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Rotavirus Group A (RVA) is a primary pathogen that causes viral diarrhea in humans and animals. Porcine rotaviruses (PoRVs) are widely epidemic in pig farms in China, causing great economic losses to the swine industry. In the past 30 years, the G5 RVA had been the main epidemic genotype in pig farms worldwide. However, G9 RVA is an emerging genotype that is gradually becoming prevalent in humans and animals. To explore its potential mechanism, we isolated G9P[23] and G5P[23] rotaviruses, named 923 H and NG523 respectively, from diarrheal samples and compared the growth curves and virulence of two strains. In vitro experiments revealed that pig small intestine epithelial cells were more susceptible to 923 H strain. In vivo experiments showed that 923 H strain was more virulent than NG523 strain, causing more severe damage to piglets. The viral load of G9 infection groups in intestinal and extra-intestinal tissues was higher than that of G5 infection group. Histopathological examination showed cell degeneration, necrosis and nuclear condensation in the jejunum of G9 RVA infection group as well as more inflammatory cell infiltration and tissue destruction in the lung of G9 RVA infection group. Our results indicate that 923 H strain is more pathogenic than NG523 strain, which provides new insights into the widespread epidemic of G9 RVA in pig farms., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts of interest with respect to the research authorship and/or publication of this article., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Group A rotaviruses (RVA) and group C rotaviruses (RVC) are important enteric pathogens in swine. Comprehensive studies investigating porcine rotaviruses in Canada are necessary to enhance understanding of the frequency, impacts, and dynamics of these infections in swine herds. This study aims to estimate the prevalence of RVA and RVC, describe circulating strains, and assess the association of rotaviruses with diarrhea at the piglet, litter, and batch levels in Canadian farrowing swine productions. A matched case-control study was conducted on farrowing farms within an integrated production system experiencing a diarrheic episode. Rectal swabs from 94 diarrheic piglets and 127 healthy piglets were collected and subjected to VP7 and VP4 gene amplification of RVA and RVC using RT-PCR. Results indicated a 45.4 % and 27.4 % prevalence for RVA and RVC in piglets, respectively. A significant association between RVC and diarrhea (odds ratio = 7.1; p = 0.02) was identified at the batch level, while RVA detection did not show a significant relationship with diarrhea. Molecular characterization of various RVA and RVC strains detected in this study described at least four different RVA strains and three different RVC strains circulating on farms within the integrated production system. This study estimates the prevalence of RVA and RVC and describes the main viral strains in swine herds experiencing an episode of neonatal diarrhea. While it also highlights the importance of RVC in piglet diarrhea when detected in a batch, results from his study warrant the implementation of additional prevention measures and regular surveillance for the control of both RVA and RVC in swine herds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2025. Published by Elsevier B.V. All rights reserved.)

Background: Despite global rotavirus vaccination efforts, rotavirus remains a leading cause of childhood deaths from acute gastroenteritis. Post-vaccination studies in India, particularly in eastern India, have been limited, despite high prevalence of rotavirus in this region prior to vaccine introduction. This study was conducted to assess the impact of rotavirus vaccine on the epidemiology of rotavirus and other enteric viruses, as well as the changes in the diversity of rotavirus strains among children (≤5 years) with acute gastroenteritis., Methods: A total of 877 stool samples from children hospitalized with acute diarrhea during 2022-2023, were screened for enteric viruses using multiplex PCR. Rotavirus positive samples were genotyped by sequencing and phylogenetic analysis of VP4 and VP7 genes were done., Results and Discussion: Out of 877 diarrheal cases, 47 % tested positive for at least one enteropathogenic virus. Rotavirus was most prevalent (25.9 %), followed by norovirus (11.4 %), adenovirus-F (10.6 %), and astrovirus (5.3 %). Among mixed infections, rotavirus and norovirus co-infections were the most common. Rotavirus infection was highest in children aged 12-24 months, while other enteric viruses were more common in the 6-24 month age group. Clinical severity was higher among rotavirus-infected patients compared to those infected with other enteric viruses. The G3P[8] genotype of rotavirus predominated, with notable increase in G2P[4] and the detection of rare strains like G3P[6] and G11P[25]. G3P[6] was identified for the first time in this region showing Wa-like genome constellation. Unlike pre-vaccine period, G9 genotype was not detected. Mutations in antigenic epitope of circulating strains compared to vaccine strains may affect vaccine efficacy., Conclusion: The study highlights the persistent burden of childhood diarrhea despite rotavirus vaccination. Subtle alterations in the proportion of other enteric viruses and diversity of circulating rotavirus genotypes in the post-vaccination period were observed. Continuous long-term surveillance is required to evaluate the impact of vaccine in this region., Competing Interests: Declaration of competing interest The authors declare that they have no known financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Broadly neutralizing antibodies (bNAbs) could offer escape-tolerant and lasting protection against viral infections and therefore guide development of broad-spectrum vaccines. The increasing challenge posed by viral evolution and immune evasion intensifies the importance of the discovery of bNAbs and their underlying neutralization mechanism. Here, focusing on the pivotal viral protein VP4 of rotavirus (RV), we identify a potent bNAb, 7H13, exhibiting broad-spectrum neutralization across diverse RV genotypes and demonstrating strong prevention of virus infection in female mice. A combination of time-resolved cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography (cryo-ET) analysis reveals a counterintuitive dynamic process of virus inactivation, in which 7H13 asymmetrically binds to a conserved epitope in the capsid-proximal aspect of VP4, triggers a conformational switch in a critical residue-F418-thereby disrupts the meta-stable conformation of VP4 essential for normal viral infection. Structure-guided mutagenesis corroborates the essential role of the 7H13 heavy chain I54 in activating F418 switch and destabilizing VP4. These findings define an atypical NAbs' neutralization mechanism and reveal a potential type of virus vulnerable site for universal vaccine and therapeutics design., Competing Interests: Competing interests: The authors declare that they have no conflicts of interest., (© 2025. The Author(s).)

Viruses engage in a variety of processes to subvert host defenses and create an environment amenable to replication. Here, using rotavirus as a prototype, we show that calcium conductance out of the endoplasmic reticulum by the virus encoded ion channel, NSP4 , induces intercellular calcium waves that extend beyond the infected cell and contribute to pathogenesis. Viruses that lack the ability to induce this signaling show diminished viral shedding and attenuated disease in a mouse model of rotavirus diarrhea. This implicates nonstructural protein 4 (NSP4) as a virulence factor and provides mechanistic insight into its mode of action. Critically, this signaling induces a transcriptional signature characteristic of interferon-independent innate immune activation, which is not observed in response to a mutant NSP4 that does not conduct calcium. This implicates calcium dysregulation as a means of pathogen recognition, a theme broadly applicable to calcium-altering pathogens beyond rotavirus.

Species A rotaviruses (RVs), which belong to the family Sedoreoviridae and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.

Previous systematic literature reviews of rotavirus genotype circulation in Europe and the Middle East are limited because they do not include country-specific prevalence data. This study documents country-specific evidence on the prevalence of rotavirus genotypes in Europe and the Middle East to enable more precise epidemiological modeling and contribute to the evidence-base about circulating rotavirus genotypes in the post-vaccination era. This study systematically searched PubMed, Embase and Scopus for all empirical epidemiological studies that presented genotype-specific surveillance data for countries in Europe and the Middle East published between 2006 and 2021. The STROBE checklist was used to assess the quality of included studies. Proportional meta-analysis was conducted using the generic inverse variance method with arcsine transformation and generalized linear-mixed models to summarize genotype prevalence. Our analysis estimated the genotype prevalence by country across three date categories corresponding with rotavirus seasons: 2006-2010, 2011-2015, 2016-2021. A total of 7601 deduplicated papers were identified of which 88 studies were included in the final review. Rotavirus genotypes exhibited significant variability across regions and time periods, with G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and, to a lesser extent G12P[8], being the most prevalent genotypes through different regions and time-periods. Uncommon genotypes included G3P[9] in Poland, G2P[6] in Iraq, G4P[4] in Qatar, and G9P[4] as reported by the European Rotavirus Network. There was high genotype diversity with routinely identified genotypes being G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]; there was high variability across time periods and regions. Continued surveillance at the national and regional levels is relevant to support further research and inform public health decision-making.

