Exploring the role of rotavirus, adenovirus, and norovirus in gastroenteritis among children in Iraq: an epidemiological and molecular study

Background and aim: Acute gastroenteritis (AGE) is a major cause of morbidity and mortality among children <5 years, with rotavirus A (RVA), adenovirus (AdV), and norovirus (NoV) being key viral agents. Co-infections may contribute to worse outcomes and viral evolution. Data on RVA co-infections and VP6 molecular characteristics in Iraq are limited. This study investigated the epidemiology of the three viruses and characterized RVA VP6 to address this gap.

Materials and methods: From September 2022 to August 2023, 170 stool samples were collected from children with AGE admitted to AL-Batool Teaching Hospital. All samples  were tested for RVA, AdV, and NoV by RT-PCR. VP6 subgrouping and phylogenetic analyses were performed on RVA-positive samples, and homology modeling with molecular dynamics simulations assessed structural impacts of VP6 amino acid changes.

Results: RVA, AdV, and NoV rates were 45%, 6%, and 3%, respectively. Overall, 86% of samples were positive for one or more viruses, and co-infections were detected in 4.1%, predominantly RVA–AdV. Rural residence and children aged 0–12 months were significantly associated with RVA infection, with seasonal peaks in winter and spring. VP6 subgroup II predominated (84.2%) and was associated with severe diarrhea. All VP6 sequences clustered within the DS-1-like (I2) lineage, sharing >98% identity with regional strains. Key substitutions (I38L, F63L, R117G) increased VP6 monomer stability without affecting trimer assembly.

Conclusion: Although co-infections were infrequent, their clinical relevance and the high viral loads observed underscore the need for ongoing surveillance of enteric viruses in Iraqi children with AGE. This study provides the first molecular and structural characterization of RVA VP6 in Iraq, highlighting the evolutionary stability of the DS-1–like backbone and its importance for genomic surveillance and vaccine development.

1 Introduction

Acute gastroenteritis (AGE) is still the most frequent gastrointestinal disease worldwide. It continues to result in a significant public health burden, especially in children under five years of age in low- and middle-income countries (LMIC) (1) (1). Annually, young children are affected by an estimated 3–5 million cases of AGE worldwide and approximately 12% of childhood deaths are attributed to AGE. Although AGE can arise from bacterial, parasitic, or fungal pathogens, viruses account for more than 70% of pediatric cases (1), with group A rotavirus (RVA), adenovirus (AdV), and norovirus (NoV) recognized as the principal etiological agents (2).

RVA, a member of the Sedoreoviridae family, remains the leading viral cause of severe AGE in children. The RVA genome comprises 11 double-stranded RNA segments encoding six structural (VP1–VP4, VP6, VP7) and five or six nonstructural proteins (NSP1–NSP5/6) (3, 4). Based on VP6 serology and sequence analysis, twelve rotavirus groups (A–L) have been identified, with RVA being the predominant group infecting humans (5, 6). Despite global declines in RVA-associated hospitalizations and mortality following vaccine introduction, with deaths decreasing from 528,000 to 128,500 annually (7–9), RVA continues to circulate at high levels. In countries without universal vaccination, detection rates may reach ~38%, compared with ~23% in vaccinated populations (10).

Human adenoviruses (HAdVs), non-enveloped DNA viruses of the genus Mastadenovirus, are also important causes of pediatric AGE. While species F (types 40/41) are the canonical enteric types, species A, B, C, D, and G have also been implicated in childhood gastroenteritis (11). Globally, HAdVs account for approximately 1–32% of AGE cases, with wide variation due to differences in diagnostic tools, population immunity, and geographic distribution (12, 13).

Noroviruses (NoVs) are the second most common viral cause of pediatric AGE after RVA and are responsible for an estimated 70,000–200,000 deaths annually, mostly in LMICs (14). Their genome contains three ORFs encoding VP1, VP2, and several nonstructural proteins (15). Among the 10 known norovirus genogroups (GI–GX), GI and GII are predominant in human infections and GII strains are consistently found as the dominant circulating genogroup worldwide (4, 18).

