Group A rotaviruses (RVAs) are the leading cause of viral diarrhoea across various host species, including mammals and birds. The VP7 and VP4 proteins of these viruses play critical roles in determining genotype specificity, influencing viral infectivity and host adaptation. This study employed machine-learning techniques to classify RVA genotypes based on the molecular and physicochemical properties of these proteins. A dataset of 94 VP7 and 68 VP4 protein sequences was collected from various host species. Seven machine-learning algorithms-Naïve Bayes (NB), logistic regression (LR), decision tree (DT), random forest (RF), k-nearest neighbour (kNN), support vector machine (SVM), and artificial neural network (ANN)-were used for genotype classification. Feature subsets were configured using ranking-based attribute selection, and classification performance was evaluated using accuracy (ACC), precision, recall, Matthews' correlation coefficient (MCC), and the area under the curve (AUC). kNN demonstrated the highest classification accuracy for both VP7 (ACC = 97.87 %) and VP4 (ACC = 100 %), outperforming NB, LR, DT, RF, SVM, and ANN. For VP7 sequences, key properties influencing genotype classification included hydrophobicity, normalised van der Waals volume, and leucine composition. For VP4, polarity, normalised van der Waals volume, and polarizability were the most significant factors. In summary, the genotype-specific molecular features of VP7 and VP4 proteins served as reliable markers for RVA classification. Our findings highlight the potential of machine-learning approaches to predict RVA genotypes based on the physicochemical properties of amino acids, providing valuable insights into the molecular mechanisms that drive viral evolution, host specificity, and immune evasion.

Rotavirus C (RVC) is an important cause of gastroenteritis in humans and pigs and has also been detected in cattle, ferrets, minks, and dogs. Incidental zoonotic transmissions have been described. In contrast to rotavirus A (RVA), a complete genotyping system for RVC has not yet been established due to limited or incomplete sequence data. In this study, 138 complete nucleotide sequences for VP7 (G-type) and 97 complete nucleotide sequences for VP4 (P-type) of porcine RVC-positive samples have successfully been generated and genotyped. Together with available sequences from the NCBI database, phylogenetic analyses were conducted, cut-off values were re-evaluated, and the current classification system was adapted. Pairwise identity frequency analyses revealed a new cut-off value of 82% instead of the previous 85% for the G-type and confirmed the current cut-off value of 85% for the P-type. This resulted in the identification of 21 G-types and 39 P-types, including 4 new G-types and 10 new P-types. The results of the investigations expand the existing knowledge about the genetics of RVC and demonstrate the enormous diversity of porcine RVC sequences in particular.IMPORTANCEThis article provides a new sequence data set of porcine rotavirus C (RVC) strains. The extended full-length analysis of RVC G-types and P-types enabled us to review the current classification system. According to the guidelines of the rotavirus classification working group (RCWG), the results led to a new cut-off value of RVC G-types and required the reclassification of numerous RVC G-types. In addition, several new genotypes have been found. The present work closes the aforementioned knowledge gap and provides important, comprehensive data for RVC genetic diversity.

Rotavirus infection represents a major etiology of severe diarrheal disease in neonatal and weaned piglets, causing substantial economic burdens to the global swine industry. Lactobacillus plantarum, a ubiquitous probiotic in natural ecosystems, has demonstrated multifaceted biological functions. The stimulator of the interferon gene (STING) is involved in type I interferon (IFN-I) mediated host antiviral innate immunity, which is a pivotal adaptor in response to the microbial DNA/RNA-activated signaling pathways. Emerging evidence suggests that certain probiotic strains can activate the STING-dependent pathway to induce IFN-I responses. In the present study, we successfully isolated a strain of Lactobacillus plantarum (designated LP1)from porcine intestinal contents and investigate its potential to counteract porcine rotavirus (PoRV) infection via modulation of antiviral signaling pathway.

