Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China.

Rotavirus group A (RVA) is a main pathogen causing diarrheal diseases in humans and animals. Various genotypes are prevalent in the Chinese pig herd. The genetic diversity of RVA lead to distinctly characteristics. In the present study, a porcine RVA strain, named AHFY2022, was successfully isolated from the small intestine tissue of piglets with severe diarrhea. The AHFY2022 strain was identified by cytopathic effects (CPE) observation, indirect immunofluorescence assay (IFA), electron microscopy (EM), high-throughput sequencing, and pathogenesis to piglets. The genomic investigation using NGS data revealed that AHFY2022 exhibited the genotypes G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, using the online platform the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). Moreover, experimental inoculation in 5-day-old and 27-day-old piglets demonstrated that AHFY2022 caused severe diarrhea, fecal shedding, small intestinal villi damage, and colonization in all challenged piglets. Taken together, our results detailed the virological features of the porcine rotavirus G9P[23] from China, including the whole-genome sequences, genotypes, growth kinetics in MA104 cells and the pathogenicity in suckling piglets.

Molecular epidemiology of rotavirus among children in Western Canada: Dynamic changes in genotype prevalence in four consecutive seasons.

Rotavirus molecular surveillance remains important in the postvaccine era to monitor the changes in transmission patterns, identify vaccine-induced antigenic changes and discover potentially pathogenic vaccine-related strains. The Canadian province of Alberta introduced rotavirus vaccination into its provincial vaccination schedule in June 2015. To evaluate the impact of this program on stool rotavirus positivity rate, strain diversity, and seasonal trends, we analyzed a prospective cohort of children with acute gastroenteritis recruited between December 2014 and August 2018. We identified dynamic changes in rotavirus positivity and genotype trends during pre- and post-rotavirus vaccine introduction periods. Genotypes G9P[8], G1P[8], G2P[4], and G12P[8] predominated consecutively each season with overall lower rotavirus incidence rates in 2016 and 2017. The demographic and clinical features of rotavirus gastroenteritis were comparable among wild-type rotaviruses; however, children with G12P[8] infections were older (p < 0.001). Continued efforts to monitor changes in the molecular epidemiology of rotavirus using whole genome sequence characterization are needed to further understand the impact of the selection pressure of vaccination on rotavirus evolution.

Detection and full-genotype determination of rare and reassortant rotavirus A strains in Nizhny Novgorod in the European part of Russia.

Reassortant DS-1-like rotavirus A strains have been shown to circulate widely in many countries around the world. In Russia, the prevalence of such strains remains unclear due to the preferred use of the traditional binary classification system. In this work, we obtained partial sequence data from all 11 genome segments and determined the full-genotype constellations of rare and reassortant rotaviruses circulating in Nizhny Novgorod in 2016-2019. DS-1-like G3P[8] and G8P[8] strains were found, reflecting the global trend. Most likely, these strains were introduced into the territory of Russia from other countries but subsequently underwent further evolutionary changes locally. G3P[8], G9P[8], and G12P[8] Wa-like strains of subgenotypic lineages that are unusual for the territory of Russia were also identified. Reassortant G2P[8], G4P[4], and G9P[4] strains with one Wa-like gene (VP4 or VP7) on a DS-1-like backbone were found, and these apparently had a local origin. Feline-like G3P[9] and G6P[9] strains were found to be phylogenetically close to BA222 isolated from a cat in Italy but carried some traces of reassortment with human strains from Russia and other countries. Thus, full-genotype determination of rotavirus A strains in Nizhny Novgorod has clarified some questions related to their origin and evolution.

Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

Characterization of an avian rotavirus A strain isolated from a velvet scoter (Melanitta fusca): implication for the role of migratory birds in global spread of avian rotaviruses.

Avian G18P[17] rotaviruses with similar complete genome constellation, including strains that showed pathogenicity in mammals, have been detected worldwide. However, it remains unclear how these strains spread geographically. In this study, to investigate the role of migratory birds in the dispersion of avian rotaviruses, we analysed whole genetic characters of the rotavirus strain RK1 that was isolated from a migratory species of birds [velvet scoter (Melanitta fusca)] in Japan in 1989. Genetic analyses revealed that the genotype constellation of the RK1 strain, G18-P[17]-I4-R4-C4-M4-A21-N4-T4-E4-H4, was highly consistent with those of other G18P[17] strains detected in various parts of the world, supporting the possibility that the G18P[17] strains spread via migratory birds that move over a wide area. Furthermore, the RK1 strain induced diarrhoea in suckling mice after oral gastric inoculation, indicating that at least some of the rotaviruses that originated from migratory birds are infectious to and pathogenic in mammals. In conclusion, it was demonstrated that migratory birds may contribute to the global spread of avian rotaviruses that are pathogenic in mammalian species.

