Molecular characterization of human respiratory syncytial virus strains circulating among hospitalized children in Jordan

Abstract

Background

Human Respiratory Syncytial Virus (HRSV) is a primary cause of severe pediatric respiratory infections, particularly in infants and young children, often resulting in hospitalization. The virus possesses a high degree of mutagenic potential, contributing to significant antigenic diversity, which complicates immune responses and poses challenges for vaccine development and disease management. This study was conducted in Jordan from 2022 to 2023 to epidemiologically determine the prevalence and molecular characteristics of RSV.

Methods

A total of 288 nasopharyngeal (NP) swabs were collected from hospitalized children at Prince Hamza Hospital, Amman, Jordan. All samples were screened for common viral and bacterial respiratory pathogens using PCR. A partial segment of the G gene of RSV was amplified for molecular characterization and phylogenetic tree analysis.

Results

Viral and/or bacterial infection was identified in 71.9% (207/288) of the tested specimens. Among these, 35 samples (12.2%, 35/288) tested positive for RSV. Specific subgroup PCR analysis identified (25, 71.4%) RSV-A, (4, 11.4%) RSV-B, and (6, 17.1%) could not be identified using our set of primers. Phylogenetic tree analysis revealed that RSV-A ON1 and RSV-B BA9 genotype strains predominate in Jordan. We observed multiple substitutions in our studied sample which would drive variation in the level of antigenicity and pathogenicity of RSV. Glycosylation sites identified were consistent with previously reported studies.

Conclusion

This study provides updated epidemiological data on the strains circulating in Amman, Jordan and their molecular characteristics. Continuous RSV surveillance informs vaccine development, guides public health interventions, and enables timely administration of prophylactic treatments, reducing the burden of RSV-related illness.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-024-10185-7.

Introduction

Human Respiratory syncytial virus (HRSV) is considered a major threat to human health, particularly among children [1]. Globally, HRSV is responsible for around 33 million acute lower respiratory tract infections annually in children under five years of age, causing approximately 3.2 million hospitalizations and 100,000 to 200,000 deaths, with the majority of fatalities occurring in developing countries [2]. Moreover, over the past decade, HRSV infections have increasingly affected older adults aged 65 and above [3]. HRSV is mainly transmitted via nasal or oral secretions from infected individuals, and so far, its animal reservoir or an intermediate host has not been identified [4]. People can contract HRSV directly through exposure to large respiratory droplets or indirectly by handling surfaces contaminated with the virus, such as toys, cribs, pacifiers, strollers, and highchairs [5]. It is noteworthy that, due to the virus’s antigenic diversity, an initial HRSV infection does not provide lasting immunity, accounting for the relatively higher reinfections rate among both children and adults [6]. Despite limited treatment options for HRSV infection, advancements in understanding the virus’s biology have led to the development of the world’s first HRSV vaccines, RSVPreF3 (Arexvy, GSK) and (Abrysvo, pfizer), and mResvia (Moderna) which target elderly individuals (60 years and older) and pregnant women in their 32–36 weeks gestation [7–9]. Moreover, In July 2023, the FDA approved a new prophylactic drug, Nirsevimab, for infants and young children with severe HRSV infections [10].

In 2022, HRSV was renamed as human orthopneumovirus (HOPV), belonging to the Pneumoviridae family of enveloped RNA viruses, which primarily affects the respiratory system [11, 12]. It is a negative-sense, single-stranded virus with a genome length of approximately 15.2 kb, comprising 10 genes encoding 11 proteins: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L [13]. HRSV strains are classified into two main subtypes, HRSV-A and HRSV-B, based on antigenic and genetic differences, with HRSV-A generally being more common [14]. These subtypes display notable amino acid differences, particularly in the attachment glycoprotein (G). The glycoprotein (G) and fusion (F) proteins are essential for the virus’s ability to attach to and enter host cells [15]. The G protein, which is 300 amino acids long, consists of three main domains: an N-terminal cytosolic domain, a transmembrane domain, and an ectodomain. The ectodomain encompasses two hypervariable mucin-like domains (MLD1, MLD2), a conserved central domain (CCD), and a heparin-binding domain (HBD) [16]. The heavy glycosylation of N-linked and O-linked sugars in the G protein results in high amino acid variation among HRSV genes, making it an important target for vaccine and antiviral agents’ development [17]. Sequencing the second variable region or C-terminal region of the G protein is considered the best approach for studying HRSV evolution and genotyping. The F protein of HRSV is a type I membrane protein that is conserved across HRSV A and B antigenic subgroups. It exists in at least two conformations: the metastable pre-fusion (PreF) and the stable post-fusion (PostF) [18]. The PreF conformation contains unique antigenic epitopes, specifically site Ø and site V, which are responsible for inducing nearly 90% of F-specific neutralizing antibodies in humans, compared to the PostF conformation​ [19]. A recent study indicated that levels of anti-G antibodies in adults significantly decrease over time, which may contribute to ongoing susceptibility to RSV infections throughout life [20]. Therefore, to consider optimal protective efficacy of RSV vaccines, it is crucial to diversify the antigens utilized in their formulation.