LP1 exhibited superior tolerance to simulated gastrointestinal conditions (pH 3.0 and 0.3% bile salts) compared with other isolated Lactobacillus strains. In vitro adhesion assays demonstrated that LP1effectively colonized porcine intestinal epithelial cells (IPEC-J2) without inducing cytotoxicity or apoptosis. Animal experiments also confirmed the protective effect of LP1 in mice against rotavirus, by reducing body weight loss, promoting viral clearance in feces, and alleviating intestinal mucosal damage. Mechanistic investigations identified STING-IRF3 pathway activation as the pivotal antiviral mechanism. Both phosphorylation of STING and IRF3 in LP1-treated IPEC-J2 cells accompanied by upregulated transcription and secretion of IFN-β and interferon-stimulated genes (ISGs). Consistent findings were observed in intestinal tissues of LP1-protected mice with STING pathway activation correlating with reduction in viral titers. Crucially, STING inhibitor (C-170) administration could reverse LP1-mediated antiviral effects.

LP1 exerts potent anti-PoRV activity in both murine models and porcine intestinal epithelial (IPEC-J2) cells through STING-IRF3 signaling axis-mediated IFN-β production.

Rotavirus A (RVA), a member of the Sedoreoviridae family, is significant intestinal pathogen that cause diarrhea in both piglets and humans. During of an outbreak that struck nursing piglets with diarrhea, a human-porcine reassortment rotavirus, named as RVA/Pig-wt/China/HB05/2023/G9P[23] (hereafter referred to as HB05), was identified. This specific strain was found to be prevalent in pig farms in several regions, including Hebei, Liaoning, Sichuan, Zhejiang and Henan, and caused significant economic losses from March to August 2023. To further explore the evolutionary diversity of HB05, a comprehensive analysis of all gene segments was conducted. The genome constellation was identified as G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Nucleotide sequence identity and phylogenetic analyses indicated that the NSP3 gene of HB05 is most closely related to the corresponding genes of Human strains, with the highest homology at 95.45% identity. The other genes (VP1-VP4, VP6-VP7, NSP1-NSP2, NSP4-NSP5) exhibited the closest relationship to porcine strains, with the highest homology ranging from 94.79 to 98.89% similarity. Therefore, it is likely that HB05 originated from genetic reassortment between porcine and human rotaviruses. The pathogenicity study performed on 3-day-old piglets revealed that severe diarrhea manifested 8 h post-infection after oral inoculation with the PoRV HB05 strain at a dose of 2 × 10^5.5 TCID50/mL per piglet. To our knowledge, this marks the first report of a prevalent and highly virulent human-porcine reassortment G9P[23] rotavirus A (RVA) strain identified in mainland China. This finding provides valuable insights into the evolutionary traits of the G9P[23] strain and suggests a possible risk of cross-species transmission.

Rotavirus alphagastroenteritidis is the leading cause of acute gastroenteritis worldwide in young humans and animals. In 2023-2024, a relatively high rotavirus detection rate (34.5%) was detected in children with diarrhea in Caracas. All rotavirus strains were typed as P[8], using a multiplex RT-PCR assay, while the G-type was not identified. This unusual pattern, not previously observed in Venezuela, prompted the VP7 gene sequencing of nineteen strains, which displayed a high sequence identity (99.3-100%) compatible with the G3 genotype. These strains clustered into a well-supported lineage IX encompassing human reassortants of equine-like G3P[8] strains described elsewhere, showing a very close genetic relationship (99.0-99.9%). Old G3 rotavirus isolates obtained from diarrheic samples in the past were included in the analysis and grouped into lineage I together with ancestral reference G3 strains. The novel G3P[8]s carry amino acid changes in VP7-neutralizing epitopes, compared with the RotaTeq-WI78-8-vaccine strain. Full genome sequencing of a representative strain revealed a genotype constellation including an equine-like G3P[8] in a DS-1-like backbone (I2-R2-C2-M2-A2-N2-T2-E2-H2), confirming the role of animal strains as a source of diversification, and the importance of unceasingly revising molecular typing strategies and vaccine efficacy to guarantee their success.