Risk factors for bovine rotavirus infection and genotyping of bovine rotavirus in diarrheic calves in Bangladesh.

Bovine rotavirus (BRV) is considered the leading cause of calf diarrhea worldwide, including Bangladesh. In this study we aimed to identify risk factors for BRV infection and determine the G and P genotypes of BRV strains in diarrheic calves. Fecal samples were collected from 200 diarrheic calves in three districts between January 2014 and October 2015. These samples were screened to detect the presence of BRV using rapid test-strips BIO K 152 (RTSBK). The RTSBK positive samples were further tested by polyacrylamide gel electrophoresis and the silver staining technique to detect rotavirus dsRNA. Risk factors were identified by multivariable logistic regression analysis. The G and P genotypes of BRV were determined by RT-PCR and sequencing. A phylogenetic tree was constructed based on the neighbor-joining method using CLC sequence viewer 8.0. About 23% of the diarrheic calves were BRV positive. The odds of BRV infection were 3.8- (95% confidence interval [95% CI]: 1.0-14.7) and 3.9-times (95% CI:1.1-14.2) higher in Barisal and Madaripur districts, respectively, than Sirrajganj. The risk of BRV infection was 3.1-times (95% CI: 1.5-6.5) higher in calves aged ≤ 5 weeks than those aged >5 weeks. Moreover, the risk of BRV infection was 2.6-times (95% CI:1.1-5.8) higher in crossbred (Holstein Friesian, Shahiwal) than indigenous calves. G6P[11] was the predominant genotype (94.4%), followed by G10P[11] (5.6%). The BRV G6 strains were found to be closest (98.9-99.9%) to Indian strains, and BRV G10 strains showed 99.9% identities with Indian strain. The VP4 gene of all P[11] strains showed >90% identities to each other and also with Indian strains. The most frequently identified BRV genotype was G6P[11]. About 23% of calf diarrhea cases were associated with BRV. To control disease, high-risk areas and younger crossbred calves should be targeted for surveillance and management. The predominant genotype could be utilized as the future vaccine candidate or vaccines with the dominant genotype should be used to control BRV diarrhea in Bangladesh.

m6A modifications regulate intestinal immunity and rotavirus infection.

N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3ΔIEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3ΔIEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.

Detection of a novel species A, DS-1-like, G4P[6] rotavirus strain from a Brazilian child with gastroenteritis.

Rotaviruses belonging to species A (RVA) remain among the most common causes of severe gastroenteritis in children aged <5 years, leading to substantial morbidity and mortality worldwide. Genome reassortment events between two human strains or human and animal strains represent one of the mechanisms which appear to generate the broad genetic variability of circulating. According to a nucleotide, sequence-based classification system, RVA strains are currently classified into three genotype constellations including Wa-like (genogroup I), DS-1-like (genogroup II), and AU-like (genogroup III). The present study reports the detection of an unusual RVA G4P[6] strain (coded as strain HSE005), which might have originated from a natural reassortment event between human and animal RVA strains. Molecular characterization of this isolate showed that it belonged to genogroup II, genotype G4P[6]. In addition, two genes (VP3 and NSP4) of this strain denoted evidence of reassortment events involving strains of distinct zoonotic evolutionary origins. Therefore, we propose that a new G4P[6] strain was identified, highlighting a possible first zoonotic transmission including a reassortment event that involved the VP3 gene.

Do Rotavirus Strains Affect Vaccine Effectiveness? A Systematic Review and Meta-analysis.

