We collected 3,375 nasopharyngeal swabs from subjects with ILI symptoms from eight countries throughout Central and South America. We performed direct RT-PCR for HRVs and HEVs and sequenced all positive samples (n = 632) (Figure 1 ). Our subjects had a median age of 3 years, ranging from less than 1 month to 25 years, an interquartile range of 1 to 8 years, and a male/female ratio of 1.2:1.





Figure 1. Sample collection sites grouped by climatic/geographic similarities. Most of the sample-collection sites (dots) throughout Latin America were grouped into six regions (colors) by their climatic and geographic similarities: latitude, longitude, altitude (meters above sea level), rainy season, and the Köppen climate classification were considered. Lima, Peru, was not considered for the temporal distribution analysis because it could not be grouped in to one of the six regions. Most of the sample-collection sites (dots) throughout Latin America were grouped into six regions (colors) by their climatic and geographic similarities: latitude, longitude, altitude (meters above sea level), rainy season, and the Köppen climate classification were considered. Lima, Peru, was not considered for the temporal distribution analysis because it could not be grouped in to one of the six regions.





Overall, HRVs and HEVs were identified in 16% (548 samples) and 3% (84 samples) of the ILI cases, respectively. Among the HRVs, HRV-A was the most represented species (9% of ILI cases), followed by HRV-C (6%) and HRV-B (1%). Although the number of ILI samples collected among countries varied considerably (Figure 2 , lower panel) we found no statiscally significant geographic differences in the proportions of HEV, HRV-A, HRV-B, and HRV-C.



Figure 2. Percentage of HRV and HEV by age and by country. The percentage of human enteroviruses (HEV) and of each human rhinovirus species (HRV-A, HRV-B, and HRV-C) in samples from subjects with influenza like illness is shown by age (upper panel) and by country (lower panel). The total number of samples collected for each age group and country is shown above each percentage bar (bold). The percentage of human enteroviruses (HEV) and of each human rhinovirus species (HRV-A, HRV-B, and HRV-C) in samples from subjects with influenza like illness is shown by age (upper panel) and by country (lower panel). The total number of samples collected for each age group and country is shown above each percentage bar ().



p <0.05 for both). However, using the two proportion z-test, we noted no difference in proportions of specific HRV species or HEVs per total ILI cases among different age groups. Nevertheless, the risk of detecting HRV-C in children younger than 5 years with ILI was 1.59 (C.I. 1.17-2.17; p <0.05) compared to older children and young adults (525 years).

HRVs were identified significantly more frequently in children younger than 1 year (24%) compared to those between 1 and 5 years of age (15%) or older than five years (13%) (Figure 2 , upper panel), a statistically significant finding (<0.05 for both). However, using the two proportion z-test, we noted no difference in proportions of specific HRV species or HEVs per total ILI cases among different age groups. Nevertheless, the risk of detecting HRV-C in children younger than 5 years with ILI was 1.59 (C.I. 1.17-2.17;<0.05) compared to older children and young adults (525 years).

Certain pre-existing respiratory conditions, such as rhinitis or chronic bronchitis, were found more often in those with HRV isolated (O.R. = 1.14 [95% C.I. 1.09  1.81]; ×2 = 7.82; p-value < 0.05). Furthermore, the presence of these pre-existing conditions specifically increased the risk for detection of HRV-C (O.R. = 1.71 [95% C.I. 1.18  2.45]; ×2 = 9.17; p < 0.05); asthma was a condition that doubled the risk of HRV-C detection (O.R. = 2.07 [95% C.I. 1.08  3.79]; ×2 = 6.19; p <0.05).



In addition, we detected multiple HEV serotypes, including EV-D68, EV-C99, EV-C104, EV-C109, and EV-B110. We also identified EV-A71 in two participants, both from the department of Tumbes, Peru, although from different cities. The first subject, a one year-old girl from the city of Tumbes (same name as the department), presented on April 27, 2011, with fever, rhinorrhea, cough, and erythema on pharyngeal examination. The second, a two year-old girl from the city of Zarumilla, presented on May 10, 2011, with fever, malaise, rhinorrhea, cough, and weight loss. Neither subject had rash, gastrointestinal manifestations, convulsions, change in consciousness, or other neurological deficits. Lastly, two polioviruses were detected and were related to the Sabin-1 polio vaccine strain.

Coxsackieviruses comprised the majority of the HEV group (65% of the HEVs identified), showing a variety of types: 9 for coxsackievirus A and 5 for coxsackievirus B (Figure 3 ).We also identified EV-A71 in two participants, both from the department of Tumbes, Peru, although from different cities. The first subject, a one year-old girl from the city of Tumbes (same name as the department), presented on April 27, 2011, with fever, rhinorrhea, cough, and erythema on pharyngeal examination. The second, a two year-old girl from the city of Zarumilla, presented on May 10, 2011, with fever, malaise, rhinorrhea, cough, and weight loss. Neither subject had rash, gastrointestinal manifestations, convulsions, change in consciousness, or other neurological deficits. Lastly, two polioviruses were detected and were related to the Sabin-1 polio vaccine strain.





Figure 3. Number of human enteroviruses detected. Number of ILI samples (n = 84) in which human enteroviruses (HEVs) were detected divided into species (A-D) and for each species divided into types (EV = enterovirus, CV = coxsackievirus, E = echovirus, and PV = poliovirus). Number of ILI samples (n = 84) in which human enteroviruses (HEVs) were detected divided into species (A-D) and for each species divided into types (EV =CV = coxsackievirus, E = echovirus, and PV = poliovirus).