The present study was conducted to monitor the genotype diversity of circulating species A rotavirus (RVA) in Iran. A total of 300 faecal specimens were collected from children under 5 years of age hospitalized for acute gastroenteritis between October 2023 and October 2024. G3P[8] represented 72.91% (70/96) of all RVA-positive samples, further subdivided into equine-like G3P[8]-I2-E2 DS-1-like and human G3P[8]-I1-E1 Wa-like. A retrospective genetic analysis of G3P[8] strains isolated from 2015 to 2017 was also performed and showed that G3P[8] strains belong to the G3P[8]-E1-I1 Wa-like genetic pattern, which is typically similar to human G3P[8] Wa-like strains in this study. The emergence of equine-like G3P[8] DS-1-like strains in Iran may not be related to selection pressure from rotavirus vaccination, but rather to cross-border migration of rotavirus strains due to population movements.

In the post-rotavirus (RVA) vaccination era, uncommon and zoonotic strains have emerged as causative agents of acute gastroenteritis in humans, including the equine-like G3P[8] strains. First identified in 2013, this strain has quickly spread worldwide, reaching the position of the most prevalent genotype in many countries, including Brazil. Here, we report full genotype characterization and phylogenetic analysis of two equine-like G3P[8] strains detected in Goiás, a state in the Cerrado biome of the Brazilian Midwestern region, during the year of 2019. The strains were detected in different socioeconomic and demographic contexts: GO-MR from an asymptomatic adult living in a rural traditional community and GO-H5 from a symptomatic child from the state capital, with access to safe drinking water and essential sanitation services. These strains also displayed different backbone constellations considering the NSP2 gene segment (G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for GO-MR and G3-P[8]-I2-R2-C2-M2-A2-N1-T2-E2-H2 for GO-H5). Furthermore, significant mutations in the main epitope sites of the VP7 and VP8* proteins of the detected strains, and other Brazilian G3P[8] viruses, were found with the comparison to RV1 and RV5 vaccine proteins, indicating a potential ability of these viruses to evade vaccine protection, which may contribute to their prevalence both nationally and globally. In summary, this study corroborates the genetic diversity of equine-like G3P[8] DS-1-like strains circulating worldwide, highlights the epidemiological importance of adults as reservoirs of RVA and shows the substantial differences between these emerging strains and the currently used anti-RVA vaccines, which may partially explain their predominance due to potential evasion of vaccine-induced protection.

Porcine rotavirus is one of the most important pathogens causing diarrhea in newborn piglets, and the genome of this virus contains 11 double-stranded RNA segments, which are easy to be recombined among strains to produce new strains with different antigenic properties. The reverse genetics system is an informative tool for studying virus biology. Recently, adaptable plasmid-based reverse genetics systems were developed for the porcine rotavirus OSU strain; however, such systems have not been developed for epidemic porcine rotavirus genotypes in China. In this study, we successfully established a reverse genetic system based on an epidemic strain of porcine rotavirus JXAY01 isolated in recent years, which was characterized by a specific genotype constellation: G5-P[23]-I12-R1-C1-M1-A8-N1-T7-E1-H1. 11 gene segments of porcine rotavirus JXAY01 were cloned into plasmid vectors similar to the SA11 system. JXAY01 genome segment plasmids were co-transfected with 10 complementary SA11 genome plasmids, and 11 monoreassortant strains were successfully rescued. Viral replication analyses of the parental SA11 strain and the monoreassortant strains showed that the structural protein replacement monoreassortants had reduced cell proliferation compared with the parental SA11 and non-structural protein replacement strains. The recombinant rJXAY01 strain could be successfully rescued using 11 pRG-JXAY01 plasmids. Whole genome sequencing showed 12 amino acid differences between the isolate JXAY01 and the recombinant rJXAY01, but there was no significant difference in their in vitro replication ability. This study reports the reverse genetic system, which lays the foundation for further understanding of porcine rotavirus molecular biology and novel vaccine development.