Post rotavirus vaccine introduction in Mozambique (September 2015), we documented a decline in rotavirus-associated diarrhoea and genotypes changes in our diarrhoeal surveillance spanning 2008-2021. This study aimed to perform whole-genome sequencing of rotavirus strains from 2009 to 2012 (pre-vaccine) and 2017-2018 (post-vaccine). Rotavirus strains previously detected by conventional PCR as G2P[4], G2P[6], G3P[4], G8P[4], G8P[6], and G9P[6] from children with moderate-to-severe and less-severe diarrhoea and without diarrhoea (healthy community controls) were sequenced using Illumina MiSeq ® platform and analysed using bioinformatics tools. All these G and P-type combinations exhibited DS-1-like constellation in the rest of the genome segments as, I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that strains from children with and without diarrhoea clustered together with other Mozambican and global strains. Notably, the NSP4 gene of strains G3P[4] and G8P[4] in children with diarrhoea clustered with animal strains, such as bovine and caprine, with similarity identities ranging from 89.1 to 97.0% nucleotide and 89.5-97.0% amino acids. Our findings revealed genetic similarities among rotavirus strains from children with and without diarrhoea, as well as with animal strains, reinforcing the need of implementing studies with One Health approach in our setting, to elucidate the genetic diversity of this important pathogen., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: Previous Ethics Committee approval was granted by the Mozambican National Bioethics Committee, Ministry of Health, Mozambique, through the reference numbers 11/CNBS/07; IRB 00002657 and 209/CNBS/15; IRB00002657. All the legal guardians/next of kin of the children who participated in this study, signed a written informed consent form., (© 2024. The Author(s).)

Rotavirus group A (RVA) is a major cause of pediatric acute gastroenteritis (AGE). Vaccination is an effective public health strategy and Angola implemented it in 2014. This hospital-based study aimed to estimate the prevalence of RVA infection and the severity of AGE in children under five years of age treated at six hospitals in Luanda Province. Between April 2021 and May 2022, 1251 fecal samples were screened by an immunochromatographic rapid test (SD Bioline). Data on socio-demographic profile, nutritional status, and clinical assessment were obtained. The association of RVA infection and AGE severity with possible risk factors was evaluated with a binary logistic regression model. Overall, the detection rate was 57.8% and girls tend to be more often infected than boys (55.2%). Infection was more common in the youngest group (1 to 6 months, 60.3%). Important sources of RVA infection were drinking water kept in tanks (57.9%) and private sanitary facilities with piped water (61%). Surprisingly, according to the Vesikari Scale score, the most severe symptoms were observed in children vaccinated with two doses (80.7%). RVA prevalence remains high despite vaccination, and further studies should address the association between infection sources and disease severity, as well as the causes underlying vaccine (un)effectiveness.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution., Competing Interests: The authors declare no conflict of interest.

Introduction: This study, conducted in China prior to RotaTeq's launch, examined the epidemiological, molecular, and evolutionary features of the G1P[8] genotype RVA in children admitted with diarrhea, to aid in evaluating its efficacy and impact on G1P[8] RVA in China., Methods: Data from the Chinese viral diarrhea surveillance network were collected from January 2016 to December 2018. RVA strains identified as the G1P[8] genotype were subjected to whole-genome sequencing. Neutralizing epitope, amino acid selection pressure, and evolution dynamics analyses on VP7 and VP4 were performed using BioEdit v.7.0.9.0 and PyMOL v.2.5.2, four algorithms (MEME, SLAC, FEL, and FUBAR) in the Datamonkey online software, and the MCMC model in BEAST v. 1.10.4, respectively. Phylogenetic and identity features of 11 genes were assessed by DNAStar and MEGA v.7., Results: Results showed that the detection rate of G1P[8] in China from 2016 to 2018 was generally low with significant seasonality. The whole genome of G1P[8] of four 2016 childhood diarrhea specimens was successfully sequenced. Phylogenetic and neutralizing epitope analysis showed that Rotavin-M1 might have better protection on G1P[8] prevalent in China than Rotarix and RotaTeq. Two conserved N-glycosylation sites on VP7 of Chinese G1P[8] might affect the protective effect of the vaccine. Evolution rate and selection pressure analysis identified the possibility of rapidly evolving and adapting to the new environment introduced by vaccines of G1P[8], whereas positive selection specific to VP4 indicated the potential tendency to select for dominant traits. Identity and phylogeny analysis showed that Chinese G1P[8] from before 2018 was generally stable with possible genetic recombination among local strains., Discussion: These findings not only are of great significance for predicting the prevalence of G1P [8] in China, but also provide data reference for evaluating rotavirus vaccine efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Peng, Wang, Shahar, Xiong, Zhang, Pang, Wang, Kong, Li and Duan.)

Group A rotaviruses (RVAs) are a major cause of acute gastroenteritis in children under 5 years of age worldwide. Herein, the genetic sequences of 11 RNA segments from three uncommon G9P[4] RVA strains found in the stool samples of children under 5 years of age in Iran were analyzed using next-generation sequencing (NGS) technology. The genomic constellations of these three uncommon G9P[4] strains indicated the presence of the double and quadruple reassortants of two G9P[4] strains, containing the VP7/NSP2 and VP7/VP2/NSP2/NSP4 genes on a DS-1-like genetic background, respectively. The genome of one strain indicated a Wa-like genetic backbone in a single-reassortant with the VP4 of the DS1-like human strains. With the exception of VP1, VP2, VP7, NSP2, NSP3, and NSP4 genes, which clustered with RVA of human origins belonging to cognate gene sequences of genogroup 1/2 genotypes/lineages, the remaining five genes (VP8/VP4, VP3, VP6, NSP1, NSP5) displayed direct evidence of recombination. It is presumed that the presence of uncommon G9P[4] strains in Iran is not linked to vaccination pressure, but rather to the high prevalence of RVA co-infection or the direct import of these uncommon RVA reassortants strains from other countries (especially those that have implemented RV vaccination)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Inter-genogroup reassortment of Rotavirus A (RVA) strains has highlighted the spread of unusual RVA strains worldwide. We previously reported the equine-like G3 RVA as the predominant strain in Indonesia in 2015-2016. However, since July 2017, typical human genotypes G1 and G3 have replaced these strains completely. To understand how dynamic changes in RVA occur in Indonesia, we performed a detailed epidemiological study. A total of 356 stool specimens were collected from hospitalized children in Sidoarjo, Indonesia between 2018 and 2022. Whole-genome sequencing was performed for all 26 RVA-positive samples using next-generation sequencing. Twenty-four samples were determined to be the unusual RVA G9P[4], while two were G9P[6]. Detailed analysis revealed that seven G9P[4] strains had the typical DS-1-like backbone, while the other strains exhibited a double-reassortant profile (G9-N1) on the DS-1-like backbone. The Bayesian evolutionary analyses suggested that the Indonesian G9P[4] strains share a common ancestor with previously reported G9P[4] strains in the VP7 and VP4 genes. G9P[4] DS-1-like strains were identified as the predominant genotype in Indonesia in 2021 for the first time. These results suggest that the G9P[4] strains were generated from the previous G9P[4] strains that had undergone further intra-reassortments with the other circulating strains., (© 2024 Wiley Periodicals LLC.)

