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Epidemiology and genetic characterization of respiratory syncytial virus in children with acute respiratory infections: Findings from the influenza sentinel surveillance network in Central African Republic, 2015 to 2018

Abstract Background and aims Respiratory syncytial virus (RSV) is one of the main viral pathogens causing acute respiratory infections in children under 5 years of age but has seldom been studied in Central African Republic (CAF). Taking advantage of the national influenza surveillance network in CAF, this study aimed at providing the first insights into RSV prevalence and seasonality over 4 years of surveillance and the clinical manifestations of RSV in this population in CAF. Methods A total of 3903 children under 5 years matching the influenza‐like illness (ILI, 68.5%) or severe acute respiratory infection (SARI, 31.5%) case definitions were recruited from January 2015 to December 2018. The presence of RSV viral RNA in nasopharyngeal samples was assessed by RT‐PCR, followed by RSV‐A and RSV‐B typing and Sanger sequencing on a subset of samples. Phylogenetic analyses were carried on partial G protein sequences. Associations between RSV and demographic or clinical manifestations were investigated by statistical analyses. Results RSV prevalence was significantly higher in infants <6 months (13.4%), in hospitalized children (13.3% vs 5.5%) and in male patients (9.5% vs 6.4%). An overall prevalence of RSV of 8.0% in the period of 2015 to 2018 was shown, with significant annual (6.4%‐10.6%) and seasonal (12.7% in rainy season vs 3.0% in dry season) fluctuations. While RSV seasons in 2015, 2016, and 2018 were relatively similar, 2017 showed deviations from the overall patterns with significantly higher RSV circulation and an outbreak peak 3 to 5 months earlier. Concomitant circulation of RSV‐A and RSV‐B with an alternating predominance of RSV‐A and RSV‐B strains and temporal RSV‐A genotype replacement from NA1 to ON1 was observed. Conclusion This study represents the first in‐depth epidemiological analysis of RSV in CAF and provides first insights into RSV genetic diversity and seasonality in the country.


| INTRODUCTION
Respiratory syncytial virus (RSV), first characterized in 1956, 1 often causes lower respiratory tract infections in infants. It is the second leading cause of bronchiolitis and pneumonia, particularly during the winter months in temperate countries and during the rainy season in tropical countries. [2][3][4][5][6] RSV was estimated to be responsible for more than 90 million deaths worldwide between 1995 and 2010, including 20 million deaths in sub-Saharan Africa alone. 7,8 RSV belongs to the order Mononegavirales, the family Pneumoviridae, the genus Orthopneumovirus and the species Human orthopneumovirus. It is an enveloped virus, with a linear, negativesense RNA genome of about 15 000 base pairs (bp). The genome contains 10 genes that code for 11 proteins. 5 Among them, the surface glycoproteins G and F are the main targets of neutralizing antibodies. 9 The F protein governs the fusion of the viral envelope with the cell membrane. The glycoprotein G is involved in the attachment of the virus to the CX3CR1 chemokine receptor expressed on epithelial cells and facilitates the penetration of the virus into the host cell. 10 The variability of the G protein facilitates evasion of the immune response, allowing reinfections throughout life and complicates vaccine development. 11 Epidemiological and molecular studies on the antigenic and genetic variability of the G protein have classified RSV into two highly divergent phylogenetic sub-groups A and B. 3,12,13 To date, 15 RSV-A genotypes and 37 RSV-B genotypes have been defined. [14][15][16][17] In the RSV-A subgroup, the ON1 genotype which contains a 72 nucleotide (nt) duplication in the G protein, 2,4

was first described in 2010 in
Canada and is currently the most frequent RSV-A genotype. In RSV-B, BA genotypes, first detected in 1999, have now become the predominant RSV-B circulating worldwide and carry a 60 nt duplication. 18,19 In Central African Republic (CAF), respiratory infections represent the most common cause for seeking medical care. 20 A preliminary study reported RSV in 3.0% (10/329) of children aged 0 to 15 years enrolled from January to December 2010. 21 Figure S1.
Within the sentinel surveillance, the WHO criteria for ILI (measured fever or history of fever ≥38 C and cough with onset within the last 10 days) or SARI (measured fever or history of fever ≥38 C and cough with onset within the last 10 days requiring hospitalization) 22 were adopted. The study population included outpatients and inpatients under the age 5 who met the ILI and SARI case definitions, recruited from January 2015 to December 2018 at the five sentinel sites after obtaining consent.

| Sample and data collection
For each participant, a nasopharyngeal swab was collected then placed in a labeled tube with 3 mL of Universal Transport Medium (Copan, Italy). Samples were stored at 4 C or sent immediately in a refrigerated box to the National Reference Centre for Influenza for diagnosis. Each sample was then aliquoted into four 1.5 mL Eppendorf tubes for influenza virus culture, extraction of nucleic acids, and longterm storage at À80 C.
Social, demographic, clinical, and epidemiological data were recorded for each patient using a standardized questionnaire. The total number of consultations per site per year for any cause as well as for ILI symptoms were retrieved from the national database hosted at the Institut Pasteur de Bangui. This database is populated by standardized reports provided weekly by the sentinel sites.

