Complete genomic analysis of rabbit rotavirus G3P[22] in China

A rabbit rotavirus Z3171 isolate from diarrheic rabbits was identified and sequenced. The genotype constellation of Z3171 is G3-P[22]-I2-R3-C3-M3-A9-N2-T1-E3-H3, which is different from the constellation observed in previously characterized LRV strains. However, the genome of Z3171 differed substantially from those of the rabbit rotavirus strains N5 and Rab1404 in terms of both gene content and gene sequence. Our study suggests that either a reassortment event occurred between human and rabbit rotavirus strains or there are undetected genotypes circulating in the rabbit population. This is the first report of detection of a G3P[22] RVA strain in rabbits in China.


Introduction
Rotaviruses (RVs) are a major cause of acute gastroenteritis in young children and in a wide variety of animals [1]. They belong to the family Sedoreoviridae and have a genome consisting of 11 segments of dsRNA coding for six structural proteins (VP1-VP4, VP6, and VP7) and six nonstructural proteins (NSP1-NSP6) [2]. RVA strains are classified into G and P genotypes based on their VP7 and VP4 genes, of the 11 segments of a rabbit RV strain isolated from the intestine of a deceased rabbit in China in 2020.

Virus isolation
When a juvenile rabbit in China died of acute enteritis in 2020, a rectal swab was collected for a routine bacterial and enteritis-related virological examination, and a rotavirus was detected using RT-PCR. Briefly, the supernatant of the swab sample was treated with penicillin-streptomycin solution (Solarbio, China) and trypsin, and the rabbit rotavirus RVA/Rabbit/CHN/Z3171/2020/G3P [22] (Z3171) was isolated successfully by tissue culture in MA-104 cells.

RT-PCR and nucleotide sequencing
RV dsRNA was isolated from the viral pellet using a QIAamp Viral RNA Micro Kit, following the manufacturer's protocols (QIAGEN, The Netherlands). Fifty µL of RNase-free water was used to elute the total RNA, which was then frozen at -80°C for later use.
The VP1-VP4, VP6-VP7, and NSP1-NSP5 regions were amplified using previously described primers (Supplementary Table S1) [10][11][12]. A OneStep RT-PCR Kit (QIAGEN) was employed for the RT-PCR assay. The RT-PCR program was as follows: a reverse transcription step at 50°C for 30 min, initial denaturation at 95°C for 5 min, 35 cycles of amplification at 95°C for 20 s, 45°C for 30 s (VP4, VP6, VP7, and NSP2-NSP5), or 46°C for 30 s (VP1-VP3, and NSP1), and 72°C for 3 min, and a final extension step at 74°C for 10 min. Each PCR product was purified following the protocol provided by the manufacturer of the Gel Extraction Kit (Axygen, China) and cloned into the vector pMD18-T (TaKaRa, Dalian, China). Sequencing was carried out commercially in an ABI 3730 DNA Analyzer in triplicate reactions using M13 forward and M13 reverse primers (Sangon, Shanghai, China).
The SeqBuilder module of the DNASTAR software package (Lasergene, USA) was used to assemble the sequences of the genome segments from the individual sequence reads. Open reading frames (ORFs) were identified using the same module, and amino acid sequences were deduced from the nucleotide sequences.