Rotavirus (RV) mainly infects mature intestinal epithelial cells and impairs intestinal absorption function, which leads to the death of infected cells and eventually fatal diarrhea. Ferroptosis is a novel regulatory cell death pattern, which can be caused by virus infection. 1α,25-hydroxyvitamin D 3 (1,25D 3 ) has an anti-RV infection effect and can regulate ferroptosis. However, whether RV infection can induce ferroptosis, and whether 1,25D 3 can inhibit RV infection by regulating ferroptosis has not yet been studied. Present study shows that RV infection or erastin treatment induces IPEC-J2 cell death, which results in mitochondrial shrinkage, decreased mitochondrial membrane potential (MMP) and glutathione (GSH) content, increased MMP, intracellular Fe 2+ , reactive oxygen species (ROS), and malondialdehyde (MDA) contents. Meanwhile, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and deferoxamine (DFO) treatment can effectively reverse the increase of intracellular Fe 2+ , ROS and MDA levels induced by RV infection. Moreover, RV infection increases activating transcription factor 3 (ATF3) mRNA and protein expressions, and inhibited SLC7A11 and glutathione peroxidase 4 (GPX4) expressions, which was partially alleviated by siATF3. 1,25D 3 treatment significantly eliminates RV induced ferroptosis via ATF3-SLC7A11-GPX4 axis. Therefore, these results reveals that RV infection induces ferroptosis in IPEC-J2 cell and 1,25D 3 alleviates RV induced ferroptosis by regulating the ATF3-SLC7A11-GPX4 axis., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P [1] C73, G10P [11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV., Competing Interests: Conflict of interest All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Equine rotavirus A (ERVA) can cause foal diarrhoea and the most common ERVA genotypes are G3P[12] and G14P[12]. Since the introduction of a monovalent killed G3P[12] vaccine, infection in neonates has decreased. We aimed to determine the dynamics and longevity of maternally derived anti-G3P[12] neutralizing antibodies (NAbs) in foals and what, if any, cross-reactivity exists between maternally derived NAbs against G14P[12]. Serum samples were collected from 50 mare-foal pairs before each vaccination and up to 6 months post-foaling for mares and up to 7 months of age for foals. These samples were then used for virus-neutralization antibody assays with both G3P[12] and G14P[12] viruses. We observed that vaccination of mares could increase their serum NAb titers. Pre-nursing serum samples of foals collected at birth before the first nursing contained no detectable NAbs. In contrast, post-nursing serum samples of foals showed a significant amount of NAb levels, thereby confirming that these NAbs are passed through the mare's colostrum. Our study demonstrated that there is variation in the ratio of NAbs transferred from the serum of mares to the serum of their foals. Results also confirmed evidence of cross-reactivity between maternal antibodies in the serum of G3P[12] vaccinated dams and G14P[12]. Heterologous (G14P[12]) NAb titers were about 2- to 4-fold lower than homologous (G3P[12]) titers in colostrum, milk, and serum samples of both mares and their foals. Our data demonstrate that G3 and G14 NAbs in the serum of foals decreased steadily over time with the lowest point measured at approximately 4 months of age., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To evaluate the use of a multiplex real-time polymerase chain reaction (RT-PCR) test in detecting three viruses namely Rotavirus, Norovirus (genotypes 1 and 2), and Astrovirus that cause gastroenteritis in children under the age of five years., Study Design: A cross-sectional study. Place and Duration of the Study: Department of Virology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from January to July 2023., Methodology: A total of 87 children having acute diarrhoea and less than 5 years of age were included in this study from the outpatient clinic. Multiplex PCR was performed for the detection of three viruses: Rotavirus, Norovirus, and Astrovirus in stool samples of patients using a commercially available PCR kit. The data were analysed using Statistical Package for Social Sciences (SPSS) version 27:00., Results: Out of total 87 children, aged 2.5 ± 1.5 years, 56 (64.3%) were positive for multiplex RT-PCR and 31 (35.6%) were negative. Rotavirus was identified in 24 (27.5%) children as the most common cause of acute diarrhoea, followed by Norovirus in 20 (22.9%), and Astrovirus in 7 (8%) patients, while co-infection with multiple viruses occurred in 5 (5.7%) of the cases., Conclusion: This study revealed viral aetiology as a significant cause of acute diarrhoea in children. Multiplex PCR in the healthcare system can make it easier to identify, treat, and control the upsurge of diarrhoea. Prompt diagnosis of viral causes can lead to the prevention of unnecessary use of antibiotics., Key Words: Multiplex polymerase chain reaction, Rotavirus, Norovirus, Astroviridae, Diarrhoea.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The LLR RVA vaccine, which licensed in 2000 and widely used in China, significantly reduced rotavirus disease burden in China. With the changing of RV circulating strains and the emergence of new genotypes, the LLR vaccine against RVGE needed to be upgraded. In this study, we aimed to establish an RG system for the RVA vaccine strain LLR (G10P[15]). Transfection with plasmids expressing 11 genomic RNA segments of LLR along with the pCMV/868CP helper plasmid, resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the LLR strain was successfully established. Furthermore, the plasmid-based reverse genetics system was used to generate lamb RVA reassortants with VP4 or VP7 genes derived from human RVA strains in China, which were not previously adapted to cell culture. We were able to rescue the six VP7 (G1, G2, G3, G4, G8, and G9) mono-reassortants, but no VP4 (P[4] or P[8]) mono-reassortant was rescued. The six VP7 reassortants covered all G-genotypes currently circulating in China and stably replicated in MA104 cells, which should be exploited as the next-generation rotavirus vaccines candidates in China. Furthermore, the LLR RG system in this study will be a useful vaccine vector for intestinal pathogens such as norovirus and Vibrio cholerae., (© 2024 Wiley Periodicals LLC.)

Post-translational modifications (PTMs), as epigenetic modifications, are significant in the interaction between virus and its host. However, it is unclear whether rotavirus (RV) causes changes in both the host cell epigenetic protein modification and the regulatory mechanism of viral replication. Here, we analyzed the proteome of Caco-2 cells to determine if acetylation modification occurred within the cells after RV infection. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein involved in glycolysis, was deacetylated at lysine 219 via histone deacetylase 9 (HDAC9) in 50 h after the RV infection. Remarkably, the deacetylation of GAPDH promoted RV replication. Finally, we found that glycolysis was alterable in Caco-2 cells by RV or the deacetylation of GAPDH lysine 219, using the Seahorse XF Glycolysis Stress Test. In conclusion, our results demonstrate for the first time that RV infection promoted deacetylation of GAPDH at lysine 219 in order to increase its own viral replication in Caco-2 cells., Competing Interests: Declarations Conflict of interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part., (© 2024. The Author(s).)

