Human transmission and outbreaks of feline‐like G6 rotavirus revealed with whole‐genome analysis of G6P[9] feline rotavirus

Group A rotaviruses (RVAs) are generally highly species‐specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole‐genome analysis of these G6P[9] strains identified the genotype constellation G6‐P[9]‐I2‐R2‐C2‐M2‐A3‐N2‐T3‐E3‐H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human‐wt/JPN/KF17/2010/G6P[9], previously detected in a 3‐year‐old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline‐origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline‐like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

Group A rotaviruses (RVAs) are significant etiological agents of severe diarrhea in both young children and animals worldwide.RVA belongs to the family Reoviridae, and its genome consists of 11 double-stranded RNA segments, which encode six structural proteins (VP1-4, VP6, and VP7) and six nonstructural proteins (NSP1-6). 1 Although RVAs are generally highly species-specific, some strains infect across species. 2,3Animal RVAs serve as a potential reservoir for genetic exchange with human RVAs and can infect humans either by direct virion transmission or by contributing genes to reassortants.
One genotype constellation has been identified as G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 (feline/human-like, FRV-1/AU-1-like), which is occasionally reported in humans, as mentioned above.The other genotype constellation is G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6 (feline/caninelike, Cat97/CU-1-like), which has also been detected in humans and dogs. 14In the United Kingdom, multiple RVA strains with the G6P [9]   genotype have also been reported in shelter cats, although no genomic sequence data for these strains are yet available. 19 2012 and 2013, rectal swab samples were collected from 61 asymptomatic cats housed in Japanese public health facilities to test for RVAs.Three G6P [9] RVA strains and two G3P [9] RVA strains were identified.In this study, whole-genome analysis of these five strains was used to assess the relationships between these feline strains, human RVA strains, and those of other animals from Japan and throughout the world.

| Sample collection
Rectal swab samples were obtained from 61 shelter cats at the Mie Prefecture Health Center during the periods of June-August 2012 and June-August 2013 (Figure 1).At the time of sample collection,

Mie Prefecture
Cat/Mie20120003f/G3P [9]  Cat/Mie20120016f/G3P [9]  Cat/Mie20120017f/G6P [9]  Cat/Mie20120022f/G6P [9]  Cat/Mie20120049f/G6P [9]   Tochigi Prefecture Human/KF17/G6P [9]   100km F I G U R E 1 Locations of Mie Prefecture and Tochigi Prefecture, Japan Mie Prefecture, where the current investigation was conducted, is highlighted in red, and Tochigi Prefecture, where the Human/KF17/G6P [9] strain was first detected, is shaded in blue.none of the cats exhibited symptoms of gastroenteritis.The swab samples were collected and thoroughly agitated in 1 mL of Minimum Essential Medium (Thermo Fisher Scientific).The supernatant obtained after centrifugation was used as the test specimen and stored at −80°C until further analysis.The test specimens were analyzed with direct sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products.Any remaining samples were subsequently analyzed using next-generation sequencing (NGS).

| RNA extraction
Viral RNA was extracted from 200 μL of each specimen and eluted in 50 μL of elution buffer from the High Pure Viral RNA Kit (Roche Molecular Diagnostics), according to the manufacturer's instructions.

| RT-PCR and GP genotyping
Reverse transcription was performed with SuperScript™ III Reverse Transcriptase (Invitrogen) at 50°C for 60 min, followed by 15 min at 94°C.PCR (25 μL reaction volume) was performed with PrimeSTAR ® GXL DNA Polymerase (Takara Bio), with the following thermal cycling conditions: 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min.The PCR products were analyzed with electrophoresis on a 1% agarose gel stained with ethidium bromide.1][22] The G and P genotypes were determined by the size of the PCR product when the conventional G and P primers were used. 23,24

| Nucleotide sequencing
The PCR products were purified with the Wizard ® SV Gel and PCR Clean-Up System (Promega) and sequenced with the BigDye™ Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) on a SeqStudio Genetic Analyzer (Applied Biosystems), according to the manufacturers' instructions.The sequencing files were analyzed with SnapGene v.5.2.5 (GSL Biotech).