HRSV subtypes can be classified through antigenic methods, focusing on the F and G proteins that elicit neutralizing antibodies, as well as genotypic methods, which analyze variations in the nucleotide sequences of the G gene [21]​. This dual approach enhances our understanding of HRSV diversity and its epidemiological significance. As of today, at least 16 genotypes for HRSV-A (GA1–7, SAA1, NA1–4, CB-A, and ON1-3) and 37 genotypes for HRSV-B (GB1–6, GB12, GB13, SAB1–4, URU1, URU2, CB1, THB, BA1–14, BA-C, BA-CCA, BA-CCB, JAB1, NZB1, and NZB2) have been identified, demonstrating variable pathogenicity and co-circulation during epidemics [22]. Epidemiological studies have reported fluctuating predominance between HRSV-A and HRSV-B in seasonal outbreaks, which correlates with variations in clinical outcomes. For example, in some seasons, HRSV-B has been associated with milder infections, while HRSV-A has led to outbreaks with higher rates of severe disease [23]. The most prevalent HRSV-A strain worldwide is the ON1 genotype, while the most prevalent HRSV-B strain is the BA genotype [24, 25]. The ON1 genotype first emerged in Ontario, Canada, in 2010 and was characterized by a 72-nucleotide duplication in the G glycoprotein gene, which has contributed to its rapid spread and dominance globally [26]. The BA genotype, which includes several sub-genotypes such as BA9, first emerged in Buenos Aires, Argentina, in 1999. It is distinguished by a 60-nucleotide duplication in the G glycoprotein gene, which has similarly facilitated its global spread and prevalence [27]. Both the ON1 and BA genotypes have become the dominant strains in many parts of the world, largely replacing earlier genotypes due to their enhanced ability to evade immune responses [28].

The emergence of new genotypes can lead to recurrent HRSV infections and significant epidemics. Recently, the prevalence of the HRSV-A ON-1 genotype has significantly increased [29]. The predominance of HRSV-A over HRSV-B varies geographically and seasonally. For instance, in many regions globally, HRSV-A has often been reported as the more prevalent subgroup [29]. A study from Japan highlighted the dominance of the HRSV-A ON1 genotype, which rapidly spread worldwide after its identification in 2010 [30]. Seasonal changes can also shift the dominance between subtypes, as observed in numerous reports globally, showing that while one subtype may dominate during one season, the other may take over in subsequent years [31, 32].

This study aimed to further examine the predominant HRSV genotypes in Jordan by analyzing the G gene of HRSV-A and HRSV-B strains circulating during the 2022/2023 seasonal epidemics. In continuation of previous investigations conducted in Jordan, this study also sought to explore the epidemiology of circulating HRSV genotypes and assess the genetic variability of the G gene. Importantly, this is not the first study to characterize RSV in Jordan, but rather a recent effort building on earlier studies by Trovão et al. (2021) and Khuri-Bulos et al. (2018) which characterized the circulating genotypes in Jordanian children around ten years ago [33, 34].

Materials and methods

Study population

This study was conducted at a referral hospital in Amman from October 2022 till February 2023. The study enrolled 288 patients admitted to the pediatric ward at Prince Hamza Hospital (PHH) aged 0–5 years, as well as children and adult patients aged 6–70 years visiting the ENT clinic, all exhibiting symptoms of respiratory tract infections. These symptoms included persistent fever, sore throat, runny nose, sneezing, coughing, and difficulty breathing. Nasopharyngeal flocked swabs (ZYBIO, China) were used to collect samples, which were then placed in sterile tubes containing 3 ml of cryoprotectant solution. The samples were either processed immediately or stored at -20ºC for DNA extraction. Throughout the study, all CDC recommendations concerning appropriate and safe aseptic techniques for sample collection and transportation protocols were strictly followed. This study received Institutional Review Board (IRB) approvals from The Hashemite University (No. 3/7/2020/2021) and PHH (No. MH/ 517/2022) in accordance with established guidelines. Informed consent forms were signed by all guardians of participating children. Patient demographics, clinical presentation, and health status were obtained utilizing a standardized questionnaire.

Nucleic acid extraction and reverse transcription

Oropharyngeal/nasopharyngeal swabs were collected from 288 patients and transported to the laboratory at The Hashemite University School of Medicine. Nucleic acid extraction was performed from the swabs using the QIAamp MinElute Virus Spin Kit (Qiagen, Germany), in accordance with the manufacturer’s protocol. The extracted nucleic acids were eluted in 200 µl of AE buffer and stored at -80 °C for future analysis. For reverse transcription, RNA templates were converted into complementary DNA (cDNA) using the QuantiTect Reverse Transcription Kit (Qiagen, Germany), following the manufacturer’s guidelines. The resulting cDNA was then tested for HRSV detection and sequencing via polymerase chain reaction (PCR).

Initial evaluation of nasopharyngeal specimens for prevalent respiratory viruses

To identify the viral etiology responsible for the respiratory symptoms observed in patients, we utilized multiplex RT-PCR assays that target a wide range of pathogens. Specifically, we employed the FTD Respiratory Pathogens 33 (Fast-track Diagnostics, Germany), which is designed to detect 21 viruses, 11 bacteria, and a single fungus, as previously described [35]. The FTD assay is a comprehensive one-step RT-PCR kit containing primer-probe mixtures that enable the simultaneous detection of 33 respiratory pathogens. These include human respiratory syncytial viruses (A and B), human metapneumoviruses (A and B), human parainfluenza viruses (types 1–4), human rhinovirus, human adenovirus, human coronaviruses (NL63, 229E, OC43, HKU1), influenza viruses (A, swine H1N1, B, and C), enterovirus, human parechovirus, human bocavirus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Streptococcus pneumoniae, non-type b Haemophilus influenzae, Haemophilus influenzae type b, Staphylococcus aureus, Moraxella catarrhalis, Bordetella species, Klebsiella pneumoniae, Legionella pneumophila/longbeachae, Salmonella species, Pneumocystis jirovecii, and equine arteritis virus, the latter of which served as an internal control.