In Countries with poor hygiene environment and weak medical care system, co-infection with several enteric viruses is common. Co-infection can contribute to greater clinical severity, reduced vaccine effectiveness, and an increased likelihood of viral evolution. This evolutionary potential arises from mechanisms such as recombination and reassortment, which are more likely to occur when multiple viral strains infect the host simultaneously (19). However, the biological interactions among co-infecting viruses remain poorly understood. In Iraq, only a limited number of studies have examined viral etiologies of AGE, and comprehensive analyses of RVA, AdV, and NoV co-infections are scarce.

Although this study investigates all three major enteric viruses, special emphasis was placed on RVA due to its continued predominance in Iraqi children and persistent circulation despite vaccine introduction in 2012. Importantly, nearly all Iraqi rotavirus surveillance to date has focused on VP7 (G typing) and VP4 (P typing), leaving the genomic backbone, particularly VP6, largely uncharacterized (16, 17). VP6, the highly conserved intermediate capsid protein, serves as a key molecular marker for subgrouping and genogroup classification (>98% amino acid identity among strains) and has gained increasing interest as a potential target for next-generation vaccine design (18, 19). Despite being internal to the virion, VP6 becomes exposed during cell entry, enabling VP6-specific antibodies to mediate intracellular neutralization through TRIM21-dependent mechanisms and pore-blocking activity (20).

To address this major knowledge gap, the present study investigates the prevalence, demographic patterns, and co-infection profiles of RVA, AdV, and NoV among hospitalized children with AGE in Diyala Province, Iraq. Furthermore, it provides the first molecular and structural characterization of circulating Iraqi RVA strains using VP6. By integrating epidemiological data, VP6 subgrouping, phylogenetic analysis, and computational structural modeling, this study offers new insights into viral circulation in a post-vaccine population and highlights the value of VP6 as a complementary marker for genomic surveillance and vaccine development.

2 Materials and methods

2.1 Ethical approval and study design

This hospital-based, cross-sectional study was conducted at the College of Medicine, University of Diyala, in collaboration with the Pediatric Department of Al-Batool Teaching Hospitals, from September 2022 to August 2023. Ethical approval was obtained from the Ethics Committee of the College of Medicine, University of Diyala (Approval Code: 2024RFA889).

2.2 Sample collection

As part of a routine epidemiological investigation of viral agents causing acute gastroenteritis (AGE), 170 stool specimens were collected from unvaccinated children under five years of age who were hospitalized with AGE in Diyala, Iraq, between September 2022 and August 2023. Samples were stored at −20°C until analysis.

Demographic and clinical information, including age, sex, residence, water source, and clinical presentation, was obtained from hospital records.

2.3 Sample processing

Approximately 0.1 g of solid stool or 100 µL of liquid stool was diluted in 1 mL of phosphate-buffered saline (PBS) to prepare a 10% suspension. The mixture was vortexed for 30 s and incubated at room temperature for 10 min. After centrifugation at 2500 rpm for 5 min, the clarified supernatant was transferred to sterile tubes and stored at −20 °C for downstream analysis (3).

2.4 Viral DNA/RNA extraction

Viral RNA/DNA was extracted from the supernatant of a 10% (w/v) stool suspension prepared in PBS using a commercial viral RNA/DNA extraction kit (GeneAll, South Korea), according to the manufacturer’s instructions and the spin-column protocol (3).

2.5 Detection of rotavirus A by real-time PCR

Viral RNA extracted from stool specimens was first reverse-transcribed into complementary DNA (cDNA). Briefly, 10 µL of RNA was mixed with 1 µL random hexamer primers and 1 µL dNTP mix, heated at 65°C for 5 min, and immediately chilled on ice. The reverse transcription reaction was then prepared using 4 µL 5× RT buffer, 1 µL RNase inhibitor, and 1 µL reverse transcriptase, and incubated at 42°C for 30–45 min, followed by enzyme inactivation at 70°C for 10 min.