Phylogenetic analysis
The BLAST (Basic Local Alignment Search Tool) program at the National Center for Biotechnology Information (NCBI, National Institutes of Health, Maryland) was used to conduct searches for similarities in nucleotide and protein sequences.
The complete nucleotide sequence data of the 11 segments of strain Z3171 reported in the present study were deposited in the GenBank database under the accession numbers OM416829 to OM416839.
Using nucleotide and deduced amino acid sequences of the various rotavirus gene segments obtained from the Gen-Bank database, phylogenetic and molecular evolutionary studies were performed using MEGA version 7.0 (https:// www.megasoftware.net). The GTR + I + G model was predicted to be the best to describe the substitution patterns for the nucleotide sequences, and phylogenetic trees were constructed by the maximum-likelihood method with 1000 bootstrap replicates.
Sequences of human, pig, bovine, simian, and rabbit RVA strains were used for phylogenetic analysis based on each of the 11 genome segments to examine the evolutionary history of isolate Z3171 and how it is related to 1 3 129 Page 2 of 6 Complete genomic analysis of rabbit rotavirus G3P [22] in China Table 1 Genotypes of all 11 segments of isolate Z3171, which were sequenced in this study, and those of the reference LRV, human, and animal RVA strains. The percent nucleotide sequence identity between Z3171 and other rotavirus strains is also shown.   Shaded cells indicate strains with the same genotype as Z3171.
"-" : no sequence data available for analysis.
Green indicates the same genotype as the isolate. from humans. The four RNA-dependent RNA polymerase motifs in VP1 [20] were found to be conserved among the core proteins, as were the basic region, two leucine zippers (amino acids 536 to 557 and amino acids 665 to 686) and five straight blocks of conserved acidic and basic amino acid residues in VP2 [21]. Important for translation, replication, and packaging of RVs, the nonstructural rotavirus proteins are restricted to infected cells. Z3171 is related to genotypes A9, N2, T1, and E3 based on its NSP1-NSP4 gene sequence similarity to the human strains M2-102 (91.8% identity), Omsk08-442 (95.5%), LL4260 (96.5%), and R3265 (98.1%). Genetic variations were found in the NSP3 and NSP4 genes. T1 has been found in a variety of species, including humans, pigs, and cows [22,23]. The NSP5 segment of strain Z3171 grouped with bovine strain DQ-75 (98.4%) and human stain K8 (94%), indicating that it is related to genotype H3 ( Table 1 and Supplementary Fig. S1). It has been established that DQ-75 shares its ancestry with human RVs. The length of the NSP5 gene of Z3171 is 667 nt. The NSP5 genes of major RVs in the NCBI database range in length from 594 to 667 nucleotides. The NSP5 open reading frame (ORF) encodes 198 amino acids (Supplementary Fig. S2). The NSP5 segments of only four strains (human rabbit-like strain B4106, BE5028, rabbit rotavirus N5, and Rab1404) were 1034-1044 nt in length, and 368-nucleotide repeats were detected, with several nucleotide insertions and deletions in the repeat region. B4106 and BE5028 are considered to be the result of interspecific transmission between humans and rabbits. No repeat sequences were found in isolate Z3171.