The eight multiplex RT-PCR reactions were prepared according to the manufacturer’s instructions. Each PCR reaction was conducted in a final volume of 25 µl, where 10 µl of the extracted nucleic acid samples were combined with 15 µl of the master mix provided in the kits. The thermal profile for the multiplex real-time RT-PCR was as follows: incubation at 50 °C for 15 min, initial denaturation at 94 °C for 1 min, followed by 40 cycles of denaturation at 94 °C for 8 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. A sample was deemed positive for a microorganism if a sigmoidal curve with a cycle threshold (CT) value of < 40 was observed. An internal control (IC) was included in the extraction process alongside the specimens and used in each PCR run, along with positive and negative controls provided by the manufacturers.

Amplification of G protein encoded gene of RSV-A and RSV-B

Samples positive for HRSV-A and -B, identified using the FTD kit, were further subjected to amplification via Nested PCR utilizing the following pair of outer primers: RSVA-G513-F 5’-AGTGTTCAACTTTGTACCCTGC-3’ and RSVA-F131-R 5’-CTGCACTGCATGTTGATTGAT-3’ to determine whether it was subtype A or B. In the first-round PCR, 5 µl of cDNA were added to a 25 µl reaction mixture containing 1 µl of each primer, the run was performed at 94˚C for 5 min, followed by 40 cycles consisting of 94˚C for 30 s, 53˚C for 30 s, and 72˚C for 1 min, followed by a final extension step at 72˚C for 10 min. Five microliters of the first-round PCR product served as the template for the second-round PCR. The second-round PCR was carried out in a 50 µl reaction mixture using the following pair of inner primers: RSVA-G606-F 5’-AACCACCACCAAGCCCACAA-3’ and RSV-F22-R 5’-CAACTCCATTGTTATTTGCC-3’. The cycling conditions were the same as for the first-round PCR, except for the annealing temperature, which was set at 54.6˚C. The amplicon with the expected band size of both outer (666 base pairs) and inner (464 base pairs) primer sets of HRSV-A was visualized by electrophoresis on a 2% agarose gel and visualized using UV transillumination (Alpha Innotech, USA).

Samples that tested negative for RSV-A above were subjected to further analysis using RSV-B specific primers: BGF 5’-GCAGCCATAATATTCATCATCTCT-3’ and BGR 5’-TGCCCCAGRTTTAATTTCGTTC-3’. Five microliters of cDNA were added to a 50 µl reaction mixture that was then incubated in a thermal cycler under the following cycling conditions: 94˚C for 5 min, followed by 40 cycles of 94˚C for 30 s, 52.3˚C for 1 min, and 72˚C for 1 min, with a final extension step at 72˚C for 10 min. The amplified product, with an expected band size of 801 bp, was visualized using 1% agarose gel electrophoresis and the gel documentation system (Alpha Innotech, USA).

DNA sequencing

DNA sequencing was conducted as follows: First, the PCR products were purified using a commercially available DNA purification kit (Axen PCR DNA kit, Macrogen Inc., Korea) in accordance with the manufacturer’s instructions. Next, the same primers used for the PCR (RSVA-G606-F and RSV-F22-R for RSV-A, and BGF and BGR for RSV-B) were employed to sequence the DNA in both the forward and reverse directions using the Nextera XT DNA Library Preparation Kit on Illumina NovaSeq 6000 sequencing system (Macrogen Inc., Korea). The nucleotide sequences corresponding to the second hypervariable region of the G gene, obtained through this process, were submitted to GenBank and assigned accession numbers PP903836– PP903857.

Sequence analysis

Nucleotide sequences were edited and aligned using BioEdit Software version 7.2. Consensus sequences were generated by aligning forward and reverse sequences. Sequences were then compiled and aligned using Clustal W version 2.0. Reference sequences for the HRSV G gene subgroups A and B were retrieved from GenBank (https://www.ncbi.nlm.nih.gov) as of May 2024. Alignment focused on the second variable region of the G gene. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) method to infer evolutionary relationships among HRSV strains with 1000 bootstrap replications, implemented in MEGA 11. Deduced amino acid sequences were analyzed with BioEdit Software version 7.2, comparing them to reference strains from GenBank to identify amino acid substitutions, deletions, and insertions in the G protein. Potential N-linked and O-linked glycosylation sites in the second variable region of the G protein were predicted using the NetNGlyc 1.0 server (NetNGlyc-1.0 - redirect (dtu.dk)) and the NetOGlyc 4.0 server (NetOGlyc 4.0 - DTU Health Tech - Bioinformatic Services), respectively. Glycosylation sites were characterized based on the following criteria: potential N-linked glycosylation sites were identified by the presence of asparagine (N)-X-Serine (S)/Threonine (T) (where X is any amino acid except proline (P)), while potential O-linked glycosylation sites were indicated by the presence of serine (S) and threonine (T) residues.

Results

Determination of circulating respiratory viruses

First, we have documented the frequencies of several respiratory viruses and bacteria over a period of five months. A total of 288 samples were initially screened using the FTD respiratory PCR kit. The overall detection rate for common respiratory viruses and bacteria was 71.9% (207 out of 288 specimens) (Table 1). Thirty-five specimens (12.2%) tested positive for HRSV making it the third highest detected virus. The frequencies of respiratory viruses were as follows: Influenza B (IFB) at 16.0% (46/288), Influenza A (IFA) at 14.2% (41/288), HRSV at 12.2% (35/288), followed by influenza C (IFC), Human rhinovirus (HRV), and Adenovirus (AdV) at 7.3% (21/288), 7.3% (21/288), and 5.6% (16/288), respectively (Table 1). In terms of bacterial infections S. aureus was the most common isolated pathogen at 11.8% (34/288), followed by S. pneumoniae at 11.5% (33/288), H. influenzae (mostly type B), and M. catarrhalis 8.3% (24/288), and 6.3% (18/288), respectively (Table 1). All 35 HRSV-positive samples were selected for HRSV subtyping and genotyping. A total of 29 samples (29/35) were deemed either HRSV-A and/or HRSV-B viruses and were further selected for sequencing and phylogenetic analysis. We were unable to determine the genotype of the remaining six HRSV-FTD-positive specimens due to the failure to amplify any bands using HRSV-A and -B specific primers. Additionally, limited funding restricted the possibility of conducting further molecular analyses. Therefore, they were excluded from further analysis to ensure the integrity and reliability of the results. It is noteworthy that most HRSV-positive samples (80%, 28/35) were associated with coinfections with other respiratory viruses and/or bacteria (Table 2).