By cell culture/immunofluorescence (as well as real time PCR for influenza viruses), we detected other respiratory viruses in 11% of the HRV-positive samples and 11% of the HEV-positive samples. The most commonly detected viruses were adenovirus, influenza virus A and parainfluenza virus 1 (Figure 4 ).



Figure 4. Second virus detected in HRV/HEV positive samples.Number of ILI samples (n = 67) where a second virus was detected in HRV-positive and HEV-positive samples. Number of ILI samples (n = 67) where a second virus was detected in HRV-positive and HEV-positive samples.





Although we found a higher frequency of cough, rhinorrhea, and dyspnea in subjects with HRV-C compared to those with etiologies other than HRV and HEV, this finding was not significantly more common when comparing HRV-C with HRV-A or HRV-B. HEV showed a significantly lower frequency of cough than HRV-C and the non-HRV/non-HEV group. Finally, there was no statistically significant difference in the rate of hospitalization for HRV-C (20%) compared to all other viruses. Other frequencies and comparisons are on Table 1

Table 1. Clinical manifestations of HRV and HEV





We analyzed the temporal distribution of the three HRV species and HEV across six regions, grouped by similar climatic and geographical characteristics. Figure 1 shows the collection sites that were selected and grouped into regions for this analysis. Figure 5 depicts the percentage of each HRV species and HEV per total ILI samples collected and shows that HRV was present in tropical regions (Central America, northern and tropical forest regions) all year long. In no region was HRV or HEV activity detected more often in the rainy season or the higher temperature season. However, HRV-C accounted for a higher percentage (in most cases > 5%) of ILI cases north of the equator (first two panels) over the months of September 2010 to January 2011 and dropped in the following months, while the opposite was seen for the sites south of the equator (last four panels) where the detection of HRV-C increased during the months of April 2011 to July 2011.



Figure 5. Temporal distribution of HRV and HEV infections by region.The percentage of viral infections (positive samples/total collected ILI samples per region in Figure The percentage of viral infections (positive samples/total collected ILI samples per region in Figure 4 ) detected monthly is shown for HEV and each HRV species. The total ILI samples collected per month in each region is represented by the continuous purple line. The rainy season (RS) is depicted by a red dotted line.







For the 632 HRV and HEV sequences from Latin America, separate phylogenetic trees were inferred for two regions: the 5UTR and the VP4/VP2 protein-coding region (Figure 6 ). A lack of detectable spatial patterns in the data is shown for the VP4/VP2 coding region (upper left tree), with all viruses detected in all countries (data not shown).



Figure 6. Phylogenetic analyses of HRV and HEV. This illustrates that recombination events are much more prevalent in the untranslated regions (UTRs) compared with a translated region (VP4/VP2). Separate alignments of the coding (VP4/VP2; 464 nt) and untranslated (5UTR; 555 nt) regions sequences were constructed using MUSCLE v.3.8.31. Maximum likelihood phylogenetic trees were inferred separately for the non-coding and coding regions using PhyML v.3.0 using a general-reversible substitution model with gamma-distributed among-site rate variability. Samples are labeled by following format: Sample code / Country of collection / Month- Year of collection. Phylogenetic trees were colored by HRV species: HRV-A, HRV-B, HRV-C, and four types of HEV (HEV-A, HEV-B, HEV-C and HEV-D). In addition, four HRV-C clades show different recombination events and these are denoted individually as HRV-C.I to IV. This illustrates that recombination events are much more prevalent in the untranslated regions (UTRs) compared with a translated region (VP4/VP2). Separate alignments of the coding (VP4/VP2; 464 nt) and untranslated (5UTR; 555 nt) regions sequences were constructed using MUSCLE v.3.8.31. Maximum likelihood phylogenetic trees were inferred separately for the non-coding and coding regions using PhyML v.3.0 using a general-reversible substitution model with gamma-distributed among-site rate variability. Samples are labeled by following format: Sample code / Country of collection / Month- Year of collection. Phylogenetic trees were colored by HRV species: HRV-A, HRV-B, HRV-C, and four types of HEV (HEV-A, HEV-B, HEV-C and). In addition, four HRV-C clades show different recombination events and these are denoted individually as HRV-C.I to IV.





The RDP, BOOTSCAN, and GENECONV algorithms available in the RDP were used to identify recombinant viruses within the coding and 5UTR regions, separately. No recombination events were detected within the coding regions, but recombination was observed in the 5UTR region for eight rhinoviruses (by at least two of the three methods, p < 0.05).



Recombination between the coding and 5UTR regions was identified by inferring separate phylogenies for the coding and 5UTR sequences and identifying major incongruencies in tree topology.



The lower right tree depicting the 5UTR region shows that recombination events occur and their outcomes are circulating in this region. HRV-C frequently acquired the 5UTR region of HRV-A by recombination processes, including multiple different clades, but this was not observed with HRV-B or any of the HEVs. Members of the HEV-A and HEV-B clades also seemed to undergo recombinant events at the 5UTR region.



17,27,

The HRV-C recombinant strains accounted for the majority of HRV-C viruses detected (Table 2 ) as only clade I contained the HRV-C with no recombination events. In Figure 5 , 5UTR analysis, we have labeled four HRV-C clades as they show different recombination events. We show one previously published isolate that is most representative of each clade (Table 2 ) [ 6 28 ] which allowed placement of known isolates like the NAT045 isolate in clade HRV-C.II and the Antwerp HRV 98/99 isolate in clade HRV-C.IV to better understand the variability of the HRV-C strains and to compare to other typing proposals [ 29 ].