The frequency of HRV identified in our subjects with ILI is similar or greater to what has been found for influenza virus [30, 31], human metapneumovirus [32, 33], respiratory syncytial virus [30, 31], and adenovirus [31, 34] in the region, emphasizing the potential clinical and public health importance of this virus. Although differences in subject recruitment strategies prevent exact comparison between our studies and others, our HRV detection proportions are similar to pediatric respiratory disease studies from North America and Asia which had proportions that varied between 7.7% and 17.4% [7, 17, 35]. In South America, three separate studies from Brazil observed HRV detection proportions with a broader range, 15.9% to 46.7%, in children with ARI [22, 23, 25].
To further characterize the genetic diversity of these viruses, we sequenced all HRVs and HEVs identified. HRV-A and HRV-C species accounted for the majority of the HRV viruses (58% and 38%, respectively), and showed, in both cases, a great variety of serotypes, including more than 60 for HRV-A. The relative low percentage of HRV-B detected (4% of total HRVs) could relate to the fact that this virus has been shown to present with no fever  and our inclusion criteria required a fever ≥38°C, and thus, we could have underestimated the presence of this virus in the population. However, others have noted similar compositions of the three HRV species, with HRV-B accounting for only 4-11% of all HRVs recovered from subjects with respiratory complaints [7, 17, 22, 35]. Among HEVs, coxsackieviruses, both A and B, were the most predominant. We also identified low numbers of EV-D68, which has been associated with respiratory disease by others , although never in the countries of our study. In addition, we identified two cases of EV-A71, an agent traditionally associated with hand-foot-and-mouth disease, as well as severe neurological and cardiac complications [36, 37]. EV-A71 has also been infrequently linked with respiratory disease in Canada , Taiwan , and Australia , although never in Latin America. While the prior reports were associated with focal EV-A71 outbreaks, no outbreak in Peru was reported during our study period. Nevertheless, the fact our surveillance spanned multiple Latin American countries over a one-year period and the only two EV-A71 cases occurred within two weeks of each other in towns separated by a mere 25 kilometers suggests a possible unidentified localized outbreak. Other similarities between our EV-A71 respiratory cases and those of prior reports include age < 5 years and mild symptoms.
Our study shows that children younger than 1 year had a higher proportion of HRV infection than older children and young adults, which is consistent with previous studies [7, 19, 41]. Children 5 years or younger had a slightly higher risk of infection by HRV-C compared with those older than 5. However, no differences in the prevalence among age groups were detected for HEVs throughout our network.
A second virus was detected in approximately 11% of HRV-positive samples, much lower than a rate (37%) reported by others . We used two methods of detection, PCR for influenza viruses and cell culture for all other viruses (including influenza virus), perhaps allowing for higher identification proportions of influenza and most likely underestimating the number of secondary infections. However, we did not observe more severe symptoms in patients with another respiratory virus identified, a finding that is consistent with what has been reported previously .
Several studies have suggested differences in illness severity between HRV species, including a specific association of HRV-C with wheezing and asthma exacerbation [6, 7, 35]. In agreement with these studies, our results showed a higher frequency of dyspnea in subjects with HRV-C compared with other viruses; in addition, we noted an association of HRV-C detection with pre-existing respiratory diseases, in particular with asthma.
Our phylogenetic analyses showed the presence of almost all known serotypes of HRV-A and HRV-B, as well as the presence of HRV-C. HRV-C variants are not yet formally classified into types, although there have been some proposed typing methods . We show that recombinant events occur at the 5’UTR  consistent with others who reported picornaviruses having recombination breakpoints restricted to non-structural regions of their genome . We showed that the circulating HRV-C recombinants accounted for the majority of all HRV-C species, illustrating how much this virus varies over a very short period of time.
Limitations of our study include a lack of uniformity in samples collected among countries and the fact that we collected samples over a period of only one year, preventing definitive characterization of the seasonality of HRV and HEV in Latin America. Although an analysis spanning three to five years would have been ideal, we analyzed the temporal distribution of the three HRV species and HEVs by aggregating collection sites by their climatic and geographic similarities. As has been previously described in other continents [13, 43], HRVs and HEVs showed a similar “year-long” temporal distribution throughout Central and South America during the one-year period of collection. We did not detect any seasonality for specific HRV-A or HRV-B species, although HRV-C species seemed to possess opposite seasonal trends on either side of the equator. Others have noted seasonal differences among the individual HRV species, including high rates of HRV-A in April in the U.S.  and low rates in summer in China , high rates of HRV-B during winter in Australia  and China , and high rates of HRV-C in October in the U.S.  and winter in China ; like ours, these studies collected samples for only a one or two-year time span. Another important limitation of our study was that the identification of HRV and HEV did not unequivocally imply causality, a factor that would have been obviated with the inclusion of age-matched asymptomatic controls.
Although we are not the first to demonstrate a high prevalence of HRV in children with ILI, our results provide new perspectives into this virus’s global reach and additional insight into its clinical and phylogenetic characteristics in this underreported area of the world. While proven preventive and treatment strategies exist for other common respiratory viruses, including respiratory syncytial virus (pharmacotherapy, passive immunization), adenovirus (active immunization), and influenza virus (pharmacotherapy, active immunization), no HRV vaccine or antiviral currently exists [44, 45]. Our high prevalence of HRV and HEV identification in young Latin Americans indicates that pharmacotherapy against influenza virus or the common bacterial etiologies of respiratory disease should not be presumptive, but guided by diagnostic confirmation when available in this population. Despite the current limitations of rapid diagnosis and clinical management of HRV or HEV respiratory disease, we hope that surveillance such as ours will provide further impetus for the development of rapid diagnostic methods, vaccines, and antivirals, as well as further elucidation of what populations are most at risk and what types of HRV and HEV species are most worth targeting.