Search

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.

Human enteric viruses, such as norovirus, adenovirus, rotavirus, and enterovirus, are crucial targets in controlling biological contamination in water systems worldwide. Due to their small size and low concentrations in water, effective virus concentration and detection methods are essential for ensuring microbial safety. This paper reviews the typical and innovative methods for concentrating and detecting human enteric viruses, highlights viral contamination levels across different water bodies, and discusses the removal efficiencies of virus through various treatment technologies. The application and current gaps of quantitative microbial risk assessment (QMRA) for evaluating the risks of human enteric viruses is also explored. Innovative methods such as digital polymerase chain reaction and isothermal amplification show promise in sensitivity and convenience, however, distinguishing between infectious and non-infectious viruses should be a key focus of future detection techniques. The highest concentrations of human enteric viruses were detected in wastewater, ranging from 10 3 to 10 6 copies/L, while drinking water showed significantly lower concentrations, often below 10 2 copies/L. QMRA studies suggest that exposure to human enteric viruses, whether through contaminated drinking water, occupational contact, or accidental wastewater discharge, could result in a life expectancy of 1.96 × 10 -4 to 4.53 × 10 -1 days/year., 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.)

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.)

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.)

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).)

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl 2 with or without acid rinse by H 2 SO 4 and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl 2 using electronegative filters with acid rinse by H 2 SO 4 combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water., Competing Interests: Declaration of Competing Interest All authors declare that there is no conflict of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.)

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea-rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis., 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.)

Background: Quantitative molecular assays are increasingly used for detection of enteric viruses., Methods: We compared the clinical severity using the modified Vesikari score (mVS) of enteric viruses detected by conventional assays (enzyme immunoassays [EIAs] for rotavirus and adenovirus 40/41 and conventional polymerase chain reaction for astrovirus, sapovirus, and norovirus) and a quantitative molecular assay (TaqMan Array Card [TAC]) among children aged 0-59 months in the Global Enteric Multicenter Study. For rotavirus and adenovirus 40/41, we compared severity between EIA-positive and TAC-positive cases assigned etiologies using different cycle threshold (Ct) cutoffs., Results: Using conventional assays, the median mVS (interquartile range) was 10 (8-11) for rotavirus, 9 (7-11) for adenovirus 40/41, 8 (6-10) for astrovirus, sapovirus, and norovirus GII, and 7 (6-9) for norovirus GI. Compared with rotavirus EIA-positive cases, the median mVS was 2 and 3 points lower for EIA-negative/TAC-positive cases with Ct <32.6 or Ct ≥32.6 and <35, respectively (P < .001). Adenovirus 40/41 EIA-positive and EIA-negative/TAC-positive cases were similar, regardless of Ct cutoff., Conclusions: Quantitative molecular assays compared with conventional assays, such as EIA, may influence the severity of identified cases, especially for rotavirus. Cutoffs to assign etiology for quantitative assays should be considered in the design and interpretation of enteric virus studies., Competing Interests: Potential conflicts of interest. D. S. reports employment and shares with GSK Vaccines, outside the submitted work. A. K. M. Z. was involved in this work as the site principial investigator of the GEMS study at the Aga Khan University during 2008–2012; she is currently the President of Gender Equality at the Bill & Melinda Gates Foundation and has not influenced this analysis from her position there. K. L. K. received funding from the Bill & Melinda Gates Foundation for part of the submitted work and outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

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).)

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.

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: 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.

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.)

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.)

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.)

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 [...].

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).)

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).)

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.)

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 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.)

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.

