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New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses.

Rhinoviruses and enteroviruses Enteroviruses
Viruses which live in the gastrointestinal tract. Coxsackie viruses, viruses that cause hand-foot-mouth disease, are an enterovirus.

Mentioned in: Hand-Foot-and-Mouth Disease
 are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus picornavirus

Any of a group of the smallest known animal viruses. (Pico refers to their small size, rna to their core of RNA.) This group of spheroidal viruses includes viruses that attack the vertebrate intestinal tract and often invade the central nervous system as well
 evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 assays. We then sequenced the 5' untranslated region, capsid capsid /cap·sid/ (kap´sid) the shell of protein that protects the nucleic acid of a virus; it is composed of structural units, or capsomers.

cap·sid
n.
 protein VP1, and protease protease /pro·te·ase/ (pro´te-as) endopeptidase.

pro·te·ase
n.
Any of various enzymes, including the proteinases and peptidases, that catalyze the hydrolytic breakdown of proteins.
 precursor 3CD regions of virus-positive samples. Subsequent phylogenetic phy·lo·ge·net·ic
adj.
1. Of or relating to phylogeny or phylogenetics.

2. Relating to or based on evolutionary development or history.
 analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus enterovirus /en·tero·vi·rus/ (en´ter-o-vi?rus) any virus of the genus Enterovirus. enterovi´ral
Enterovirus /En·tero·vi·rus/ (en´ter-o-vi?rus 
 genotype associated with respiratory symptoms and acute otitis media Acute otitis media
Inflammation of the middle ear with signs of infection lasting less than three months.

Mentioned in: Myringotomy and Ear Tubes

acute otitis media 
, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents.  sites, thereby demonstrating that rhinoviruses can recombine re·com·bine
v.
To undergo or cause genetic recombination; form new combinations.
 in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

Human rhinoviruses (HRVs) and enteroviruses (HEVs) are leading causes of infection in humans. These 2 picornaviruses share an identical genomic organization Organisms have a vast array of ways in which their respective genomes are organized. A comparison of the genomic organization of six major model organisms shows size expansion with the increase of complexity of the organism. , have similar functional RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 secondary structures, and are classified within the same genus (www.ictvonline.org/virusTaxonomy.asp) because of their high sequence homology homology (hōmŏl`əjē), in biology, the correspondence between structures of different species that is attributable to their evolutionary descent from a common ancestor.  (1). However, despite their common genomic features, these 2 groups of viruses have different phenotypic characteristics. In vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body.

in vi·vo
adj.
Within a living organism.



in vivo adv.
, rhinoviruses are restricted to the respiratory tract respiratory tract
n.
The air passages from the nose to the pulmonary alveoli, including the pharynx, larynx, trachea, and bronchi.


Respiratory tract 
, whereas enteroviruses infect primarily the gastrointestinal tract gastrointestinal tract
n.
The part of the digestive system consisting of the stomach, small intestine, and large intestine.


Gastrointestinal tract 
 and can spread to other sites such as the central nervous system. However, some enteroviruses exhibit specific respiratory tropism tropism (trōp`ĭzəm), involuntary response of an organism, or part of an organism, involving orientation toward (positive tropism) or away from (negative tropism) one or more external stimuli.  and thus have properties similar to rhinoviruses (2-5). In vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.

in vi·tro
adj.
In an artificial environment outside a living organism.
, most HRVs and HEVs differ by their optimal growth temperature, acid tolerance, receptor usage, and cell tropism. The genomic basis for these phenotypic differences between similar viruses is not yet fully understood.

HRVs and HEVs are characterized by [approximately equal to]100 serotypes. Recently, molecular diagnostic tools have shown that this diversity expands beyond those predefined serotypes and encompasses also previously unrecognized rhinovirus rhinovirus

Any of a group of picornaviruses capable of causing common colds in humans. The virus is thought to be transmitted to the upper respiratory tract by airborne droplets.
 and enterovirus genotypes. As an example, a new HRV HRV Croatia (ISO Country code)
HRV Heart Rate Variability
HRV Human Rhinovirus
HRV Heat Recovery Ventilator
HRV High Resolution Visible
HRV Haute Resolution Visible
HRV Hypersonic Research Vehicle
HRV Hercules Recovery Vehicle
 lineage named HRV-C was recently identified and now complements the 2 previously known A and B lineages (6-8) (N.J. Knowles, pers. comm.). The C lineage has not only a distinct phylogeny (9-16) but is also characterized by specific cis-acting RNA structures (17).