Table 1.

Respiratory pathogen frequencies identified via fast track diagnostics real time PCR kit (FTD) (n = 288). Influenza A virus, Influenza B virus, Influenza C virus, Influenza A (H1N1) virus, Human Parainfluenza viruse-1, Human Parainfluenza viruse-2, Human Parainfluenza viruse-3, Human Parainfluenza viruse-4, Human coronavirus NL63, Human coronavirus 229E, Human coronavirus OC43, Human coronavirus HKU1, Human metapneumovirus A and B, Human rhinovirus, Human respiratory syncytial viruses A and B, Human adenovirus, Enterovirus, Human parechovirus, Human bocavirus, Pneumocystis jirovecii fungus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae type B, Staphylococcus aureus, Moraxella catarrhalis, Klebsiella pneumoniae, Legionella pneumophilae / Legionella longbeacheae, Haemophilus influenzae.

Pathogen	Positive n (%)
IBV	46 (16.0)
IAV	41 (14.2)
HRSV A & B	35 (12.2)
S. aureus	34 (11.8)
S. pneumoniae	33 (11.5)
H. influenzae	24 (8.3)
ICV	21 (7.3)
HRV	21 (7.3)
H. influenzae B	21 (7.3)
M. catarrhalis	18 (6.3)
HAdV	16 (5.6)
Bordetella spp.	16 (5.6)
C. pneumoniae	15 (5.2)
EV	13 (4.5)
HPeV	9 (3.1)
HMPV A & B	8 (2.8)
HBoV	7 (2.4)
HCoV 229E	6 (2.1)
HPIV-3	3 (1.0)
HCoV NL63	2 (0.7)
HCoV HKU1	2 (0.7)
IAV(HINI)	2 (0.7)
HPIV-2	1 (0.4)
HPIV-4	1 (0.4)
P. jirovecii	1 (0.4)
K. pneumoniae	1 (0.4)
HPIV-1	0
HCoVOC43	0
M. pneumoniae	0

Table 2.

Classification of RSV based on type and RSV coinfections observed in each patient

RSV type	RSV positive case No.	IAV	IBV	ICV	HPIV-3	HCoV NL63	HRV	HPeV	HMPV	HAdV	S. pneumonia	C. pneumonia	S. aureus	M. catarrhalis	P. jirovecii	L. pneumophila	H. influenza
RSV A	3	√					√						√				√
	6			√							√	√
	8										√						√
	9						√
	11
	13											√					√
	15
	16
	17
	18			√			√						√
	19			√			√					√		√	√		√
	20			√			√
	23										√
	24										√
	31				√						√	√
	33										√	√		√
	34												√
	49												√
	55																√
	67									√	√
	72										√			√			√
	73		√														√
	74		√								√
	76			√				√				√
	78
	84																√
RSV B	202	√	√										√			√
	218												√
	258
Undetermined	3						√				√
	5					√					√
	7											√
	10
	59
	66								√								√

Demographic and clinical profile of patients with HRSV infections

Among the patients that tested positive for HRSV, 42.9% (15 out of 35) were females (Table 3). The HRSV-positive patients were categorized into three age groups: 0–5 years, 6–18 years and ≥ 19 years. As expected, most cases of HRSV infections were observed in children under 5 years of age (91.4%). The median duration of hospital stay for children with HRSV infections was 6 days. The most common clinical symptoms observed: cough (97.1%), difficulty breathing (85.7%), and nasal discharge (68.6%), and fever (60.0%) (Table 3). However, no significant differences (p > 0.05) were found in age groups and clinical characteristics between HRSV-A and HRSV-B positive patients.

Table 3.

Demographic and clinical characteristics of the HRSV positive patients

Variable	N = 35	%
Gender
Male	20	57.1%
Female	15	42.9%
Age
0–5	32	91.4%
6–18	0	0
≥ 19	3	8.6%
Residential city
Amman	22	62.9
Zarqa	13	37.1
Symptoms during sample collection
Cough	34	97.1
Difficulty breathing	30	85.7
Nasal discharge and congestion	24	68.6
Fever	21	60.0
Nausea	19	54.3
Vomiting	19	54.3
Sore throat	6	17.1
Chills	5	14.3
Diarrhea	5	14.3
Myalgia	4	11.4
Health status
Healthy	32	91.4
Allergy	3	8.6