Rotavirus A (RVA) is one of the major viral causes of acute gastroenteritis in calves globally. Bovine RVA can represent a public health concern as it is capable of zoonotic transmission. We assessed the burden, genotype distribution, and zoonotic potential of RVA among calves in Amhara National Regional State, Ethiopia. A multi-center cross-sectional study was conducted involving a total of 266 calves. Clinical data and fecal samples were collected by trained veterinarians. Total RNA was extracted using QIAamp viral RNA mini kit and RVA was detected by One-step qRT-PCR. Amplification and Sanger sequencing of VP7 and VP4 genes were performed to determine G-types and P-types of the circulating RVA, respectively. The prevalence of RVA among calves was 41/266 (15.4 %, 95 % CI = 11.3 %-19.5 %). The circulating G-types were G6, G8, and G10, while the circulating P-types were P[1], P[4], P[8], P[11], P[13] and P[14]. G10P[11] (37.5 %) followed by G6P[14] (18.8 %), and G6P[1] (12.5 %) were the dominant G/P combinations circulating among calves in the study area. The circulating bovine RVA strains including human-like bovine (GxP[4] and GxP[8]) and porcine-like bovine (G8P[13]) P-genotypes identified in calves were closely related to RVA strains globally reported from bovine, human, caprine, porcine, and other hosts. Our data reveal that the prevalence of RVA in calves is significant with diverse genotypes circulating in the study area with a potential for zoonosis and/or reverse zoonosis. Hence, continuous surveillance of the circulating RVA genotypes is crucial to curb the RVA-associated morbidity and mortality in cattle and human populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Porcine rotavirus A (RVA) is one of the major etiological agents of diarrhea in piglets and constitutes a significant threat to the swine industry. A molecular epidemiological investigation was conducted on 2422 diarrhea samples from Chinese pig farms to enhance our understanding of the molecular epidemiology and evolutionary diversity of RVA. The findings revealed an average RVA positivity rate of 42% (943/2422), and the study included data from 26 provinces, primarily in the eastern, southern and southwestern regions. Genetic evolutionary analysis revealed that G9 was the predominant genotype among the G-type genotypes, accounting for 25.32% of the total. The VP4 genotypes were P[7] (36.49%) and P[23] (36.49%). The predominant genotypic combinations of RVA were G9P[23] and G9P[7]. Eleven RVA strains were obtained via MA104 cell isolation. A rat model was established to assess the pathogenicity of these strains, with three strains exhibiting high pathogenicity in the model. Specifically, the RVA Porcine CHN HUBEI 2022 (Q-1), RVA Porcine CHN SHANXI 2022 (3.14-E), and RVA Porcine CHN HUBEI 2022 (5.11-U) strains were shown to cause diarrhea in the rats and damage the intestinal villi during the proliferation phase of the infection, leading to characteristic lesions in the small intestine. These data indicate that continuous monitoring of RVA can provide essential data for the prevention and control of this virus.

The causes of diarrhea after 10 years of rotavirus vaccination in Rwanda were investigated with real-time polymerase chain reaction in 496 children with diarrhea and 298 without. Rotavirus was detected in 11% of children with diarrhea (odds ratio, 2.48; P = .002). Comparison of population attributable fractions (PAFs) shows that Shigella (PAF, 11%) and enterotoxigenic Escherichia coli producing labile toxin (PAF, 12%) have replaced rotavirus as the main causative agents. The PAF for rotavirus had declined from 41% prevaccination to 6.5% postvaccination, indicating that rotavirus has become one among several similarly important causes of childhood diarrhea in Rwanda. A rotavirus genotype shift to G3P[8] points at the importance of continued genotype surveillance., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Rotavirus-associated gastroenteritis is a common health problem in children, different variations of rotavirus genotypes differ according to geographic locations and the practice of wide-scale vaccination. Therefore, the present study aimed to detect both the G and P genotypes of rotavirus in children ≤ 5 years old in one center in Egypt as a cross-sectional study, to correlate the genotypes with various demographic and clinical data in infected children and to evaluate the common mixed genotypes G and P in infected children., Method: The cross-sectional study included children with acute gastroenteritis ≤ 5 years old from January 2023 till March 2024 recruited from Mansoura University Children's Hospital, Egypt based upon laboratory diagnosis by exclusion of bacterial and protozoa pathogens. The stool samples were obtained from each child and subjected to detection of rotavirus antigen by enzyme-linked immunosorbent assay (ELISA) followed by genotypes identification of G and P genotypes by nested polymerase chain reaction (PCR)., Result: A nested PCR study for rotavirus genotypes revealed that G1 was the most common genotype (24.7%) followed by G2 (21.1%), G3 (20%), G9 (20%), and G4 (14.1%). The genotyping of the P genotype revealed that P9 was the commonest genotype (24.7%), followed by P4 (21.2%), P10 (20%), P8 (17.6%) and P6 (16.5%). The commonest combined genotypes of G and P were G1P4 (85.7%), G3P8(88.2%), followed by G2P6 (77.8%) and G9P9(76.5%) and G4P9 (66.7%) followed by G4P10 (33.3%), G9P10(23.5%), G2P10(22.2%), G1P10 (14.3%), G3P10(11.8%). The distribution was significant (P = 0.001). The positive rotavirus antigen was more frequently detected in females (55.3%) than males (44.7%, Odd ratio 0.2, 95% CI 0.22-0.71, P = 0.001). There was a significant association between the summer season and positive rotavirus antigen (P = 0.001) and rural residence of the patients (Odd ratio 6,9 95%CI 3,5-13.5, P = 0.001). The significant associated clinical sign with positive rotavirus antigen was fever (Odd ratio 3,3, 95%CI 1,8-6.05, P = 0.001). The genotypes G and P were significantly associated with positive rotavirus antigen as all cases positive by antigen had been detected by nested PCR with the commonest genotypes G4 (24.7%, P = 0.001) and genotype P9 (24.7%, P = 0.001)., Conclusion: The present study highlights the common genotypes of rotavirus at one center in Egypt, G1, G2, and G3 were the commonest G genotypes. As regard genotype P the commonest genotypes were P9, P4, and P10. The commonest combined genotypes were G1P4, G3P8, G2P6. There was no effect of the practice of rotavirus vaccination at limited rates at private health sections as the rotavirus is still a major pathogen of acute gastroenteritis in children. There is a need for the inclusion of rotavirus vaccination in the national program of children vaccination in Egypt., Competing Interests: Declarations Ethics approval and consent to participate All methods were performed by the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards. The ethical approval of the study was obtained from the ethical committee of Mansoura Faculty of Medicine (R. 24.06.2665) and written Informed consent was obtained from the parent of each child. Consent for publication Not applicable. Competing interests There is no conflict of interest for any of the authors., (© 2024. The Author(s).)

