Feline Rotavirus Research Articles

Development of a Quadruplex RT-qPCR for the Detection of Feline Kobuvirus, Feline Astrovirus, Feline Bufavirus, and Feline Rotavirus

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis.

Whole Genome Sequences of the Wildtype AU-1 Rotavirus A Strain: The Prototype of the AU-1-like Genotype Constellation.

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.

Genomic analysis of two rare human G3P[9] rotavirus strains in Ningxia, China

G3P (Matthijnssens et al., 2008b [9]) is a rare combination of human rotavirus VP7/VP4 genotypes with a complex evolutionary pattern but limited related studies. Detailed genomic characterisation and genetic evolutionary analyses of G3P (Matthijnssens et al., 2008b [9]) rotaviruses have helped to enhance our understanding of rotavirus diversity. For the first time, we detected two human G3P (Matthijnssens et al., 2008b [9]) Rotavirus A (RVA) strains, RVA/Human-tc/CHN/2020999/2020/G3P (Matthijnssens et al., 2008b [9]) and RVA/Human-wt/CHN/23582009/2023/G3P (Matthijnssens et al., 2008b [9]), in diarrhoea patients from the Ningxia region of China, and carried out a whole-genome analysis of these strains. 2,020,999 and 23,582,009 have identical gene constellations: G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3, and this genotypic constellation was reported first time in China. They are closely related in 11 genome segments. The genotypes of these two strains are different from the human RVA strains L621 and E2451, which are only G3P (Matthijnssens et al., 2008b [9]) strains reported so far in China, but are identical to those of the Thai feline strain Meesuk and the Korean human strain CAU12–2-51.Phylogenetic analysis showed that the VP6, VP1-VP3, and NSP2 genes of the two strains in this study clustered with human/bovine and feline/bovine rotavirus strains to form a sublineage distinct from the common DS-1-like G2 human rotavirus. In contrast, the VP7, VP4, NSP1, and NSP3-NSP5 gene segments were closely associated with human/feline rotavirus and feline rotavirus strains. These findings suggest that the evolutionary origin of the G3P (Matthijnssens et al., 2008b [9]) human rotavirus found in Ningxia, China, is consistent with the Meesuk and CAU12–2-51 strains， may have arisen through reassortment between uncommon human/bovine， feline/bovine rotavirus strains and human/feline， feline rotaviruses. However, VP1-VP2 gene segments did not have the same lineage as strains Meesuk and CAU12–2-51, suggesting that these genes might be derived from additional reassortment event.

A G3P[9] rotavirus strain with an unusual genome constellation in a diarrheic cat in Thailand.

Rotavirus infection can cause diarrhea in many animal species. A 2-year-old indoor female Siamese cat with bloody mucoid diarrhea tested positive for rotavirus (RV) group A by real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequent conventional RT-PCR amplification of the 11 RV segments and sequencing revealed a G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3 genome constellation. Phylogenetic analysis showed that the VP4, VP7, NSP1, NSP3, NSP4, and NSP5 genes were closely related to those of human feline-like rotaviruses, while the VP1, VP2, VP3, VP6, and NSP2 genes were genetically closest to those of human bovine-like rotaviruses, suggesting that genetic reassortment had occurred. The uniqueness of this G3P[9] feline rotavirus strain expands our knowledge about feline rotaviruses.

Evidence for presumable feline origin of sporadic G6P[9] rotaviruses in humans

Species A rotaviruses are highly diverse and impose a substantial burden to human and animal health. Interspecies transmission between livestock, domestic animals and humans is commonly observed, but spread of animal-like rotaviruses within the human population is limited.During the continued monitoring of rotavirus strains in Germany, an unusual G6P[9] rotavirus strain was detected in feces of a child. The complete rotavirus coding sequences revealed a unique G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E2-H3 genotype constellation. The virus was phylogenetically related to feline G3P[9] strains and other human G6P[9] rotaviruses of presumable zoonotic origin. Analysis of primer binding sites of G6 specific genotyping revealed further evidence of a G6P[9] feline reservoir. Moreover, substantial deficits of conventional semi-nested PCR genotyping approaches in detecting contemporary G6P[9] were revealed. Rotavirus strain GER29-14 most likely resulted from a direct or recent interspecies transmission from a cat to human. Further studies could assess nucleic acid sequences and genotype constellations of feline rotavirus to confirm the likely feline origin of sporadic human G6P[9] strains.