Genetic characterization and phylogenetic analysis

All 29 (25 HRSV-A and 4 HRSV-B positive samples) PCR amplicons were sequenced, yielding high-quality sequences for 22 HRSV-A (22/25) and two HRSV-B (2/4) samples that underwent further analysis. We could not determine the genotype of the remaining five HRSV-positive specimens due to poor-quality sequence data or unusual sequence patterns that could not be reliably interpreted. These five samples either produced incomplete, ambiguous sequence results or failed to generate any usable sequence data, likely due to factors such as low viral load or RNA degradation. Therefore, they were excluded from further analysis to ensure the integrity and reliability of the results. The sequences from 24 samples were thoroughly cleaned, edited, and aligned with reference genotypes for accurate comparison. Importantly, none of the tested samples showed coinfection with both HRSV-A and HRSV-B. Through sequence and phylogenetic analysis of a partial G gene sequence, it was determined that the HRSV-A strains in our study belonged to the ON1 genotype. These strains exhibited an average nucleotide sequence similarity of 97% among themselves. They were closely related to the ON1 genotype that was first discovered in Ontario, Canada, in 2010 (Fig. 1). Specifically, the sequence homology between the HRSV-A ON1 strains in our study and the ON1 reference strain from Canada (ON 67-1210, GenBank number: JN257693) was found to be 95.0% at the nucleotide level and 91.3% at the amino acid level. All 22 sequences of the HRSV-A ON1 genotype closely matched previously isolated ON1 strains from Amman (KX655699.1 and MK109777.1), with sequence identities ranging from 92 to 94%. This ON1 genotype is characterized by a 72-nucleotide and 24-amino acid insertion and duplication in the C-terminal region of the G gene’s second hypervariable region (Fig. 2). This duplication, which starts after nucleotide 777 in the G gene (according to the reference strain JN257693), was consistently present in all 22 samples (Fig. 2). The average pairwise distance between the G gene sequences of our Jordanian samples and the ON1 prototype reference was calculated to be 0.078.

Fig. 1.

Phylogenetic analysis of the G gene of HRSV group A was performed using sequences collected in Amman, Jordan, in 2023. In this analysis, sequences from Jordan identified in our study are highlighted with a red square, while reference strains are labeled with their respective accession numbers. The analysis included a total of 179 nucleotide sequences. To ensure accuracy, all ambiguous positions were removed from each sequence pair using the pairwise deletion option. The final dataset comprised 367 nucleotide positions. Evolutionary analyses were carried out using MEGA11

Fig. 2.

Sequence alignment of deduced amino acids in HRSV-A. The sequence alignment of the second hypervariable region of the G protein of the HRSV-A group for the ON1 strains in this study, corresponding to the prototype ON1 strain from Canada (GenBank accession number JN257693). The amino acid positions range from 227 to 321 of the G protein. Identical residues are indicated by dots. The 24 amino acid duplicated region is framed in red boxes

The HRSV-B strains (n = 2) belonged to the BA genotype and clustered with strains previously assigned to the BA-9 genotype, which features a 60-nucleotide duplication (Fig. 3). The sequence homology between the HRSV-B sequences, and the BA reference strain (isolated originally from Argentina, AY333364) ranged from 94.6 to 95.0% at the nucleotide level and 91.7–93.9% at the amino acid level (Fig. 3). All sequences exhibited an insertion of 60 nucleotides after position 719 and a deletion of CCAAAA at position 475 (affecting P159 and K160) in all HRSV-B samples (Fig. 4). The average pairwise distance of variation in the G gene between all HRSV-B samples and other references, including the prototype, was 0.087. Both HRSV-B strains in our study were closely related to strains previously reported in Houston, Texas and Seattle, Washington (PP882660.1 and PP770458.1). These two strains did not bear any similarity to the previously reported Jordanian HRSV-B strain (MK109775.1).

Fig. 3.

Phylogenetic analysis of the G gene of HRSV group B was conducted using sequences collected in Amman, Jordan, in 2023. In this analysis, the Jordanian sequences identified in our study are marked with a red square, and reference strains are labeled with their corresponding accession numbers. The analysis included 54 nucleotide sequences. To maintain accuracy, all ambiguous positions were excluded from each sequence pair using the pairwise deletion option. The final dataset consisted of 729 nucleotide positions. Evolutionary analyses were performed using MEGA11

Fig. 4.

Sequence alignment of deduced amino acids in HRSV-B. Sequence alignment of the deduced amino acids in HRSV-B was performed for the second hypervariable region of the G protein. This analysis focused on the BA strain from our study and compared it to the BA reference strain from Argentina (accession number AY333364). The amino acid positions range from 85 to 312 of the G protein. Identical residues are indicated by dots. Amino acid deletions are seen at positions 159, 160, and 261 (green boxes). The 20 amino acid duplicated region is framed in red boxes

Deduced amino acid sequence analysis of the G gene

In this study, a partial G gene protein sequence was investigated in most of the ON1 strains with a length of 87 amino acids. None of our samples had insertion or deletion mutations in the investigated area (227–313). Comparing the ON1 amino acid sequences in this study with the reference Canada ON1 strain, we observed the following amino acid changes: L274P in all 22 samples (100%), L298P/S in all 22 samples (100%), Y304H in 21 samples (95.5%), L247I, V303A, and L310P in 16 samples (72.7%), N242D, P275Q, and G284D in 15 samples (68.2%). The nucleotide and amino acid sequence variability between our sequences and the ON1 reference strain are detailed in Fig. 2 and Table 4. Additionally, a partial G gene protein sequence was investigated in the BA strains of HRSV-B with a length of 237 amino acids. The BA strains in HRSV group B showed variations within the G gene protein length due to the deletion mutation P159 and K160. More than 25 mutations in the G gene sequences were identified compared to the reference strain (AY333364, Argentina). Both HRSV-B samples had the same following substitutions: L105P, T107A, Y112H, R136T, T138S, deletion of P159 and K160, I200T, K218T, I229T, S247P, deletion of E261, V271A, I281T, T290I, T312I. The nucleotide and amino acid sequence differences between our sequences and the BA reference strain are shown in Fig. 4 and Table 5.

