Animal Rotavirus Strains Research Articles

Immunological and molecular detection of rotavirus genotype in calves with gastroenteritis in Diyala-Iraq

Aim: To explore the prevalence of rotavirus infection along with the molecular detection and genotyping of group A rotavirus (RVA) among bovine calves up to 5 months old in Diyala province-Iraq. Methods: This is a cross sectional study conducted in Diyala province-Iraq during the period of 2019-2020. One hundred bovine calves with age range of 1-5 months were included in the study. All were suffering acute gastroenteritis. Serum anti-rotavirus IgM and IgG plus fecal rotavirus Ag were tested for using ELISA techniques. Stool samples positive for rotavirus Ag were submitted for reverse transcription PCR (RT-PCR) for G and P genes, followed by sequencing and genotyping thereafter. Statistical analysis was done using SPSS version 25 and P values ≤ 0.05 were considered significant. Results: The positivity rate of anti-rotavirus IgM was 80% (P = 0.0001), and that of anti-rotavirus IgG was 79% (P = 0.0001). The rotavirus stool antigen was detected in 68% of calves (P = 0.01). A total of 45 stool samples which were positive for rotavirus Ag were submitted for RT-PCR; 13 (28.9%) were positive and 32 (71.1%) were negative (P = 0.084). 10 PCR positive samples were used for sequencing and genotyping and indicated that all investigated strains belonged to G1P[8] genotype. Conclusion: The current strains analyzed belonged to the G1P[8] RVA genotypes, affirming that employment of VP7 gene polymorphism accurately yielded uniform phylogenetic distances amongst investigated rotavirus strains and that there were no noticeable assortment events between human and animal rotavirus strains in Diyala province.

Rotavirus in Calves and Its Zoonotic Importance.

Rotavirus is a major pathogen responsible for diarrheal disease in calves, resulting in loss of productivity and economy of farmers. However, various facets of diarrheal disease caused by rotavirus in calves in the world are inadequately understood, considering that diarrheal disease caused by rotavirus is a vital health problem in calves that interrupts production benefits with reduced weight gain and increased mortality, and its potential for zoonotic spread. The pathological changes made by rotavirus are almost exclusively limited to the small intestine that leads to diarrhea. It is environmentally distributed worldwide and was extensively studied. Reassortment is one of the important mechanisms for generating genetic diversity of rotaviruses and eventually for viral evolution. So, the primary strategy is to reduce the burden of rotavirus infections by practicing early colostrum's feeding in newborn calves, using vaccine, and improving livestock management. Rotaviruses have a wide host range, infecting many animal species as well as humans. As it was found that certain animal rotavirus strains had antigenic similarities to some human strains, this may be an indication for an animal to play a role as a source of rotavirus infection in humans. Groups A to C have been shown to infect both humans and animals. The most commonly detected strains in both human and animals are G2, G3, G4, and G9, P [6]. Therefore, this review was made to get overview epidemiology status and zoonotic importance of bovine rotavirus.

Rotavirus NSP1 Inhibits Type I and Type III Interferon Induction.

Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or β-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium.

Epidemiological and molecular study of Rotavirus infection among human and animal in Karbala and Basrah provinces

This study was aimed to determine the prevalence of rotaviruses in Karbala and Basrah provinces, the genetic reassortment of human and animal rotavirus strains and the novel strains. Rotaviruses were detected by Immunochromatography Test (ICT) then the positive samples were tested by reverse transcription PCR (RT-PCR) using specific primers to VP7 gene, this gene is responsible for VP7 antigen which is responsible for stimulation the immune system to produce neutralizing antibodies. The VP7 gene was implemented in rotavirus vaccine of Iraqi immunization program. The RT PCR results showed that 56.3% (27/48) of samples positive in children under five years of age in Karbala province while Basrah province revealed 58.5% (31/53) positive samples in children whereas samples taken from calves revealed 43.1% (22/51) and 45.5% (25/55) positive samples in Karbala and Basrah provinces respectively. The sequencing of human and animal samples revealed that there was genetic reassortment between human and animal strains while in comparing with international strains there was closely related with Indian and Pakistani human strains. Necessarily for further bioinformatics studies are needed to study the genetic alterations with viral proteins analysis of rotavirus.