Full genotype constellations of six feline Rotavirus A strains isolated in Japan in the 1990s including a rare A15 NSP1 genotype.

Full genome sequencing of six feline Rotavirus A (RVA) strains isolated in Japan in the 1990s revealed three genotype constellations, one of which had a unique constellation of G3-P[3]-I3-R3-C3-M3-A15-N3-T3-E3-H6. Genotype A15, carried by RVA/Cat-tc/JPN/FRV348/1994/G3P[3], is a rare NSP1 genotype, and only one human and one canine RVA strains have thus far been reported to carry this genotype. The other three G3P[3] strains (FRV72, FRV73, and FRV303) possessed a constellation of I3-R3-C2-M3-A9-N2-T3-E3-H6, whereas two G3P[9] strains (FRV317 and FRV384) possessed a constellation of I3-R3-C3-M3-A3-N3-T3-E3-H3.

Whole genome characterization of a G6P[5] rotavirus A strain isolated from a stray cat in Japan

The whole genome of an unusual G6P[5] rotavirus A strain named FRV537, which was isolated from a stray cat in Japan, was characterized to determine its species of origin. The genotype constellation of FRV537 was G6-P[5]-I2-R2-C2-M2-A13-N2- T6-E2-H3. No known feline rotavirus has this genotype constellation; the Japanese equine strain OH-4 is the only known strain that does. While FRV537 shares the same genotype with some feline rotaviruses in all genes except those encoding VP4 and NSP1, none of these genes are closely related to those of known feline rotaviruses. By contrast, G6P[5] is almost exclusively present in bovine rotaviruses. The VP7 and VP4 genes of FRV537 formed a lineage with typical bovine rotaviruses with high bootstrap values. As to the internal capsid and nonstructural gene constellation, three bovine rotavirus strains had a constellation identical to that of FRV537. Moreover, each of the genotypes of FRV537 was found to be a common bovine genotype. In addition to the high nucleotide sequence identities between FRV537 and bovine rotaviruses in each genome segment (≥95%), phylogenetic analysis revealed a close relationship to bovine/artiodactyl rotaviruses. Thus, the molecular and phylogenetic evidence suggests that FRV537 isolated from a stray cat was of bovine rotavirus origin.

Human G3P[9] rotavirus strains possessing an identical genotype constellation to AU-1 isolated at high prevalence in Brazil, 1997-1999.

Rotavirus (RV) A is a very common cause of acute diarrhoea in infants and young children worldwide. Most human strains are classified into two major Wa-like and DS-1-like genotype constellations, whilst a minor third strain, AU-1, was described in 1989 among human RV isolates from Japan. AU-1 demonstrates a high degree of homology to a feline RV, FRV-1, which suggests interspecies transmission of feline RV. However, there has been no subsequent report of RVs possessing the AU-1 genotype throughout all 11 genes of the genome. Between March 1997 and December 1999, 157 RV-positive stool samples were collected from Brazilian children, and 16 of the RVs (10.2 %) were P[9] genotype. We analysed eight strains by almost full-genome sequencing. These eight strains were divided into two groups: five AU-1-like and three Wa-like strains. Four of the five AU-1-like strains had the AU-1-like genotype constellation throughout the 11 genes. The remaining AU-1-like strain was considered to be a reassortant strain comprosed of nine, two and one genes from the AU-1-like, Wa-like and G9 strains, respectively. The three Wa-like strains were considered to be reassortants comprising seven to eight genes and three to four genes from Wa-like and non-Wa-like strains, respectively. This report of human G3P[9] RV strains possessing the AU-1 genotype constellation throughout all genes demonstrates the stability and infectivity of the AU-1-like strain with its original genotype over distance and time.

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.

Correction: Whole-Genome Analysis of a Rare Human Korean G3P[9] Rotavirus Strain Suggests a Complex Evolutionary Origin Potentially Involving Reassortment Events between Feline and Bovine Rotaviruses

Correction: Whole-Genome Analysis of a Rare Human Korean G3P[9] Rotavirus Strain Suggests a Complex Evolutionary Origin Potentially Involving Reassortment Events between Feline and Bovine Rotaviruses

Whole-Genome Analysis of a Rare Human Korean G3P[9] Rotavirus Strain Suggests a Complex Evolutionary Origin Potentially Involving Reassortment Events between Feline and Bovine Rotaviruses