Table 4.

Nucleotide and amino acid variations between our RSV-A Jordanian strains and the ON1 prototype

Position site	ON 1 Nucleotide	Variant nucleotide	Amino acid change	Codon change	Protein effect	Frequency (%)
724	A	G	N242D	AAC to GAC	Substitution	15/22 (68.2)
742	C	A	L248I	CTC to ATC	Substitution	16/22 (72.7)
749	C	T	S250F	TCC to TTC	Substitution	1/22 (4.5)
772	C	T	H258Y	CAC to TAC	Substitution	1/22 (4.5)
808	T and C	C and T	S270P	TCC to CCT	Substitution	6/22 (27.3)
817	T	C	Y273H	TAT to CAT	Substitution	6/22 (27.3)
821	T	C	L274P	CTA to CCA	Substitution	22/22 (100)
824	C	A	P276Q	CCA to CAA	Substitution	15/22 (68.2)
850	G and T	A and C	G284D	GGT to GAC	Substitution	15/22 (68.2)
893	T	C	L298P	CTA to CCA	Substitution	16/22 (72.7)
893	C and T	T and C	L298S	CTA to TCA	Substitution	6/22 (27.3)
908	T	C	V303A	GTC to GCC	Substitution	16/22 (72.7)
910	C	T	Y304H	CAT to TAT	Substitution	21/22 (95.5)
911	A	C	Y304P	CAT to CCT	Substitution	1/22 (4.5)
929	T	C	L310P	CTA to CCA	Substitution	16/22 (72.7)
941	T	C	L314P	CTA to CCA	Substitution	22/22 (100)
956	C	T	T319I	ACC to ATC	Substitution	1/22 (4.5)
958	A	G	T320A	ACC to GCC	Substitution	22/22 (100)
961	A	G	K321E	AAA to GAA	Substitution	1/22 (4.5)

Table 5.

Nucleotide and amino acid variations between our RSV-B Jordanian strains and the BA prototype

Position site	BA Nucleotide	Variant nucleotide	Amino acid change	Codon change	Protein effect	Frequency (%)
298	A	G	S to G	AGC to GGC	Substitution	1/2 (50)
314	T	C	L to P	CTC to CCC	Substitution	2/2 (100)
319	A	G	T to A	ACC to GCC	Substitution	2/2 (100)
331	T	C	Y to H	TAC to CAC	Substitution	2/2 (100)
391	G	A	A to T	GCA to ACA	Substitution	1/2 (50)
407	G	C	R to T	AGA to ACA	Substitution	2/2 (100)
410	C	T	T to I	ACC to ATC	Substitution	1/2 (50)
412	A and C	T and T	T to S	ACC to TCC/ TCT	Substitution	2/2 (100)
505–507				CCA	Deletion	2/2 (100)
508–510				AAA	Deletion	2/2 (100)
599	T	C	I to T	ATA to ACA	Substitution	2/2 (100)
611	A	C	N to T	AAC to ACC	Substitution	1/2 (50)
619	C	T	P to S	CCC to TCT	Substitution	1/2 (50)
646	C and A	T and C	P to S	CCA to TCC	Substitution	1/2 (50)
653	A	C	K to T	AAA to ACA	Substitution	2/2 (100)
656	T	C	L to P	CTA to CCA	Substitution	1/2 (50)
668	T	C	L to P	CTG to CCG	Substitution	1/2 (50)
686	T and C	C and T	I to T	ATC to ACT	Substitution	2/2 (100)
739	T	C	S to P	TCA to CCA	Substitution	2/2 (100)
774	A	C	K to N	AAA to AAC	Substitution	1/2 (50)
783				GAA	Deletion	2/2 (100)
793	A	T	T to S	ACC to TCC	Substitution	1/2 (50)
796	T and A	C and G	S to P	TCA to CCG	Substitution	1/2 (50)
809	T	C	V to A	GTG to GCG	Substitution	2/2 (100)
812	T	C	L to P	CTC to CCC	Substitution	1/2 (50)
826	T	C	S to P	TCA to CCA	Substitution	1/2 (50)
839	T	C	I to T	ATC to ACC	Substitution	2/2 (100)
856	T	C	H to Y	TAC to CAC	Substitution	1/2 (50)
859	C	T	S to P	CCA to TCA	Substitution	1/2 (50)
863	C	T	T to I	ACC to ATC	Substitution	2/2 (100)
932	C	T	T to I	ACC to ATC	Substitution	2/2 (100)

Analysis of N- and O-Glycosylation sites in amino acid sequence

Analyzing the N-glycosylation sites revealed eight sites in the HRSV-A ON1 reference strain JN257693, whereas our partially sequenced strains had only one conserved N-glycosylation site at position 237 (NTT) across all samples. For our HRSV-B sequences, three N-glycosylation sites were predicted. All three N-glycosylation sites (at 86, and 296, 310, NIT, NST and NST respectively) reported in the BA prototype were conserved across our two samples. An amino acid substitution at position 258 (K/N) resulted in a gain of a potential N-glycosylation site (NHT) in one sample. The number of predicted O-glycosylation in the ON1 strains varied from 38 to 41. All amino acid positions with O-glycosylation potential in the prototype ON1 strain were also predicted as O-glycosylation sites in the Jordanian strains. Additionally, some strains exhibited predicted O-glycosylation sites not found in the prototype ON1 strains such as S307 and S311. The number and positions of predicted O-glycosylation sites in the BA strains varied, ranging from 41 to 44 sites. Two of these predicted O-glycosylation sites, resulting from the mutations I220T, K218T, I228T, and I281T, were found in our strains but not in the reference strain.

