Molecular epidemiology of Enteroviruses and Rhinoviruses in patients with acute respiratory infections in Yaounde, Cameroon

Abstract Background Acute respiratory infections (ARI) are associated with a huge morbidity and mortality worldwide. Rhinoviruses (RVs) and Enteroviruses (EVs) are recognized as leading causes of ARI. Objectives The present study describes the molecular epidemiology of RVs and EVs in Cameroon over a 3‐year surveillance period. Methods From September 2011 to October 2014, nasopharyngeal swabs were collected from patients with influenza‐like illness (ILI) and severe acute respiratory infections (SARI). Two sub‐genomic regions of the EVs and RVs were targeted for molecular characterization. These included the most conserved 5′‐untranslated region (5′UTR) and the viral protein 4/viral protein 2 transition region (VP4/VP2). Results A total of 974 samples were collected. Children ≤5 years accounted for 85.7% (835/974) of all participants. Among them, 160 (16.4%) were positive for RVs and/or EVs. RVs and/or EVs were significantly more identified in ILI compared to SARI patients (P = .015). Both viruses co‐circulated all year long with a marked increase of occurrence during rainy and cold season. All RV species were found to circulate in Cameroon, with 6, 10 and 6 virus types belonging to the RV‐A, RV‐B and RV‐C, respectively. EV species identified comprised EV‐A (1 Coxsackie virus A5), EV‐B (1 Coxsackie virus A9 and 2 Coxsackie virus B1) and EV‐C (1 EV‐C117). Conclusions This study indicates a strong year‐round occurrence of EV and RV associated respiratory infections in Cameroon. Molecular characterization identified a wide variety of RVs and EVs in patients with ARI in Cameroon.


| INTRODUC TI ON
Acute respiratory infections (ARI) constitute a major cause of morbidity and mortality worldwide. Although global mortality due to lower respiratory tract infection (LRTI) and upper respiratory tract infection (URTI) underwent a reduction of 21.1% and 42.1%, respectively, from 2007 to 2017, these infections still caused more than 2.5 million deaths in 2017. 1 The burden of LRTI was worsened even more with the emergence of COVID-19 in late 2019 which has already caused more than 2.4 million deaths worldwide in less than a year. 2 Respiratory viruses are also responsible for huge economic losses due to the use of health care resources and absenteeism at work and school. [3][4][5] Respiratory viruses, including Rhinoviruses (RVs) and Enteroviruses (EVs), are the most common cause of ARI.
Rhinoviruses are the main cause of URTI worldwide with common cold being their main clinical presentation. 6,7 With the development of molecular detection assays, RVs have recently been shown to be also involved in a significant proportion of more severe LRTIs such as wheezing, bronchiolitis, asthma exacerbation and chronic obstructive pulmonary disease. [8][9][10] Rhinoviruses have also been reported in apparently healthy individuals, possibly due to the prolonged viral shedding period. 11,12 Enteroviruses, on the other hand, have a broader tropism and can cause a wide range of human infections including acute respiratory infections, meningitis, encephalitis, gastroenteritis, acute flaccid paralysis or conjunctivitis. [13][14][15] A new strain of EV-D68 re-emerged, circulated worldwide and caused particularly severe acute respiratory infections (SARI) and acute flaccid myelitis. 16 Enteroviruses and RVs belong to the Picornaviridae family and the Enterovirus genus. According to the International Committee on Taxonomy of Viruses (ICTV), the Enterovirus genus comprises 15 species EV-A to L and RV-A to C. 17 Based on the sequence diversity of the structural protein coding genes, individual viruses of the EV species are further assigned into virus types. Rhinoviruses consist of more than a hundred virus types. Virus type assignments based on the nucleotide sequence of the VP4/VP2 region correlate with the type assignment based on the VP1 region of EVs and RVs. 18,19 All RV and EV species have a global distribution and a yearround circulation pattern with occasional peaks. 20 In West Africa, [21][22][23] Southern Africa, 24,25 Northern Africa, 26 were informed of the study objectives, constraints, risks and benefits of participating in the study and those who provided their informed consent were enrolled. We included outpatients and inpatients with acute respiratory infections. A case of ARI was considered as any patient with a fever <5 days in addition to a cough and/or sore throat. Participants who did not provide their consent were not included.

| Laboratory analysis
We collected a nasopharyngeal swab from all participants in a tube containing 1 mL of viral transport medium. Sample was Second round PCR amplicons were sequenced using the BigDye Terminator 1.1 ® kit (Thermo Fisher Scientific) according to the manufacturer's recommendations.

| Sequence analysis
Sequences were assembled and edited using EDITSEQ of the software Seqman™ II Lasergene (DNA). RV and EV reference sequences were retrieved from the GenBank database. Multiple sequence alignments were carried out using the CLUSTAL W method 47 in the MEGA software version 6. 48 Originally, we used the neighbour-joining method with a 1000-bootstrap replicates to generate an extended phylogenetic tree with all known EV types in one hand and all known RV types in the other hand (Figures S1 and S2). Then, the EV and RV sequence data sets were downsized by omitting most branches containing no studied sequences.
Reduced data set was used for maximum likelihood phylogenetic analyses using the best fit evolution model which was General Time Reversible with a Gamma distribution and assuming that a certain fraction of sites was invariable on an evolutionary level (GTR + G + I). The reliability of the tree nodes was evaluated by 1000 bootstrap resampling. Nucleotide sequences obtained in the present study were submitted to GenBank under the registration numbers MN508757 to MN508783.

| Statistical analysis
We collected clinical and sociodemographic data from participants using a standardized form. We analysed the data using R software version 3.6.0. We described the categorical variables in number and percentage and the continuous variables in mean and standard deviation. We used the chi-square test to compare categorical variables and linear regression for continuous variables, and P values < .05 were considered statistically significant.

| Viral identification
Of the 974 swabs tested, 160 (16.4%) were positive for RVs and/or EVs ( Table 1). The rate of RV/EV positivity in inpatients vs outpatient was significantly different (P = .015). The rate of RV/EV positivity with respect to the age (P = .023) and year of recruitment was also significantly different (P = .002). In contrast, no significant difference in RV/EV positivity was found with respect to age group (P = .291) or sex (P = .604). The most common symptoms recorded in participants were rhinorrhoea and cough. RVs and EVs were found to circulate throughout the year (Figure 1). Apart from fatigue (P = .030) and headaches (P = .004), all other symptoms recorded were not significantly associated with RV/EV infections.

| Genotyping
Overall, 43 RV/EV positive samples were available for molecular characterization in this study. The 5′UTR-VP4-VP2 region was am-     [63][64][65] It is noteworthy that we identify no EV-D68 isolate despite the epi- In conclusion, the findings of the present study suggest an important level of annual circulation of respiratory RV/EV in Cameroon.
Molecular characterizations have revealed a large diversity of RV/EV strains.

ACK N OWLED G EM ENTS
We thank all laboratory and hospital staff involved data and respiratory samples collection used for this study.

CO N FLI C T O F I NTE R E S T
None.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/irv.12851.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.