| NGS
6][27] Briefly, a 200-bp fragment library was constructed for each sample with the NEBNext Ultra II RNA Library Prep Kit for Illumina v4.0 (New England Biolabs), according to the manufacturer's instructions.Each library was purified with Sera-Mag™ Select magnetic beads (Cytiva).The DNA concentrations were determined with a Qubit™ 4 fluorometer and the Qubit™ dsDNA HS Assay Kit (Invitrogen).A 151-cycle pairedend-read sequencing run was conducted on an iSeq.100 desktop sequencer (Illumina) with the iSeq.100 Reagent Kit v2 (300 cycles).
The sequence data were analyzed with the CLC Genomics Workbench v8.0.2 (CLC Bio), and the sequences of the strains were deposited in the GenBank/EMBL/DDBJ databases under accession numbers LC790331-LC790380).

| Genotyping and phylogenetic analysis
The genotype of each of the 11 genome segments was determined with the Rotavirus A Genotype Determination online classification tool (https://www.rivm.nl/mpf/typingtool/rotavirusa/).All 11 segments of each complete genome were separately aligned with MUSCLE. 28Phylogenetic trees were constructed with the maximum likelihood (ML) method and 1000 bootstrap replicates with the MEGA 11 software (https://www.megasoftware.net).Representative reference sequences were selected for comparison from GenBank, available on the National Center for Biotechnology Information website (https://www.ncbi.nlm.nih.gov/nucleotide/).Nucleotide and amino acid identities were calculated using Clustal W online alignment tool (https://www.genome.jp/tools-bin/clustalw).
The VP3 (M2) and NSP1 (A3) trees showed distinct feline-like and bovine-like lineages, with all feline strains belonging to the former and all bovine strains to the latter (Figure 3C,D).On the VP3 Phylogenetic trees of the VP7 (G6), VP4 (P [9]), VP6 (I2), and NSP4 (E3) genes of the study strains and representative RVA strains (Panels A−D) show the phylogenies of the VP7 (G6), VP4 (P [9]), VP6 (I2), and NSP4 (E3) genes, respectively.The phylogenetic trees were constructed with the maximum likelihood method with 1000 bootstrap replicates, using the MEGA 11 software package.Bootstrap values of ≥70% are indicated at each node.The genetic distance (nucleotide substitutions per site) is indicated at the bottom of the trees.Strain Mie20120017f is represented by a black filled square, the other feline study strains by a black square, the KF17 strain by a gray filled square, the FRV-1/AU-1-like and Cat97/CU-1like feline strains by red filled squares, other feline strains by red filled triangles, bovine strains by green filled circles, the bovine-origin feline strain FRV537 by a red filled circle, the AU-1 strain by a magenta filled rhombus, and the RotaTeq strain by an orange filled triangle.Nucleotide identities between strain Mie20120017f and other strains are shown in parentheses.