In this study, we screened a large number of persons with acute respiratory diseases by using assays designed to overcome the diversity of both rhinoviruses and enteroviruses circulating in humans. Whenever possible, we systematically sequenced 5. untranslated region (UTR UTR Untranslated Region (genetics)
UTR Unicode Technical Report
UTR Unique Taxpayer Reference (UK Inland Revenue)
UTR Unable to Reach
UTR Unable to Reproduce
UTR University Technical Representative
), capsid protein VP1, and protease precursor 3CD regions of strains. Our goals were 1) to characterize the diversity of circulating rhinoviruses and, to a lesser extent, enteroviruses, to identify putative new picornavirus variants, and 2) to assess whether recombination may drive HRV evolution, which has not been shown in natural human infections (18).

Materials and Methods

RNA Extraction, Reverse Transcription-PCR, and Real-Time PCR

Reverse transcription-PCR (Superscript Any letter, digit or symbol that appears above the line. For example, 10 to the 9th power is written with the 9 in superscript (109). Contrast with subscript.  II; Invitrogen, Carlsbad, CA, USA) was performed on RNA extracted by using the HCV HCV
abbr.
hepatitis C virus


HCV 1 Hepatitis C virus, see there 2. Human coronavirus. See Coronavirus.
 Amplicor Specimen Preparation kit (Roche, Indianapolis, IN, USA), TRIzol (Invitrogen), or the QIAamp Viral RNA Mini kit (QIAGEN, Valencia, CA, USA). Real-time PCR specific for HRV-A, HRV-B, and HEV HEV
abbr.
hepatitis E virus



HEV

hemagglutinating encephalomyelitis virus of pigs.
 (19), and a generic panenterhino real-time PCR (forward primer 5.-AGCCTGCGTGGCKGCC-3., reverse primer 5.-GAAACACGGACACCCAAAGTAGT-3., and probe 5-FAM-CTCCGGCCCCTGAATGYGGCTAA-TAMRA3.), were performed in several cohort studies (Table).

Clinical Specimens

Picornavirus-positive samples were detected from patients enrolled in cohort studies in different regions of Switzerland during 1999-2008. The main characteristics of these populations, type of respiratory specimens, and screening methods are shown in the Table. The rhinovirus serotypes used for 3CD sequencing were obtained from the American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for  (Manassas, VA, USA).

PCR and Sequencing

Sequencing was performed directly from the clinical specimen except for samples selected by routine isolation methods on human embryonic (HE) primary fibroblast fibroblast /fi·bro·blast/ (fi´bro-blast)
1. an immature fiber-producing cell of connective tissue capable of differentiating into chondroblast, collagenoblast, or osteoblast.

2.
 cell lines (Table) or for HRV reference serotypes. Primers used to amplify the 5.-UTR and the VP1 and 3CD regions are listed in online Technical Appendix 1 Table 1A (available from www.cdc.gov/EID/content/15/5/719-Techapp1.pdf).

Full-length genome sequences of CL-1231094, a related clinical strain of enterovirus, and partial sequences of CL-Fnp5 and CL-QJ274218 were obtained as follows. RNA extracted by using the QIAamp Viral RNA Mini kit (QIAGEN) plus DNase treatment or with Trizol was reverse transcribed with random-tagged primer FR26RV-N and amplified with the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA) with a specific forward primer and FR20RV reverse primer (online Technical Appendix 1 Table 1B) (23). Amplification products were separated by electrophoresis on agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments.
 gels and fragments (0.6-2.5 kb) were extracted by using the QIAquick Gel Extraction In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis.  kit (QIAGEN). Purified products were cloned by using the TOPO TOPO Tri-N-Octylphosphine Oxide
TOPO Topographic/Topography
TOPO Trioctyl-Phosphine Oxide
ToPo Torposten (German Military Gate Post)
TOPO Tunable Optical Parametric Oscillator
 TA cloning kit (Invitrogen).

