Phylogenetic analysis of human rhinoviruses collected over four successive years in Sydney, Australia.

Background Human rhinoviruses (HRV) cause a wide spectrum of disease, ranging from a mild influenza‐like illness (ILI) to severe respiratory infection. Molecular epidemiological data are limited for HRV circulating in the Southern Hemisphere. Objectives To identify the species and genotypes of HRV from clinical samples collected in Sydney, Australia, from 2006 to 2009. Methods Combined nose and throat swabs or nasopharyngeal aspirates collected from individuals with ILI were tested for HRV using real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). Sequencing data of 5′UTR and VP4/VP2 coding regions on RT‐PCR‐positive specimens were analysed. Results Human rhinoviruses were detected by real‐time PCR in 20.9% (116/555) of samples tested. Phylogenetic analysis of 5′UTR and VP4/VP2 on HRV‐positive samples was concordant in the grouping of HRV A and B species but not HRV C species. Eighty per cent (16/20) of sequences that grouped as HRV C in the VP4/VP2 tree clustered as HRV A, alongside some previously described C strains as subspecies C/A. Discordant branching was seen within HRV A group: two sequences clustering as A in the VP4/VP2 tree branched within the C/A subspecies in the 5′UTR tree, and one sequence showed identity to different HRV A strains in the two genes. The prevalence of HRV C and C/A species was greater in paediatric compared to adult patients (47.9% vs 25.5%, P = .032). Conclusion Human rhinoviruses are a common cause of respiratory infections, and HRV C is present in the Southern Hemisphere. Sequencing of multiple HRV regions may be necessary to determine exact phylogenetic relationships.


| INTRODUCTION
Human rhinoviruses (HRV) are a diverse virus group, currently known to contain 167 serotypes. 1 Along with human enteroviruses (HEV), HRV belong to the Picornaviridae family, 2 although they are phylo genetically unrelated to HEV despite similarities in genome organization and structure. Human rhinoviruses are generally associated with the common cold and mild upper respiratory infections, 3 but can also cause severe respiratory infections in immunocompromised hosts (including lung and hematopoietic stem cell transplant recipients) or patients with chronic pulmonary diseases. [4][5][6][7] Unlike respiratory syncytial virus (RSV) and influenza viruses, HRV can cause respiratory illness throughout the year, but peak incidence occurs in early autumn and spring in temperate climates. 6 Early molecular analyses of the HRV capsid protein coding regions clustered different serotypes into two distinct species, HRV A and HRV B. 8 In 2007, a new HRV genetic variant (subsequently designated HRV C) was identified in patients with severe pneumonia from the United States of America (USA), Germany, Hong Kong, Australia and China. [9][10][11][12][13] Severe respiratory disease and asthma exacerbations in children were observed. 11,14 HRV C has been further divided into two subspecies, HRV Cc and HRV Ca. 15 HRV C strains are difficult to grow in cell lines known to support the growth of other rhinoviruses, 11 although two HRV C isolates have been propagated in nasal epithelial cell cultures. 16 There are limited molecular epidemiological data on HRV circulating in the Southern Hemisphere, including Australia. This study aimed to identify the species and genotypes of HRV from clinical samples collected in Sydney, Australia, over four consecutive years by analysing the nucleotide homology in the 5′UTR, VP4 and part of the VP2 capsid protein coding regions.

| Primers and probes
Primers used to amplify all picornaviruses, 18 the HRV-specific probes 5′UTR and VP4-VP2 gene sequencing primers are listed in Table 1.
The RT-PCR probe sequences were designed to include most HRV sequences available in GenBank ® in 2007, and sequences generated from the present study.

| RNA extraction
RNA was prepared directly from combined NTS or NPA using Roche High Pure RNA kits (Roche, Mannheim, Germany).

| Real-time reverse-transcriptase PCR (RT-PCR)
The cDNA was reverse-transcribed from 10 μL of specimen RNA using 100 units of SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 4 μL used for real-time PCR as described previously. 19 Amplification was performed in glass capillaries on

| Sequencing PCR
Human rhinoviruses RT-PCR-positive samples were then tested using UTR primers EV140 and EV170 to amplify a 395-bp product. 20 Where RNA was available, samples were amplified with primers RHI3A and EVP4R, generating a 638-bp fragment that included part of 5′UTR, all of VP4 and part of VP2 regions. 18

| Phylogenetic analysis
Using BLAST ® , nearly all the sequences showed >98% similarity to one or more HRV sequences with partial cds available in GenBank ® .
These were approximately 390-420 bp and did not cover the full sequence from 5′UTR to VP2 of our study samples, and were not included in the construction of the ML tree.
As the data size was large, it was not possible to include each one of the HRV prototype sequences in the tree construction. Figure 1 shows the ML tree for the 5′UTR for all 93 sequences, and Figs 2 and 3 show the ML trees, respectively, of VP4/VP2 and 5′UTR/VP4/VP2 regions of the 65 samples.    in group C with VP4/VP2 analysis also clustered in group C in the 5′UTR along with reference strain C-EU840728; 16 of the 20 HRV C samples grouped as HRV A, along with reference strains C-X2-EF077280, C-EU840952, C-DQ875932, C-JQ994498, C-EF077279, C-EF582387 and C-GQ223227. Four clades that grouped as C species in the VP4/VP2 analysis segregated into the A genogroup in the 5′UTR analysis (Figs 1 and 2).

| 5′UTR
Discordant branching was seen in the following and indicated by a circle  Figure 1). Samples with asterisk (*) denote strains that grouped as C in the VP4/VP2 region, but grouped with HRV A species. Samples with only 5′UTR sequences are denoted by ^. Discordant branches in the VP4/VP2 and 5′UTR sequences are indicated by • Sample MS06-3 and HRV A12 (EF173415) clustered in a clade more related to C-DQ875932 (subspecies Ca). The two samples showed between 80% and 82% ID to C-DQ875932 strain and samples in that cluster and similar identity to A15 and its cluster in the UTR tree, but in the VP4/VP2 tree the two samples showed 61% identity to C-DQ875932 and lower to other C strains and 62% and 80% identity to A 15 and 1B, respectively.