Molekularne značajke i zoonotski potencijal rotavirusa

Rotaviruses are one of the most important pathogens causing acute gastroenteritis in humans and animals. Among nine species,rotavirus A is particularly emphasised considering its prevalence and pathogenicity. Even today, apart from the substantial morbidityand hospitalisation rates in countries with high hygiene standards, it causes considerable mortality in developing countries. Suchimpact is especially evident if available vaccines are not included in the national immunisation program. Due to the nature of theirgenome, the evolution of rotaviruses is quite rapid owing to the accumulation of point mutations and genome reassortment. Therefore,the high heterogeneity and the fact that nowadays there are 36 G genotypes and 51 P genotypes of rotavirus A, is not surprising.When direct interspecies transmission occurs, rotaviruses are mostly not able to efficiently infect or transmit in the new host. However,genome reassortment can result in the generation of new, potentially emergent, human-animal reassortants. Thus, it is necessaryto continuously monitor the animal rotavirus strains in order to get complete perspective of the potential animal reservoirs thatcould be responsible for the introduction of novel rotaviruses in human population or rotavirus genes in the genetic backbone ofhuman strains. Systematic investigation of rotavirus molecular characteristics in a certain region is of special importance before theintroduction or during the implementation of national immunisation program. Comprehensive research that would evaluate theanimal reservoirs and the zoonotic potential of rotavirus A strains in Croatian ecosystem is currently in progress.Key words: ROTAVIRUS; GENOTYPE; ZOONOSES; CROATIA

Detection and Whole-Genome Analysis of a Zoonotic G8P[14] Rotavirus Strain Isolated from a Child with Diarrhea.

ABSTRACTThe group A rotavirus strain SI-2987/09 was detected in a child with severe diarrhea. The whole-genome analysis revealed the G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genome constellation, which reflects the zoonotic transmission of the strain most probably from the ungulate group. This was also confirmed by the high nucleotide identity to those animal rotavirus strains.

Glycan-specificity of four neuraminidase-sensitive animal rotavirus strains

Group A rotaviruses (RVAs) are divided into neuraminidase (NA)-sensitive and NA-insensitive strains depending upon their binding affinity to the VP8* domain in the terminal sialic acids (SAs) of cell surface carbohydrates. Although NA-sensitive strains are known to use terminal SAs as an attachment factor, the exact nature of this attachment factor is largely unknown. Here we show that the specific linkage of SA-containing glycan to glycoprotein or glycolipid is an attachment factor used by NA-sensitive porcine G9P[7] PRG9121 and G9P[23] PRG942, bovine G6P[1] NCDV, and canine G3P[3] strains. Infectivity of porcine G9P[7] and G9P[23] strains was markedly blocked by α2,3-linkage and α2,6-linkage inhibitors, indicating that these strains bind to both α2,3- and α2,6-linked SAs. However, the infectivity of bovine G6P[1] and canine G3P[3] strains was significantly reduced by α2,6-linkage inhibitor but not by α2,3-linkage blockers, demonstrating a predilection of these strains for α2,6-linked SAs. The infectivity of four NA-sensitive strains was equally reduced by inhibitors of lipid membrane and N-linked glycoprotein but not by an inhibitor of O-linked glycoprotein, indicating that these strains utilize both glycolipid and N-linked glycoprotein. Our study demonstrates that four NA-sensitive animal strains could have a strain-dependent binding preference toward α2,6-linked SAs (P[1] NCDV and P[3] CU-1 strains) or both α2,3- and α2,6-linked SAs (P[7] PRG9121 and P[23] PRG942 strains) to the glycolipid and N-linked glycoprotein.

Whole genome sequencing of a rare rotavirus from archived stool sample demonstrates independent zoonotic origin of human G8P[14] strains in Hungary

Genotype P[14] rotaviruses in humans are thought to be zoonotic strains originating from bovine or ovine host species. Over the past 30 years only few genotype P[14] strains were identified in Hungary totaling<0.1% of all human rotaviruses whose genotype had been determined. In this study we report the genome sequence and phylogenetic analysis of a human genotype G8P[14] strain, RVA/Human-wt/HUN/182-02/2001/G8P[14]. The whole genome constellation (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) of this strain was shared with another Hungarian zoonotic G8P[14] strain, RVA/Human-wt/HUN/BP1062/2004/G8P[14], although phylogenetic analyses revealed the two rotaviruses likely had different progenitors. Overall, our findings indicate that human G8P[14] rotavirus detected in Hungary in the past originated from independent zoonotic events. Further studies are needed to assess the public health risk associated with infections by various animal rotavirus strains.

Substantial Receptor-induced Structural Rearrangement of Rotavirus VP8*: Potential Implications for Cross-Species Infection.