Minipreps were prepared from individual colonies and clones with the largest inserts were chosen for sequencing. Sequences obtained were used to design a new forward primer (online Technical Appendix 1 Table 1) to advance toward the 3. end of the genome. PCR products of 3. genomic ends were obtained by using the BD Smart Race cDNA amplification kit (Becton Dickinson BD (NYSE: BDX), is a medical technology company that manufactures and sells medical devices, instrument systems and reagents. Founded in 1897 and headquartered in Franklin Lakes, New Jersey, BD employs 27,000 people in nearly 50 countries. , Franklin Lakes, NJ, USA) according to according to
prep.
1. As stated or indicated by; on the authority of: according to historians.

2. In keeping with: according to instructions.

3.
 manufacturer's instructions. All PCR products were purified by using microcon columns (Millipore, Billerica, MA, USA) and sequenced by using the ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.


(Application Binary Interface) A specification for a specific hardware platform combined with the operating system.
 Prism 3130XL DNA Sequencer A DNA sequencer is an instrument used to automate the DNA sequencing process.

DNA sequencers have become more important due to large genomics projects and the need to increase productivity.
 (Applied Biosystems, Foster City, CA, USA). Chromatograms were imported for proofreading Proofreading traditionally means reading a proof copy of a text in order to detect and correct any errors. Modern proofreading often requires reading copy at earlier stages as well.  with the vector NTI NTI NewTech Infosystems (software company, Irvine, California)
NTI Nuclear Threat Initiative
NTI National Transit Institute (New Brunswick, New Jersey)
NTI Nunavut Tunngavik Incorporated
 Advance 10 program (Invitrogen). Overlapping fragments were assembled with the contigExpress module of the vector NTI Advance 10.

Sequence Analysis, Phylogeny, and Bootscanning of Recombinants

Alignments were constructed by using MUSCLE (24) with a maximum of 64 iterations. (For detailed analyses, see http://cegg.unige.ch/picornavirus.) Multiple FastA was converted into PHYLIP PHYLIP Phylogeny Inference Package (genetics software)  format (for tree building) with the EMBOSS em·boss  
tr.v. em·bossed, em·boss·ing, em·boss·es
1. To mold or carve in relief: emboss a design on a coin.

2.
 program Seqret (25). Trees were built with PhyML (26) by using the general time reversible model, BIONJ for the initial tree, and optimized tree topology and branch lengths. Trees with <50 species and larger trees used 16 and 8 rate categories, respectively. Transition/transversion ratios, proportions of invariant (programming) invariant - A rule, such as the ordering of an ordered list or heap, that applies throughout the life of a data structure or procedure. Each change to the data structure must maintain the correctness of the invariant.  sites, and shape parameters of the [gamma] distribution were estimated.

To investigate the hypothesis of recombination and map the breakpoints, we adapted the bootscanning method (27) as follows. The alignment was sliced into windows of constant size and fixed overlap and a 100replicate maximum-likelihood (using HRV-93 as an outgroup) was computed for each window. From each tree, the distance between the candidate recombinant and all other sequences was extracted. This extraction yielded a matrix of distances for each window and for each alignment position. A threshold was defined as the lowest distance plus a fraction (15%) of the difference between the highest and lowest distances. The nearest neighbors of the candidate recombinant were defined as sequences at a distance smaller than this threshold. This distance ensured that the nearest neighbor, as well as any close relative, was always included. Possible recombination breakpoints thus corresponded to changes of nearest neighbors. Serotypes included in this analysis represented serotypes close to CL-013775 and CL-073908 on the basis of 5.-UTR and VP1 phlyogenetic trees (online Technical Appendix 2 Figure 1, panels A, B, available from www.cdc.gov/EID/content/15/5/719-Techapp2.pdf), as well as serotypes close to CL-135587 on the basis of VP1 and 3CD phlyogenetic trees (online Technical Appendix 2 Figure 1, panels B, C) and whose full-length sequence was available.

Distance matrices were computed from alignments with the distmat program in EMBOSS (http://bioweb2.pasteur.fr/docs/EMBOSS/embossdata.html) by using the Tamura distance correction. This method uses transition and transversion trans·ver·sion
n.
Eruption of a tooth in a position normally occupied by another.


transversion,
n eruption of a tooth in the wrong position
 rates and takes into account the deviation of GC content from the expected value Expected value

The weighted average of a probability distribution. Also known as the mean value.
 of 50%. Gap and ambiguous positions were ignored. Final values were then converted to similarity matrices by subtracting each value from 100.

