Subtypes A and B of HRSV were found co-circulating among children in the apparently healthy category as well as among children seeking medical care due to respiratory infection. This is not surprising, as co-circulation of HRSV in a community is a well-known and common occurrence both in the tropical and temperate regions of the world, as well as in developed and developing countries (Aamir et al., 2013; Fall et al., 2016; Gimferrer et al., 2015; Scott et al., 2004).
HRSV subtype A was the predominant subtype detected in Ibadan, Nigeria. To the best of our knowledge, there is no previous report published on HRSV subtype detection in any part of Nigeria, to which comparison of this study could be made. However, the subtype A predominance has similarly been reported in Vietnam (Tran et al., 2013)(Tran et al., 2013), Kenya (Scott et al., 2004)(Scott et al., 2004) and Philippines (Malasao et al., 2015) among other places(Malasao et al., 2015). Available evidence in the literature (Dong et al., 2015; Mlinaric-galinovic et al., 2009, 2012; Peret et al., 1998) shows that the predominant subtype varies and alternate from time-to-time as well as place to place(Dong et al., 2015; Mlinaric-galinovic et al., 2009; Mlinaric-galinovic et al., 2012; Peret et al., 1998). One possible explanation for the alternation in predominance is the development of specific immunity against a specific RSV type that is prevalent in a given area at the preceding year. Interestingly, subtype B was the predominant subtype detected among the apparently healthy children attending PHCs. This is in concordance with the findings of Tran et al., (2012) that subgroup B infected children were admitted to the hospital less often than subgroup A infected children. Although 8 of 41 (19.5%) samples detected to be HRSV-positive by the matrix gene amplification could not be successfully subtyped, most of which were those collected at SHF from children seeking medical care due to respiratory infection, we can confidently draw inference from the available data that subtype A was predominant among children seeking medical care due to respiratory infection. This showed that HRSV A was more associated to hospital visits and admission than HRSV B. All the participants with disease severity score ?8 were those infected with HRSV subtype A. This may be an indication that infection with subtype A resulted in more severe disease than that of subtype B. The result of the severity scoring further corroborates the result of the HRSV subtype detected among the few children diagnosed by clinicians and hospitalized on account of bronchopneumonia and bronchiolitis. All HRSV detected from participants with these clinical diagnoses belong to the subtype A. The number of participants diagnosed of bronchopneumonia and bronchiolitis are very small hence we cannot conclude that HRSV subtype B may not also be implicated in such clinical outcome. However, our report agrees with most studies (McConnochie et al., 1990; Mufson et al., 1988; Taylor et al., 1989; Tran et al., 2013; Walsh et al., 1997) that HRSV subtype A is associated with more severe clinical disease than subtype B.
Overall, the prevalence of HRSV among the study participants was 17.7%. This is an indication that HRSV is still a significant cause of respiratory infection in Nigeria as previously reported (Faneye et al., 2014; Odaibo et al., 2013; Olaleye et al., 1992). This prevalence is lower than the prevalence gotten from a previous study conducted in Ibadan and carried out in the same department (Odaibo et al., 2013). A prevalence of 35.4% was reported by Odaibo et al. (2013). The difference in prevalence may be due to the fact that oropharyngeal and nasopharyngeal swab samples were collected in our study, whereas they used nasopharyngeal wash. The detection of respiratory viruses in patient samples has been shown to be highly dependent on the source of the clinical specimens among other factors (Abu-Diab et al., 2008). Nasopharyngeal wash and aspirates have generally been considered to be superior to swab specimens for detection of respiratory viruses (Treuhaft et al., 1985; Masters et al., 1987; Loens et al., 2009). However, obtaining a wash: is more difficult than obtaining a swab, usually unpleasant to the patient, requires specialized training and equipment, and may not be feasible in an outpatient or field setting. Although the rate of RSV detection is also dependent on the sensitivity of the detection method, the PCR method employed in this study is known to be more sensitive than the serological assays used by the previous studies in Nigeria, hence the lower prevalence detected in this study could not be attributable to the method used. The difference in prevalence may also be due to change in climatic and environmental conditions as well as change in severity of RSV epidemics from time to time as shown by the varying prevalence rates in previous studies in Nigeria (Nwankwo et al., 1988; Olaleye et al., 1992). Prevalence similar to that reported in this study was also reported among children less than 5 years in Kenya (Bigogo et al., 2013) as well as Sudan (Khalil et al., 2015)(Bigogo et al., 2013). Our HRSV detection rate is distantly higher than the(Odaibo et al., 2013; Olaleye et al., 1992; Robertson et al., 2004) 0.41% positivity detected by Akinloye et al., (2011) among relatively similar population in Ibadan. This may be due to the fact that our PCR for HRSV detection was done using two different sets of primers that target the conserved M and N gene of the virus. Varying HRSV positivity/prevalence has been recently reported from different parts of Africa, including 60.4% from Ghana (Obodai et al., 2014), 21.2% from Madagascar (Razanajatovo et al., 2011), 11.9% from Gabon (Lekana-Douki et al., 2014), 11.4% from Senegal (Fall et al., 2016) and 5.7% from Cameroon (Njouom et al., 2012). The discrepancies in the reported prevalence could be linked to variation in technical approaches, geographical differences in virus burden, the number of patients tested, the periods during which samples were collected as well as the type of sample collected and even the period and length of the study.