RVA was detected using a SYBR Green–based qRT-PCR targeting a 200 bp fragment of the VP6 gene. Each 20 µL reaction contained 10 µL SYBR Green Master Mix, 0.5 µL forward primer (5′-CTCAGCTGATGGAGCGACTA-3′), 0.5 µL reverse primer (5′-CTGCTACCGCTGGTGTCATA-3′), 5 µL cDNA, and 4 µL nuclease-free water (21). Amplification was performed on an ABI 7500 Real-Time PCR System as follows: 45°C for 10 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. A sample was considered positive for RVA when the Ct value was ≤ 35, which is a widely accepted diagnostic threshold for SYBR Green–based RVA assays. This cutoff corresponds to the limit of reliable quantification reported in previous RVA detection studies and minimizes false positives associated with late-cycle nonspecific fluorescence (22).

2.6 Detection of adenovirus

Adenovirus detection was performed using a validated conventional PCR assay targeting a 301 bp region of the hexon gene, using primers Hex1deg and Hex2deg (Table 1) (23).

Table 1

Table 1. List of primers used in this study.

Reaction components and amplification steps were standardized to match the format described for RVA, except for annealing temperatures, which were optimized according to the original protocol. PCR products were electrophoresed on 1.5% agarose gels stained with ethidium bromide and visualized under UV illumination.

This conventional protocol represents the routine and validated adenovirus method used in our diagnostic laboratory and regional surveillance studies, and was the only available assay at the time of sample processing.

2.7 Detection of norovirus

NoV genogroups GI and GII were detected using a probe-based real-time PCR assay on a Rotor-Gene™ system (Qiagen, USA). Primer–probe sets (COG1F/COG1R/Ring1 for GI; COG2F/COG2R/Ring2 for GII) are listed in Table 1 (24).

Each 20 µL reaction contained cDNA template, 4× CAPITAL™ qPCR Probe Master Mix (Biotech Rabbit, Germany), 500 nM of each primer, and 100 nM of each probe. Thermal cycling followed the manufacturer’s recommendations: initial denaturation at 95°C for 2–3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Amplification curves and Ct values were automatically recorded and interpreted according to established NoV diagnostic criteria.

2.8 VP6 genogrouping by conventional and multiplex PCR

VP6 genogrouping was performed using conventional RT-PCR followed by multiplex PCR. The initial RT-PCR used forward primer (5′-GGCTTTTAAACGAAGTCTTC-3′) and reverse primer (5′-GGTCACATCCTCTCACTA-3′). Multiplex PCR employed the same forward primer with genogroup-specific reverse primers: HG1-R (5′-GAAATGTAAAACCAGTTC-3′) for Genogroup I and HG2-R (5′-CTACTCCATTTCTTTGAGAC-3′) for Genogroup II. Amplicon sizes were 480 bp (I1) and 351 bp (I2).

2.9 Sequencing and phylogenetic analysis

Selected VP6-positive amplicons were Sanger-sequenced by Macrogen Inc. (Seoul, South Korea) using the ABI BigDye Terminator v3.1 Kit and ABI 3500 Genetic Analyzer. Sequences were analyzed using BioEdit (v7.1) (25) and confirmed by BLASTn. Phylogenetic trees were constructed in MEGA 6.06 (26) using the neighbor-joining method with 1000 bootstrap replicates, and the trees were visualized in iTOL (27). Final sequences were submitted to GenBank, and accession numbers were obtained.

2.10 Computational tools

Molecular modeling and visualization were performed using MOE 2024.06 (Chemical Computing Group, Montreal, Canada) (28). Structural validation was conducted using the SAVES v6.1 web server (29). Molecular dynamics (MD) simulations were carried out with GROMACS 2025.0 (30), and visualizations were prepared with PyMOL v0.99beta06 (DeLano Scientific LLC) (31).