[FIGURE 1 OMITTED]

Results

Screening of Persons with Respiratory Tract Infections Persons enrolled in several cohorts of children and adults with respiratory infections (Table) were screened for picornavirus by culture isolation on HE cell lines, real-time PCR specific for HRV-A and HRV-B (19), or by a panenterhino real-time PCR designed to theoretically detect all rhinoviruses and enteroviruses with publicly available sequences. Of 1,592 respiratory samples tested by real-time PCR, 248 were virus positive (Table). The 5.-UTR sequences were obtained for 77 real-time PCR or culture-positive samples and VP1 and 3CD sequences for 48 of these (Table; online Technical Appendix 1 Table 2). In parallel, the 3CD sequences were identified for all reference serotypes. The results of this screening are summarized in online Technical Appendix 1 Table 2, and all sequences are available from GenBank (accession nos. EU840726-EU840988).

On the basis of these results, respiratory infections caused by HRV-B might be less frequent than those caused by HRV-A, and HRV-A infections are distributed among the whole library of reference serotypes. A specific real-time PCR used to detect enteroviruses in respiratory specimens from some of the cohorts studied indicated that these viruses are rare in children (2.5% vs. 6.3% for HRV) and even rarer or absent in adults (0% vs. 24% for HRV) (28).

Phylogeny and Molecular Epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases,  of 5'-UTR

To include all 99 HRV reference strains and new divergent rhinoviruses described recently by Lee et al. (13), we reconstructed a phylogenetic tree (online Technical Appendix 2 Figure 1, panel A) on the basis of a sequence of 280 nt in the 5.-UTR. This sequence provided a correct clustering of HRV-A, HRV-B, and HEV strains according to the accepted whole-genome phylogeny (online Technical Appendix 2 Figure 1, panel D) (15) but did not resolve appropriately the phylogeny of the 4 HEV species and the HRV-A and HRV-C viruses. The condensed con·dense  
v. con·densed, con·dens·ing, con·dens·es

v.tr.
1. To reduce the volume or compass of.

2. To make more concise; abridge or shorten.

3. Physics
a.
 tree version (Figure 1, panel A) enabled us to identify 2 groups phylogenetically phy·lo·ge·net·ic  
adj.
1. Of or relating to phylogeny or phylogenetics.

2. Relating to or based on evolutionary development or history: a phylogenetic classification of species.
 distant from all previously known HRVs and HEVs. The first group, referred to as HRV-C., contained some of our clinical samples and rhinoviruses sequenced by Lee et al. (13). The second group was a new clade clade Cladus, subtype Genetics A branch of biological taxa or species that share features inherited from a common ancestor; a single phylogenetic group or line. See Inheritance, Species.  and was named EV-104. This clade included 8 clinical samples collected in different regions of Switzerland without direct epidemiologic links (online Technical Appendix 1 Table 2).

Identification of HRV-C Viruses by Sequencing of HRV Viruses with Divergent 5'-UTRs

Characterization of HRVs newly identified during 2006-2008 showed that they all belong to the same HRV-C species (9-16). Recently, Lee et al. (13) identified another cluster of viruses (HRV-C.; Figure 1, panel A) and suggested that this group was phylogenetically distinct from all other HRVs on the basis of analysis of their 5.-UTR sequences. To define the phylogeny, we adapted a previously described method (23) to complete the genome sequence directly from our clinical strains (CL-Fnp5 and CLQJ274218) that showed a similar divergent 5.-UTR (online Technical Appendix 2 Figure 1, panel A). A condensed version (Figure 1, panel B) of the phylogenetic tree based on VP1 sequences (online Technical Appendix 2 Figure 1, panel B) indicated that CL-Fnp5 clustered with the new HRV-C clade, a finding further confirmed by CL-QJ 274218 partial sequences. This finding supports the view that new HRVs variants described since 2006 (9-16) all belong to the same lineage.

New Divergent Lineage of HEV Species C

As shown in Figure 1, panel A, the panenterhino realtime PCR enabled detection of a new HEV strain phylogenetically distinct from all previously known HEV species and associated with respiratory diseases. Enterovirus-specifi c real-time PCRs or reference VP1 primer sets routinely used to type enteroviruses (primers 222 and 224 and nested primers AN88 and 89) (29,30) did not amplify this new genotype. We could not grow this virus on HeLa and HE cell lines. Consequently, we applied the method described above to complete the genome sequence directly from the CL-1231094 (EU840733) clinical specimen. VP1 and fulllength genome sequences showed that, albeit divergent at the 5.-UTR level, this new variant belonged to the HEV-C species (Figure 1, panels B, C). Full-length genome phylogenetic tree (Figure 2) and VP1 protein identity plots (online Technical Appendix 2 Figure 2) with all members of the HEV-C species indicated that this virus represents a new HEV-C genotype that shares 68%, 66%, and 63% nucleotide and 77%, 75%, and 68% amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins.  sequence identity, respectively, with coxsackieviruses A19 (CVA CVA
abbr.
cerebrovascular accident


CVA,
n See accident, cerebrovascular.