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.

Rotavirus alphagastroenteritidis is the major causative agent of acute gastroenteritis in both children under the age of 5 and young mammals and birds globally. RVAs are non-enveloped viruses with a genome comprising 11 double-stranded RNA segments. In 2008, the Rotavirus Classification Working Group pioneered a comprehensive and complete RVA genome classification system, establishing a specific threshold, which measures the genetic distances between homologous genes. The aim of this study was to perform an updated systematic analysis of the genetic variability across all RVA genes. Our investigation involved assessing the established cutoff values for each RVA genome segment and determining the need for any updates. To achieve this objective, multiple sequence alignments were constructed for all 11 genes and one for each genotype with discrepancies. Also, pairwise distances along with their cutoff values were evaluated. The analyses provided insights into the current relevance of cutoff values, which remain applicable for the majority of genotypes. In conclusion, this study fortifies the current classification system by highlighting its robustness and accurate genotyping of Rotavirus alphagastroenteritidis.

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.

Canine circovirus (CCV) has been detected globally, but its genotyping remains unevenly characterized. To comprehend the evolution and genotyping of CCV, 887 rectal swabs of dogs were collected during 2018-2023. According to p-distance/frequency histograms and phylogenetic trees based on complete genome sequences, the 21 newly obtained Chinese and 84 reference CCV strains were mainly divided into 7 subtypes (CCV-1a, CCV-1b, CCV-1c, CCV-1d, CCV-1e, CCV-2a, and CCV-2b). Among the 21 newly obtained CCVs, 9 strains belonged to CCV-1d, 2 strains belonged to CCV-1b, and the remaining strains belonged to CCV-1c. Recombination analysis indicated that recombination events occurred between CCV strains of different subtypes, hosts, and countries. The CCV capsid protein features 19 variable sites, with 2 sites (T58Q, P239A) displaying regional specificity and 3 (Q57T, E150Q, and T195Q) manifesting subtype specificity. Therefore, this genotyping analysis provides a reference for the molecular characteristics of CCV strains found globally.

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).

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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_ROTA_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.

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.

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.

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.

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.

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.

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.

Group A rotaviruses (RVAs) are widespread in humans and many animal species and represent the most epidemiologically important rotavirus group. The aim of the study was the identification of the genotype pattern of human RVA strains circulating in Poland, assessment of their phylogenetic relationships to pig RVAs and identification of reassortant and zoonotic virus strains. Human stool samples which were RVA positive (n = 166) were collected from children and adults at the age of 1 month to 74 years with symptoms of diarrhoea. Identification of the G and P genotypes of human RVAs as well as the complete genotype of reassortant and zoonotic virus strains was performed by the use of an RT-PCR method. The G (G1-G4, G8 or G9) and/or P (P[4], P[6], P[8] or P[9]) genotypes were determined for 148 (89.2%) out of 166 RVA strains present in human stool. G1P[8] RVA strains prevailed, and G4P[8] (20.5%), G9P[8] (15.7%) and G2P[4] (13.3%) human RVA strains were also frequently identified. The full genome analysis of human G4P[6] as well as pig G1P[8] and G5P[6] RVAs revealed the occurrence of porcine-human reassortants and zoonotic RVAs. Detection of G4P[6] in pigs confirms their role as a reservoir of zoonotic RVAs.