2.11 Homology modeling of VP6

The wild-type VP6 sequence (UniProt ID: P18610) was modeled using MOE 2024.06 with the RVA VP6 crystal structure (PDB ID: 1QHD, 1.95 Å) as a template. Mutations (I38L, F63L, N76S, R117G, D130E) and subgroup variants were introduced via residue substitution with local energy minimization. Trimer modeling preserved the Zn²⁺ and Ca²⁺ ions, which are critical for structural stability.

2.12 Model quality assessment

Models were validated using PROCHECK, VERIFY3D, and ERRAT (SAVES v6.1) (32). Ramachandran plots, non-bonded interaction scores, and sequence-structure compatibility were assessed. Only models with favorable Ramachandran statistics and high ERRAT scores were used for MD simulations.

2.13 Molecular dynamics simulations

MD simulations were used to evaluate the dynamic behavior of wild-type and mutant VP6 proteins. Systems were prepared with CHARMM-GUI using CHARMM36m or AMBER03 force fields (32). Proteins were solvated in TIP3P water boxes, neutralized with 0.15 M NaCl, and equilibrated under NVT and NPT ensembles. Production runs were performed for 100 ns. Trajectories were analyzed for root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonds, radius of gyration (Rg), and solvent-accessible surface area (SASA).

2.14 Statistical analysis

Statistical analysis was performed using the chi-square (χ²) test and independent-samples t-tests to assess group differences. Multivariate analysis was conducted using multiple logistic regression to identify independent associations. A P-value of less than 0.05 was considered statistically significant for all analyses. All statistical computations were executed using IBM SPSS Statistics software (version 22; SPSS Inc., Chicago, Illinois, USA).

3 Results

3.1 Demographic characteristics

A total of 170 hospitalized children with acute gastroenteritis were enrolled in this study, with a mean age of 20.18 ± 16.48 months (range: 1–60 months). The cohort included 117 males (69%) and 53 females (31%), with mean ages of 18.75 ± 15.13 and 21.19 ± 16.54 months, respectively. Sampling was performed in all seasons; specimens were mainly collected in winter (86.73%), then in autumn (66.66%), spring (77.77%), and summer (41.66%). Age analysis revealed that infants (0–12 months) accounted for the majority of cases (53.22%). All children presented with diarrhea (watery or mucoid). The most common associated clinical feature was fever, reported in 59% of cases. Further demographic and clinical characteristics of the study population are summarized in Table 2.

Table 2

Table 2. Demographic and clinical characteristics of children with acute gastroenteritis included in the study.

3.2 Prevalence of RVA, AdV, and NoV

Among the 170 stool samples analyzed, 84.8% (91/170) tested positive for at least one of the three enteric viruses assessed. RVA was the most commonly detected pathogen, identified in 45% (76/170) of samples. AdV and NoV were detected in 6% (10/170) and 3% (5/170) of cases, respectively. These findings indicate that RVA was the predominant viral agent associated with acute gastroenteritis in children during the study period, while AdV and NoV circulated at much lower levels. A detailed distribution of virus positivity is provided in Table 2.

3.3 Demographic risk factors and their correlation with clinical disease severity in viral gastroenteritis cases

Analysis of demographic and clinical factors revealed that age and place of residence were significantly associated with RVA infection (p < 0.001). Infants aged 0–12 months had the highest infection rate, accounting for 53.22% of RVA-positive cases. Children living in rural areas were more positive (54.90%) than those in urban areas (18.4%). No significant correlations were found with RVA infection in relation to gender, maternal education level or source of drinking water. All RVA positive cases were found to have diarrhea (a characteristic of rotaviral gastroenteritis), and none of them was later clinically diagnosed as having intussusceptions. Other commonly reported symptoms were fever (65%), vomiting (67.03%), abdominal pain (61.11%), and dehydration (62.5%). A Seasonality analysis demonstrated a significant increase peak during winter for RVA infections (86.73%) followed by spring (77.77%) and autumn (66.66%), and the lowest detection rate was in summer (41.66%). These results indicate different age- and seasonal patterns of RVA activity in Diyala province. Additional demographic and clinical data are presented in Table 2.

