Introduction

Rotavirus is one of the most common viruses causing gastroenteritis worldwide [1] and primarily transmitted through the fecal-oral route, usually from direct contact between people. Rotavirus infection primarily causes diarrhea and vomiting symptoms by damaging intestinal epithelial cells and activating the enteric nervous system through NSP4 enterotoxin, but there are no specific anti-rotavirus drugs to alleviate the course and severity of the diarrhea [2]. It has estimated that rotavirus infection contributes to more than a hundred thousand deaths among children under 5 years of age worldwide in 2016 [3] and to significant losses to livestock production [4]. In China, there are reports of rotavirus positive cases in Henan, Hunan, Fujian and other regions [2]. Rotavirus infection accounted for 19.1% of the monitoring diarrhea cases in China from 2009 to 2020 [5]. The detection rate of rotavirus in medical facilities in China, whether in outpatient or inpatient settings, surpasses that of other Asian countries [6] and raises the substantial disease burden [7].

Rotaviruses are double-stranded RNA viruses, containing 11 segments of double-stranded RNA, and belong to the family Sedoreoviridae [8]. Rotavirus had 6 viral structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and 6 non-structural proteins (NSP1-NSP6) [9]. Until now, according to the antigenicity and sequence diversity of VP6, rotavirus is divided into 12 different species (RVA-RVL) [10]. Group A-C rotaviruses are associated with acute gastroenteritis in humans. Group A rotavirus is the main cause of gastroenteritis in infants and its pathogenicity and detection rate rank first worldwide [11]. Group B rotavirus is known to cause severe diarrhea, mainly in adults [12]. Rotavirus C (RVC) was first identified in piglets with diarrhea in 1980 [13], followed by identification in humans in 1982 [14]. Subsequently, RVC has been found to infect all age groups and considered to be an important cause of diarrhea [15, 16]. Currently, RVC has been documented in numerous instances of both sporadic cases and fulminant gastroenteritis worldwide, including regions such as Argentina, Italy, China, Japan, India and South Korea [17,18,19,20,21,22].

Rotavirus serotypes are specified by a dual serotyping system, serotypes G (glycoprotein) and P (protease-sensitive) [23]. P serotype specificity designates the spike protein (VP4) and G serotype specificity refers to the outer layer protein (VP7). VP4 and VP7 elicit serotype-specific and serotype-cross-reactive neutralizing antibody responses [24, 25]. VP7 has the ability to constitute the outer surface of viral particles. VP4 can bind to molecules on the cell surface, promoting virus entry into the cell and determining virus toxicity [26]. So far, 42 G-genes and 58 P-genes have been described globally [27]. The most common G forms in humans are G1-G4 and G9, and the P forms are P[4], P[6] and P[8] [28]. The G-P combinations G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] accounted more than 80% of the global circulating genotype [29]. G9P [8] is the common genotype in different age groups, and G9P[8] is more common in children than in adults [30].

RVC strains have been detected in both humans and animals [31,32,33,34]. However, the genetic characteristics of RVC are limited and only a few published reports contain genetic and molecular epidemiological data for RVC strains. In this study, we report the genetic characteristics of RVC isolated from an acute diarrhea outbreak in China in 2015. The whole genome of the RCV isolate was obtained, and the phylogenetic and comparative analyses were performed to investigate the possible evolutionary origin.

Materials and methods

Sample collection and processing

An outbreak of gastroenteritis occurred at a school located in Hebei Province of China. Twenty-two patients were admitted and samples from patients were collected, including anal swabs, blood, stool and nasal swabs. Samples of anal and stool swabs were divided into two parts; one was used for bacteria culture and the left were for pathogen detection.

Pathogen screen and identification

Genomic DNA and RNA were extracted using the TIANamp virus DNA/RNA Kit (TIANGEN, DP315, China) according to the manufacturer’s instructions. Multiplex RT-PCR was performed using Seeplex® RV12 ACE and Seeplex® PneumoBacter assay kits (Seegene, Seoul, South Korea) [35]. All samples were screened for suspected pathogens of Adenovirus, Rotavirus, Norovirus, Sapovirus, Astrovirus, Shigella and Salmonella. Rotavirus-positive samples were further typed by PCR and sanger sequencing with primers specific for the VP7 and VP4 genes (Supplementary Table S1). PCR products were sent for sanger sequencing and BLAST was performed against NCBI database for genotyping.