Segmented RNA viruses are capable of exchanging genome segments via reassortment as a means of immune evasion and to maintain viral fitness. Reassortments of single-genome segments are common among group A rotaviruses. Multiple instances of co-reassortment of two genome segments, GS6(VP6) and GS10(NSP4), have been documented in surveillance. Specifically, a division between NSP4 genotypes has been observed in the NSP4 double-layered particle (DLP)-binding domain. A previously hypothesized mechanism for this co-reassortment has been suggested to be the interaction between VP6 and NSP4 during DLP transport from viroplasms for particle maturation. In this study, we used sequence analysis, RNA secondary structure prediction, molecular dynamics and reverse genetics to form a hypothesis regarding the role of the NSP4 DLP-binding domain. Sequence analysis showed that the polarity of NSP4 DLP-binding domain amino acids 169 and 174 is clearly divided between E1 and E2 NSP4 genotypes. Viruses with E1 NSP4s had 169A/I or 169S/T with 174S. E2 NSP4s had 169R/K and 174A. RNA secondary structure prediction showed that mutation in both 545 (aa169) and 561 (aa174) causes global structure remodelling. Molecular dynamics showed that the NSP4/VP6 interaction stability is increased by mutating both aa positions 169 and 174. Using reverse genetics, we showed that an R169I mutation alone does not prevent rescue. Conversely, 174A to 174S prevented rescue, and rescue could be returned by combining 174S with 169I. When compared to rSA11 NSP4-wt, both rSA11 NSP4-R169I and rSA11 NSP4-R169I/A174S had a negligible but significant reduction in titre at specific time points. This study suggests that amino acid 174 of NSP4 may be essential in maintaining the VP6/NSP4 interaction required for DLP transport. Our results suggest that maintenance of specific polarities of amino acids at positions 169 and 174 may be required for the fitness of rotavirus field strains.

Enteric avian rotavirus (ARV) is the etiological agent of several health problems that pose a global threat to commercial chickens. Therefore, to avoid these widespread epidemics and high mortality rates, only vaccine and strict biosecurity are required.

The present study employs computational techniques to design a unique multi-epitope-based vaccine candidate that successfully activates immune cells against the ARV by combining adjuvant, linker, and B and T-cell epitopes. Starting, homologous sequences in the various ARV serotypes were revealed in the NCBI BLAST database, and then the two surface proteins (VP4 and VP7) of the ARV were retrieved from the UniprotKB database. The Clustal Omega server was then used to identify the conserved regions among the homologous sequences, and the B and T-cell epitopes were predicted using IEDB servers. Then, superior epitopes-2 MHC-1 epitopes, 2 MHC-2 epitopes, and 3B-cell epitopes-were combined with various adjuvants to create a total of four unique vaccine candidates. Afterward, the designed vaccine candidates underwent computational validation to assess their antigenicity, allergenicity, and stability. The vaccine candidate (V2) that demonstrated non-antigenicity, a high VaxiJen score, and non-allergenicity was ultimately chosen for molecular docking and dynamic simulation.

Although the V2 and V4 vaccine candidates were highly immunogenic, V2 had a higher solubility rate. The predicted values of the aliphatic index and GRAVY value were 30.4 and 0.417, respectively. In terms of binding energy, V2 outperformed V4. Being successfully docked with TLRs, V2 was praised as the finest. After adaptation, the sequence's 50.73 % GC content outside of the BglII or ApaI restriction sites indicated that it was equivalently safe to clone. The chosen sequence was then inserted into the pET28a(+) vector within the BglII and ApaI restriction sites. This resulted in a final clone that was 4914 base pairs long, with the inserted sequence accounting for 478 bp and the vector accounting for the remainder.

The immune-mediated simulation results for the selected vaccine construct showed significant response; thus, the study confirmed that the selected V2 vaccine candidate could enhance the immune response against ARV.

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.

Group A rotaviruses (RVAs) are major causes of severe gastroenteritis in infants and young animals. To enhance our understanding of the relationship between human and animals RVAs, complete genome data are necessary. We screened 92 intestinal and stool samples from diarrheic piglets by RT‒PCR targeting the VP6 gene, revealing a prevalence of 10.9%. RVA was confirmed in two out of 5 calf samples. We successfully isolated two porcine samples using MA104 cell line. The full-length genetic constellation of the two isolates were determined to be G9-P[23]-I5-R1-C1-M1-A8-N1-T7-E1-H1, with close similarity to human Wa-like and porcine strains. Sequence analysis revealed the majority of genes were closely related to porcine and human RVAs. Phylogenetic analysis revealed that these isolates might have their ancestral origin from pigs, although some of their gene segments were related to human strains. This study reveals evidence of reassortment and possible interspecies transmission between pigs and humans in China.