A rare human rotavirus, G3P[9] strain RVA/Human-tc/KOR/CAU12-2-51/2013/G3P[9], was isolated from the stool of a 9-year-old female hospitalized with acute watery diarrhea in August 2012 in South Korea using a cell culture system, and its genome was analyzed. The complete genomic constellation of the CAU12-2-51 strain revealed a novel genotype constellation for human rotavirus, G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. Phylogenetic analysis revealed that the CAU12-2-51 strain originated from feline- and bovine-like reassortment strains. The genes encoding VP4, VP7, NSP1, NSP3, NSP4, and NSP5 were related to human/feline-like and feline rotavirus strains, whereas the remaining five genes encoding VP1, VP2, VP3, VP6, and NSP2 were related to the human/bovine-like and bovine rotavirus strains. This novel strain was identified for the first time, providing evidence of feline/bovine-to-human transmission of rotavirus. The data presented herein provide information regarding rotavirus diversity and evolution.

Feline fecal virome reveals novel and prevalent enteric viruses

Humans keep more than 80 million cats worldwide, ensuring frequent exposure to their viruses. Despite such interactions the enteric virome of cats remains poorly understood. We analyzed a fecal sample from a single healthy cat from Portugal using viral metagenomics and detected five eukaryotic viral genomes. These viruses included a novel picornavirus (proposed genus “Sakobuvirus”) and bocavirus (feline bocavirus 2), a variant of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus. Feline astroviruses (mamastrovirus 2) are the closest known relatives of the classic human astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7% (astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal samples. Co-infections were frequent with 40% (4/10) of infected cats shedding more than one of these five viruses. Our study provides an initial description of the feline fecal virome indicating a high level of asymptomatic infections. Availability of the genome sequences of these viruses will facilitate future tropism and feline disease association studies.

Comparison of different RT-qPCR assays for the detection of human and bovine group A rotaviruses and characterization by sequences analysis of genes encoding VP4 and VP7 capsid proteins

The aim of this study was to compare the performance of four RT-qPCR assays for the detection of human and bovine group A rotaviruses and to characterize the positive samples by sequence analysis of VP4 and VP7 genes. RNA extracted from eight human rotavirus strains, and a panel of 33 human and 25 bovine faecal samples was subjected to different RT-qPCR detection systems. Among these assays, only RT-qPCR primers and probe systems B and C were able to detect all human rotavirus strains from cell culture solutions and faecal samples. However, the results showed that the system C was generally more sensitive by one or two logs than the other RT-qPCR assays tested. With the bovine faecal samples, the most efficient RT-qPCR systems were B and A with the detection in 100 and 92% of samples tested, respectively. Human group A rotavirus G1P[8] and bovine G6P[11] were the most frequently used strains identified in this study. A G3P[9] strain, closely related to a feline rotavirus isolated in the USA, was also discovered in a human rotavirus infection. The RT-qPCR system B was the only TaqMan assay evaluated in this study able to detect rotavirus RNA in all positive human and bovine faecal samples. Utilization of only one RT-qPCR for the detection of human and bovine group A rotaviruses and the possibility of human infection by a feline rotavirus strain.

Multiple reassortment and interspecies transmission events contribute to the diversity of feline, canine and feline/canine-like human group A rotavirus strains

RNA–RNA hybridization assays and complete genome sequence analyses have shown that feline rotavirus (FRV) and canine rotavirus (CRV) strains display at least two distinct genotype constellations (genogroups), represented by the FRV strain RVA/Cat-tc/AUS/Cat97/1984/G3P[3] and the human rotavirus (HRV) strain RVA/Human-tc/JPN/AU-1/1982/G3P3[9], respectively. G3P[3] and G3P[9] strains have been detected sporadically in humans. The complete genomes of two CRV strains (RVA/Dog-tc/ITA/RV198-95/1995/G3P[3] and RVA/Dog-tc/ITA/RV52-96/1996/G3P[3]) and an unusual HRV strain (RVA/Human-tc/ITA/PA260-97/1997/G3P[3]) were determined to further elucidate the complex relationships among FRV, CRV and HRV strains. The CRV strains RV198-95 and RV52-96 were shown to possess a Cat97-like genotype constellation. However, 3 and 5 genes of RV198-95 and RV52-96, respectively, were found in distinct subclusters of the same genotypes, suggesting the occurrence of reassortment events among strains belonging to this FRV/CRV/HRV genogroup. Detailed phylogenetic analyses of the HRV strain PA260-97 showed that (i) 8 genome segments (VP3, VP4, VP6, VP7 and NSP2-5) clustered closely with RV198-95 and/or RV52-96; (ii) 2 genome segments (VP1 and VP2) were more closely related to HRV AU-1; and (iii) 1 genome segment (NSP1) was distantly related to any other established NSP1 genotypes and was ratified as a new NSP1 genotype, A15. These findings suggest that the human strain PA260-97 has a history of zoonotic transmission and is likely a reassortant among FRV/CRV strains from the Cat97 and AU-1-like genogroups. In addition, a potential third BA222-05-like genogroup of FRV and HRV strains should be recognized, consisting of rotavirus strains with a stable genetic genotype constellation of genes also partially related to bovine rotavirus (BRV) and bovine-like rotaviruses. The detailed phylogenetic analysis indicated that three major genotype constellations exist among FRV, CRV and feline/canine-like HRV strains, and that reassortment and interspecies transmission events contribute significantly to their wide genetic diversity.