BACKGROUND: Rotavirus causes 215,000 deaths from severe childhood diarrhea annually. Concerns exist that a monovalent vaccine (RV1) and a pentavalent vaccine (RV5) may be less effective against rotavirus strains not contained in the vaccines. We estimated the vaccine effectiveness (VE) of RV1 and RV5 against severe rotavirus gastroenteritis caused by vaccine (homotypic) and nonvaccine (partially and fully heterotypic) strains. METHODS: After conducting a systematic review, we meta-analyzed 31 case-control studies (N = 27,293) conducted between 2006 and 2020 using a random-effects regression model. RESULTS: In high-income countries, RV1 VE was 10% lower against partially heterotypic (P = 0.04) and fully heterotypic (P = 0.10) compared with homotypic strains (homotypic VE: 90% [95% confidence intervals (CI): 82-94]; partially heterotypic VE: 79% [95% CI: 71-85]; fully heterotypic VE: 80% [95% CI: 65-88]). In middle-income countries, RV1 VE was 14-16% lower against partially heterotypic (P = 0.06) and fully heterotypic (P = 0.04) compared with homotypic strains (homotypic VE: 81% [95% CI: 69-88]; partially heterotypic VE: 67% [95% CI: 54-76]; fully heterotypic VE: 65% [95% CI: 51-75]). Strain-specific RV5 VE differences were less pronounced, and primarily derived from high-income countries. Limited data were available from low-income countries. CONCLUSIONS: Vaccine effectiveness of RV1 and RV5 was somewhat lower against nonvaccine than vaccine strains. Ongoing surveillance is important to continue long-term monitoring for strain replacement, particularly in low-income settings where data are limited.

Reduction in Severity of All-Cause Gastroenteritis Requiring Hospitalisation in Children Vaccinated against Rotavirus in Malawi.

Rotavirus is the major cause of severe gastroenteritis in children aged <5 years. Introduction of the G1P[8] Rotarix® rotavirus vaccine in Malawi in 2012 has reduced rotavirus-associated hospitalisations and diarrhoeal mortality. However, the impact of rotavirus vaccine on the severity of gastroenteritis presented in children requiring hospitalisation remains unknown. We conducted a hospital-based surveillance study to assess the impact of Rotarix® vaccination on the severity of gastroenteritis presented by Malawian children. Stool samples were collected from children aged <5 years who required hospitalisation with acute gastroenteritis from December 2011 to October 2019. Gastroenteritis severity was determined using Ruuska and Vesikari scores. Rotavirus was detected using enzyme immunoassay. Rotavirus genotypes were determined using nested RT-PCR. Associations between Rotarix® vaccination and gastroenteritis severity were investigated using adjusted linear regression. In total, 3159 children were enrolled. After adjusting for mid-upper arm circumference (MUAC), age, gender and receipt of other vaccines, all-cause gastroenteritis severity scores were 2.21 units lower (p < 0.001) among Rotarix®-vaccinated (n = 2224) compared to Rotarix®-unvaccinated children (n = 935). The reduction in severity score was observed against every rotavirus genotype, although the magnitude was smaller among those infected with G12P[6] compared to the remaining genotypes (p = 0.011). Each one-year increment in age was associated with a decrease of 0.43 severity score (p < 0.001). Our findings provide additional evidence on the impact of Rotarix® in Malawi, lending further support to Malawi's Rotarix® programme.

Genomic characterization of an African G4P[6] human rotavirus strain identified in a diarrheic child in Kenya: Evidence for porcine-to-human interspecies transmission and reassortment.

Human rotavirus strains having the unconventional G4P[6] genotype have been sporadically identified in diarrheic patients in different parts of the world. However, the whole genome of only one human G4P[6] strain from Africa (central Africa) has been sequenced and analyzed, and thus the exact origin and evolutionary pattern of African G4P[6] strains remain to be elucidated. In this study, we characterized the full genome of an African G4P[6] strain (RVA/Human-wt/KEN/KCH148/2019/G4P[6]) identified in a stool specimen from a diarrheic child in Kenya. Full genome analysis of strain KCH148 revealed a unique Wa-like genogroup constellation: G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1. NSP3 genotype T7 is commonly found in porcine rotavirus strains. Furthermore, phylogenetic analysis showed that 10 of the 11 genes of strain KCH148 (VP7, VP4, VP6, VP1-VP3, NSP1, and NSP3-NSP5) appeared to be of porcine origin, the remaining NSP2 gene appearing to be of human origin. Therefore, strain KCH148 was found to have a porcine rotavirus backbone and thus is likely to be of porcine origin. Furthermore, strain KCH148 is assumed to have been derived through interspecies transmission and reassortment events involving porcine and human rotavirus strains. To our knowledge, this is the first report on full genome-based characterization of a human G4P[6] strain from east Africa. Our observations demonstrated the diversity of human G4P[6] strains in Africa, and provide important insights into the origin and evolutionary pattern of zoonotic G4P[6] strains on the African continent.