| DISCUSSION
In this study, the complete genome sequence of one representative G6P [9] feline RVA strain from among a set of three G6P [9] and two G3P [9] strains collected from asymptomatic shelter cats was determined.The G3P [9] strains, Mie20120003f and Mie20120016f, both showed typical FRV-1/AU-1-like genotype constellations.
Notably, previously detected feline RVA strains with typical FRV-1/ AU-1-like genotypes were isolated from cats in Japan in 1986 and 1994 (FRV-1, FRV317, and FRV384), and they continued to circulate in Japanese cats in the 2010s. 34,35is is the first report of a whole-genome analysis of feline G6P the prevalence of multiple G6P [9] RVA strains in shelter cats in the United Kingdom in 2012. 19However, the genetic sequences of these G6P [9] strains were not registered, raising questions about their similarity to strain Mie20120017f described in the present study.
Furthermore, G6P [9] strains detected in humans, such as those detected in Hungary in 1998 (e.g., Hun7 and Hun8), share high nucleotide identity (>98%) with strain Mie20120017f in their VP7 and VP4 genes. 36These data suggest that G6P [9] strains similar to Mie20120017f may have been circulating among European cats since the 1990s.
Interestingly, this study shows that Mie20120017f shares high nucleotide identity (>98.1%)across all 11 gene segments with strain KF17/G6P [9], which was detected in a 3-year-old girl with acute gastroenteritis in Tochigi Prefecture, Japan, in 2010. 29Similarly, strain PG05/G6P [9], detected in a 1-year-old in Italy in 2011, and strain GER29-14/G6P [9], detected in an 8-year-old with acute gastroenteritis in Germany in 2014, share high nucleotide identity with Mie20120017f in multiple genetic segments. 30,31Despite the lack of any apparent contact with animals in the medical histories of these children, [29][30][31] these findings strongly suggest that they were infected with feline RVA.Previous studies have suggested that the direct transmission of RVA from cats to humans may involve contact between them.[57/1,727]), 13,19,37 indicating that humans may unknowingly contract feline RVA from stray cats in their environments.
Intriguingly, a recent study reported that in 2018-2019, G6P [8]   strains were identified in Brazil, and whole-genome analyses revealed that these strains had a DS-1 backbone. 45However, an analysis of stool samples from patients in Brazil with RVA gastroenteritis between 2020 and 2022 showed a significant shift in prevalence.
In 2020, equine-like G3P [8] strains dominated at 11.3% (13/115), but by 2022, G6P [8] accounted for 83.5% (96/115) of strains. 337][48] This suggests that the RVA strains currently circulating in Brazil are characterized by an equinelike G3 lineage backbone, with only the VP7 gene undergoing monoreassortment with a feline-like G6.A survey conducted among pet, sheltered, and stray cats in Brazil during 2018-2019 detected RVA in 12.6% (13/103) of cases, 49 indicating an environment conducive to reassortment between feline RVA and human RVA.Like the global spread of the equine-like G3 strain in humans after its first detection in Australia in 2013, 43,[50][51][52] vigilance is required to monitor the potential future spread of feline-like G6 strains among humans.
The VP4 protein plays a crucial role in virus attachment to host cells, with different VP4 genotypes identified among host species. 53line RVA strains were classified as P [9] and P [3] strains until the 1990s, but since 2000, P [9] strains have predominated (Table 1).
Consistently, all five feline RVA strains identified in this study were of the P [9] genotype.Phylogenetic analysis revealed two genetically distinct lineages of VP4 P [9] circulating within feline RVAs, showing approximately 93-94% nucleotide identity between lineages.Notably, human P [9] strains are present in both lineages, albeit sporadically reported. 10,29,54Previous research suggests that the VP8* protein of P [9] RV binds strongly to type A histo-blood group antigens (HBGAs) compared to the VP8* protein of typical human P [8] RV. 53 This observation implies that the presence of type A HBGA may facilitate P [9] RVA infection, supported by reports of P [9] RVA detection in children with blood type A. 10,30 However, documented cases of human-to-human transmission of P [9] RVA strains are lacking, warranting further investigation into human infections with P [9] RVAs.

| CONCLUSION
A whole-genome analysis of RVA strain Mie20120017f revealed a novel genotype constellation among cats, G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3, making it the third feline RVA strain in circulation, after the FRV-1/AU-1-like and Cat97/CU-1-like strains.This study also revealed that G6 RVA strains, which have been sporadically reported in humans worldwide, originate from feline sources.The investigation of RVAs in animals not only extends our understanding of these infections in animals but also clarifies the previously unidentified origins of RVA infections in humans.These findings indicate that a feline-like G6P [8] human strain was circulating in Brazil in 2022, underscoring the need for ongoing surveillance to monitor potential global outbreaks.

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I G U R E 3 (Continued).