The prevalence of HRSV vary sharply in the two groups of participants in this study. The participants attending primary health centers (PHCs) were adjudged apparently healthy by their parents / guardian and were only brought to the hospital to receive vaccine, unlike their counterparts that presented at the secondary health facility (SHF) purposely for care on respiratory tract illness. It is therefore not surprising that the prevalence of HRSV (34.6%) among children seeking care for respiratory infection was significantly higher than the 8.7% obtained among apparently healthy children. The prevalence of HRSV among those seeking care for respiratory tract infection showed that HRSV infection is a major reason for hospital visits among children aged 5 years and below in Nigeria. Reports from other parts of the world has also shown that HRSV infection is a major reason for hospital visit in developing (Okiro et al., 2012) as well as developed (Iwane et al., 2004) countries.(Okiro et al., 2012) Laboratory diagnosis as well as treatment regimen for respiratory infection in Nigeria and perhaps in other developing countries more often focus on bacterial etiological agents than viral agents. The prevalence of HRSV among those seeking care for respiratory tract illness therefore brings to fore, among other things, the need to test for virus in addition to the bacterial screening, for better management of the patients.
Phylogenetic analyses showed that two genotypes of HRSV-A: ON1 and NA2 were circulating among children in Ibadan, with ON1 genotype being the predominant of the subtype A detected. Genetic analyses have shown that the ON1 which evolved from genotype NA1 (Hirano et al., 2014); has signature 72nt insertion at the C – terminal end of the attachment glycoprotein and was first reported in Ontario in Canada in the year 2012 (Eshaghi et al., 2012). The prototype of the ON1 genotype is the strain ON67-1210A with the characteristic 72nt insertion in G, translating into 24 additional amino acids, 23 (QEETLHSTTSEGYLSPSQVYTTS) of which are exact duplicates of the preceding amino acids at positions 261–283. Unlike the prototype strain, ON1 strains detected in Ibadan Nigeria did not have their nucleotide insertions translated into duplicate of the amino acid at positions 261-283. The ON1 genotype has been increasingly detected in many countries (Cui et al., 2013; Agoti et al., 2014; Ren et al., 2014; Tabatabai et al., 2014; Fall et al., 2016) since it was first reported in Canada, and is now replacing other HRSV A genotypes in different parts of the world. Like its ancestor that emerged in 2004 and became predominant in many areas within a period of 10 years (Forcic et al., 2012; Tan et al., 2012; Cui et al., 2013; Etemadi et al., 2013; Pretorius et al., 2013; Fernanda de-Paris, Beck et al., 2014), in no distant time, the ON1 genotype may become the predominating HRSV-A globally, going by the reports from different parts of the world (Agoti et al., 2014; Kim et al., 2014; Panayiotou et al., 2014 Duvvuri et al., 2015). As at September 2015, when the last sample identified as ON1 in this study was collected, sequence information available in GenBank showed that this genotype had been reported in about twenty-one countries that spread across almost all the continents of the world (Duvvuri et al., 2015) and may be circulating undetected or unreported in many other countries.