Rotavirus-cell binding is the essential first step in rotavirus infection. This binding is a major determinant of rotavirus tropism, as host cell invasion is necessary to initiate infection. Initial rotavirus-cell interactions are mediated by carbohydrate-recognizing domain VP8* of the rotavirus capsid spike protein VP4. Here, we report the first observation of significant structural rearrangement of VP8* from human and animal rotavirus strains upon glycan receptor binding. The structural adaptability of rotavirus VP8* delivers important insights into how human and animal rotaviruses utilize the wider range of cellular glycans identified as VP8* binding partners. Furthermore, our studies on rotaviruses with atypical genetic makeup provide information expected to be critical for understanding the mechanisms of animal rotavirus gene emergence in humans and support implementation of epidemiologic surveillance of animal reservoirs as well as future vaccination schemes.

Full genomic characterization of a novel genotype combination, G4P[14], of a human rotavirus strain from Barbados

Since 2004, the Pan American Health Organization (PAHO) has carried out rotavirus surveillance in Latin America and the Caribbean. Here we report the characterization of human rotavirus with the novel G–P combination of G4P[14], detected through PAHO surveillance in Barbados. Full genome sequencing of strain RVA/Human-wt/BRB/CDC1133/2012/G4P[14] revealed that its genotype is G4-P[14]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The possession of a Genogroup 1 (Wa-like) backbone distinguishes this strain from other P[14] rotavirus strains. Phylogenetic analyses suggested that this strain was likely generated by genetic reassortment between human, porcine and possibly other animal rotavirus strains and identified 7 lineages within the P[14] genotype. The results of this study reinforce the potential role of interspecies transmission in generating human rotavirus diversity through reassortment. Continued surveillance is important to determine if rotavirus vaccines will protect against strains that express the P[14] rotavirus genotype.

Identification of novel Ghanaian G8P[6] human-bovine reassortant rotavirus strain by next generation sequencing.

Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide. The monovalent rotavirus vaccine Rotarix was introduced into the national Expanded Programme on Immunization (EPI) in Ghana in May 2012. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVAs in circulation pre-vaccine introduction. The common bovine rotavirus VP7 genotype G8 has been sporadically detected in Ghanaian children, usually in combination with the VP4 genotype P[6]. To investigate the genomic constellations and phylogeny of RVA strains in circulation prior to vaccine introduction, the full genomes of two unusual G8P[6] strains, GH018-08 and GH019-08, detected during burden of disease surveillance, were characterized by Illumina MiSeq sequencing. The Ghanaian isolates, GH018-08 and GH019-08, exhibited the unusual, previously unreported genotype constellation G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Phylogenetic analyses confirmed that 10 out of the 11 genes of GH018-08 and GH019-08 were identical/nearly identical, with significant variation detected only in their VP1 genes, and clearly established the occurrence of multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. These findings highlight the contribution of reassortment and interspecies transmission events to the high rotavirus diversity in this region of Africa, and justify the need for simultaneous monitoring of animal and human rotavirus strains.

Randomized placebo-controlled clinical trial of immunoglobulin Y as adjunct to standard supportive therapy for rotavirus-associated diarrhea among pediatric patients

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1, G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3h later showed a significant dose-dependent reduction in frequency (p<0.05) and duration (p<0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n=26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p=0.004), mean duration of intravenous fluid administration (p=0.03), mean duration of diarrhea from day of admission (p<0.01) and mean duration of rotavirus clearance from stool from day of admission (p=0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients.

Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen

As with many other viruses, the initial cell attachment of rotaviruses, major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans1–4. The distally located VP8* domain of the rotavirus spike protein VP45 mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus (HR) strains bind to glycans with internal Sia such as GM13. Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies1,3,6,7, it is not yet known how VP8* of HRs interacts with Sia and whether their cell attachment necessarily involves sialoglycans. We found that VP8* of a HR strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-Atype antibodies as well as significantly enhanced in CHO cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of HR. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelial and on red blood cells8, and are recognized as susceptibility and cell attachment factors for gastric pathogens like H. pylori9 and noroviruses10. Our crystallographic studies show that the A-type HBGA binds to the HR VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific HR strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population.