Rotaviruses A (RVA) are a major cause of acute viral gastroenteritis in humans worldwide and are responsible for about two million hospitalizations per year. They can also infect other mammals such as pigs, calves, goats, lambs, and horses, in which they are also considered a major cause of viral diarrhea. While RVA is well studied in humans and domestic animals, its occurrence in wild ruminants is not well known. The RVA genome is a double-stranded RNA consisting of 11 segments, and genotyping is based on the VP7 (G) and VP4 (P) segments. Currently, there are 42G genotypes and 58P genotypes. RVA has a high mutation rate, and some combinations of G and P genotypes can infect different animal species, leading to speculation about the potential for zoonotic transmission.

A total of 432 fecal samples were collected from roe deer, red deer, chamois, mouflon and Alpine ibex in Slovenia between 2017 and 2021. To investigate the presence of RVA in wild ruminants, real-time RT-PCR was used. Positive samples were subjected to next generation sequencing (NGS) using RIP-seq method.

In total, 7 samples were RVA positive. Complete genomes were determined and phylogenetically analyzed for all 7 RVAs. Four different genotype constellations were present in 7 positive RVA animals: G8-P[14]-I2- R2-C2-M2-A3-N2-T6-E2-H3, G6-P [14]-I2-R2-C2-M2-A11-N2-T6-E2-H3, G10-P [15]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and G10-P [15]-I2-R2-C2-M2-A11- N2-T6-E2-H3. Genotypes G6P[14] and G10P[15] were found in both roe deer and red deer, representing the first confirmed occurrence of RVA in red deer. In addition, genotype G8P[14] was found in chamois, representing the first known case of positive RVA in this species. Some of these genotypes have also been found in humans, indicating the potential for zoonotic transmission.

In the present study, first, rotaviruses that caused acute gastroenteritis in children under five years of age during the time before the vaccine was introduced in Iran (1986 to 2023) are reviewed. Subsequently, the antigenic epitopes of the VP7 and VP4/VP8 proteins in circulating rotavirus strains in Iran and that of the vaccine strains were compared and their genetic differences in histo-blood group antigens (HBGAs) and the potential impact on rotavirus infection susceptibility and vaccine efficacy were discussed. Overall data indicate that rotavirus was estimated in about 38.1 % of samples tested. The most common genotypes or combinations were G1 and P[8], or G1P[8]. From 2015 to 2023, there was a decline in the prevalence of G1P[8], with intermittent peaks of genotypes G3P[8] and G9P[8]. The analyses suggested that the monovalent Rotarix vaccine or monovalent vaccines containing the G1P[8] component might be proper in areas with a similar rotavirus genotype pattern and genetic background as the Iranian population where the G1P[8] strain is the most predominant and has the ability to bind to HBGA secretors. While the same concept can be applied to RotaTeq and RotasIIL vaccines, their complex vaccine technology, which involves reassortment, makes them less of a priority. The ROTASIIL vaccine, despite not having the VP4 arm (P[5]) as a suitable protection option, has previously shown the ability to neutralize not only G9-lineage I strains but also other G9-lineages at high titers. Thus, vaccination with the ROTASIIL vaccine may be more effective in Iran compared to RotaTeq. However, considering the rotavirus genotypic pattern, ROTAVAC might not be a good choice for Iran. Overall, the findings of this study provide valuable insights into the prevalence of rotavirus strains and the potential effectiveness of different vaccines in the Iranian and similar populations.

Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.

