Transmission of Respiratory Syncytial Virus genotypes in Cali, Colombia

Abstract Background Colombia's climatological variety, added to pathogen diversity, creates local niches for infectious diseases. In Bogotá, respiratory syncytial virus causes 30%‐52% of the cases of respiratory infections. In coastal or inter‐Andean cities with higher temperature and longer dry seasons, frequency of this virus is 7%‐13%. By 2017, increased hospitalizations due to airway infections occurred in regions whose weather is differently influenced by “El Niño Southern Oscillation” than in Bogotá, although microbial diversity might have also been involved. Methods For Cali, an inter‐Andean city with warm tropical weather, records of respiratory syncytial virus from 2014 to 2018, in children two years old or younger, were analyzed, and genotypes transmitted during 2016‐2017 were identified based on partial sequences of glycoprotein G. Results Most cases of respiratory syncytial virus in Cali occur in the first semesters, with peaks expressed around March‐April, without a clear association with pluviosity. Unlike the biannual rotating pattern of Bogotá, co‐circulation of types A and B was detected. As years pass, transmission seasons are becoming longer and frequencies of the virus augment. The viral genotypes identified follow international trends with dominance of Ontario and Buenos Aires clades. Similar to other isolates in these clades, viruses from Cali exhibit glycosylation variability that may account for their fitness. Conclusions The pattern of respiratory syncytial virus transmission in Cali differs from that in Bogotá. Its epidemiology is shifting and will remain so with the advent of novel respiratory diseases. This may impact the introduction of vaccination schemes for these or other respiratory viruses.


| INTRODUC TI ON
Respiratory syncytial virus (RSV) is one of the main causes of acute respiratory infection (ARI) among infants and the elderly. This virus is particularly associated to the admission into intensive care units in children under the age of two years, who develop either influenza-like illness (ILI) or severe acute respiratory infection (SARI). [1][2][3] Environments with low humidity and high temperatures favor the survival of RSV virions, while during the winter or rainy seasons, the number of infections increases, since more people remain indoors. 4,5 In temperate zones, this virus has seasonal transmission with onsets matching the coldest weeks of winter. 1,6,7 In equatorial countries, RSV is involved in ARI cases all year round, with outbreaks in those rainy seasons of high humidity and low temperature.
RSV has been classified in two types (RSV-A and RSV-B), based on genetic differences in the region encoding glycoprotein G, a protein that facilitates binding of the virus to epithelial cells of the airway. 7 Within these types, 11 RSV-A (GA1-GA7, NA, SAA, ON, LBA) and 23 RSV-B genotypes (GB1-GB4, SAB1-SAB4, URU1, URU2, BA1-BA12, THB) have been established. 9,10 New RSV genotypes arise by mutations and duplications in the glycoprotein G gene and spread according to their antigenicity and virulence; these properties are also influenced by substitutions in the fusion protein. 8,10,11 In Colombia, RSV is endemically transmitted throughout the year, with higher occurrence during the rainy seasons. [12][13][14] RSV-A and RSV-B circulate in a biannual rotational dynamic characterized by an increase in cases and hospitalizations when RSV-A predominates. 15 This observation results from a couple of studies and whether the prediction accommodates to demographics and climatic diversity across the country is yet to be established. 12,14,16 Recently, 37 samples collected between 2000 and 2009 were genotyped for RSV. 17 Researchers found genotypes RSV-A-GA2, RSV-A-GA5, and RSV-B-BA circulating in the country; however, data are scattered at random dates within this period, and most samples come from a single location.
New variants of RSV-A with duplications in glycoprotein G are replacing genotypes like those found in Colombia by Avila and coworkers.
Among these, RSV-A-NA1 was associated with severe bronchiolitis in children, 18 and RSV-A-ON1 has been linked to higher severity of ILI, to a sooner onset for hospitalization, 19 and to longer stays in intensive care units. 20 Genotype RSV-A-ON, initially detected in Ontario (Canada) in 2010, is believed to have virulence comparable to its more likely predecessors, genotypes RSV-A-GA2 and RSV-A-NA1, 19,21,22 but with a higher dispersal capacity. 23,24 In spite of its broad distribution during recent years, no official record has been established on the presence of genotype RSV-A-ON in Colombia.
According to the National Epidemiological Surveillance System (SIVIGILA), onsets of RSV transmission in Cali, main city in Valle del Cauca, coincide with those reported nationwide. 13,25 However, for 2016 and 2017, nationwide proportions of RSV in virus-positive samples were 51% and 67%, while in Cali the RSV percentages among virus positives were 50% and 51%. 26,27 In 2017, increase of ILI-SARI and augment of RSV and influenza A frequencies was observed in Valle del Cauca. 25 Although many of these differences are attributable to methodological variations in RSV detection, microclimatic and virus-derived factors may also account for RSV prevalence across the Colombian territory. This work deepens the analysis of the epidemiology of ILI-SARI cases associated with RSV among infants in Cali, by reviewing the virus circulation patterns in 2014 -2018, and detecting genetic variants circulating in the city in years 2016-2017.

| ARI cases related to RSV in Cali
To construct a RSV circulation curve in children in Cali, younger than

| Genotype assignment and variability in selected samples
The

| Relationship between RSV subtype and symptoms
The association between the main clinical manifestations and RSV type was analyzed by comparing proportions between independent groups. Qualitative variables (fever, coughing, etc) were compared using Fisher's exact test with Bonferroni correction. Student's test was used for the comparison of quantitative data (ie, length of stay in intensive care unit); values were taken as different when the corresponding test showed a probability value lower than 0.05.