Background: Infection is the cause of diarrhoea, and rotaviruses and adenoviruses are important pathogens in children., Methods: A retrospective study was conducted on 144,067 children with diarrhoea between 2014 and 2023 in China. We used the colloidal gold method to detect intestinal adenovirus and rotavirus A antigens in faeces. The epidemiological characteristics of these viruses and the impact of meteorological factors on them were analysed before and after coronavirus disease 2019 (COVID-19) pandemic., Results: During this decade, the positive rate of adenovirus infection was 6.41%, while the positive rate of rotavirus A infection was 11.81%, higher than that of adenovirus infection. The positive rate of adenovirus and rotavirus A coinfection was 1.92%. The positive rates of adenovirus, rotavirus A and coinfection showed a fluctuating trend, and suddenly decreased in 2020. There was an apparent decrease of positive rate of rotavirus A, with a decrease of 57.27%, during 2020-2023. Surprisingly, the positive rate of adenovirus infection exceeded that of rotavirus A infection in 2021 and 2023. During the COVID-19 pandemic, the proportion of female patients and children over two years of age infected with adenovirus or rotavirus A increased, while the proportion of cases in winter decreased. In addition, we found that the positive rate of rotavirus A infection was related to average temperature and sunshine, and the positive rate of adenovirus and rotavirus A coinfection was only related to sunshine. However, these correlations disappeared during the COVID-19 pandemic., Conclusions: This study revealed the recent prevalence of adenovirus and rotavirus A infections in children with diarrhoea in south-central China and provided a theoretical basis for the prevention and control of viral diarrhoea., Competing Interests: Declarations Ethics approval and consent to participate The project was approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center, and the ethical number is 2021-24B01. The data used in this study was anonymized and there was no known risk to participants. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Rotaviruses are the major etiological agents of gastroenteritis and diarrheal outbreaks in plenty of mammalian species. The genus Rotavirus is highly diverse and currently comprises nine genetically distinct species, and four of them (A, B, C, and H) are common for humans and pigs. There is a strong necessity to comprehend phylogenetic relationships among rotaviruses from different host species to assess interspecies transmission, specifically between humans and livestock. To reveal the genetic origin of rotaviruses from Russian pig farms, nanopore-based metagenomic sequencing was performed on the PCR-positive specimens., Methods: Samples were selected among the cases submitted to routine diagnostic or monitoring studies to the Laboratory of Biochemistry and Molecular Biology of "Federal Scientific Center VIEV" (Moscow, Russia). The selected positive samples were genotyped using nanopore sequencing method., Results: Five porcine RVA isolates were completely sequenced, and genotype analysis revealed various porcine G/P genogroups: G2, G3, G4, G5, G11 and P[6], P[7], P[13], P[23], P[27] with a typical backbone constellation I5-R1-C1-M1-A8-N1-T1/7-E1-H1. The RVB isolate was detected in combination with RVA in a rectal swab from a diseased pig in Krasnoyarsk Krai. It was characterized by the following genogroups: G15-P[X]-I11-R4-C4-M4-A8-N10-T4-E4-H7. The first complete porcine RVC genome from Russia was obtained with genomic constellation G6-P[5]-I14-R1-C1-M1-A7-N9-T6-E1-H1, and the phylogenetic analysis revealed putative novel genotype group for the VP6 gene-I14. Additionally, the first porcine kobuvirus isolate from Russia was phylogenetically characterized., Conclusions: The applied nanopore sequencing method successfully genotyped the RV isolates and additionally revealed co-circulated species. The study demonstrates high genetic variability of Russian RVA isolates in VP4/VP7 genes and phylogenetically describes local RVB and RVC. Complete characterization of genomic segments is a crucial methodology in tracing the rotavirus's evolution and evaluating interspecies transmissions., Competing Interests: Declarations Ethical approval and consent to participate The animal study protocol was approved by the local Ethical and Animal Welfare Committee of the Federal State Budget Scientific Institution “Federal Scientific Center VIEV”, (Moscow, Russia) (Approval number 92/22 from 9 February 2021) for studies involving animals. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Porcine epidemic diarrhea virus (PEDV) and rotavirus has posed a significant threat to the pig industry annually across different nations, resulting in huge economic losses. The frequent co-infection of these two viruses in clinical settings complicates the process of differential diagnoses. Rapid and accurate detection of PEDV and rotavirus is in great demand for timely diarrhea disease prevention and control. In this study, tris stabilized AuNPs were prepared and a sensitive lateral flow immunoassay (LFIA) sensor was developed for the simultaneous and rapid detection of PEDV and rotavirus on site. After the system optimization, the established LFIA can simultaneously identify PEDV and rotavirus with limits of detection (LOD) of 1.25 × 10 3 TCID 50 mL -1 and 3.13 × 10 2 pg mL -1 , respectively. When applying for clinical samples, the LFIA show a concordance of 95 % and 100 % to reverse transcript polymerase chain reaction (RT-PCR) for PEDV and rotavirus respectively. Therefore, this LFIA can qualitatively detect PEDV and rotavirus in 18 min with high sensitivity and accuracy without any sophisticated equipment and operation, making it a promising candidate for the early diagnosis of PEDV or/and rotavirus diarrhea on site., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Hailan Chen reports financial support was provided by Bama County for Talents in Science and Technology. Qianlian Su reports financial support was provided by Project for Enhancing Young and Middle-aged Teacher's Research Basis Ability in Universities in Guangxi. Hui You reports financial support was provided by Guangxi Bagui Scholar Program. Jiakang He reports financial support was provided by Guangxi Key R&D Program Project.]., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rotavirus species A (RVA) is a major cause of acute viral gastroenteritis in young humans and diverse animal species. The study of the genetic characteristics of RVAs that infect rabbits (Oryctolagus cuniculus) (lapine strain [LRV]) has been limited, and, to date, the most common and epidemiologically important combinations of G/P genotypes in rabbits have been reported to be G3 P[14] and G3 P[22]. In this study, a rotavirus species A detected from an outbreak of enteritis in a Mexican commercial rabbitry was genotypically characterized. Based on sequence and phylogenetic analysis of the VP7 and VP4 genes, the strain identified in this study (C-3/15) demonstrated a G3 P[8] genotype of rotavirus, which had not previously been reported in rabbits. Moreover, both genes were closely related to human, not lapine, rotaviruses. The G3 genotype has been reported in a wide variety of hosts, including humans and rabbits, whereas the P[8] genotype has only been reported in humans. Because this combination of genotypes has never been identified in rabbits, it is proposed that the finding presented here is possibly the result of an interspecies transmission event. This is the first work to study the molecular characteristics of rotaviruses in rabbits in Mexico, as well as the identification of human G3 and P[8] genotypes in a rabbit with enteric disease.

Background: Viral enteric infections are illnesses caused by several types of viruses that primarily affect the gastrointestinal system. Globally, three major viruses associated with gastroenteritis, including norovirus (NoV), rotavirus A (RVA), and human adenovirus (HAdV), have been widely recognized. Accordingly, a rapid and sensitive diagnostic tool, such as immunochromatographic (IC) test, for diarrheal virus detection is required and helpful for rapid diagnosis., Methods: The IP-Triple I IC test kit for simultaneous triple virus detections of NoV, RVA, and HAdV was evaluated for its efficacy by using stool specimens that were known positive for these viruses by real-time RT-PCR or PCR, which was used as the gold standard method., Results: The results revealed that the IP-Triple I IC test kit exhibited a very high level of specificity (100%) for NoV, RVA, and HAdV detections, while the sensitivity was slightly different for these three viruses. The sensitivity of detection for RVA was 86.7%, whereas those for NoV and HAdV were 70.6% and 76.2%, respectively. This IP-Triple I IC kit could detect common antigens of a wide range of NoV, RVA, and HAdV genotypes (NoV GII.2, GII.4, GII.6, GII.7, GII.10, GII.17, RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8], HAdV-C1, -C2, -C5, -A12, -F40, and -F41)., Conclusions: Our results revealed that the IP-Triple I IC test kit is highly effective for simultaneous, direct detection of common antigens of several genotypes of NoV, RVA, and HAdV in stool specimens and useful for screening and rapid diagnosis of diarrheal viruses during the seasonal outbreak.

Using metagenomic sequencing we identified equine rotavirus group B (ERVB) of ruminant origin in foal diarrhea outbreaks in the 2021 foaling season. To further investigate ERVB occurrence and determine its environmental stability, we collected mare and foal fecal samples from different farms in Central Kentucky during the 2022 foaling season. The RT-qPCR-based analyses showed that ERVB genome was detected in 16.67 % (42/252) of surveyed mare samples and 26.56 % (34/128) of foal samples. Furthermore, 94.12 % (16/17) of collected soil samples and 100 % (13/13) of water samples obtained from the ERVB-positive farm premises also tested weakly positive. In addition, ERVB genome fragments were detected in 58.33 % (7/12) of indoor samples collected from the equipment/barn/hospital wards during the outbreak period. Finally, the seroprevalence study showed 87 % (113/130) of surveyed horse serum samples were positive for ERVB antibodies. Despite unsuccessful attempts in ERVB cultivation, phylogenetic analyses showed that fecal ERVB strains representing 2022 and 2023 foal diarrhea outbreaks, like 2021 strains, were more closely related to ruminant rotavirus B than other viruses. Further sequence analyses revealed that none of the three viral capsid proteins, the primary targets of virus-neutralizing antibodies, exhibited notable mutations among ERVB strains circulated over the past three years. Our data demonstrated that ERVB was widespread in horses on affected farms with extreme stability in the farm environment. These findings continue to support the need for future surveillance of ERVB in horses and the surrounding environment, and the development of effective countermeasures to protect horses against this new viral disease., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