Human astroviruses (HAstVs) are considered important causative pathogens of acute gastroenteritis (AGE) in children under 5 years of age worldwide, along with group A rotavirus (RVA), norovirus (NoV), and enteric adenovirus (EAdV). The present study was aimed to both detect HAstV and its co-infections and investigate genetic analysis of circulating HAstV and co-infected virus in hospitalized children under 5 years of age with AGE in Iran. Accordingly, a sum of 200 stool specimens were screened by PCR for HAstV during 2021-2022. The HAstV was found in 0.5% of 200 specimens (n = 1) while was co-infected with RVA. The genetic and phylogenetic analysis indicated HAstV1 genotype, which clustered with viruses from lineage 1b, which has not been previously reported in Iran. The detected RVA strain belonged to G1 lineage II/P[8]-lineage III, which has been reported previously in Iran as the most common strain. The further genetic analysis of RVA VP6 and NSP4 demonstrated an atypical genotype pattern G1P[8]-I1-E2, as a mono-reassortant of a Wa-like genogroup, which appeared to be reassorted with the NSP4 gene of E2 genotype of the G2P[4] DS-1 genogroup. Although the clinical outcomes of the AGE-causing viruses co-infection is not yet entirely clear, it seems that future studies will be helpful to merge clinical and epidemiological data of co-infecting viruses for a more accurate medical and clinical relevance in symptomatic children.

Mozambique introduced the Rotarix® vaccine into the National Immunization Program in September 2015. Following vaccine introduction, rotavirus A (RVA) genotypes, G9P[4] and G9P[6], were detected for the first time since rotavirus surveillance programs were implemented in the country. To understand the emergence of these strains, the whole genomes of 47 ELISA RVA positive strains detected between 2015 and 2018 were characterized using an Illumina MiSeq-based sequencing pipeline. Of the 29 G9 strains characterized, 14 exhibited a typical Wa-like genome constellation and 15 a DS-1-like genome constellation. Mostly, the G9P[4] and G9P[6] strains clustered consistently for most of the genome segments, except the G- and P-genotypes. For the G9 genotype, the strains formed three different conserved clades, separated by the P type (P[4], P[6] and P[8]), suggesting different origins for this genotype. Analysis of the VP6-encoding gene revealed that seven G9P[6] strains clustered close to antelope and bovine strains. A rare E6 NSP4 genotype was detected for strain RVA/Human-wt/MOZ/HCN1595/2017/G9P[4] and a genetically distinct lineage IV or OP354-like P[8] was identified for RVA/Human-wt/MOZ/HGJM0644/2015/G9P[8] strain. These results highlight the need for genomic surveillance of RVA strains detected in Mozambique and the importance of following a One Health approach to identify and characterize potential zoonotic strains causing acute gastroenteritis in Mozambican children.

In the 2010s, several unusual rotavirus strains emerged, causing epidemics worldwide. This study reports a comprehensive molecular epidemiological study of rotaviruses in Japan based on full-genome analysis. From 2014 to 2019, a total of 489 rotavirus-positive stool specimens were identified, and the associated viral genomes were analyzed by next-generation sequencing. The genotype constellations of those strains were classified into nine patterns (G1P[8] (Wa), G1P[8]-E2, G1P[8] (DS-1), G2P[4] (DS-1), G3P[8] (Wa), G3P[8] (DS-1), G8P[8] (DS-1), G9P[8] (Wa), and G9P[8]-E2). The major prevalent genotype differed by year, comprising G8P[8] (DS-1) (37% of that year's isolates) in 2014, G1P[8] (DS-1) (65%) in 2015, G9P[8] (Wa) (72%) in 2016, G3P[8] (DS-1) (66%) in 2017, G1P[8]-E2 (53%) in 2018, and G9P[8] (Wa) (26%) in 2019. The G1P[8]-E2 strains (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1) isolated from a total of 42 specimens in discontinuous years (2015 and 2018), which were the newly-emerged NSP4 mono-reassortant strains. Based on the results of the Bayesian evolutionary analyses, G1P[8]-E2 and G9P[8]-E2 were hypothesized to have been generated from distinct independent inter-genogroup reassortment events. The G1 strains detected in this study were classified into multiple clusters, depending on the year of detection. A comparison of the predicted amino acid sequences of the VP7 epitopes revealed that the G1 strains detected in different years encoded VP7 epitopes harboring distinct mutations. These mutations may be responsible for immune escape and annual changes in the prevalent strains.