CVA

cerebrovascular accident.

CVA Cerebrovascular accident, see there
19), A22, and A1, the closest serotypes. This new virus was named EV-104 (www.picornastudygroup.com/types/enterovirus_genus.htm).

Specific primers (Ent_P1.29/P2.13 and Ent_P3.30/ P3.32; online Technical Appendix 1 Table 1C) were then designed to amplify the VP1 and 3D regions of the 7 other samples of this cluster collected from children with acute respiratory tract infections and otitis media Otitis Media Definition

Otitis media is an infection of the middle ear space, behind the eardrum (tympanic membrane). It is characterized by pain, dizziness, and partial loss of hearing.
. VP1 nucleotide homology among these strains was 94%-98%, except for 1 distantly related sample (74%-76%), which may represent an additional genotype. Additional sequencing is ongoing to verify this assumption.

At the 5.-UTR level, the strain described by Lee et al. (13) and EV-104 diverged from other members of HRV-C and HEV-C species, respectively. Thus, the 5.-UTR-based phylogeny was inconsistent with that based on VP1 sequences and suggested possible recombination events (Figure 1, panels A, B). Because the 5.-UTR is the target of most molecular diagnostic assays, this sequence divergence needs to be taken into account in future studies.

Recombination Events between 5'-UTR, VP1, and 3CD Genome Regions

Other studies have provided sequences of clinical strains, but genetic characterization was often limited to 1 genomic region. Our goal was to sequence 3 genomic regions for each analyzed strain to determine definitively whether recombination events could represent a driving force for the evolution of rhinoviruses in their natural environment. Although recombination events have been suggested for reference serotypes, they have never been shown for circulating clinical strains (18,31,32). In contrast, recombination is well established as a driving force of enterovirus evolution. Thus, we completed the 5.-UTR, VP1, and 3CD sequences of 43 clinical strains by using a pool of adapted and degenerated primers (online Technical Appendix 1 Table 1A).

[FIGURE 2 OMITTED]

Independent phylogenetic trees (online Technical Appendix 2) and similarity matrices were constructed for the 3 genomic regions. Since the last common ancestor and as depicted on the distance matrices and highlighted by boxplots of maximum-likelihood branch length distributions (online Technical Appendix 2 Figure 3), there are more mutations fixed in the VP1 region than in the 3CD region, and more in the 3CD region than in 5.-UTR, which is indicative of a variable rate of evolution in these regions. Accordingly, VP1 sequences enabled genotyping of all but 3 clinical strains analyzed (online Technical Appendix 2, Figure 1, panel B). These strains may represent rhinovirus genotypes only distantly related to predefined reference serotypes. In contrast, genotyping based on 3CD and 5.-UTR was less accurate, as expected. These results confirmed that molecular typing of rhinoviruses, similarly to other picornaviruses, must use capsid sequences.

Phylogeny of the 5.-UTR, VP1, and 3CD of reference serotypes showed many incongruities caused by insufficient tree resolution or recombinant viruses as previously proposed (18,31). As an example, 2 VP1 clusters including HRV-85/HRV-40 and HRV-18/HRV-50/HRV-34 (online Technical Appendix 2 Figure 1, panel B) were reorganized as HRV-85/HRV-18/HRV-40 and HRV-50/HRV-34, respectively, on 3CD (online Technical Appendix 2 Figure 1, panel C). The differential cosegregations between these virus strains suggested recombination events. When available, full-length genome sequence bootscanning applied to all serotypes will give an estimate of the number of reference strains with mosaic genomes.