Background: Kenya introduced a monovalent rotavirus vaccine administered orally at 6 and 10 weeks of age into her National Immunization Program in July 2014. The study evaluated the long-term impact of the vaccine on hospitalization for all-cause and rotavirus-specific acute gastroenteritis (AGE) and strain epidemiology in Kenya., Methods: Data on all-cause and rotavirus-specific AGE and strain distribution were derived from an eleven-year hospital-based surveillance of AGE among children aged <5 years at Kiambu County Teaching and Referral Hospital (KCTRH) in Central Kenya between 2009 and 2020. Fecal samples were screened for group A rotavirus using ELISA and genotyped using multiplex semi-nested RT-PCR. Trends in all-cause and rotavirus-related AGE and strain distribution were compared between the pre-vaccine (July 2009-June 2014), early post-vaccine (July 2014-June 2016) and late post-vaccine (February 2019-October 2020) periods., Results: Rotavirus-specific AGE was detected at 27.5% (429/1546, 95% CI: 25.5-30.1%) in the pre-vaccine period; 13.8% (91/658, 95% CI: 11.3-16.6%) in the early post-vaccine period (July 2014-June 2016); and 12.0% (229/1916, 95% CI: 10.6-13.5%) in the late post-vaccine period (February 2019-October 2020). This amounted to a decline of 49.8% (95% CI: 34.6%-63.7%) in rotavirus-specific AGE in the early post-vaccine period and 53.4% (95% CI: 41.5-70.3%) in the late post-vaccine period when compared to the pre-vaccine period. All-cause AGE hospitalizations declined by 40.2% (95% CI: 30.8%-50.2%) and 75.3% (95% CI: 65.9-83.1%) in the early post-vaccine and late post-vaccine periods, respectively, when compared to the pre-vaccine period. G3P [8] was the predominant strain in the late post-vaccine period, replacing G1P[8] which had predominated in the pre-vaccine and early post-vaccine periods. Additionally, we detected considerable proportions of uncommon strains G3P[6] (4.8%) and G12P[6] (3.5%) in the post-vaccine era., Conclusion: Rotavirus vaccination has resulted in a significant decline in all-cause and rotavirus-specific AGE, and thus, provides strong evidence for public health policy makers in Kenya to support the sustained use of the rotavirus vaccine in routine immunization. However, the shift in strain dominance and age distribution of rotavirus AGE in the post-vaccine era underscores the need for continued surveillance to assess any possible vaccine-induced selective pressure that could diminish the vaccine effectiveness over time., 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. Published by Elsevier Ltd.)

The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics., (© 2024. The Author(s).)

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.

Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of -50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

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).)

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.

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.)

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., 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.)

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP4 86-175aa (NSP4*) and VP4 26-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine., 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.)

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a 'minigenome' or 'minireplicon' assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis -acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a 'first-generation' plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

A total of 490 diarrhoeic samples from calves aged between 0 and 6 months were screened for the presence of different G- and P-genotypes of rotavirus circulating in bovines in the Kashmir Valley. Of the 490 diarrhoeic samples, Group A rotavirus was detected in 68 (13.87%) samples by polymerase chain reaction (PCR) followed by RNA-PAGE. Genotyping analysis revealed G10, G6, G3, P[11] and P[5] to be the predominant types. The most common types of combinations detected were G10P[11] (27.90%) and G6P[11] (20.60%). The prevalence rate of G10 and P[11] decreased from 60% to 36.76% and 100%-69.11%, respectively. Genotypes G6, G3, P[1] and P[5], which were not previously reported, were detected and unusual combinations such as G6P[11], G3P[11], G10P[5], G3P[5], G6P[1], G6P[5], G6+G8P[11] were also observed for the first time. Fluctuations in the predominant types, emergence of new types and possible genetic reassortment events suggest an unstable epidemiological situation and the need for continuous surveillance of the circulating types to ensure the suitability of the vaccination programme. The present data suggests G10, G6, P[11] and P[5] genotypes could be incorporated in the polyvalent vaccine to offer increased protection against bovine rotavirus infection in India.

