Canine Rotavirus Strains Research Articles

Molecular evolution and genetic characteristics of G3P[3] group A canine rotavirus isolated in Wuhan, China.

Rotaviruses can infect multiple animal species and have the potential for cross-recombination based on the segmented genome characteristics. To study the intra-host recombination and zoonotic potential of group A canine rotavirus (CRV), 438 samples were collected from domestic dogs in six animal hospitals and from stray dogs from October 2019 to May 2021 in Wuhan, China. Seven of the samples were positive (7/438) for group A CRV from which a CRV strain was successfully isolated in MA-104 cells. The genotype of the isolated strain was characterized by whole-genome sequencing showing that the genotype was group A CRV G3P[3]. According to the Rotavirus Classification Working Group (RCWG), the genomic constellation of the isolated CRV was G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6, which belongs to the AU-1-like group with gene segments of AU-1-like and Cat 97-like strains. Based on the phylogenetic analysis of the 11 gene segments, we found that the different segments of the isolated group A CRV were closely related to several reassortment rotaviruses from different animal sources and bat strains. Based on the analysis of the molecular evolution and genetic characteristics, we concluded that the isolated strain might be a reassortment strain. These data further enrich our understanding of rotavirus molecular evolution and genetic characteristics in China.

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*).

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate-binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus-cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*64-224 of canine rotavirus strain K9 interacts with N-acetylneuraminic and N-glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase-sensitive animal rotaviruses from pigs (CRW-8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N-glycolyl- over the N-acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.

Whole genotype constellation of prototype feline rotavirus strains FRV-1 and FRV64 and their phylogenetic relationships with feline-like human rotavirus strains.

Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.

Rare G3P[3] rotavirus strain detected in Brazil: Possible human–canine interspecies transmission

BackgroundAn unusual strain of human rotavirus G3P[3] (R2638 strain) was detected from a 1-year-old child patient during the epidemiological survey of rotavirus in the state of São Paulo, Brazil in 2011. ObjectiveThe aim of this study was to carry out sequence analyses of the two outer capsid proteins (VP4 and VP7) of the R2638 strain detected in order to obtain further information of the genetic relationships between human and animal rotaviruses. Study designRotavirus G3P[3] was detected using a commercial immunoenzymatic assay, SDS-PAGE, and genotyped by RT-PCR. The analysis of the genetic relationship between human and animal rotaviruses was carried out by sequencing the VP7 and VP4 genes. ResultsThe VP7 gene of the R2638 strain displayed the highest nucleotide identity to the canine strains A79-10 (96.6%) and CU-1 (96.2%) isolated in USA. The VP4 sequence showed the highest nucleotide identity to P[3] canine rotavirus strain RV52/96 isolated in Italy at 94.1%. Furthermore, the VP4 genes of P[3] strains could be discriminated into two phylogentically distinct clusters. ConclusionThe present study reinforces the hypothesis that animal's rotaviruses might be able to cross the species barriers, and the lack of systematic surveillance of rotavirus infection in small animals hinders the ability to establish firm epidemiologic connections. Moreover, in 2006 rotavirus vaccine was included in the Brazilian Immunization Program, and selective vaccine pressure could increase the circulation of uncommon strains. This is the first report of G3P[3] in over 20-year period of monitoring in Brazil.

Isolation and genetic characterization of two G3P5A[3] canine rotavirus strains in Italy

Two strains of canine rotavirus were isolated from pups with clinical signs of gastroenteritis. Both strains were identified by polymerase chain reaction (PCR) as G3P5A[3], although restriction endonuclease analysis of the PCR amplicons revealed a genetic difference between the two isolates in the VP7 gene. The isolation in Italy of canine rotaviruses displaying the same VP7 and VP4 specificities as in the USA and in Japan, suggests that the G3 and P5A[3] types are highly conserved among canine rotavirus strains.

Human rotavirus HCR3 possesses a genomic RNA constellation indistinguishable from that of feline and canine rotaviruses.

Infection and spread of group A rotaviruses under natural conditions are mostly limited to one animal host species. However, rare molecular evidence exists for interspecies transmission by whole virions of animal rotaviruses to humans. Human rotavirus strain HCR3, which was isolated in 1984 from a healthy infant in Philadelphia, U.S.A. was shown by RNA-RNA hybridization to form 11 hybrid bands with feline rotavirus strain FRV64 and canine rotavirus strains CU-1 and K9, but not with rotaviruses commonly found in humans. Thus, HCR3 was concluded to be originally a rotavirus circulating in cats and dogs and accidental interspecies transmission by whole virions to humans was likely to have occurred in the past.