F I G U R E 3
Maximum-likelihood tree of the 5′UTR/VP4/VP2 region, showing relationships of clinical strains, newly described strains and prototype strains of HRV (constructed as described for Fig. 1). Samples with asterisk (*) denote strains that grouped as C in the VP4/VP2 region, but grouped with HRV A species in 5′UTR analysis. Discordant branches in the VP4/VP2 and 5′UTR sequences are indicated by •
Within HRV A, identity at > 86% was seen with known HRV reference strains as shown in Table 2

| 5′UTR/VP4/VP2 region
The grouping of samples as C and CA in the 5′UTR/VP4/VP2 analysis was very similar to that seen in the 5′UTR analysis. The discordant branching pattern seen with samples MS06-3 and HRV A12 (EF173415), MS07-18 and MS08-7 and CS09-2 (indicated with •) in the 5′UTR ML tree was not seen; it was in agreement with that seen in the VP4/VP2 ML tree.

| Prevalence of HRV C according to age groups
In The distribution of the different HRV subtypes is shown in Table 3.
Both HRV A and C or C/A variant were detected in higher numbers than HRV B. Human rhinoviruses C or C/A variant was detected more In our study, the sequences grouped into three phylogenetically distinct species: A (52.6%), C/CA (37.6%) and B (9.6%). However, there was discordance between proposed phylogeny groups when sequences from the 5′UTR and VP4/VP2 coding regions were analysed. Sixteen of the 20 samples that clustered as HRV C in the VP4/ VP2 ML tree segregated as A in the 5′UTR analysis, as did some of the early, well-characterized HRV C strains from New York (EU840952 and DQ875932), 23,26 San Francisco (EF077279 and EF077280), 25 Hong Kong Special Administrative Region (EF582387) 11 and China (GQ223227). 15 Twelve of 28 sequences with only UTR sequences also segregated along with the above-mentioned reference C strains, branching in two major subgroups within HRV A. These have been labelled in this study as C/A, clustering with C strains, but grouping as A in the 5′UTR region: these formed 30% of the clinical sequences. Huang et al. 15 reported similar clustering of field strains in their study and designated the above C strains (EU840952, DQ875932, EF077279, EF077280, GQ223227 and EF582387) as Ca, a subspecies of HRV C that clustered differently to HRV A, HRV B and HRV C in the UTR. They further showed that HRV Ca subspecies were formed from interspecies recombination in the 5′UTR region. Similar inconsistent clustering of field strains as compared to VP4/VP2 was also reported in the 3D polymerase-coding region as well as 5′UTR region. 30 Three discordant branching events were seen in our analyses. MS07-18 (identity to A65) in one branch and MS06-3 along with HRV A12 in another branch segregated as A in the VP4/VP2 analysis, but localized within the Ca subspecies in the 5′UTR analysis. Sample CS09-2 clustered with HRV A89 with 96% relatedness in the UTR region but differed in the VP4/VP2 region. These three samples may represent recombinants as reported by Palmenberg et al. and Kim et al. 31,32 There is no designated region within the HRV genome that is  26,34 The clustering of HRV C as C and C/A when 5′UTR region was included in the analysis (Figs 1 and 3) in our study may suggest that they are true C species that showed recombination with A in the 5′UTR.
In this study, the 5′UTR primers and the primers chosen to amplify the entire VP4 and partial VP2 region produced amplicons with overlapping sequences that resulted in an approximately 900bp continuous sequence. We used a single set of primers for each of the two PCR assays but did not use cloning, which has been used in other studies. 24 were collected from patients with respiratory illness who were sick enough to warrant testing or in many cases hospitalization, but our data are insufficient to attribute clinical severity to any of the HRV

species.
It is possible that HRV C or the C/A variants may cause exacerbation of respiratory infections in infants that require presentation to ED compared to HRV A or B, and this may contribute to the higher percentage of HRV C or C/A infection in the paediatric population. One of the early reports of HRV C-QPM variant severity was from Australian samples collected in 2003 from children with lower respiratory infection. 37 Other reports have observed the association between HRV C variants and asthmatic wheeze and severe lower respiratory infections. 10,11,23,27,38 Xiang et al. 35 reported that the clinical manifestations of HRV A and C are similar, and co-infection with RSV and HRV in infants increases the severity of infection. Both HRV A and C are more virulent than HRV B in infants and HRV virulence is greater in winter, although peak infection rates occur in spring and fall. 9 In conclusion, the present study shows that sequencing of one region alone is insufficient for determining the lineage of the HRV variants. The presence of many diverse strains has become apparent, and it is likely that more will emerge.
Genotypic assignment and identification of HRV types will facilitate monitoring of emerging novel variants, and investigations into type-associated differences in disease epidemiology, transmission and outcomes.

ETHICS STATEMENT
Samples used in this study were not collected for this study per se, but as part of a study assessing the use of face mask in controlling respiratory virus transmission in households following approval by the local institutional review board or for laboratory diagnosis of patients with an influenza-like illness. The present study does not involve the reporting of patient data, and no patient intervention occurred with the obtained results.