Rare Rotavirus Strains in Children with Severe Diarrhea, Malaysia

Rare Rotavirus Strains in Children with Severe Diarrhea, Malaysia

A feline rotavirus G3P[9] carries traces of multiple reassortment events and resembles rare human G3P[9] rotaviruses

The full-length genome sequence of a feline G3P[9] rotavirus (RV) strain, BA222, identified from the intestinal content of an adult cat, was determined. Strain BA222 possessed a G3-P[9]-I2-R2-C2-M2-A3-N1-T3-E2-H3 genomic constellation, differing substantially from other feline RVs. Phylogenetic analyses of each genome segment revealed common origins with selected animal and zoonotic human RVs, notably with rare multi-reassortant human G3P[9] RVs (Ita/PAI58/96 and Ita/PAH136/96). Altogether, the findings suggest that feline RVs are genetically diverse and that human RVs may occasionally originate either directly or indirectly (via reassortment) from feline RVs.

Molecular characterization of rotaviruses in a Japanese raccoon dog ( Nyctereutes procyonoides) and a masked palm civet ( Paguma larvata) in Japan

Group A rotaviruses infect and cause diarrhea in humans and a wide range of mammals. Previous studies have suggested that some strains can cross the species barrier to infect humans ( Martella et al., 2010). However, there are few reports on infection and characterization of rotaviruses in wild animals. To estimate what types of rotaviruses infect wild animals, we investigated infection of rotaviruses in wild animals living in urban areas in Japan between 2003 and 2008. Of 145 fecal specimens obtained, we detected rotaviruses in one sample from a Japanese raccoon dog ( Nyctereutes procyonoides) (RAC-DG5) and in one sample from a masked palm civet ( Paguma larvata) (MP-CIVET66) by RT-semi-nested PCR. Sequence analyses of the VP4 and VP7 genes of RAC-DG5 and MP-CIVET66 strains revealed that these strains belong to G3bP[9] genotype. Furthermore, phylogenetic analyses showed that RAC-DG5 and MP-CIVET66 strains were closely related to human and feline rotaviruses, suggesting interspecies transmission from humans or cats. To our knowledge, this is the first report on the detection and characterization of rotaviruses in a Japanese raccoon dog and masked palm civet. These findings show that wild animals constitute a potential zoonotic risk of rotaviruses.

Whole genome sequence and phylogenetic analyses reveal human rotavirus G3P[3] strains Ro1845 and HCR3A are examples of direct virion transmission of canine/feline rotaviruses to humans

Rotaviruses, the major causative agents of infantile diarrhea worldwide, are, in general, highly species-specific. Interspecies virus transmission is thought to be one of the important contributors involved in the evolution and diversity of rotaviruses in nature. Human rotavirus (HRV) G3P[3] strains Ro1845 and HCR3A have been reported to be closely related genetically to certain canine and feline rotaviruses (RVs). Whole genome sequence and phylogenetic analyses of each of these 2 HRVs as well as 3 canine RVs (CU-1, K9 and A79-10, each with G3P[3] specificity) and 2 feline RVs (Cat97 with G3P[3] specificity and Cat2 with G3P[9] specificity) revealed that (i) each of 11 genes of the Ro1845 and HCR3A was of canine/feline origin; (ii) canine and feline rotaviruses with G3P[3] specificity bore highly conserved species-specific genomes; and (iii) the Cat2 strain may have evolved via multiple reassortment events involving canine, feline, human and bovine rotaviruses.

Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999.

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (n = 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.

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.