Species Specificity and Interspecies Relatedness in VP4 Genotypes Demonstrated by VP4 Sequence Analysis of Equine, Feline, and Canine Rotavirus Strains

We determined the nucleotide and deduced amino acid sequences of the VP4 genes of five equine, two feline, and two canine rotavirus strains. A high degree of homology (>97.0%) was found among the VP4 amino acid sequences of the equine strains H2, FI-14, and FI23. Equine strain L338 has a distinct VP4 amino acid sequence from those of the other equine strains (78.1% or less homology), and the L338 VP4 exhibited more than 17.0% divergence at the amino acid level from those of rotavirus strains published so far. The VP4 amino acid sequence of equine strain H1, which showed low homology with those of other equine strains, shares >95.4% homology to those of porcine strains OSU and YM. VP4 amino acid sequences of feline strain Cat97 and canine strains CU-1 and K9 showed a high degree of homology (96.8 to 97.2%) to one another, and were found to be quite similar (96.0-97.0% homology) to that of a human HCR3 strain recently characterized. Feline strain Cat2, whose VP4 sequence is distinct from that of strain Cat97, has a VP4 similar to those of human strains K8 and AU-1 (97.8 and 97.5% homologies at amino acid level, respectively). Thus, the VP4 sequences of rotaviruses showed species specificity and interspecies relatedness.

A new serotype of the outer capsid protein VP4 shared by an unusual human rotavirus strain Ro1845 and canine rotaviruses

The VP4 protein of human rotavirus (HRV) strain Ro1845 and canine rotavirus strains K9 and CU-1 exhibited greater than 98% amino acid identity within their group, but showed less identity with VP4 proteins of other HRV and animal rotavirus strains, the simian rotavirus strain RRV VP4 being most similar to them (90% amino acid identity). To exclude the possibility that these three strains were members of the RRV VP4 serotype P3, neutralization studies were performed using antisera to reassortant viruses containing the VP4 gene from each of Ro1845, CU-1 and RRV. The result established close antigenic similarity among the VP4 proteins of Ro1845, K9 and CU-1 and revealed only a marginal degree of similarity between the VP4 proteins of these three strains and that of strain RRV. These sequence and serological data suggest that the VP4 proteins of Ro1845, K9 and CU-1 represent a new P serotype which we propose to assign P13.

Analysis of gene selection in reassortant formation between canine rotavirus K9 and human rotaviruses with different antigenic specificities

A number of antigenic mosaic reassortants which have neutralization proteins VP4 and VP7 derived from different parental strains were analysed in order to study gene selection in reassortant formation between animal and human rotaviruses (HRV). These reassortants were isolated from mixed infection of MA-104 cells with canine rotavirus strain K9 (subgroup I and G serotype 3) and HRV strains (with subgroup I or II antigen and G serotype 1-4, 9 or 12 antigen), through repeated selections with anti-VP4 and anti-VP7 neutralizing monoclonal antibodies directed specifically at HRV and K9, respectively. By serological and genomic analyses, all the isolated clones were found to be antigenic mosaic reassortants possessing VP4 of K9 and VP7 of HRV. In the reassortants between strain K9 and one of the six strains of subgroup II HRV, a single or a few genotypes with particular constellations of RNA segments were predominant, with only a few RNA segments including gene 4 (encoding VP4) being derived from K9. In contrast, in the reassortants between strain K9 and any one of the subgroup I HRV, more than nine different genotypes were identified and various RNA segments, except for segments 8 and 10, were derived from K9. These findings indicated that the RNA segments of K9 might be reassorted more readily with those of subgroup I HRV than with those of subgroup II HRV, suggesting the possible existence of functional mechanisms which determine the extent of diversity of genome selection depending on the pairs of parent strains in the reassortant formation.

Hemagglutinin activity of two distinct genogroups of feline and canine rotavirus strains.

Genomes of hemagglutinating strains of feline and canine rotaviruses, were much more closely related to each other than to non-hemagglutinating strains. The Cat2 feline rotavirus appears to derive from reassortment between hemagglutinating and non-hemagglutinating strains.