3.4 Co-infections RVs, AdVs, and NoVs

Co-infections involving more than one viral pathogen were identified in 4.1% (7/170) of the study participants, while the majority (84.8%) had single-virus infections. The RVA–AdV combination was the most frequently observed co-infection, occurring in 2.9% of cases. No co-infections involving AdV–NoV were detected during the study period. The distribution of single and mixed infections is detailed in Table 3.

Table 3

Table 3. Patterns of co-infection among detected enteric viruses (RVA, AdV, and NoV) in children with acute gastroenteritis.

3.5 VP6 genogroup distribution

VP6 genogrouping of the 76 RT-qPCR-confirmed samples revealed a clear predominance of subgroup II (SG II), identified in 64 samples (84.2%), while subgroup I (SG I) was detected in 10 samples (13.2%). Two samples (2.6%) amplified both SG I and SG II targets, suggesting possible mixed infections or non-typical variants with overlapping subgroup-specific sequences, highlighting the molecular heterogeneity of circulating RVA strains.

SG II strains were also significantly associated with diarrhea (p < 0.0001), indicating a higher potential for pathogenicity than SG I strains. These findings indicate that SG II is the predominant VP6 lineage in this population and may contribute to the clinical severity of RVA infections in children under 5 years old.

3.6 VP6 gene sequencing and phylogenetic analysis

To further characterize the VP6 genogroups, representative strains from each subgroup were sequenced. For SG II, two strains exhibited four nucleotide substitutions at positions 15 (A/T), 99 (A/G), 171 (T/G), and 215 (T/C), with only the substitution at position 215 resulting in a non-synonymous change (F72S). These sequences were submitted to GenBank under accession numbers PP350763.1 and PP350764.1 (Figure 1).

Figure 1

Figure 1. Multiple sequence alignment of VP6 gene fragments from SGI and SGII rotavirus A strains. The sequences include the reference strains EU372725 (SGI) and EU372727 (SGII), along with the Iraqi strains PP350761.1 and PP350762.1 (SGI), and PP350763.1 and PP350764.1 (SGII).

Conserved nucleotides shared across all sequences are highlighted with a gray background. At the same time, variable positions are shown on a white background, with bases color-coded by type (adenine in green, thymine in red, cytosine in blue, and guanine in black). The primers used to amplify the VP6 region are underlined beneath the reference sequences. This alignment highlights conserved and divergent regions, showing the existence of synonymous and non-synonymous substitutions between strains. The contrast between conserved and variable sites visualized on the sequences provides a global description of sequence similarity and helps to detect genetic divergence within and between SGI and SGII genogroups.

For SG I, two strains displayed six nucleotide substitutions in comparison with the reference strain (PP861507.1), including four non-synonymous substitutions (F63L, R117G, N76S and D130E) which indicate a higher sequence divergence in comparison with SG II. These sequences were submitted under the accession numbers PP350761.1 and PP350762.1. A higher number of non-synonymous substitutions seen for SG I denotes higher molecular variability and thus presages for more flavor in downstream evolutionary analyses (Figure 2).

Figure 2

Figure 2. Nucleotide and deduced amino acid sequences of VP6 amplicons from S1 (PP350761.1) and S2 (PP350762.1) were aligned with the reference sequence PP861507.1. Red asterisks (*) under the alignment indicate base substitutions. Amino acid substitutions are bolded in red to highlight group-specific variations.

Phylogenetic analysis revealed that all the Iraqi isolates formed a monophyletic branch within the DS-1-like (I2) genogroup, sharing>98.5% nucleotide identity with Southeast Asian reference strains, including RVA/Human-wt/IDN/SOEP144/2016/G3P[8]. This clustering suggests strain homogeneity within Iraq and illustrates cross-border regional strain circulation (Figure 3).