All the HRSV-B viruses found in this study belong to the BA genotype which was first reported in Buenos Aires, Argentina in 1999 (Trento et al., 2003) with characteristic 60 nucleotides (20 amino acids) duplication in the C-terminal end of the G glycoprotein. Similar to the ON1 genotype, the BA genotype has been detected in many countries nearly predominating and replacing previously circulating genotypes of HRSV subtype B including the SAB1, SAB2 and SAB3 previously detected in some parts of Africa (Venter et al., 2001; Scott et al., 2004; Shobugawa et al., 2009; Dapat et al., 2010; van Niekerk and Venter 2011; Panayiotou et al., 2014 Esposito et al., 2015;). Although there are no prior data on the genotypes of HRSV circulating in Nigeria, based on reports from other countries, we could also say that the BA genotype is most likely replacing previously circulating genotypes of HRSV B in Ibadan and possibly other parts of Nigeria. The BA genotype has shown high diversity resulting in emergence of different lineages, including the BA1 – BA12, and the recently classified BA-CCA and BA-CCB (Zheng et al., 2017). The isolates of the BA genotype appeared to be the most divergent of the genotypes detected in this study as shown by the ‘between isolates’ p – distance. Phylogenetic analysis showed that the BA genotypes from this study clustered in three different groups. Group 1 (NGR/OA31/15-RSVB, NGR/OR01/15-RSVB, NGR/OR75/15-RSVB and NGR/OR79/15-RSVB), Group 2 (NGR/OA01/15-RSVB, NGR/OA04/15-RSVB, NGR/OA19/15-RSVB, NGR/OA30/15-RSVB, NGR/OA36/15-RSVB and NGR/OR27/15-RSVB), Group 3 (NGR/OA10/15-RSVB). The group one appeared in same cluster with the BA genotype isolated from Vietnam and previously assigned to the sub-genotype BA9 (Tran et al., 2013). Although the group 1 also appeared in the same clade with isolates previously assigned to lineage BA11, nucleotide blast results further showed their 99% similarity with the BA9 lineage including those isolated from China (GenBank accession number KT781359). The Nigeria isolates in the group 2 appeared in the same cluster with the BA genotype from North-Eastern China recently designated as the BA-CCA and BA-CCB (Zheng et al., 2017). However, nucleotide blast of the sequences of the group 2 in GenBank showed their high relatedness to viruses isolated from China, and assigned to the BA9 lineage, with accession number KT781370 and KT781379 (Unpublished). Genotype overlap and disagreement in assigning to a particular sub-genotype or lineage is a common occurrence in the BA genotype. This often results due to the different reference sequences used by different studies. For example, Bose et al (2015) reported that sequences previously classified as belonging to the BA4 lineage were intermixed with those reported as belonging to the BA7, BA8, BA9, and BA10 lineages. The BA9 lineage was first identified during the 2006-2007 season in Niigata, Japan (Dapat et al., 2010) and was subsequently reported to be the predominating HRSV-B globally in the 2009-2010 season (Ohno et al., 2013). Since the time of the first report of the BA9, it has been detected in more than twenty-three countries across different continents of the world (Haider et al., 2018). Whereas Haider et al. suggested that the global transmission of the BA9 lineage of the BA genotype could be associated with travelers, the accumulation of positively selected mutations especially at the region of the twenty amino acid insertions could also confer a fitness advantage on the BA9. This HRSV-B genotype has also been reported in West Africa, and so far, it is the only HRSV-B genotype reported in this region (Fall et al., 2016). The third group of the BA genotype found in this study consist of a single isolate that did not form cluster with any of the reference BA genotype on the phylogenetic tree, perhaps pointing to the likelihood of the emergence of a distinct/new BA sub-genotype. It is however not surprising that the isolate did not appear in similar clades with other isolates from this study as the said isolate had seven unique amino acid substitutions (at positions 231, 243, 254, 260, 269, 279 and 294) which were not found in any of the other isolates. Raghuram et al. (2015) made a similar report where two of the BA sequences from their study could not be assigned to any of the BA sub-genotypes. The length of the attachment glycoprotein of the BA genotype from Nigeria differ from one isolate to the other. Variability in the attachment glycoprotein of HRSV subtype B is known to often result from mechanism which include: amino acid substitution, insertion, deletion, duplication, and change in stop codon usage (Zlateva et al., 2005; Zhan et al., 2010). Whereas all the isolates had the 60 nucleotides (translating into 20 amino acids) insertions characteristic of the BA genotype, and there were no deletions, the variations in the glycoprotein length resulted from the variability in their stop codon usage. The isolates using ‘TAA’ as stop codon had 312 amino acid lengths while those using ‘TAG’ had 319 amino acids. This is not an unexpected occurrence as BA genotypes are known to be highly variable in their stop codon usage, with previous reports of the use of TAA and TAG (Tran et al., 2013; Bose et al., 2015; Raghuram et al., 2015) as well as CAG (Trento et al., 2003) stop codons usage. Premature stop codons in RSV has been suggested to have drastic antigenic changes on the attachment glycoprotein, perhaps by affecting the protein folding pattern thereby leading to absence of reactivity with most anti-G antibodies (Rueda et al., 1991). The isolates with 312 amino acids could therefore be more evasive to antibody neutralization. The stop codon variability observed among the BA isolates were not found in the ON1 isolates. This among other reasons may account for the relative conservation known with the ON1 genotype unlike the variability in the BA leading to the multiple BA lineages like BA1 – BA12 (Zlateva et al., 2005, Zheng et al., 2017)