Genetic heterogeneity of wild-type G4P[6] porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd

Group A rotavirus (RV-A) with short electropherotype was identified by ss-PAGE in a neonatal diarrhea outbreak at a Brazilian pig farm where the sows were regularly vaccinated with a commercial vaccine containing OSU (G5P[7]) and Gottfried (G4P[6]) porcine RV-A (PoRV-A) strains. The ss-PAGE positive stool samples ( n = 20) were characterized as P[6] genotype by multiplex-nested-RT-PCR assay. The nucleotide analysis of the VP4 gene (VP8*) state that the viruses clustered in P[6] lineages that are also shared by RV-A strains identified in human hosts. Nucleotide analysis of the VP7 gene identified different lineages in G4 including a new lineage tentatively designated IX. The immunological pressure induced by commercial vaccine with a rotavirus containing a G4P[6] genotype of porcine origin (Gottfried strain) might have allowed the selection of PoRV-A strains with characteristics found in RV-A strains isolated of human hosts, such as P[6]-Ie and If, and promoted the selection or emergence of RV-A strains with a new lineage of the G4 genotype. The characterization of PoRV-A strains with unusual genotypes described in this study highlight the importance of surveys on the relationship between human and animal rotavirus strains.

Genomic characterization of human rotavirus G10 strains from the African Rotavirus Network: Relationship to animal rotaviruses

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. The aim of this study was to elucidate the genetic and evolutionary relationships of short fragments of all 11 gene segments of G10 HRV strains identified in West Africa through the African Rotavirus Network (ARN) system. During 1998–2004 surveillance within the ARN, we identified 5 G10 P[8] HRV strains. Fragments of all 11 gene segments of these G10 strains were sequenced. Phylogenetic and sequence analyses of each gene segment revealed high nucleotide similarities amongst the ARN strains (97–100%) except in the case of the VP1(85–96%) and NSP2 genes (87.8–99.7%) where some strains were divergent. All genes of the ARN strains were classified as Wa-like (genotype 1) with the exception of their VP7 gene of all strains (genotype G10) and the VP6 gene of a single strain, 6755/2002/ARN (DS-1 like, genotype 2). While classified as Wa-like, the NSP2 genes of four of the ARN strains occupied a distinct sub-lineage related to simian strain Tuch, while the NSP2 of strain 6755/2002/ARN and NSP5 genes of all strains were closely related to the cognate genes of both human and animal strains belonging to the Wa-like genogroup. Although these findings help to elucidate the evolution of ARN G10 strains, additional sequence studies of cognate animal rotavirus genes are needed to determine irrefutably the specific origin of those genes relative to both human and animal rotavirus strains.

Rotaviruses from Canadian farm samples

Animal rotavirus (RoV) strains detected in Canadian swine and dairy cattle farms were characterized by sequence analysis of viral protein 4 (VP4), VP6, VP7 and non-structural protein 4 segments from 15 RoV strains. Some porcine strains were found to contain a mixture of segments typical of human and animal viruses. One strain represented a novel VP6 genotype "I14", G2-P[27]-I14. Other strains detected in porcine samples represented multiple different segment types. These results illustrate the active evolution of animal RoV strains and underline the need for surveillance of both animal and human strains in public health-monitoring programs.

Genomic diversity among group A rotaviruses from diarrheic children, piglets, buffalo and cow calves of Madhya Pradesh

Diarrheal disease continues to be a global health problem, particularly among young ones in developing nations. Amongst several viral and non-viral agents associated with diarrhea, group A rotavirus has been recognized as the major etiological agent of childhood gastroenteritis in human infants as well as several animal species throughout the world. During this study, a total of 181 diarrheic stool samples collected from children, piglets, buffalo and cow calves of Madhya Pradesh, central India were analyzed by electrophoretic mobilities of the 11 segments of dsRNA by polyacrylamide gel electrophoresis (PAGE). This technique revealed prevalence of rotavirus among different species (human-26.09%, pig-25.71%, buffalo-23.61% and cattle-21.43%). Prevalence of existence of circulating 8 different electropherotypes of group A rotaviruses indicated high genomic diversity among rotaviruses in this geographical region. Majority of the electropherotypes from humans and animals were of long pattern (75%) than short electropherotypes (9.09%). Same electropherotype was found to exist either only in a single species or in more than one species implicating the possibility of cross species transmission of the rotavirus strains. As it was found that certain animal rotavirus strains had electropherotypic similarities to some human strains, speculation increased about whether animals play a role as a source of rotavirus infection in humans or vice-versa. There is a need for further detailed study on the molecular characterization of rotaviruses which would have important implication in vaccine evaluation program.

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: Relationship to animal rotaviruses

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996-2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT-PCR amplicons) of all 11-gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88-100% nt and 91-100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83-99% and 97-99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains.

Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains.

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.