Figure 3

Figure 3. Phylogenetic tree of the VP6 nt sequences of Iraqi HRV strains, indicating their genetic relationship to strains representing the SG I genogroup. The trees were created using neighbor-joining analysis in MEGA 11 with 1000 bootstrap replicates. The red five-pointed star denotes the VP6 sequences of the Iraqi strains detected in this study.

These findings further support the prevalence of the I2 genogroup among VP6 strains circulating in Iraq, demonstrate the high level of sequence conservation, and highlight the need for continued monitoring of the genetic diversity and evolutionary dynamics of RVA in the region.

3.7 Homology modeling and structural evaluation

A structural model of the VP6 monomer was generated using the high-identity template 1QHD (99% sequence identity to the target sequence, UniProt P18610). Homology modeling and energy minimization produced high-quality models for the wild type (WT), five single-point mutants, and two representative variants (S1, SG I; S2, SG II). Structural validation using PROCHECK, ERRAT, and VERIFY3D (SAVES v6.1) showed strong model quality for all variants, with ERRAT scores of 88.56–89.54, VERIFY3D scores >82.5, and >87% of residues located in favored Ramachandran regions (Supplementary Table S1).

Overall, the mutations introduced minimal global structural deviation, supporting the use of these models for subsequent structural comparison and dynamic analyses.

3.8 Structural description of VP6 and variant comparison homology modeling

VP6 adopts a conserved bilobed fold consisting of an α-helical N-terminal domain (residues 1–230) and a β-barrel C-terminal domain forming the outer capsid layer (Figure 4). Structural alignment of WT, S1, and S2 models showed very low RMSD values (0.10–0.19 Å), and alignment with the 1QHD crystal structure yielded a global RMSD of 0.802 Å (Supplementary Figure S1), confirming overall structural conservation.

Figure 4

Figure 4. Cartoon representation of the VP6 monomer highlighting its structural and sequence features. The left panel shows the 3D structure of the VP6 monomer, with its N- and C-terminal domains displayed and colored by secondary structure elements (α-helices in red, β-strands in yellow). The right panel illustrates the same monomer colored by amino acid sequence order, with annotated α-helices. The central zoom-in focuses on the N-terminal domain, highlighting the locations of amino acid substitutions, which are color-coded by sequence position and clearly labeled.

The five amino acid differences observed among the Iraqi isolates were all located in the N-terminal α-helical region (Supplementary Table S2) and were largely solvent-exposed. These mutations are therefore unlikely to alter the VP6 core fold but may subtly modify surface topology relevant to molecular interactions or antigenicity. Both S1 and S2 maintained the canonical structural organization, highlighting the evolutionary stability of the VP6 backbone.

3.9 Structural and physicochemical impacts of mutations

Global biophysical parameters, including molecular mass (~44.8 kDa), radius of gyration (~28.4 Å), and hydrodynamic radius (~28.8 Å), were highly comparable between WT, S1, and S2 (Supplementary Table S3), indicating preservation of overall compactness. Surface area calculations showed only minor increases in total and hydrophobic solvent-accessible surface areas in S2, consistent with a slightly more solvent-exposed conformation. Electrostatic properties, isoelectric points, and net charge also remained similar across all models.

Local residue-level analyses (Supplementary Table S4) revealed limited structural perturbations. S1 displayed modest localized rearrangements, whereas S2 exhibited a higher number of residues with altered exposure, suggesting slightly greater flexibility. Despite these local changes, both variants preserved the overall VP6 trimeric architecture. These findings support the conclusion that naturally occurring substitutions in the circulating Iraqi RVA strains primarily influence localized surface features rather than the global fold.