Similarly, the noncoding region, VP1, and 3CD trees showed major phylogenetic incongruities for 3 clinical isolates (online Technical Appendix 2 Figure 1). Two of these isolates (CL-013775 and CL-073908) were typed as HRV67 on the basis of VP1 sequence and were closest to this serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon.

se·ro·type
n.
See serovar.

v.
 in 3CD, whereas the 5.-UTR cosegregated with HRV-36 (see 5.-UTR recombinant; online Technical Appendix 2 Figure 1, panels A-C A-C Air Conditioning ). These viruses were isolated by cell culture from 2 epidemiologically linked cases and thus represented transmission of the same virus. To confirm the recombination, we completed the sequencing by obtaining the 5.-UTR, VP4, and VP2 sequences (EU840918 and EU840930) and compared them with HRV-36, HRV-67, and other closely related serotypes. Bootscanning analysis (Figure 3, panel A) enabled mapping of the recombination site within the 5.-UTR, just before the polyprotein start codon. Sequence alignment mapped recombination breakpoints more precisely between positions 524 and 553 with reference to HRV-2 (X02316).

[FIGURE 3 OMITTED]

The other incongruent in·con·gru·ent  
adj.
1. Not congruent.

2. Incongruous.



in·congru·ence n.
 isolate (CL-135587) was typed as HRV-76 on the basis of VP1 sequence and was closest to this serotype in the 5.-UTR, but 3CD cosegregates with HRV-56 (3C recombinant; online Technical Appendix 2 Figure 1, panels B, C). Similarly, we completed the full-length sequence of this isolate (EU840726) and HRV-56 (EU840727). The same approach enabled mapping of the recombination site at the N terminus of protein 3C between positions 1511 and 1523 with reference to HRV-2 (Figure 3, panel B). These results demonstrate that recombination occurs among clinical rhinoviruses. In our analysis of 40 rhinovirus-positive samples collected over 9 years (3 additional samples were duplicates of 2 different viruses; online Technical Appendix 1 Table 2) for 3 genomic regions, 2 of the analyzed viruses appeared to be recombinants. The 2 documented recombinations occurred in members of the HRV-A species. The design of this study and technical issues (e.g., inability to sequence low viral loads) limited the ability to calculate a recombination rate, particularly for HRV-B and HRV-C.

Discussion

Our genomic analysis of picornaviruses associated with upper or lower respiratory diseases in adults and children indicates that rhinoviruses circulating in the community are widely diverse. The large number of circulating genotypes supports the view that rhinoviruses do not circulate by waves or outbreaks of a given dominant genotype, which might explain the high frequency of reinfection reinfection /re·in·fec·tion/ (-in-fek´shun) a second infection by the same agent or a second infection of an organ with a different agent.

re·in·fec·tion
n.
 during short periods. As expected, the observed variability is higher for surface capsid proteins, the targets of most immune pressure, and this region remains the only accurate one for genotyping and defining phylogeny. Technical constraints such as the limited amount of clinical specimens, the use of different screening methods, and the need to sequence an unknown target of extreme variability might have limited the representativeness of our sequence collection. Therefore, our study should not be considered as an exhaustive epidemiologic analysis of rhinoviruses and enteroviruses associated with respiratory diseases.

By using a systematic approach, we have identified a new enterovirus genotype (EV-104) that has a divergent 5.-UTR. Undetectable by conventional methods, EV-104 could be detected by using a more generic real-time PCR assay designed to match all known available rhinovirus and enterovirus sequences. Such diagnostic tools have and will lead to constant discovery of new picornavirus genotypes (9-14,16,33-36). These genotypes may represent viruses, in most instances, that have remained undetected because of insensitive cell cultures or overly restrictive molecular tools. In addition, enterovirus genotypes causing respiratory infections, such as EV-68 and CV-A21, might be underrepresented un·der·rep·re·sent·ed  
adj.
Insufficiently or inadequately represented: the underrepresented minority groups, ignored by the government. 
 because enteroviruses are usually searched for in fecal specimens (37).

EV-104 belongs to the HEV-C species: CV-A19, CVA22, and CV-A1 are its closest serotypes. These HEV-C subgroup viruses are genetically distinct from all other serotypes of the species. These viruses show no evidence of recombination with other HEV-C strains and, similar to EV-104, do not grow in cell culture (29). On the basis of our epidemiologic data, we conclude that EV-104 was found in 8 children from different regions of Switzerland who had respiratory illnesses such as acute otitis media or pneumonia. Future studies using adapted detection tools will provide more information on the range of this virus. On the basis of its genomic features and similarities with coxsackieviruses and poliovirus poliovirus /po·lio·vi·rus/ (pol´-e-o-vi?rus) the causative agent of poliomyelitis, separable, on the basis of specificity of neutralizing antibody, into three serotypes designated types 1, 2, and 3. , EV-104 could theoretically infect the central nervous system (2,38). Detection of new subtypes of picornaviruses indicates that viruses with new phenotypic traits could emerge, and conclusions on tropism of new strains should be substantiated by extensive experimental or clinical investigations (39).