| Surveillance of RSV infections in Cali
Surveillance for ILI-SARI in the city has improved during the last five years, with the consequent augment in RSV reports. By 2014, these data were almost absent, but in the following years reports increased by four to eightfold. Between 2014 and 2018, a total of 6,440 ILI-SARI cases were reported to SIVIGILA from Valle del Cauca, 4,083 of them autochthonous from Cali ( Figure 1). Almost half of these cases,

| RSV-A and RSV-B genotypes in Cali 2016 -2017
Genotype of RSV-A detected during both years was ON, while RSV-B sequences belong to the BA clade (see Figure 4A and 4B).

| Amino acid profile in the hypervariable region of glycoprotein G
As observed in Figure 5A, the fragment of glycoprotein G sequenced for these samples revealed the duplication characteristic of ON variants (residues 282-306). There were 12 variable sites compared to five in the original region (residues 259-289). However, the variation per site is low, as ten of the variable positions detected in the duplication are unique to one sequence. Residues prone to be N-glycosylated occur before this duplication, and four of them (N237, N242, N251, and N255) are highly conserved among isolates from Cali, ON variants and representatives of NA1 genotype; an additional site with potential for N-glycosylation occurs in a few isolates from Cali. Amino acids with potential for O-glycosylation are placed near or in the duplication itself; except for LDAB-369, which has two of these residues, isolates from Cali have four or more residues that cross the threshold for O-glycosylation potential, similar to KJ710391-NA1 and the ON variants referenced.  Figure 5B shows the duplication characteristic of the BA clade and how it is involved in the diversification of glycoprotein G. All clades contain conserved sites for O-glycosylation at the c-terminal, while N-glycosylation sites, also conserved, are located before and after the duplicated region. Interestingly, the duplicated region in these variants is not accumulating residues prone to glycosylation and variability is low, if compared to the duplicated stretch in RSV-A variants.

| RSV genotype and clinical manifestations
About one third of samples positive for RSV correspond to cases whose medical records were available (Supplementary Table 1).
Overall, RSV is causing mild to moderate infections that manifest with cough, nasal congestion, and respiratory difficulty. Hospitalization was required in 75% of these cases. Males were slightly more prone  Table 2).

| D ISCUSS I ON
RSV transmission pattern in Cali is differentiable from that described in national conglomerates and data exclusively from Bogotá, although some characteristics of the main transmission season are common. In Bogotá, where historically more data are captured by SIVIGILA, 34 RSV prevalence ranges between 30% and 52%, with onsets correlated with the first rainy season, during the months of March-May. 16 Bogotá has an average temperature of 13.1°C and relative humidity ranging from 77% to 83%, whereas some other main cities in the country are coastal or inter-Andean, with average temperatures above 20°C and longer dry seasons. Prevalence of RSV in the warmest cities ranges between 7% and 13%, without a clear link to rainy seasons. 16 According to oceanic Niño indexes, a variable that uses changes of oceanic temperature in the Pacific area influenced by El Niño Southern Oscillation to define warm ("el niño") and cold ("la niña") intervals, the most recent warm period was recorded for 2016, fol- The pattern of transmission of RSV observed in Cali is co-circulation of the two virus types, which implies that infants are at risk of reinfection. 35 However, no cases of co-infection with multiple genotypes were observed. Putting together SIVIGILA data with RSV types detected per week, the trend is to have longer transmission seasons of both genotypes. Whether this is an effect of the above-mentioned climatological phenomenon could be evaluated by expanding RSV variability surveillance. Evidence accumulated here suggests that in Cali, RSV transmission seasons are changing to last all year round.
RSV-A samples characterized clearly belong to the contemporary ON clades. 23,33 The same was obtained for RSV-B, with variants that group to the BA9 and more recent subclades. 9 This variability has been described in different parts of the world, and therefore, in Cali the transmission of RSV does not occur as an isolated event, but rather as a result of the successful genotypes spreading throughout all continents.
We found no association of the type of virus with disease severity. However, findings are to be taken cautiously as the small number of samples analyzed here could be misleading. Similarly, associations between RSV-A-ON and SARI, as previously reported, 19,20,24 could not be evaluated with the amount of genetic variation obtained in this study.
Since glycoprotein G is one of the main antigens on the surface of RSV, its variability is directly reflected in viral fitness. In the second hypervariable region, known to support a considerable amount of carbohydrates, 37 the glycosylation potential showed no particular glycosylation profile as a characteristic of the newest RSV variants. However, a series of conserved threonine O-glycosylation sites were spotted as likely indispensable for the functioning of this domain. The duplicated region of RSV-A augments the amount of N-glycosylation sites, but this is not the case in RSV-B variants. In RSV-B variants, the duplication increased the distance between conserved glycosylation sites; whether this has structural effects, such as an improvement in the affinity of glycoprotein to membranes, or an evasion of immune response, is yet to be established.
This work represents an update of RSV epidemiology in Colombia and describes, for the first time, the circulation of ON1 genotype in the country. Unlike samples collected from Bogotá and a few other places before 2010, only RSV genotypes with duplications of glycoprotein G were found in Cali, suggesting a complete establishment of these genotypes in the country. There was no association between RSV type or genotype to the severity of SARI, which may also indicate that clinical manifestations depend on patient's risk factors that promote RSV infection complications.

ACK N OWLED G EM ENTS
Funding for this analysis comes from "Grant funds for the promotion of scientific publications," Universidad del Valle, 2017.

CO N FLI C T O F I NTE R E S T
We declare no conflict of interest.