Upon comparison of the nucleotides as well as the deduced amino acid sequences of the 2nd hypervariable region of HRSVA, it is clear that the ON1 subtype from our study vary considerably from the prototype ON1 (GenBank accession no. JN257693) due to accumulation of mutations. The substitution of amino acid proline for leucine at position 274 (L274P) was found in most of the Nigerian ON1 isolates while similar amino acid substitution at position 298 (L298P), within the duplicated region of the attachment glycoprotein of the virus was detected in all the isolates. Substitutions at these two sites (i.e L274P and L298P) have been reported to often occur concurrently (Agoti et al., 2014). Although the functional effect(s) of most of the substitutions we found are not well known, substitution L274P is often found in vaccine escape mutants (Gustavo et al., 2012) and has been well reported to be associated with resistance to neutralizing antibody (Rueda et al., 1991; Martínez, Dopazo, and Melero 1997; Botosso et al., 2009). The substitution of amino acid arginine for glycine at position 232 (G232R) was found in one of our isolates (NGR/OL63/15-RSVA). The effect of this substitution was not established in our study, however substitution at this position has been suggested to improve the probability of RSV survival (Zheng et al., 2017). Howbeit, Zheng et al. (2017) detected the substitution with glutamic acid at the position 232 (G232E) rather than the arginine substitution that we detected. Similarly, we detected the amino acid substitution of lysine for glutamic acid at position 262 (E262K) which is reported to help RSV evade human immunity, thereby enhancing viral survival (Zheng et al., 2017). One ON1 isolate (NGR/OL76/15-RSVA) had the substitution of isoleucine for threonine at amino acid position 292 (T292I) which is within the 24 amino acid insertion regions. As far as can be ascertained, this substitution has not been previously reported, therefore showing the genetic diversity of the isolate. Amino acid substitutions also occurred on some of our isolates at positions 249, 272, 274. These positions were previously predicted to bear positively selected substitutions (Agoti et al., 2014), hence these isolates may be well adapted to evade host immunity (Botosso et al., 2009; Tapia et al., 2014)It is expected that the G glycoprotein will be under immune selection pressure due to its importance in viral attachment and as a major target of neutralising antibodies. The bulk of the variation and the positive selection for amino acid changes are concentrated in two hypervariable regions of the G protein which is also known to contain multiple epitopes (Kim et al., 2014). Our analysis of the positive selections acting on the second hypervariable region of the G glycoprotein showed the different amino acid substitutions that are involved in the variability of the ON1 genotype of RSV-A and the BA9 genotype of RSV-B globally. Among all the ON1 genotypes globally, ten positive selection substitutions were detected, three of which were found in the Nigeria isolates. It is interesting to note that the three positive selection sites found in the Nigeria isolates were located within two of the common epitopes of ON1. There are about four B-cell epitopes within the 2nd hypervariable region of the ON1: aa221-242, 251-265, 272-284 and 287-299 (Kim et al., 2014). Seven of the eleven ON1 isolates (NGR/OL64/15-RSVA, NGR/OL76/15-RSVA, NGR/OL88/15-RSVA, NGR/OL96/15-RSVA, NGR/OL97/15-RSVA, NGR/OA02/15-RSVA and NGR/OA20/15-RSVA) had positively selected substitutions of proline for leucine at amino acid position 274 and histidine for tyrosine at amino acid position 297. These substitutions were within the third and fourth B cell epitopes. All the isolates (except NGR/OL93/15-RSVA) also had the substitution of proline for leucine at amino acid position 298. These sites of positive selections are under adaptive evolution in the G protein and the substitutions may enhance the sustained transmission of this genotype in Nigeria as it makes the virus better fit to evade hosts neutralising antibody. The presence of amino acid substitutions on two or more of the epitopes from this study coupled with the characteristics 72 nucleotide insertion may mean that the ON1 genotype will remain the predominant RSV-A genotype in Nigeria, just like it has been in other parts of the world (Cui et al., 2013; Agoti et al., 2014; Ren et al., 2014; Tabatabai et al., 2014; Fall et al., 2016). Six putative positive selection substitutions were found in all the available BA9 sequences used for the in-silico analysis, three of which were detected in the BA9 isolates from Nigeria. These substitutions (Leu219Pro, Leu223Pro and Asn293Tyr) as well as other substitutions (Ser247Pro, Thr270Ile, His287Tyr and Glu305Lys) found in the isolates are known to enhance the immune evasion potential of the virus and eventually its sustained transmission (Haider et al., 2018).