| Nucleic acid extraction and RSV detection by RT-PCR
Within 24 hours after sample reception, RNA was extracted from a 140 μL nasopharyngeal swab sample aliquot using the QIAamp Viral RNA Mini kit (Qiagen, USA) according to the manufacturer's instructions.
RSV was detected by a conventional multiplex one-step RT-PCR detecting human metapneumovirus, influenza A, and influenza B virus as described before. 23 Cycling conditions were as follows: 50 C for 30 minutes, 94 C for 15 minutes followed by 40 cycles of 94 C for 30 seconds, 55 C for 30 seconds, 72 C for 1 minute, and a final extension at 72 C for 10 minutes.

| RSV typing and sequencing
RSV strain characterization was performed in a second step, using a batch approach, when sufficient number of samples were accumulated. RSV positive samples were typed as RSV-A or RSV-B by targeting fusion protein F by a semi-nested PCR as described before. 24 cDNA synthesis was carried out using the SuperScript III First-Strand enzyme (Invitrogen, USA) and the antisense primer F164 (5 0 -GTTATGACACTGGTATACCAACC-3 0 ). 24 The first round amplification was carried out with 5 U/μL Taq DNA polymerase (Promega, USA) and 10 μM of each primer, ABG490 (5 0 -ATGATTWYCYTTTGAAGTGTTC-3 0 ) and F164. Two semi-nested PCRs were then performed on each sample with the AG655 (5 0 -GATCYCAAACCTCAAACCAC-3 0 ) or BG517 (5 0 -TTYGTTCCCTGTA GTATATGTG-3 0 ) sense primers and F164 antisense primer. 24 PCR products were separated by electrophoresis on a 1.5% agarose gel. Samples with a 450 bp band in the first semi-nested PCR were typed as RSV-A, and samples with a band between 585 and 645 bp in the second semi-nested PCR were typed as RSV-B. These amplicons were then purified using the QIAquick PCR Purification kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's protocol. Purified products were sequenced using the corresponding PCR primers on an ABI 3130 capillary sequencer.

| Amino-acid analysis
The nucleotide sequences were translated into amino-acid sequences using the standard genetic code implemented in BioEdit software. The amino-acid sequences were compared with those from the prototype RSV-A (ON1: JN257693, Canada; NA1: AB470478, Canada) and RSV-B strains (BA9: AY333364, Argentina). BioEdit was used to visualize the amino-acid variability in the second hypervariable part of the G protein.

| Statistical analyses
Statistical analyses were performed in SigmaPlot v12.5, using Mann-Whitney rank sum tests for continuous variables, χ 2 -test for categorical variables or z-test for low proportions. Odds ratios were calculated with χ 2 -test with the Yates correction.

| Participant enrollment
From January 2015 to December 2018, a total of 112 214 children below 5 years old attended the five sentinel sites, all causes included ( Figure S1).
Among them, 6238 (5.6%) children presented with respiratory symptoms characteristic of ILI. A total of 2672 (42.8%) of all ILI cases were enrolled in the study. Despite some fluctuations in the recruitment rate between sites and between years, the annual ILI recruitment rate across all sites was rather constant (range: 39.8%-46.9%; Figure S2). Only few ILI cases were recruited at the Paediatric Complex. Overall, 1231 children presenting with a respiratory syndrome corresponding to the SARI (31.5% of all recruited participants) case definitions were included in the study. SARI cases were mainly recruited (56.1%) at the Paediatric Complex, while between 3.8% and 16.5% of SARI cases were recruited in the other four centers. Unfortunately, no data on the overall number of SARI cases per site per year were available.

| Demographic and clinical characteristics of patients
The demographic and clinical characteristics for all enrolled patients are shown in Table 1. Slightly more nasopharyngeal samples were collected from male (51.9%) than female patients (48.1%; female:male ratio of 0.9:1). There were little differences in the age and sex distribution across the 4 years of the study, but there was some variability between study sites. Children aged between 0 and 6 months had an increased risk of presenting with more severe symptoms necessitating hospitalization compared to older children (P < .001), while females had a lower risk of presenting with SARI compared to male (OR = 0.84, P = .014; Table S1).