Discussion

A significant share of lower respiratory tract infections (LRTIs) in children and older adults are attributed to HRSV. Evidently, a previous study conducted in Jordan between March 2010 and April 2013, showed that HRSV accounted for 44.0% of viral infections among Jordanian infants with acute respiratory tract infections [33]. Similarly, in Guangzhou, China, a study spanning from September 2017 to December 2021 found HRSV in 6.2% of patients, with over 90% of those infected being under 4 years old [36]. Understanding the epidemiology and genotypic characteristics of HRSV is essential for developing effective therapeutic and preventive strategies. This knowledge is relevant to implementing suitable preventive measures, thereby reducing the significant burden that HRSV imposes on healthcare systems, particularly in causing severe morbidity and hospitalizations in children. In our study, conducted from October 2022 to February 2023 in Amman, Jordan, we focused on understanding the genetic diversity and subtype patterns of HRSV. Among the total specimens collected, 71.9% (207 out of 288) tested positive for spectrum of respiratory viruses and bacteria. The most frequently detected viruses during this period were influenza B virus (IBV), influenza A virus (IAV), and HRSV. Additionally, we observed high frequencies for other viruses such as influenza C virus (ICV), human rhinovirus (HRV), and adenovirus (AdV). Our findings are consistent with previously reported studies from China, Brazil, and Saudi Arabia [36–38]. Moreover, these findings highlight the widespread distribution of these respiratory viruses in Jordan, underscoring the importance of accurate clinical diagnosis and appropriate treatment strategies. Throughout our study, nasopharyngeal swabs were successfully used for the detection of respiratory viruses, and they provided sufficient nucleic acid yield for our molecular analyses. These results align with previous studies in the literature [35, 39, 40], specifically, our finding that 35 out of 288 specimens (12.2%) tested positive for HRSV. Among the 24 HRSV sequences analyzed, 22 (91.7%) were identified as HRSV-A subtype, while 2 (8.3%) were HRSV-B. Notably, HRSV-A and HRSV-B subtype coinfection was not detected in our studied samples consistent with previous analysis [36, 41, 42]. Conversely, numerous studies have reported coinfections of HRSV-A and HRSV-B [43, 44].

The highest detection rate of HRSV occurred among infants (< 2 years of age). It is well-established that HRSV infections are most prevalent in children under 2-years-old, with the likelihood of infection decreasing as age increases. This demographic pattern reinforces the need for targeted preventive measures and healthcare strategies to mitigate the impact of HRSV infections, particularly among vulnerable populations such as young children. In our study, and consistent with previous research, most children presented with symptoms of acute respiratory tract infections (ARTI), such as cough, shortness of breath, nasal discharge, and fever [45, 46]. We found that 159 out of 288 specimens (55.2%) tested positive for respiratory viruses, highlighting the significant role these viruses play in respiratory diseases. These results align with findings from other studies in USA, China, South Africa, and Jordan [35, 47–49].

Notably, we observed numerous co-infections involving HRSV and other respiratory viruses. The most frequently identified co-infection was between HRSV and human coronavirus (ICV). This observation is commensurate with studies from other countries, where co-infections of HRSV with other respiratory viruses are commonly reported in patients with LRTIs [36, 42]. The impact of these coinfections remains a subject of debate; while some data suggests they may worsen clinical symptoms, other studies have found no significant effect on disease severity or clinical presentation [50–53]. Hence, further investigation into viral coinfections is required. We were unable to evaluate the correlation between coinfection and disease severity in this study, as patient follow-up was not conducted to assess clinical outcomes of single versus coinfections. Therefore, the impact of these coinfections on disease severity in our cohort remains undetermined. Furthermore, our study examined HRSV-positive samples for bacterial co-infections and found significant rates of Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia in patients with HRSV infections [54–56]. Gaining a deeper understanding of the interactions between viral and bacterial pathogens in respiratory infections could offer valuable insights for disease management and treatment strategies.

Phylogenetic analysis revealed that all HRSV-A strains (n = 22) were closely aligned with a novel ON1 genotype, that was identified for the first time in Ontario, Canada in 2010 [24]. Since then, the ON1 genotype has been reported in various countries worldwide, including Jordan, Saudi Arabia, Germany, Japan, China, and other countries [36, 42, 57–60]. In Jordan, the ON1 genotype was first detected in few samples collected in Amman, the Capital, in 2010–2013 while most samples were among the GA2 genotypes (55/58, 94.8%) [60]. Our work suggests that the ON1 genotype may have become predominant in Jordan, potentially replacing the previously dominant GA2 genotype. However, we must acknowledge the limitations of our findings due to the small number of positive samples and the short study period of six months. ON1 was first detected in Jordan between 2010 and 2013, with only a few isolates identified [60]. Given Jordan’s integration into the global pattern of viral evolution and spread, we can cautiously suggest a potential shift toward ON1 dominance as observed in other studies conducted in our region [61, 62]. However, to draw more definitive conclusions, larger-scale, multicenter studies conducted over a longer time span are needed to confirm this trend. The ON1 genotype has now been detected in more than 30 countries, demonstrating its widespread distribution.

HRSV genotypes have the potential to evolve and gain competitive advantages over other strains [60, 63], indicating that new lineages may emerge because of ongoing antigenic shifts and the large, susceptible host populations. In our study, we identified several nucleotide and amino acid changes within the HRSV-A strains, underscoring the rapid evolution occurring in the G gene. Notably, we detected sequence duplications in the second variable region of the G protein, a finding that aligns with previous research [36, 42]. This region is rich in crucial antigenic epitopes and is integral to the development of new variants, particularly under immune selection pressures following natural infections [64]. The alterations in this region can extend the length of the G protein and alter its structural properties, hence, increasing the virus’s ability to attach [65, 66].