3.10 Molecular dynamics analysis of VP6 variants: monomeric and trimeric insights

MD simulations (100 ns) were performed for WT, S1, and S2 monomers to evaluate dynamic responses to amino acid substitutions (Table 4). S1 demonstrated lower RMSD and Rg values and increased hydrogen bonding relative to WT, indicating enhanced conformational stability, particularly around the F63L and R117G mutations. In contrast, S2 behaved similarly to WT, consistent with its conservative substitutions. Time-dependent trajectories showed that WT monomers exhibited greater structural drift after ~80 ns, whereas S1 and S2 converged toward more stable states (Figure 4).

Table 4

Table 4. The parameters of the molecular dynamics simulations (mean ± SD) for the trimetric wild type VP6 and S1 mutants.

To isolate mutation-specific effects, five single-point mutants were also simulated. I38L and R117G contributed most strongly to stability, while N76S and D130E produced minimal changes, mirroring S2’s behavior.

Trimeric simulations of WT and S1 (100 ns, modeled with preserved Zn²⁺/Ca²⁺ coordination; Table 5) revealed that inter-subunit contacts and metal binding stabilized the structure and largely mitigated monomer-level effects. Thus, while S1 mutations confer local stabilization in monomeric form, both S1 and S2 maintain the overall trimeric architecture, underscoring VP6’s structural resilience and functional robustness in circulating Iraqi RVA strains.

Table 5

Table 5. Molecular dynamics parameters (mean ± SD) of VP6 wild type and single-point mutants.

4 Discussion

Acute gastroenteritis (AGE) continues to be a leading cause of global morbidity and mortality, with children under five years of age in low- and middle-income countries being the worst affected (33, 34). Among the enteric viral agents, rotaviruses (RVAs), adenoviruses (AdVs), and noroviruses (NoVs) are important causes of pediatric AGE (35). In this research, we examined the frequency and clinical aspects of these viruses among hospitalized children in Diyala, Iraq, then concentrated on RVA VP6 to fill the national knowledge gap, as there are no earlier studies done in Iraq investigating either the molecular or the structural aspects of RV VP6.

We identified at least one viral agent in 84.8% of stool samples, in line with findings, RVA was detected in 45% of cases, a higher prevalence than some studies from Ramadi and Anbar (30–40%) (36) and Baghdad (~31%) (37), which could be attributed to variations in geographical location, sample size, and diagnostic assay used (17). In fact, the same kind of variation has been seen worldwide, such as in Iran, 11%–79% (38, 39). As in previous Iraqi reports (40), infants aged 1–12 months bore the brunt (p < 0.001), underscoring the contribution of waning maternal immunity and early-life susceptibility.

Co-infections were detected in 4.1% of all samples (6% of virus-positive samples), with RVA being the virus most frequently co-detected with AdV and NoV. Although we could not compare severity in our clinical cohort, previous data indicate that viral co-infections are associated with more severe disease, particularly in preterm infants (41, 42). The reported co-infection rates worldwide are highly different (0.3–45%) owing to the discrepancy in epidemiology, hygiene and testing strategies. These findings highlight the importance of multiplex diagnostics in hospitals serving young children.

Demographically, age, gender and living area, RVA positivity was significantly higher in infant (<12 months) (53.2%) and rural (54.9%) populations. Seasonal peaks in winter and spring were consistent with those reported from adjacent areas, which were attributed to regional climate conditions with lower temperatures and higher humidity, which favor viral stability and transmission (16, 43).

Molecular and phylogenetic analyses demonstrated that VP6 subgroup SG II (84.2%) was predominant and associated with more severe diarrhea, whereas subgroup SG I accounted for only 13.2% of cases, with co-infections being uncommon. All sequences grouped within the DS-1-like (I2) lineage and were >98% identical to strains from Southeast Asia, indicating cross-regional viral circulation facilitated by travel, trade, and migration. Earlier studies from Iraq also detected DS-1-like constellations, including the rare G8P[6] strains, indicating that although perhaps stable VP6 evolutionary pathways exist in Iraq, these are linked to at least regional sequences.