| Disease severity and RSV-related deaths
RSV-positive cases were more frequently detected among hospitalized patients compared to outpatients (13.3% vs 5.5%, OR = 2.62, P < .001; Table 3). RSV was associated with dyspnea (P < .001), wheezing (P < .001), chest indrawing (P < .001), and inability to feed (P = .002) but not with rhinorrhoea (P = .993), diarrhoea (P = .983), vomiting (P = .107), lethargy (P = .816), or convulsion (P = .752; Table S2). In hospitalized children, RSV was significantly more frequently detected in children with a diagnosis of bronchiolitis or bronchopneumonia compared to pneumonia (Table 3) and was associated with increased duration of hospitalization (P < .001). Among SARI patients for whom clinical data were exhaustive (n = 358), only younger age was significantly associated with increased odds of showing higher clinical scores, while sex and RSV status were not significantly associated (Table S3). In an alternative statistical model with clinical scores stratified as moderate (scores 0-2) and severe (3)(4)(5)(6), RSVpositive children had increased risk of experiencing a more severe disease (OR = 1.93; 95% CI: 1.10-3.39; P = .022; Table S4). The difference between the two models likely arose from the limited number of

| Seasonal circulation of RSV
RSV detection rates varied across years and ranged from 6.4% in 2015 to 10.6% in 2017 ( Table 2). Prevalence among all cases in 2017 was significantly higher (P = .002). However, RSV prevalence among ILI cases did not vary between years (range: 5.1%-6.5%; Table 2), while RSV prevalence among children presenting with SARI symptoms significantly increased in 2017 (P = .048, Table 2).
During the study period, RSV detections started in March-April and lasted until December (Figures 1 and 2). Sporadic detections were recorded between March-April and August and RSV circulation peaked in September (2015), October (2016), or November (2018). In addition to a significantly higher number of cases reported in 2017, the peak of RSV incidence occurred in June, 3 to 5 months earlier than during the other 3 years (Figure 2). RSV was also significantly more frequently detected in the rainy season (June-November) com-  Figure 3; Table 4). Genetic distances between strains of the same genotype ranged from 0% to 1.4% for RSV-A NA1, 0% to 6.3% for RSV-A ON1, and 0%  full protection. This allows reinfections that will then boost humoral immune response 41,42 and explains why RSV is still detected in older age groups, albeit with lower incidence (4.0%-7.5% in CAF). Our analyses also showed that male patients more frequently developed severe symptoms leading to hospitalization (33.3% vs 29.6% in female) and were more susceptible to RSV infection (9.5% vs 6.4%), as reported previously. 40,43 Hormonal influence on the immune system 44 and/or airway anatomical differences 40  Our study showed an overall prevalence of RSV of 8.0% in the period of 2015 to 2018, with significant annual (6.4%-10.6%) and seasonal (12.7% in rainy season vs 3.0% in dry season) fluctuations. While RSV circulation is high during the winter in temperate climates, 12 a high RSV incidence usually coincides with the rainy season in African countries with a tropical climate, such as in Kenya, 45 Cameroon, 46 Senegal, 34 and Ghana. 47 Also in CAF, most cases were reported in November in a study from 2010. 21 RSV seasonality likely depends on climatic factors such as relative humidity, temperature, and UV radiation that influence the infectivity of viral particles and stability of aerosols 48 or host factors such as overall increased susceptibility to infectious diseases in winter due to lower levels of vitamin D. [49][50][51] Deviations from overall seasonality patterns, as observed in 2017 in CAF where RSV prevalence in SARI cases was significantly higher with a peak 3 to 5 months earlier than usual, have been documented previously. In Germany, an earlier start of the RSV season as compared to the average tends to be linked to higher disease incidence. 12   While similar detection rates were found in the USA (6.1%) 71 or in Kenya (8%) 45 when using conventional RT-PCR, much higher detection rates were reported in Germany (23%) 12 and in Ghana (23%), 47 when using more sensitive real-time RT-PCR assays. 72 A substantial number of strains could not be further characterized, likely due to the delay between initial diagnostic and further characterization steps, while unstable local long-term storage conditions may have affected nucleic acid quality. Therefore, combining revised RSV surveillance criteria, a more sensitive real-time RT-PCR screening approach as well as optimizing conditions for strain typing will improve the sensitivity of RSV detection and characterization in the country in the future.

| CONCLUSION
This first in-depth epidemiological study on RSV in CAF showed con-

TRANSPARENCY STATEMENT
The corresponding author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

DATA AVAILABILITY STATEMENT
Unique study sequences were deposited in GenBank under the accession numbers HG993989 to HG994028. The information reported in this article is from a database used in a collaborative partnership between the Institut Pasteur of Bangui and the Central African Republic Ministry of Health. The data are not publicly available since they contain information deemed to be confidential for the identity of the included subjects.

ETHICS STATEMENT
The surveillance programme carried out in CAF was approved by the ethical committee from the Ministry of Health, Central African Republic (Decree 0277/MSPP/CAB/DGSPP/DMPM/SMEE of August 5,2002). Participants were only included in the study after obtaining verbal consent from the child's parents or legal guardians. Data were pseudo-anonymized, in strict compliance with patient privacy rights.