Phylogenetic analysis of the 2 HRSV-B strain sequences identified the BA genotype, which has a characteristic 60-nucleotide duplication first isolated in Buenos Aires, Argentina in 1999 [67]. These strains were all classified as BA9 (n = 2, 100%). These nucleotide insertions into BA genotypes were reported more than a decade before the emergence of ON1 genotypes. In Jordan, this is the first time to report the detection of BA-9 since the previous study has reported that all 27 samples belonged to the GB1 subtype of HRSV-B [68]. Since their initial discovery, BA genotypes have spread globally and evolved into 14 distinct genotypes [25]. It is suggested that these nucleotide insertions provided the BA genotypes with fitness advantages, enhancing their attachment and pathogenicity [27]. The elongation of the G protein in the ON1 and BA9 genotypes, caused by 72 and 60 nucleotide duplications respectively, has played a key role in their swift spread. It is hypothesized that these genetic modifications have provided significant fitness and evolutionary advantages, enabling these genotypes to rapidly disseminate and achieve global dominance over other strains [26].

Analysis of amino acid changes revealed variations at 22 different sites in the second variable region of the G protein (amino acids 227–321) in 22 sequences of the ON1 genotype, compared to the reference ON1 genotype of HRSV-A. The most notable changes were N242D, L248I, L274P, P275Q, L298P, V303A, Y304H, L310P, L314P, and T320A indicating significant selection pressure on the virus. For the HRSV-B genotype (BA9), amino acid substitutions were identified across both sequences (n = 2) in the second variable region of the G protein, compared to the BA reference sequences (AY333364). Additionally, there were variations in 31 different sites within the second variable region of the G protein (amino acids 75–312). The most notable changes were listed in the results section and differed from the changes reported previously in Jordan since our samples are BA-9 compared to the GB1 reported previously [68]. The deletion of CCAAAA nucleotides in both samples of HRSV-B in this study resulted in the loss of two PK amino acids, a phenomenon that has also been observed in multiple studies in different countries over the past years [42, 69]. Additionally, the mutations identified in the duplicated regions of the BA and ON1 genotypes suggest an incremental accumulation of mutations over the years.

Previous studies have highlighted the importance of N and O-linked glycans on the G protein in helping the virus evade host immune responses through modifying the attachment and antigenic properties of HRSV strains [70, 71]. Analysis of ON1 strains revealed one potential N-glycosylation site at amino acid positions 237 and a varying pattern of O-glycosylation sites with 38–41 potential sites. For genotype BA9 sequences, there were four N-glycosylation sites at positions 81, 86, 296 and 310 and 41–44 potential O-glycosylation sites. In one of the two strains an additional N-glycosylation site (NHT) was seen because of the K258N mutation which was also reported in previous studies [72]. These mutations suggest potential selection pressure and may indicate regions of interest for further study; however, since our analyses are based on RNA sequences and in silico predictions of glycosylation sites, the impact of these mutations on antigenicity and virus fitness remains speculative and would require biochemical validation to confirm.

Finally, our study faced certain limitations. Firstly, we focused on genotyping HRSV using only the second variable region of the G gene rather than conducting whole gene sequencing. Secondly, the limited number of positive HRSV cases overall in our study, particularly the small number of HRSV-B samples (22 HRSV-A and only 2 HRSV-B), necessitates careful interpretation of the results. This sample size is not sufficient to draw definitive conclusions regarding the prevalence and genetic diversity of HRSV subtypes circulating in Jordan. Future studies should involve larger, multicenter cohorts and extend over multiple years to capture a more representative sample of both HRSV-A and HRSV-B cases. Such studies would provide a more comprehensive understanding of the circulating strains, enabling more robust conclusions about HRSV epidemiology and the potential differences between subtypes.

Conclusions

In this work, we analyzed the molecular composition of the G gene of HRSV-A, revealing the genetic diversity of HRSV in Amman. This research fills a knowledge gap regarding the genetic makeup of the virus in this region. Increased travel has heightened the risk of introducing novel HRSV variants globally. Our findings indicated that the ON1 genotype of HRSV-A and the BA-9 genotype of HRSV-B were predominant in Amman during the study period, contrasting with a study from 10 years prior that found a high prevalence of the GA2 genotype of HRSV-A and GB1 of HRSV-B. Continuous and long-term surveillance programs coupled with clinical data must be initiated to better understand the pattern of seasonal distribution of circulating genotypes of HRSV and find any association between emerging genotypes and disease severity.

Authors’ contributions

Conceptualization, A.I.K.; resources and data curation, A.I.K., N.H., S.A.S., SB, H.A-S, and H.A-M.; methodology, N.H., and A.S.; writing—original draft preparation, A.I.K., S.A.S., M.R.A. and T.S.; writing—review and editing, A.I.K., T.S., S.A.S., N.H., and M.R.A.; supervision, A.I.K.; project administration, A.I.K. and T.S. All authors have read and agreed to the published version of the manuscript.

Funding

Work in Ashraf Khasawneh’s laboratory is supported by a grant provided by the Deanship of Scientific Research, The Hashemite University (No. 3/7/2020/2021).

Data availability

The data presented in this study are available from corresponding authors upon request.

Declarations

Ethics approval and consent to participate

The study was conducted in compliance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) at The Hashemite University (No. 3/7/2020/2021) and PHH (No. MH/ 517/2022) according to the established guidelines. Written informed consent was obtained from all participants or guardians prior to participation in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.