By completing the sequence of a seemingly divergent rhinovirus (13), we assigned this virus to the new HRV-C species, thus limiting currently to 3 the number of HRV species. For the sake of simplicity, we propose to consider this virus as a member of the HRV-C clade.

Finally, we demonstrated that rhinovirus evolves by recombination in its natural host. Known to be a driving force of enterovirus evolution, rhinovirus recombination among clinical strains has never been observed. Two clinical isolates of 40 viruses analyzed resulted from recombination events and their breakpoints were identified within the 5.-UTR sequence and the N terminus of protein 3C, respectively. These findings are consistent with the fact that recombination breakpoints in picornaviruses are restricted to nonstructural regions of the genome or between the 5'-UTR and the capsid-encoding region (40). Our observations provide new insight on the diversity and ability of rhinovirus to evolve in its natural host. The fact that only 2 of 40 analyzed viruses over a 9-year period were recombinants is suggestive of suggestive of Decision making adjective Referring to a pattern by LM or imaging, that the interpreter associates with a particular–usually malignant lesion. See Aunt Millie approach, Defensive medicine.  a lower recombination frequency in rhinoviruses than in other picornaviruses (32,40) and might be related, but not exclusively, to the short duration of rhinovirus infection (18,31,32). Recombination events occurred between HRV-A genotypes, but whether they can occur in species B and C remains unknown. Interspecies recombination is rare in picornaviruses and is mainly the result of in vitro experiments. For rhinoviruses, the different location of cre elements in each species might be an additional limiting constraint (17).

In summary, we have highlighted the large genomic diversity of the most frequent human respiratory viral infection viral infection,
n an infection by a pathogenic virus. A virus acts on the cell nucleus, taking over the genetic material within the nucleus and replicating itself.
. Our phylogenetic analysis has characterized circulating strains relative to reference strains and has identified a previously unknown enterovirus genotype. We have shown that recombination also contributes to rhinovirus evolution in its natural environment.

DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.3201/eid1505.081286

Acknowledgments

We thank Rosemary Sudan for editorial assistance and the Swiss Institute of Bioinformatics' Vital-IT facility for boot-scanning and computing infrastructure.

This study was supported by the Swiss National Science Foundation The Swiss National Science Foundation is a science research support organization mandated by the Swiss Federal Government. The SNSF was established in 1952 as a foundation under private law. Its secretariat is based in Berne.  (grants 3200B0-101670 to L.K. and 3100A0112588/I to E.Z.), the Department of Medicine of the University Hospitals of Geneva Geneva, canton and city, Switzerland
Geneva (jənē`və), Fr. Genève, canton (1990 pop. 373,019), 109 sq mi (282 sq km), SW Switzerland, surrounding the southwest tip of the Lake of Geneva.
, the University of Geneva The University of Geneva (Université de Genève) is a university in Geneva, Switzerland. It was founded by John Calvin in 1559. Initially a theological seminary, it also taught law.  Dean's Program for the Promotion of Women in Science (C.T.), and the Infectigen Foundation.

References

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Streptococcus pneumoniae Microbiology A pathogenic streptococcus with 90 serotypes associated with pneumonia, bacteremia, meningitis Transmission Person to person Incidence
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Caroline Tapparel, [1] Thomas Junier, [1] Daniel Gerlach, Sandra Van Belle, Lara Turin, Samuel Cordey, Kathrin Muhlemann, Nicolas Regamey, John-David Aubert, Paola M. Soccal, Philippe Eigenmann, Evgeny Zdobnov, [1] and Laurent Kaiser [1]