Discussion
This study revealed that isolate Z3171 has a higher degree of similarity to strains from humans, monkeys, bats, and cows than to other isolates from rabbits. This suggests that RVAs are able to successfully move to and infect a new host without much difficulty crossing species barriers. Six of the Z3171 genes have the same genotype as strain B4106. All of the genomic segments of B4106 were traced back to rabbits, suggesting that this rotavirus strain was transmitted to humans as a heterologous strain instead of a reassortant virus. Over 10 years later, in Belgium, a young person with gastroenteritis was found to be infected with RV BE5028, which was similar to a strain found in rabbits. B4106 and BE5028 are considered to be the result of interspecific transmission. On the other hand, RCH272 was thought to have resulted from a direct transfer from an other RVA strains. The VP7 gene of Z3171 has the most nucleotide sequence similarity (80%-95.9% identity) to the G3 RV strain and 62.9%-95.9% identity to the corresponding genes from humans, rabbits, cats, bats, chickens, and pigeons. The Belgian human strain B4106 (94.9% identity) was more closely related to Z3171 than the LRVs (Supplementary Fig. S1 and Table 1). Comparing the VP7 antigenic region A of strains Z3171 and B4106, only one amino acid difference was found, at residues 87 (Arg-Ser). Z3171 differs from other strains (B4106, RCH272, N5, Rab1404) in antigenic region B due to the replacement of Asp with Ala at position 145 and Ala with Val at position 147. Two mutations (Val212Thr and Ala213Thr) were found in antigen region C of strain Z3171 and human strains RCH272 and R3265 compared with rabbit strains 160/01, 229/01, and 308/01. In antigenic regions D and E, no substitutions were identified. In antigenic region F, the residue at position 238 of strains Z3171, Rab1404, B4106, RCH272 was Asp, but in strains N5, 160/01, 229/01, and 308/01, it was Asn [12]. Genotype G3 is more likely than G1, G2, and G4 to be involved in interspecies transmission, since it is found in a broad variety of species, including humans. Moreover, in rabbits, G3 is the only VP7 genotype that has been identified [6]. Maximum nucleotide sequence identities of 84-90.6% were found between the VP4 genes of strain Z3171 and the Rab1404, 229/01, 160/01, 308/01, and porcine rotavirus strain A46 (Table 1). Phylogenetic analysis showed that all five of these strains belong to the P [22] genotype group ( Supplementary Fig. S1). Presently, there are three known genotypes of the rabbit VP4 gene: P [22], P [14], and P [11]. The P [22] genotype is the most common and is very dominant [13]. Humans, cattle, and goats [14], but not rabbits [15,16] host P [14] and P [11] viruses [17]. Previous studies have identified the P [22] and P [14] genotypes in rabbit RVs [6][7][8]. Conserved cysteine and proline residues [18] and a putative α2β1 integrin ligand site that may be crucial for entry into the host cell [19] are only some of the general aspects of VP4 that are also conserved in Z3171. The VP6 gene of Z3171 belongs to the I2 genotype and shows the most similarity to the human lapine-like strain B4106 (95.6% identity) and the rabbit strain 30-96 (95.0% identity), both of which were shown by Jelle et al. to have a rabbit origin [9]. (Supplementary Fig. S1). Subgroup I rotaviruses are characterized by the presence of the conserved residues 305-Ala, 310-Asn, 315-Glu, 339-Ser, 342-Met, and 348-Ser in the deduced VP6 amino acid sequences of strains Z3171 and 30/96. Clustering analysis showed that Z3171 was most strongly linked to bat strain E3198 (94.6% identity) and human strain MS2015-1-0001 (94.3% identity) based on their VP1 genes. The VP3 gene of strain Z3171 shares 95.7% nucleotide sequence identity with M3 Rhi hip from bats and 89.73% identity with M3 L621 1 3 129 Page 4 of 6 Complete genomic analysis of rabbit rotavirus G3P [22] in China manuscript.

Data Availability
The datasets generated and/or analyzed in the current study are available in the GenBank database and in the online supplementary materials. unknown host or from numerous reassortments from a range of mammalian species, perhaps because there were few reference lapine-like RV strains available at that time. Long ago, RRV seems to have undergone reassortment [7]. Z3171 shares the most nucleotide sequence similarity with RCH272 and RRV in at least seven of its eleven segments, suggesting that RRV has undergone reassortment across species [24][25][26]. It is unknown whether the epidemic strain has mutated or whether it had already been this type of circulating locally in China for a long time. NSP5 rearranges more frequently than other genes, but the reason is unclear. Based on the above observations, we believe that some segments of Z3171 are closely related to those of human rotavirus strains, indicating either that there has been reassortment between human and rabbit rotavirus strains or that there may be unidentified genotypes circulating in the rabbit population. The segmented nature of the RVA genome has led to the generation of natural genetic recombinants with new or atypical phenotypes, as well as interspecific transmission. It has been suggested that rotaviruses have a common group antigen in animals and humans and may be easily transmitted and be pathogenic in different host species [7]. Gene segments (particularly those producing neutralizing antigens) were regularly discovered in humans during surveillance investigations, suggesting that animals may be the source of the varied genetic material of human RVs [27,28]. The findings of this study not only suggest a role of various animals in the natural development of RVA but also provide evidence that the evolution of human and animal rotaviruses often overlaps, indicating the importance of ongoing monitoring of animal RVAs. Previous studies suggest that RVA can quickly jump between hosts and cause disease [7]. The findings of this study add weight to the argument that various species have played a significant part in the natural development of RVA. This emphasized the need to continue to monitor the RVA of different animals.