Experience with serotyping rotavirus strains by reverse transcription and two-step polymerase chain reaction with generic and type-specific primers

Six VP7 serotypes or G types (G1-G4, G8 and G9) occur in group A human rotaviruses. Gouvea et al. recently reported a novel G-typing method based on reverse transcription (RT)-polymerase chain reaction (PCR) amplification of the VP7 gene with type-specific primers [Gouvea, V. et al. (1990). Journal of Clinical Microbiology 28, 276-82]. When we followed their protocol, 40 (89%) of 45 faecal rotavirus specimens were typed into G1-G4 and G9. The five specimens that were untypeable by the RT-PCR method contained three G2 and two G4 rotavirus specimens which were identified by an ELISA using G type-specific monoclonal antibodies. On the other hand, the RT-PCR typing assay was able to determine the G type of the seven isolates that were untypeable by the ELISA. Of 33 faecal rotaviruses that were typed by both assays, 100% agreement of the result was observed. In addition, when applied to some animal rotaviruses, the RT-PCR method identified G3 feline and canine rotavirus strains. We conclude that further refinement of the RT-PCR assay is desirable in order to more readily type G2 and G4 strains, although this assay displayed an applicability to epidemiologic studies comparable with ELISAs.

Genetic diversity and similarity among mammalian rotaviruses in relation to interspecies transmission of rotavirus.

To address the question whether there was any molecular evidence for interspecies transmission of rotaviruses from one animal species to another, genetic relationships among human and animal rotaviruses were examined by a series of hybridization experiments in which genomic RNAs from 14 rotavirus strains derived from seven different host species were hybridized with the [32P]-labelled transcription probes prepared from 11 strains representing rotaviruses from those seven host species. In general, higher level of homology among most, if not all, of the cognate gene segments that allowed classification into the same genogroup was shared among rotaviruses recovered from the same animal species but this level of homology was not found among rotavirus strains derived from different host species. However, such a high level of homology that was usually found among rotaviruses recovered from the same animal species was detected between feline rotavirus strain Cat97 and canine rotavirus strain K9 as well as between human rotavirus strain AU-1 and feline rotavirus strain FRV-1. The sharing of closely related genetic constellation of most of the 11 gene segments (genogroup) by rotaviruses recovered from different animal species provided molecular evidence that interspecies transmission of rotaviruses occurred in nature at least recently in the evolutionary history.

Characterization of a canine rotavirus strain by neutralization and molecular hybridization assays

A canine rotavirus, RS-15, previously isolated in Japan was determined to be subgroup I and serotype 3. When compared with prototype human and animal rotavirus strains by RNA-RNA hybridization assay, the RS-15 strain showed a high degree of homology only with the canine CU-1 strain isolated in the United States of America, suggesting that canine rotaviruses constitute a distinct gene family, which we have elsewhere proposed to term "genogroup".

Analysis by plaque reduction neutralization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo

The SB-1A rotavirus recovered from a diarrheic piglet in the United States is a naturally occurring intertypic rotavirus. When studied by reciprocal neutralization tests, the SB-1A virus was similar, if not identical, to the porcine Gottfried virus (serotype 4) and the porcine OSU virus (serotype 5). Analysis of reassortant viruses prepared from the SB-1A virus and the serotype 2 human DS-1 virus revealed that the antigenic specificity of the outer capsid protein VP3 of SB-1A was shared with the OSU virus, while the antigenic specificity of another outer capsid protein, VP7, of SB-1A appeared to be shared with the Gottfried virus. This suggests that SB-1A is a naturally occurring reassortant rotavirus between OSU-like and Gottfried-like porcine rotaviruses. In addition, using a genetic approach, we found evidence that the fourth gene was responsible for the predominantly one-way cross-neutralizing reactivity between canine rotavirus strain CU-1 (serotype 3) and porcine rotavirus strains SB-1A (serotypes 4 and 5) and OSU (serotype 5). Assignment of hemagglutination function to the fourth genome segment of porcine rotaviruses SB-1A and OSU and canine rotavirus CU-1 confirmed a similar previous gene assignment established for certain rotaviruses. Analysis of single gene 4 substitution reassortants confirmed our previous finding that VP3 was as potent in stimulating neutralizing antibodies as VP7. The observations confirm the need for a binary system of rotavirus classification and nomenclature similar to that used for the influenza A viruses; in such a system the neutralization specificity of both VP3 and VP7 would be indicated.