidemiological studies and population surveillance.

Human rhinoviruses (HRVs) are a highly prevalent and diverse group of respiratory viruses. Although HRV-A and HRV-B are traditionally detected by virus isolation, a series of unculturable HRV variants have recently been described and assigned as a new species (HRV-C) within the picornavirus Enterovirus genus. To investigate their genetic diversity and occurrence of recombination, we have performed comprehensive phylogenetic analysis of sequences from the 5 untranslated region (5 UTR), VP4/VP2, VP1, and 3Dpol regions amplified from 89 HRV-C-positive respiratory samples and available published sequences. Branching orders of VP4/VP2, VP1, and 3Dpol trees were identical, consistent with the absence of intraspecies recombination in the coding regions. However, numerous tree topology changes were apparent in the 5 UTR, where >60% of analyzed HRV-C variants showed recombination with species A sequences. Two recombination hot spots in stem-loop 5 and the polypyrimidine tract in the 5 UTR were mapped using the program GroupingScan. Available HRV-C sequences showed evidence for additional interspecies recombination with HRV-A in the 2A gene, with breakpoints mapping precisely to the boundaries of the C-terminal domain of the encoded proteinase. Pairwise distances between HRV-C variants in VP1 and VP4/VP2 regions fell into two separate distributions, resembling inter-and intraserotype distances of species A and B. These observations suggest that, without serological cross-neutralization data, HRV-C genetic groups may be equivalently classified into types using divergence thresholds derived from distance distributions. The extensive sequence data from multiple genome regions of HRV-C and analyses of recombination in the current study will assist future formulation of consensus criteria for HRV-C type assignment and identification.