There were numerous sites of negative selection on both the ON1 and the BA9 isolates. The is not surprising since most amino acid mutations in RNA viruses are deleterious, hence purifying selection is the most vital in their evolution (Holmes, 2013) We also estimated negative selection for sites in the present strains. In general,negative selection plays a role in preventing the functional deterioration of variousviruses (Domingo, 2006). For example, Domingo et al. demonstrated that negativeselection in neutralization epitopes of polioviruses was involved in receptor recognitionand in the formation of altered virions (Domingo et al., 1993). The roles of negativeselection at sites in the HRSV G protein are not clear; however, it is possible that theseamino acid substitutions act in the prevention of antigenic deterioration (Domingo,2006; Hirano et al., 2014).
Four different antigenic sites have been previously identified in the HVR2 of HRSV-A (aa229-240; 250-258; 265-273 and 283-291) and numbered relative to the prototype A2 strain (Cane 1997). The NA2 isolates from this study had some point mutations on some of these antigenic sites. These may have important antigenic and immunogenic implication on the viral survival. Similarly, two ON1 isolates (NGR/064/15-RSVA and NGR/OL97/15-RSVA) have amino acid substitution of serine for glycine at position 272 which is within the antigenic site 265-273, thereby showing the antigenic diversity among the ON1 strains.
While the ON1 isolates supposedly predisposed to more severe outcome as they were predominantly detected among those presenting for care of respiratory infection at the secondary health facility, an isolate was detected among the apparently healthy participants that came for vaccination at the PHCs. It is worthy of note, that the isolate NGR/OA02/15-RSVA had an aa substitution of isoleucine for threonine at position 245 that was not found in the other isolates. It remains to be known, whether this substitution mitigate virulence and predispose to less severe outcome of infection. Although a follow up on the child from whom the specimen was collected could have better informed us as to the eventual outcome of the infection, it was not feasible to get this information due to the lapse of time between the sample collection and the availability of the sequence information.
The HRSV-B isolates could be thought to cause less severe disease outcomes as they were detected predominantly among apparently healthy participants. However, a single isolate of the subtype B (NGR/OL79/15-RSVB) was detected among those presenting for care of respiratory tract infection at the SHF. This isolate interestingly had a unique amino acid substitution of lysine for glutamic acid at position 226 (E226K) that was not found in the isolates from the apparently healthy participants. One could therefore cautiously hypothesize that the amino acid substitution could predispose to more severe outcome of infection.
N – and O – linked glycosylation of the G protein may influence the antigenicity of HRSV by masking the epitope expression thereby affecting antibody recognition and by extension aiding immune evasion (Palomo et al., 1991; Garcia-Beato et al., 1996; Khan et al., 2014; Raghuram et al., 2015). Whereas the isolates belonging to the ON1 genotype have similarity with the prototype strain in the number and sites of N – linked glycosylation, the occurrence of more sites of O – linked glycosylation on the isolates showed the diversity in the antigenicity of the ON1 genotype. In the NA2 isolates, substitutions of tyrosine for asparagine, as well as leucine for proline at positions 273 and 274 respectively culminated into the loss of an N – linked glycosylation site. Although the number of N – linked glycosylation in the isolates relative to the prototype is same, as the loss of glycosylation was compensated for by glycosylation at another site due to the substitution of threonine for proline at position 320. It remains to be known whether the alteration in the site of the N – linked glycosylation has a debilitating effect on the pathogenicity of the NA2 genotype or not. It has been shown that alteration of site(s) of glycosylation could have drastic impacts on the survival and transmissibility of HIV and hepatitis C virus both positively and negatively (Land and Braakman, 2001; Slater-Handshy et al., 2004; Vigerust and Shepherd, 2007). The fact that alteration of the site(s) of glycosylation affects viral interactions with receptor (Vigerust and Shepherd, 2007) may be one of the reasons for the low circulation of the NA2 genotype in Ibadan, Nigeria. While some BA genotypes in Nigeria have two N – glycosylation sites, others have a single glycosylation site as substitution T312N led to the loss of potential N – glycosylation sites in some isolates.