Rotavirus A (RVA) is a prevalent cause of enteric diarrhea in infants, bovine, pigs, and sheep globally. Currently, the G6P[1]-type rotaviruses are prevalent in sheep or goat in Bangladesh, Turkey, and Uganda. However, this genotype has not been reported in Chinese sheep or goat. Therefore, 12 anal swabs were collected from diarrheal sheep in Gansu Province, China, in 2023 and tested for rotavirus using reverse transcription-polymerase chain reaction (RT-PCR). Pathological sections and immunohistochemistry were used to observe pathological changes and rotavirus antigens in the duodenum, respectively. The sheep rotavirus was isolated in MA-104 cells and characterized through indirect immunofluorescence and transmission electron microscopy. The genes of the strain were obtained using the next-generation sequencing technology and analyzed phylogenetically. One sheep was positive for rotavirus by RT-PCR, and immunohistochemistry revealed numerous rotavirus antigens in the apical portion of the duodenal villi. Transmission electron microscopy revealed that the strain was characterized by virus particles that were "wheel-shaped" and measured 70-80 nm in size. The gene constellations of this strain is G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3. BLASTn and phylogenetic tree analyses suggest that this strain is likely a recombinant of human rotavirus, goat rotavirus, and bovine rotavirus. The comparison of amino acid similarities revealed three differences in the key antigenic epitopes of the VP7 and VP4 proteins between the GO34 strain and this study strain despite the identical gene constellations of the two strains. To date, this is the first report of this constellation of RVA being found in sheep.

Animal rotaviruses A (RVAs) are considered the source of emerging, novel RVA strains that have the potential to cause global spread in humans. A case in point was the emergence of G8 bovine RVA consisting of the P[8] VP4 gene and the DS-1-like backbone genes that appeared to have jumped into humans recently. However, it was not well documented what evolutionary changes occurred on the animal RVA-derived genes during circulation in humans. Rotavirus surveillance in Vietnam found that DS-1-like G8P[8] strains emerged in 2014, circulated in two prevalent waves, and disappeared in 2021. This surveillance provided us with a unique opportunity to investigate the whole process of evolutionary changes, which occurred in an animal RVA that had jumped the host species barrier. Of the 843 G8P[8] samples collected from children with acute diarrhoea in Vietnam between 2014 and 2021, fifty-eight strains were selected based on their distinctive electropherotypes of the genomic RNA identified using polyacrylamide gel electrophoresis. Whole-genome sequence analysis of those fifty-eight strains showed that the strains dominant during the first wave of prevalence (2014-17) carried animal RVA-derived VP1, NSP2, and NSP4 genes. However, the strains from the second wave of prevalence (2018-21) lost these genes, which were replaced with cognate human RVA-derived genes, thus creating strain with G8P[8] on a fully DS-1-like human RVA gene backbone. The G8 VP7 and P[8] VP4 genes underwent some point mutations but the phylogenetic lineages to which they belonged remained unchanged. We, therefore, propose a hypothesis regarding the tendency for the animal RVA-derived genes to be expelled from the backbone genes of the progeny strains after crossing the host species barrier. This study underlines the importance of long-term surveillance of circulating wild-type strains in order to better understand the adaptation process and the fate of newly emerging, animal-derived RVA among the human population. Further studies are warranted to disclose the molecular mechanisms by which spillover animal RVAs become readily transmissible among humans, and the roles played by the expulsion of animal-derived genes and herd immunity formed in the local population.