Author affiliations: University of Geneva Hospitals, Geneva, Switzerland (C. Tapparel, S. Van Belle, L. Turin, S. Cordey, P. M. Soccal, P. Eigenmann, L. Kaiser); University of Geneva Medical School, Geneva (C. Tapparel, T. Junier, D. Gerlach, S. Van Belle, L. Turin, S. Cordey, E. Zdobnov, L. Kaiser); Swiss Institute of Bioinformatics, Geneva (T. Junier, D. Gerlach, E. Zdobnov); University Hospital of Bern, Bern, Switzerland (K. Muhlemann, N. Regamey); University Hospital of Lausanne, Lausanne, Switzerland (J.-D. Aubert); and Imperial College London History
Imperial College was founded in 1907, with the merger of the City and Guilds College, the Royal School of Mines and the Royal College of Science (all of which had been founded between 1845 and 1878) with these entities continuing to exist as "constituent colleges".
, London, UK (E. Zdobnov)

Dr Tapparel is a molecular virologist virologist

microbiologist specializing in virology.
 at the University Hospitals of Geneva. Her research interests are the molecular epidemiology of picornaviruses (rhinoviruses and enteroviruses), development of new diagnostic methods, and determination of fundamental aspects of these viruses.

[1] These authors contributed equally to this article.

Address for correspondence: Caroline Tapparel, Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals, 24 Rue Micheli-du-Crest, 1211 Geneva 14, Switzerland; email: caroline.tapparel@hcuge.ch
Table. Characteristics of screened study populations and respiratory
samples, Switzerland *

Type of study (no.       Age
enrolled)                group             Patient characteristics

Respiratory infection    <1 y              Nonhospitalized children
in newborns (243)                          with initial respiratory
                                           episode with cough

Lower respiratory        Adults            Mainly
tract infection in                         immunocompromised
hospitalized patients                      patients with lower
(147)                                      respiratory tract
                                           complications and
                                           comorbidities

Acute respiratory        <17 y             Nonhospitalized children
tract infection in                         with AOM or pneumonia
children (653)

Lower respiratory        Adults            Mainly
tract infection in                         immunocompromised
hospitalized patients                      patients with lower
(485)                                      respiratory tract
                                           complications and
                                           concurrent illnesses

Acute respiratory        <12 y             Children at an emergency
tract infection in                         department with fever and
children (64)                              acute respiratory
                                           symptoms treated with
                                           antimicrobial drugs

Isolation in routine     Children          Hospitalized patients
procedures (NA)          and
                         adults

Type of study (no.       Years of          Type of
enrolled)                study             specimens

Respiratory infection    1999-2005         NPS
in newborns (243)

Lower respiratory        2001-2003         BAL, NPS
tract infection in
hospitalized patients
(147)

Acute respiratory        2004-2007         NPS
tract infection in
children (653)

Lower respiratory        2003-2006         BAL, NPS
tract infection in
hospitalized patients
(485)

Acute respiratory        2006-2007         NPS
tract infection in
children (64)

Isolation in routine     1999-2008         BAL, NPS
procedures (NA)

Type of study (no.                         No. (%)
enrolled)                PCR               positive

Respiratory infection    HRV-A and         36 (15)
in newborns (243)        HRV-B specific
                         real time for
                         the first 203
                         and
                         panenterhino
                         for 40

Lower respiratory        HRV-A and         16 (11)
tract infection in       HRV-B specific
hospitalized patients    real time
(147)

Acute respiratory        Panenterhino      121 (18)
tract infection in
children (653)

Lower respiratory        Panenterhino      52 (11)
tract infection in
hospitalized patients
(485)

Acute respiratory        Panenterhino      23 (36)
tract infection in
children (64)

Isolation in routine     HE culture        NA
procedures (NA)          isolation

Type of study (no.
enrolled)                Reference

Respiratory infection    (20)
in newborns (243)

Lower respiratory        (21)
tract infection in
hospitalized patients
(147)

Acute respiratory        (22) and
tract infection in       ongoing
children (653)           study

Lower respiratory        (21) and
tract infection in       ongoing
hospitalized patients    study
(485)

Acute respiratory        NP
tract infection in
children (64)

Isolation in routine     NP
procedures (NA)

* NPS, nasopharyngeal samples; HRV, human rhinovirus; BAL,
bronchoalveolar lavage; AOM, acute otitis media; NP, not published;
NA, not available; HE, human embryonic primary fibroblast cell line.
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Copyright 2009 Gale, Cengage Learning. All rights reserved.

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Title Annotation:RESEARCH
Author:Tapparel, Caroline; Junier, Thomas; Gerlach, Daniel; Van Belle, Sandra; Turin, Lara; Cordey, Samuel;
Publication:Emerging Infectious Diseases
Date:May 1, 2009
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