Molecular surveillance and genetic divergence of rotavirus A antigenic epitopes in Gabonese children with acute gastroenteritis.

BACKGROUND: Rotavirus A (RVA) causes acute gastroenteritis in children <5 years of age in sub-Saharan Africa. In this study, we described the epidemiology and genetic diversity of RVA infecting Gabonese children and examined the antigenic variability of circulating strains in relation to available vaccine strains to maximize the public health benefits of introducing rotavirus vaccine through the Expanded Programme on Immunization (EPI) in Gabon. METHODS: Stool samples were collected consecutively between April 2018 and November 2019 from all hospitalized children <5 years with gastroenteritis and community controls without gastroenteritis. Children were tested for rotavirus A by quantitative RT-PCR and subsequently sequenced to identify circulating rotavirus A genotypes in the most vulnerable population. The VP7 and VP4 (VP8*) antigenic epitopes were mapped to homologs of vaccine strains to assess structural variability and potential impact on antigenicity. FINDINGS: Infections were mostly acquired during the dry season. Rotavirus A was detected in 98/177 (55%) hospitalized children with gastroenteritis and 14/67 (21%) of the control children. The most common RVA genotypes were G1 (18%), G3 (12%), G8 (18%), G9 (2%), G12 (25%), with G8 and G9 reported for the first time in Gabon. All were associated either with P[6] (31%) or P[8] (38%) genotypes. Several non-synonymous substitutions were observed in the antigenic epitopes of VP7 (positions 94 and 147) and VP8* (positions 89, 116, 146 and 150), which may modulate the elicited immune responses. INTERPRETATION: This study contributes to the epidemiological surveillance of rotavirus A required before the introduction of rotavirus vaccination in the EPI for Gabonese children.

Structural and functional characterization of bovine G1P[5] rotavirus VP8* protein.

The widely used rotavirus (RV) vaccine, Rotateq, contained reassortment strains of human and bovine G1/2/3/4P[5] RVs. The functional and structural features of bovine G1P[5] VP8* were investigated. Bovine G1P[5] VP8* was identified to interact with sialic acids and sialic acid-containing glycans. In addition, P[5] VP8* recognized α-Gal histo-blood group antigens (HBGAs). Bovine G1P[5] VP8* did not hemagglutinate the tested red blood cells. The crystal structure of P[5] VP8* was determined at 1.7 Å. Structural superimposition revealed that P[5] VP8* was most close to human P[8] VP8*, while much further to VP8*s of porcine P[7] and rhesus P[3]. Sequence alignment showed that amino acids of the putative glycan binding site in P[5] VP8* were different to those in P[3]/P[7] VP8*s, indicating that P[5] VP8* may interact with glycans using different mechanism. This study provided more understanding of P[5] RV infection and the interactions of RV VP8* and glycans.

Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns.

Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, obscuring reassortment-specific epistasis and changes in substitution rates. To determine the co-segregation patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 complete RVA genomes isolated from mammalian hosts and compared the segments' geodesic distances. We found that segments 4 (VP4) and 9 (VP7) occupied significantly different tree spaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable tree spaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the weakest association with host species. Bayesian Skyride plots were generated for each segment to compare relative genetic diversity among segments over time. All segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices were calculated with respect to different host genomes. Codon usage varied by segment with segment 11 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that translational efficiency is not a barrier in RVA zoonosis.

Detection and molecular characteristics of bovine rotavirus A in dairy calves in China.