Background: Rotavirus infections are a significant cause of severe diarrhea and related illness and death in children under five worldwide. Despite the global introduction of vaccinations for rotavirus over a decade ago, rotavirus infections still result in high deaths annually, mainly in low-income countries, including Ethiopia, and need special attention. This system review and meta-analysis aimed to comprehensively explore the positive proportion of rotavirus at pre- and post-vaccine introduction periods and genotype distribution in children under five with diarrhea in Ethiopia., Methods: The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. Database sources included PubMed, Scopus, EMBASE, and Epistemonikos, focusing on studies published before November 30, 2023. The search targeted rotavirus infection and genotype distribution in Ethiopia before and after the introduction of the Rota vaccine. Data was managed using EndNote 2020 software and stored in an Excel 2010 sheet. A random-effects model determined the pooled estimate of the rotavirus infection rate at 95% confidence intervals. The Q-and I² statistics were used to assess the study heterogeneity, and a funnel plot (Egger test) was used to determine the possibility of publication bias., Results: The analysis included data from nine studies conducted in different regions of Ethiopia. The overall prevalence of rotavirus infection was significant, with a prevalence rate of approximately 22.63% (1362/6039). The most common genotypes identified before the Rota vacation introduction were G1, G2, G3, G12, P [4], P [6], P [8], P [9], and P [10]. Meanwhile, G3 and P [8] genotypes were particularly prevalent after the Rota vaccine introduction. These findings highlight the importance of implementing preventive measures, such as vaccination, to reduce the burden of rotavirus infection in this population. The identified genotypes provide valuable insights for vaccine development and targeted interventions., Conclusion: This study contributes to the evidence base for public health interventions and strategies to reduce the impact of rotavirus infection in children under five in Ethiopia. Despite the rollout of the Rota vaccination in Ethiopia, rotavirus heterogeneity is still high, and thus, enhancing vaccination and immunization is essential., (© 2024. The Author(s).)

Background: Rotavirus is the most common cause of gastroenteritis among children. Currently, four oral live-attenuated vaccines are available to prevent rotavirus infection. The World Health Organization (WHO) has recommended including rotavirus vaccination in national immunization programs; however, it has not been introduced to the Iranian national immunization program. The study aimed to assess the frequency of rotavirus gastroenteritis in the west of Iran and investigate the necessity of rotavirus vaccination. Study Design: A case series study., Methods: In this case series study, 284 cases under six years of age who presented with acute gastroenteritis from March 2021 to 2022 to a referral hospital in the west of Iran were evaluated. Data on baseline characteristics, clinical manifestations, results of stool test, ELISA for rotavirus detection, and polymerase chain reaction (PCR) test for genotyping of rotavirus-positive samples were recorded., Results: Results showed that the prevalence of rotavirus infection was 36.6%. The highest frequency was observed among children aged 6-12 months and during the autumn. According to the PCR results, G1P[8], G9P[8], G9P[4], and G1P [4] were the dominant genotypes, and 33.75% of samples were infected with multiple rotavirus genotypes., Conclusion: The study highlights the considerable prevalence of rotavirus infection among cases of acute gastroenteritis in children under six years of age who were referred to a referral hospital in the west of Iran and the high diversity of rotavirus genotypes in the targeted community. Consequently, physicians and health policymakers should prioritize strategies for the prevention and control of this infection, particularly by considering the rotavirus vaccine as a priority for the Iranian national immunization program., (© 2024 The Author(s); Published by Hamadan University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Rotavirus, a primary contributor to severe cases of infantile gastroenteritis on a global scale, results in significant morbidity and mortality in the under-five population, particularly in middle to low-income countries, including India. WHO-approved live-attenuated vaccines are linked to a heightened susceptibility to intussusception and exhibit low efficacy, primarily attributed to the high genetic diversity of rotavirus, varying over time and across different geographic regions. Herein, molecular data on Indian rotavirus A (RVA) has been reviewed through phylogenetic analysis, revealing G1P[8] to be the prevalent strain of RVA in India. The conserved capsid protein sequences of VP7, VP4 and VP6 were used to examine helper T lymphocyte, cytotoxic T lymphocyte and linear B-cell epitopes. Twenty epitopes were identified after evaluation of factors such as antigenicity, non-allergenicity, non-toxicity, and stability. These epitopes were then interconnected using suitable linkers and an N-terminal beta defensin adjuvant. The in silico designed vaccine exhibited structural stability and interactions with integrins (αvβ3 and αIIbβ3) and toll-like receptors (TLR2 and TLR4) indicated by docking and normal mode analyses. The immune simulation profile of the designed RVA multiepitope vaccine exhibited its potential to trigger humoral as well as cell-mediated immunity, indicating that it is a promising immunogen. These computational findings indicate potential efficacy of the designed vaccine against rotavirus infection., 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 Ltd. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission., 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.)

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains Rotarix TM and RotaTeq TM , multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect., Competing Interests: Declaration of competing interest Author and co-author declare no potential conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., 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.)

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.