G9P[8] has been the predominant rotavirus A (RVA) genotype in Malaysia since the 2000s. However, the overall genetic makeup and evolution of Malaysian G9P[8] strains are still unknown. Therefore, this study aimed to evaluate and characterize the complete genomes of three G9P[8] RVA strains isolated from diarrheic children under five years old in Sabah. Contrary to the classical Wa-like constellation, these strains contained a DS-1-like genotype. Two strains, namely L202 and L234, were genotype G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1, while one (KN102) was genotype G9-P[8]-I1-R1-C1-M1-A2-N1-T1-E1-H1. Phylogenetic analysis revealed that the NSP4 genes of L202 and L234 strains were closer to that of G9P[8]-E2 strains from Japan, suggesting they might share a common ancestor. The findings from this study provide new insights into the genetic characteristics of circulating G9P[8] strains in Sabah, which are important for rotavirus surveillance and potential vaccine development in the region., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Porcine group A rotavirus (PoRVA) is one of the common enteric viruses causing severe diarrhea in piglets. Although PoRVA infection has been identified to promote IL-10 production, the role of IL-10 during viral infection remains unclear. In this study, we found that elevated IL-10 levels during PoRVA infection promote viral replication by inhibiting type I interferon production and response. IL-10 treatment upregulated the expression of SOCS3 in PoRVA-infected IPEC-J2 cells, which inhibited IFN-I production by preventing the degradation of IκB and nuclear translocation of NF-κB, thereby significantly promoting PoRVA replication. Furthermore, we determined that SOCS3 also inhibited type Ⅰ interferon signaling pathway, which led to a significantly reduced ISGs after IFN-α stimulation. In PoRVA-infected cells, overexpression of SOCS3 significantly inhibits phosphorylation and heterodimerization of STAT1, thereby promoting viral replication. Finally, we demonstrated the effect of IL-10 on PoRVA replication in vivo by murine models of PoRVA infection. PoRVA replication levels were lower in the ileum of IL-10 knockout (IL-10 -/- ) mice than that in PoRVA-infected wild-type mice, but PoRVA replication levels were higher in the ileum of IFNAR knockout (IFNAR -/- ) mice than that in PoRVA-infected wild-type mice. Taken together, our findings provide information to understand the strategies of PoRVA to evade host innate antiviral immunity., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Porcine rotavirus (PoRV), a member of the Reoviridae family, constitutes a principal etiological agent of acute diarrhea in piglets younger than eight weeks of age, and it is associated with considerable morbidity and mortality within the swine industry. The G5 genotype rotavirus strain currently predominates in circulation. To develop a safe and effective porcine rotavirus vaccine, we generated an insect cell-baculovirus expression system, and successfully expressed these three viral proteins and assembled them into virus-like particles (VLPs) co-displaying VP2, VP6, and VP7. Transmission electron microscopy (TEM) analysis revealed that the VP2-VP6-VP7 VLPs exhibited a "wheeled" morphology resembling that of native rotavirus particles, with an estimated diameter of approximately 65 nm. To evaluate the immunogenicity and protective efficacy of these VP2-VP6-VP7 VLPs, we immunized BALB/C mice with four escalating doses of the VLPs, ranging from 5 to 40 μg of VLP protein per dose. ELISA-based assessments of PoRV-specific antibodies and T cell cytokines, including IL-4, IL-2, and IFN-γ, demonstrate that immunization with VP2-VP6-VP7 VLPs can effectively elicit both humoral and cellular immune responses in mice, resulting in a notable induction of neutralizing antibodies. On days 4, 6, 8, and 10 post-infection (dpi), the VLP-vaccinated group exhibited significantly reduced levels of PoRV RNA copy numbers when compared to the PBS controls. Histological examination of the duodenum, ileum, and kidneys revealed that VP2-VP6-VP7 VLPs provided effective protection against PoRV induced intestinal injury. Collectively, these findings indicate that the VLPs generated in this study possess strong immunogenicity and suggest the considerable promise of the VLP-based vaccine candidate in the prevention and containment of Porcine Rotavirus infections., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or nonfinancial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Group A rotaviruses (RVA) remain a principal cause of childhood diarrhea in the UAE, despite universal vaccine use. Monitoring genetic diversity is important for identifying prevalent genotypes and escape mutants. Although real-time polymerase chain reaction (RT-PCR) is widely used for RVA genotyping, it may not detect some new strains. This study evaluates nanopore sequencing and RT-PCR for RVA genotyping. Thirty-three RVA strains from children under 5 years presenting with diarrhea were genotyped using both methods. Thirteen strains were genotyped by RT-PCR and confirmed by nanopore sequencing. Fifteen strains were genotyped by nanopore method alone. Most PCR-genotyped strains (56%) had the VP7 G9 genotype, with G3 in five strains and G12 in two. For VP4, P8 (n = 8) and P4 (n = 7) were dominant. The most frequent combinations were G9P[8] (31%) and G9P[4] (25%). Nanopore sequencing of 28 strains revealed G3P[8] (29%) as the most prevalent, followed by G8P[8] (18%). G9P[8] and G2P[4] were present in 14% of samples with G12P[6] being the rarest (7%). Other combinations were detected in 4% the specimens with one nontypeable. Nanopore sequencing was superior to PCR in identifying diverse and emerging genotypes like G8P[8]. This method may enhance surveillance studies and guide preventive measures for RVA gastroenteritis., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia., Methods: Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed., Results: The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7% (54/147) of fecal samples. Among 54 positive samples, 13 were positive in non-diarrheal samples (n = 48, 27.1%) and 41 in diarrheal samples (n = 99, 41.4%). Genetic analyses demonstrated that all the segments of ZP18-18, except for VP4, had high nucleotide sequence identities (80.6-92.6%) with all other known RVB strains detected in pigs. In contrast, the VP4 sequence of ZP18-18 was highly divergent from other RVB strains (< 64.6% identities) and formed a distinct lineage in the phylogenetic tree. Notably, the VP8 subunit of the VP4 showed remarkably low amino acid identities (33.3%) to those of known RVB strains, indicating that the VP8 subunit of ZP18-18 was unique among RVB strains. According to the whole genome classification for RVB, ZP18-18 was assigned to a genotype constellation, G18-P[9]-I12-R4-C4-M4-A8-N10-T5-E4-H7 with the newly established VP4 genotype P[9]., Conclusions: This current study updates the geographical distribution and the genetic diversity of RVB. Given the lack of information regarding RVB in Africa, further RVB surveillance is required to assess the potential risk to humans and animals., (© 2024. The Author(s).)

Species A rotaviruses (RVA) are a major cause of acute gastroenteritis in infants and young children and in the young of various mammalian and avian species [...].

Rotavirus group C is an important cause of sporadic cases and outbreaks of gastroenteritis worldwide. Whole-Genome sequences of human rotavirus C (RVC) in public databases are limited. We performed genome sequencing to analyze a RVC outbreak of acute gastroenteritis in China. Samples from 22 patients were screened for pathogens using RT-PCR, and six samples were positive for rotavirus. Whole-Genome sequencing analysis showed that the outbreak strain SJZ217 belongs to the G4-P[2]-I2-R2-C2-M3-A2-N2-T2-E2-H2 genotype and shares almost identical genomic sequences with Chungnam isolated in Korea. Phylogenetic analysis revealed strain SJZ217 also fell into a cluster with rotavirus C strains from Japan and Europe. Reassortment in the VP4 fragment was observed. These results helped to understand the genetic diversity and possible spread of RVC strains., (© 2024. The Author(s).)

Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions. Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications. Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips. Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A. Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies μl -1 . This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR. Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.

Rotavirus A (RVA) is the main cause of acute gastroenteritis among children under the age of five globally. The unusual bat-like human RVA strains G3P[10] (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) were detected in children with acute gastroenteritis in 2005 and 2019, respectively, in the same geographical area of Northern Thailand. To elucidate the genetic backgrounds of these unusual or bat-like human RVA strains, the complete genome of these RVA strains was sequenced and phylogenetically analyzed. All eleven genome segments of these G3P[10] strains were genotyped as G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is closely related to bat G3P[10] RVA strain (RVA/Bat-tc/CHN/MYAS33/2013/G3P[10]) and bat-like human RVA strain (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]). The findings indicate that human G3P[10] RVA strains detected in this study (RVA/Human-wt/THA/CMH079/05/2005/G3P[10] and RVA/Human-wt/THA/CMH-S015-19/2019/G3P[10]) contained all eleven genome segments similar to those of bat RVA strains and appeared to be human RVA strains of bat origin. Phylogenetic analysis revealed that several genome segments of these two RVA strains were also closely related with those of other species in addition to bats and had a zoonotic transmission history. The results of this study supported the roles of interspecies transmission of RVA strains among bats and humans in the natural environment and provided convincing evidence that the evolution of human RVAs was closely interrelated with those of animal RVAs. Continuing surveillance of RVAs in humans and animals is imperative to gain a better understanding of the origin and the evolution of these viruses., (© 2024 Wiley Periodicals LLC.)

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the Proflow TM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A 260 /A 280 ) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC. Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples Support Protocol 1: The GenElute extraction method Support Protocol 2: Hybrid 1 extraction method., (© 2024 Wiley Periodicals LLC.)

Rotavirus A is a leading cause of non-bacterial gastroenteritis in humans and domesticated animals. Despite the vast diversity of bovine Rotavirus A strains documented in South Asian countries, there are very few whole genomes available for phylogenetic study. A cross-sectional study identified a high prevalence of the G6P[11] genotype of bovine Rotavirus A circulating in the commercial cattle population in Bangladesh. Next-generation sequencing and downstream phylogenetic analysis unveiled all 11 complete gene segments of this strain (BD&#95;ROTA&#95;CVASU), classifying it under the genomic constellation G6P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, which belongs to a classical DS-1-like genomic backbone. We found strong evidence of intragenic recombination between human and bovine strains in the Non-structural protein 4 (NSP4) gene, which encodes a multifunctional enterotoxin. Our analyses highlight frequent zoonotic transmissions of rotaviruses in diverse human-animal interfaces, which might have contributed to the evolution and pathogenesis of this dominant genotype circulating in the commercial cattle population in Bangladesh., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Among group A rotaviruses (RVAs), the G1 genotype is the main genotype causing diarrhea in children, but it has rarely been reported in pigs. During our epidemiological investigation, we detected G1P[7] rotavirus infection in piglets across several provinces in China and then isolated a porcine G1P[7] rotavirus strain (CN1P7). Sequencing revealed that the virus constellation was G1-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Phylogenetic analyses revealed that CN1P7 most likely emerged due to genetic reassortment among porcine, human, giant panda and dog rotavirus strains. In vivo experiments were conducted on two-day-old piglets, which revealed that the CN1P7 strain was pathogenic to piglets. The virus was shed through the digestive tract and respiratory tract. In addition to the intestine, the CN1P7 strain displayed extraintestinal tropisms in piglets. Histopathological analysis revealed that the lung and small intestine were the targets of CN1P7. This study is the first to explore the molecular and pathogenic characterization of a pig-origin G1P[7] rotavirus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: This hospital-based cross-sectional study aims to investigate the epidemiologic and clinical characteristics of rotavirus group A (RVA) infection among children with acute gastroenteritis and to detect the most common G and P genotypes in Egypt., Methods: A total of 92 stool samples were collected from children under five who were diagnosed with acute gastroenteritis. RVA in stool samples was identified using ELISA and nested RT-PCR. Common G and P genotypes were identified utilizing multiplex nested RT-PCR assays., Results: RVA was detected at a rate of 24% (22 /92) using ELISA and 26.1% (24 /92) using VP6 nested RT-PCR. The ELISA test demonstrated diagnostic sensitivity, specificity, and accuracy of 91.7%, 100%, and 97.8%, respectively. G3 was the most prevalent G type (37.5%), followed by G1 (12.5%), whereas the most commonly detected P type were P[8] (41.7%) and P[6] (8.2%). RVA-positive samples were significantly associated with younger aged children (p = 0.026), and bottle-fed (p = 0.033) children. In addition, RVA-positive samples were more common during cooler seasons (p = 0.0001). Children with rotaviral gastroenteritis had significantly more frequent episodes of diarrhea (10.87 ± 3.63 times/day) and vomiting (8.79 ± 3.57 times/day) per day (p = 0.013 and p = 0.011, respectively). Moreover, they had a more severe Vesikari clinical score (p = 0.049)., Conclusion: RVA is a prevalent cause of acute gastroenteritis among Egyptian children in our locality. The discovery of various RVA genotypes in the local population, as well as the identification of common G and P untypeable strains, highlights the significance of implementing the rotavirus vaccine in Egyptian national immunization programs accompanied by continuous monitoring of strains., (© 2024. The Author(s).)

Most human rotaviruses belong to the Wa-like, DS-1-like, or AU-1-like genotype constellation. The AU-1-like constellation, albeit minor, captured attention because its prototype strain AU-1 originated from feline rotavirus, leading to the concept of interspecies transmission of rotavirus. The AU-1 genome sequence determined by various laboratories over the years has documented two conflicting VP7 sequences in the GenBank. As culture-adaptation may introduce changes in the viral genome, the original fecal (wild-type) and the seed stock of culture-adapted AU-1 genomes were sequenced using the Illumina's MiSeq platform to determine the authentic AU-1 sequence and to identify what mutational changes were selected during cell-culture adaptation. The wild-type and culture-adapted AU-1 genomes were identical except for one VP4-P475L substitution. Their VP7 gene was 99.9% identical to the previously reported AU-1 VP7 under accession number AB792641 but only 92.5% to that under accession number D86271. Thus, the wild-type sequences determined in this study (accession numbers OR727616-OR727626) should be used as the reference. The VP4-P475L mutation was more likely incidental than inevitable during cell-culture adaptation. This was the first study in which the whole genomes of both wild-type and cultured RVA strains were simultaneously determined by deep sequencing.

Introduction: Rotavirus infection is the major cause of severe dehydrating diarrhea requiring hospitalization in young children worldwide. Due to their segmented genome, rotaviruses are capable of gene reassortment, which makes the emergence and spread of genetically novel strains possible. The purpose of this study was to search for unusual rotaviruses circulating in Nizhny Novgorod in 2021‒2023 and their molecular genetic characterization based on all genome segments., Materials and Methods: Rotavirus-positive stool samples of children were examined by PCR genotyping and electrophoresis in PAAG. cDNA fragments of each of the 11 genes ( VP1‒VP4 , VP6 , VP7 , NSP1‒NSP5 ), 570 to 850 nucleotide pairs in length were sequenced for the selected strains. The phylogenetic analysis was performed in the MEGA X program., Results: In the study period 2021‒2023, 11 G[P] combinations with a predominance of G3P[8] (59.5%) were identified. Six atypical Rotavirus А (RVA) strains were identified: 2 strains of the G2P[4] genotype (G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E2-H3, G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E3-H2) and 4 G3P[9] strains (all strains had the genotype G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3). Phylogenetic analysis based on all genes showed an evolutionary relationship between rotaviruses similar to rotaviruses of cats and dogs (BA222-like) and unusual strains of the G2P[4] genotype, for which a mixed combination of genotypes was identified and characterized for the first time., Discussion: The results obtained expand the understanding of the diversity of reassortant RVAs, as well as complement the data on the genotypic structure of the rotavirus population in Nizhny Novgorod., Conclusion: The wide genetic diversity of reassortant RVA can help rotaviruses overcome the immunological pressure provided by natural and vaccine-induced immunity. In this regard, to control the emergence of new variants and assess changes in the virulence of rotaviruses after reassortment processes, continuous molecular monitoring for circulating RVA is necessary.