To date, the only approved prophylaxis for the prevention of infection with HRSV is the passive administration of RSV-specific immunoglobulin Palivizumab. Palivizumab (Synagis; MedImmune) is an HRSV neutralizing humanized murine monoclonal antibody (MAb) reactive with a defined epitope on the HRSV fusion (F) protein (Zhao et al., 2006). There have been some reports describing mutations in the putative palivizumab binding site of palivizumab in cell culture, cotton rats and in human population (Adams et al., 2010; Boivin et al., 2008; Zhao and Sullender 2005). We found in this study that the region of antigenic site II, within which is the Palivizumab-binding site, is relatively conserved between both subtypes of HRSV (Figure 4.9). However, at amino acid position 276, there was the substitution of serine for asparagine in all of our isolates when compared to the prototype A2 strain. There are controversies over the functional effect of the observed substitution N276S on palivizumab effectiveness. While some suggest that the said substitution confer total resistance against palivizumab (Adams et al., 2010), other reports showed that the substitution cannot, as a stand-alone, confer complete resistance to Palivizumab (Zhu et al., 2012; Papenburg et al., 2012, Xia et al., 2013). They submitted that N276S predisposes to another substitution of lysine for glutamic acid or glutamine at aa position 272 (K272E) which then lead to complete resistance against palivizumab. Substitution of asparagine for serine at position 276 on the fusion glycoprotein is a more common occurrence on subtype B of HRSV than the subtype A (Adams et al., 2010). Palivizumab is not yet in use for HRSV prevention in Nigeria, it is therefore not clear whether N276S is a natural but rare mutation or it emerged under the selection pressure of palivizumab and transmitted to Nigeria.
Most of the participants in this study were within the age group 12 months and below, with the bulk of them within the age group 6 months and below (Table 4). This agrees with the known fact that children age group 12 months and below are more susceptible to respiratory infection compared to children aged above 12 months. Unlike most studies where the highest prevalence was reported in younger age groups(Fall et al., 2016; Tran et al., 2013; Weber et al., 1998), we found here that the prevalence of HRSV increased with age. This is likely due to the fact that the samples collected tapered significantly as the age group increased, together with the fact that the participants in age groups 12 months and below were mostly from the PHCs, who were apparently healthy.
Gender was not significantly associated with susceptibility to HRSV infection in our study, like in other studies (Aamir et al., 2013; Tran et al., 2013)(Aamir et al., 2013; Tran et al., 2013) although the prevalence was slightly higher in male children. Various studies suggest that male children are more susceptible to severe HRSV infection than females (Eshaghi et al., 2012; Goto-Sugai et al., 2010). Although all the two children admitted into the secondary health facility with clinical diagnosis of bronchiolitis during the course of this study were male, on the other hand, 75% of participants clinically diagnosed with bronchopneumonia were females. These small numbers of participants with severe outcomes are therefore not sufficient to make inferences on the association of gender to disease severity.
HRSV was detected in all the participants clinically diagnosed of bronchopneumonia as well as 50% of those diagnosed of bronchiolitis. This is in tandem with other reports that HRSV is the leading etiology of pneumonia and bronchiolitis globally (Khalil et al., 2015; Lamarao et al., 2012; Shay et al., 1999; Shi et al., 2017; Venter et al., 2011). All the children with clinical diagnosis of bronchiolitis and bronchopneumonia from whom HRSV were detected were 12 months and below, with 3 of the 5 (60%) being below 6 months of age. This is similar to the finding by Lamarao et al., (2012), and further lends credence to available evidence in the literature (D´Elia et al., 2005; Lee et al., 2007; Midulla et al., 2010; Piedimonte and Perez 2014; Sricharoenchai et al., 2016)(D´Elia et al., 2005; Siqueira et al., 2005; Lee et al., 2007; Midulla et al., 2010; Piedimonte and Perez, 2014; Sricharoenchai et al., 2016) suggesting that HRSV is the main pathogen responsible for bronchiolitis and pneumonia during the first 12 months of life. Illness resulting from HRSV infection in this age group is also known to be severe (Lamarao et al., 2012)(Lamarao et al., 2012).