VP6 is highly conserved, but its I-genotype (I1–I32) classification allows an appropriate lineage-level resolution similar to the ribosomal-based classification in bacteria. This confirms VP6 as a dependable marker for postvaccine genomic surveillance, especially in areas of differing vaccine efficacy.

Structural modeling and molecular dynamics simulations showed that VP6 maintains it conserved bilobed fold and trimeric conformation in the presence of sequence mutations. The S1 substitutions (F63L, R117G) enhanced local monomer stability as indicated by decreased RMSD and increased hydrogen bonding, whereas analyses of single-point suggest the S1 mutations I38L and R117G to be stabilizing. S2-region mutations (N76S, D130E) were largely without effect, as expected for the conservative substitutions. Of note, simulations of trimeric Zn²⁺- and Ca²⁺-containing models showed minimal divergence between mutant and wild-type sequences HD and between any of them and the metal-free form, suggesting that metal coordination and inter-subunit contacts also buffer local perturbations. Taken together, these results are consistent with the idea that VP6 can accommodate moderate variability on its surface while preserving the functional architecture, which is characteristic of viral structural proteins.

There are limited studies that have investigated the structure–function relationship in VP6; thus, the epidemiological, phylogenetic and computational results reported here altogether offer a more comprehensive view of the natural mutations’ effect on viral stability and possibly antigenic behavior (44).

Due to its highly conserved, stable tertiary conformation and the proven capacity to induce broad cross-reactive protection, VP6 represents a suitable candidate platform for the development of novel vaccines. VP6-based VLPs, SRIV and nucleic acid vaccines induce substantial protection by intracellular, non-neutralizing immune mechanisms that do not rely solely on antibodies present in serum (45). In contrast to G/P-centric vaccines, VP6-based platforms are less prone to antigenic drift and may not require as frequent updates.

The limitations of the present study are the relatively small number of VP6 sequences, the absence of in vitro confirmation of the MD predictions, and the confinement to one single governorate. More extensive genomic surveillance in Iraq is required to identify further diversity and potential regional distinctions. Further analyses should investigate the relationship between mutations in VP6 and the clinical severity or vaccine response and experimentally evaluate the structural consequences of most important substitutions.

5 Conclusions

This study indicates that, in Diyala, RVA is the leading viral agent of acute gastroenteritis among hospitalized children, followed by AdV and NoV. The identification of co-infections in a significant number of cases highlights the necessity for multiplex diagnosis, as single-virus assays could misestimate the real viral load. These results highlight a need to enhance national surveillance systems and to reconsider prevention policies, especially in pediatric high risk groups.

Following characterization of the epidemiology of the three major enteric viruses, we performed further analysis on RVA due to the clinical importance and high prevalence of this virus. This study is the first to present molecular and structural characterization of RVA from Iraq based on VP6. Infants aged less than 12 months and rural dwellers were predominantly affected by RVA infections, which had a distinct seasonality in winter. SG II was the predominant sub-group and was more commonly found in severe diarrhea. Phylogenetic analysis demonstrated that all VP6 sequences were grouped in the DS-1-like I2 lineage and closely related to strains from Southeast Asia.

Structural model and molecular dynamics simulation demonstrate that VP6 retains its conserved overall fold, and the observed amino acid substitutions have little effect on the stability of the monomer or the assembly of the trimer. These findings further support VP6 as the ideal molecular marker and an effective tool for future surveillance and vaccine development in Iraq.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Ethics statement

The studies involving humans were approved by Ethics Committee of the College of Medicine, University of Diyala (Approval Code: 2024RFA889). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

Author contributions

RM: Formal Analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. FF: Software, Validation, Visualization, Writing – review & editing. IL: Conceptualization, Investigation, Supervision, Writing – review & editing. AG: Supervision, Validation, Visualization, Writing – review & editing. NB-E: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The Review editor WL declared a past co-authorship with the author AG.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fviro.2025.1748665/full#supplementary-material

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