Rotaviruses (RVs) are classified into nine species, A-D and F-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions composed of main building block proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 in uninfected cells leads to the formation of viroplasm-like structures (VLSs). Although morphologically identical to viroplasms, VLSs do not produce viral progeny but serve as excellent tools for studying complex viroplasms. A knowledge gap exists regarding non-RVA viroplasms due to the lack of specific antibodies and suitable cell culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from RVJ, all NSP5 interacted with their cognate NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2., Importance: Rotaviruses (RVs) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species., Competing Interests: The authors declare no conflict of interest.

Identified as a potential reference pathogen by the WHO Guidelines for Drinking-Water Quality, Rotavirus (RV) is among the main enteric viruses that cause waterborne diseases. The aim of this study was to identify and correlate the presence of RV in collective and individual water sources of rural communities in the state of Goiás, within the seasons in which the collections were made (rainy and dry seasons). For this, 86 water samples in the dry period and 160 samples in the rainy period were collected. Concentration of water samples, extraction of viral genetic material and molecular tests were performed. When analyzing the presence of RV in the samples, taking into consideration the period studied, RV was found to be more prevalent in the dry season (54.7%) than in the rainy season (20%), showing a strong statistical association with the dry season ( p -value < 0.001). The presence of pathogenic microorganisms in water is a public risk issue, enabling the emergence of outbreaks, endemics and epidemics. In the present research, there was an association between the presence of Rotavirus and the dry period of the year when compared to the rainy period.

Background: Calf diarrhea is a major cause of morbidity and mortality in the livestock sector worldwide and it can be caused by multiple infectious agents. In Ethiopia, cattle are the most economically important species within the livestock sector, but at the same time the young animals suffer from high rates of morbidity and mortality due to calf diarrhea. However, studies including both screening and molecular characterization of bovine enteric pathogens are lacking. Therefore, we aimed to both detect and molecularly characterize four of the major enteric pathogens in calf diarrhea, Enterotoxigenic Escherichia coli (E. coli K99 +), Cryptosporidium spp., rotavirus A (RVA), and bovine coronavirus (BCoV) in calves from central Ethiopia. Diarrheic and non-diarrheic calves were included in the study and fecal samples were analyzed with antigen-ELISA and quantitative real-time PCR (qPCR). Positive samples were further characterized by genotyping PCRs., Results: All four pathogens were detected in both diarrheic and non-diarrheic calves using qPCR and further characterization showed the presence of three Cryptosporidium species, C. andersoni, C. bovis and C. ryanae. Furthermore, genotyping of RVA-positive samples found a common bovine genotype G10P[11], as well as a more unusual G-type, G24. To our knowledge this is the first detection of the G24 RVA genotype in Ethiopia as well as in Africa. Lastly, investigation of the spike gene revealed two distinct BCoV strains, one classical BCoV strain and one bovine-like CoV strain., Conclusions: Our results show that Cryptosporidium spp., E. coli K99 + , RVA and BCoV circulate in calves from central Ethiopia. Furthermore, our findings of the rare RVA G-type G24 and a bovine-like CoV demonstrates the importance of genetic characterization., (© 2024. The Author(s).)

Globally, rotavirus continues to be the leading etiology of severe pediatric gastroenteritis, and transmission of the disease via environmental reservoirs has become an emerging concern in developing countries. From August to October 2021, a total of 69 samples comprising 48 of raw and treated sewage, and 21 surface waters, were collected from four Durban wastewater treatment plants (DWWTP), and effluent receiving rivers, respectively. Rotaviruses recovered and identified from the samples were subjected to sequencing, genotyping, and phylogenetic analysis. Of the 65 (94.2%) rotavirus-positive samples, 33.3% were from raw sewage, 16% from activated sludge, 15.9% from final effluents, and 29.0% were from the receiving river samples. A total of 49 G and 41 P genotypes were detected in sewage while 15 G and 22 P genotypes were detected in river samples. G1 genotype predominated in sewage (24.5%) followed by G3 (22.4%), G2 (14.3%), G4 (12.2%), G12 (10.2%), G9 (8.2%), and G8 (6.1%). Similarly, G1 predominated in river water samples (33.3%) and was followed by G2, G4 (20.0% each), G3, and G12 (13.3% each). Rotavirus VP4 genotypes P[4], P[6], and P[8] accounted for 36.6%, 29.3%, and 9.8%, respectively, in sewage. Correspondingly, 45.5%, 31.8%, and 13.6% were detected in river samples. The G and P genotypes not identified by the methods used were 2.1% versus 24.3% and 0.1% versus 9.1% for sewage and river water samples, respectively. Sequence comparison studies indicated a high level of nucleotide identity in the G1, G2, G3, G4, G8 VP7, and P[4], P[6], and P[8] VP4 gene sequences between strains from the environment and those from patients in the region. This is the first environmental-based study on the G and P genotypes diversity of rotavirus in municipal wastewater and their receiving rivers in this geographical region. The high similarity between environmental and clinical rotavirus strains suggests both local circulation of the virus and potential exposure risks. In addition, it highlights the usefulness of sewage surveillance as an additional tool for an epidemiological investigation, especially in populations that include individuals with subclinical or asymptomatic infections that are precluded in case-based studies., (© 2024. The Author(s).)

Rotavirus A (RVA) is a leading cause of severe gastroenteritis in children worldwide, and vaccination has become a pivotal strategy to reduce the associated morbidity and mortality. This study presents a molecular characterization of RVA genotypes circulating among vaccinated children in Pakistan during the year 2019. A total of 510 stool samples were collected from children of up to five years of age presenting with acute gastroenteritis symptoms in Rawalpindi, Islamabad regions of Pakistan. The RVA antigen was detected using ELISA on these samples. RVA G/P genotyping was performed on ELISA positive samples using Multiplex semi-nested reverse transcriptase PCR. RVA was found in 130 fecal samples, with an overall prevalence of 25.4 %. G9P[8] (20 %) is the most prevalent genotype, followed by G12P[6] (17 %), G3P[8] (14 %), G1P[8] (12 %), G2P[4] (10 %), G12P[8] (7 %), G9P[6] (7 %), G3P[6] (6 %), G3P[4] (4 %) and G1P[6] (3 %) respectively. There is a statistically significant difference (p < 0.05) found in the group age (in months) of RVA gastroenteritis cases as detected by RT-PCR. The highest number of positive cases was found in the age range from 0 to 6 months, followed by 7-12 months, 13-24 months, and 25-60 months, respectively. Dehydration is statistically significantly associated (p˂ 0.05) in RVA gastroenteritis cases compared to those who tested negative. This study emphasizes the significance of maintaining a continuous surveillance system and conducting genomic analysis of RVA genotypes in children upto the age of 5 years. This is essential for tracking the circulation of RVA genotypes. The results from this research enhance our comprehension of how RVA genotypes are changing over time in Pakistan, underscoring the ongoing necessity for improving vaccine coverage and effectiveness. This, in turn, can help reduce the impact of RVA-related illnesses in children., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)