BACKGROUND: Bovine group A rotavirus (BoRVA) is a major cause of severe gastroenteritis in newborn dairy calves. Only one study has investigated the G and P genotypes among dairy calves in a few regions of China, which were G6 and P[5]. Therefore, data on the prevalence and molecular characteristics of BoRVA in dairy calves in China remains limited. OBJECTIVES: The purpose of this study was to investigate the prevalence and molecular characteristics of BoRVA in dairy calves in China. METHODS: 269 dairy calves diarrheic samples from 23 farms in six provinces in China were collected to detect BoRVA using reverse transcription polymerase chain reaction. RESULTS: 71% of samples were determined to be BoRVA-positive. Two G genotypes (G6, G10) and two P genotypes (P[1], P[5]) were identified, and G6P[1] BoRVA was the predominant strain. Moreover, the VP7 and VP4 gene sequences of these dairy calf BoRVA strains revealed abundant genetic diversity. Interestingly, eight out of 17 complete G6 VP7 sequences were clustered into G6 lineage VI and analysis showed the strains were closely related to Chinese yak BoRVA strains. CONCLUSIONS: The results of this study show that BoRVA circulates widely among dairy calves in China, and the dominant genotype in circulation is G6P[1], first report on molecular characteristics of complete P[5] VP4 genes in chinese dairy calves. These results will help us to further understand the prevalence and genetic evolution of BoRVA among dairy calves in China and, thus, prevent the disease more effectively.

Identification of a Ruminant Origin Group B Rotavirus Associated with Diarrhea Outbreaks in Foals.

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

Cross-sectional study and genotyping of rotavirus-A infections in ruminants in Kuwait.

BACKGROUND: Group A rotaviruses (RVA) are zoonotic pathogens responsible for acute enteritis in human and neonatal ruminants. This research aimed to determine the prevalence of RVA in ruminants (cattle, sheep, and goats) and investigate the circulating RVA genotypes in these animals in Kuwait. We conducted a cross-sectional study to detect RVA in ruminants, using an immunochromatography test (IC), direct sandwich ELISA test, and real-time RT-PCR (RT-qPCR) assay using fecal samples. RESULTS: A total of 400 cattle, 334 sheep, and 222 goats were examined. The prevalence of RVA was 5.3, 1.2, and 2.3%, respectively, using IC. The ELISA test detected RVA from 4.3% of cattle, 0.9% of sheep, and 1.8% of goats. There was a significant association between the occurrence of diarrhea and the presence of RVA in bovine fecal samples (p-value = 0.0022), while no statistical association between diarrhea and the presence of RVA in fecal samples of sheep and goats was observed (p-value = 0.7250; p-value = 0.4499, respectively). Twenty-three of the IC-positive samples (17 from cattle, two from sheep, and four from goats) were tested using a RT-qPCR RVA detection assay targeting the NSP3 gene. The results showed that 21 of 23 IC-positive samples tested positive by RT-qPCR. Detection of RVA genotypes revealed that G10P[11] was the predominant strain in cattle (58.8%), followed by G8P[1] (11.7%). One sheep sample was genotyped as G8P[1]. In addition, G6P[1] and G6P[14] were detected in goat samples. CONCLUSION: The present study revealed that the IC was more sensitive in detecting RVA antigen in fecal samples than the ELISA test. A higher occurrence of RVA infection was observed in cattle than in sheep and goats. This study suggests that RVA might be a risk factor of diarrhea in bovine calves less than 2 weeks old. This research also demonstrates the circulation of RVA in sheep and goat populations in Kuwait. Finally, the G10P[11] RVA genotype was the most prevalent genotype identified from cattle samples.

Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018-2019.

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia's national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12-23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Wa-1 Equine-Like G3P[8] Rotavirus from a Child with Diarrhea in Colombia.

Rotavirus A (RVA) has been considered the main cause of diarrheal disease in children under five years in emergency services in both developed and developing countries. RVA belongs to the Reoviridae family, which comprises 11 segments of double-stranded RNA (dsRNA) as a genomic constellation that encodes for six structural and five to six nonstructural proteins. RVA has been classified in a binary system with Gx[Px] based on the spike protein (VP4) and the major outer capsid glycoprotein (VP7), respectively. The emerging equine-like G3P[8] DS-1-like strains reported worldwide in humans have arisen an important concern. Here, we carry out the complete genome characterization of a previously reported G3P[8] strain in order to recognize the genetic diversity of RVA circulating among infants in Colombia. A near-full genome phylogenetic analysis was done, confirming the presence of the novel equine-like G3P[8] with a Wa-like backbone for the first time in Colombia. This study demonstrated the importance of surveillance of emerging viruses in the Colombian population; furthermore, additional studies must focus on the understanding of the spread and transmission dynamic of this important RVA strain in different areas of the country.