Ion Torrent PGM metagenome sequencing

The RNA of five positive samples were reverse transcribed into DNA. The libraries were prepared using the Ion Xpress™ Plus Fragment Library Kit (Life Technologies, NY, USA) and each library was barcoded using the Ion Xpress™ Barcode Adapters 1–16 Kit. SPRI beads-based size selection was performed according to the published New England Bioscience (NEB) E6270 protocol. The prepared libraries were sequenced on the Ion Torrent PGM using the Ion Torrent PGM Sequencing 200 Kit and Ion 318™ Chip.

Bioinformatic analysis

De novo assembly was first performed using Newbler (version 2.8). An 11-segment rotavirus from NCBI (NC 007569; The NC 007574; The NC 007547; The NC 007572; The NC 007570; The NC 007543; The NC 007544; The NC 007545; The NC 007546; The NC 007573; NC 007571) were used as a reference genome and raw reads were remapped to check the assembly. Multiple alignments were performed for each fragment and the concatenated sequence using Clustalw2. Segments were concatenated and aligned to construct a Maximum-Likelihood (ML) phylogenetic tree using RAxML (v8.2.4) with a general time reversible (GTR) model and a gamma distribution based on 1000 bootstraps. The recombination breakpoint was determined by SimPlot software v.3.5.1 and Recombination Detection Program v.4.39 [36]. The regions of recombination loci were extracted for phylogenetic tree construction. All trees were visualized using iTOL v6 [37].

Results

Sample collection and pathogen identification

A total of twenty-two patients were reported, thirteen of whom were hospitalized and nine were quarantined. Anal swabs (22), blood (22), stool (9) and nasal swabs (22) of each patient, except for inpatients who had no stools, were screened for possible pathogens. RT-PCR results showed that four anal swab samples of hospitalized patients and two stool samples of quarantined patients were positive for rotavirus. It was further identified as RVC by PCR and sanger sequencing. All other samples were negative for Adenovirus, Rotavirus, Norovirus, Sapovirus, Astrovirus, Shigella and Salmonella.

Metagenome sequencing and phylogenetic analysis

Three anal swab samples were too low in concentration for sequencing, and the left three rotavirus-positive samples were further sequenced on PGM. Samples G9 and G10 produced a total of 877,120 and 735,492 reads, respectively. The coverage of rotavirus in these samples was 77.12% and 32.02%, respectively (Supplementary Table S2). The 11 segments of G9 had a coverage of 57.8–99.8% with mean depths of 2× ~ 7×, while G10 had a coverage up to 54.8% with low average depths. SJ217 obtained 978,144 reads, 4,188 reads of which matched the reference genome. The complete genome of SJZ217 was successfully assembled with a mean depth ranging 13× ~ 71×. The segments (VP1-4, VP6-7 and NSP1-5) of SJ217 have been deposited in GenBank under accession number KY865344-KY865454 (Supplementary Table S3).

Phylogenetic analysis

To determine the genetic relationship between SJZ217 and RVC from around the world, phylogenetic trees were constructed for 11 genome segments and the concatenated sequences. The phylogenetic tree of concatenated sequences showed that there exist three major clusters. Sequences of non-human origin form one cluster, with branches labeled in green. The branches marked in blue encompass strains from European and Asian regions. Meanwhile, the strains labeled in red mostly originate from Asia, predominantly including South Korea and Japan. Strain SJZ217 (marked in red) was clustered with human origin sequences in a clade rather than animal origin. And SJZ217 belonged to the G4P[2] genotype and clustered with strain Chungnam [38] isolated from Korea in 2014 (Fig. 1).

Fig. 1
figure 1

The phylogenetic tree of concatenated sequences showed that SJZ217 clustered with strain Chungnam isolated from Korea in 2014. The detectable human RVC sequences and seven non-human origin RVC sequences in the NCBI database are included in the phylogenetic analysis. The SJZ217 strain is represented by red color and non-human origin RVC clades are colored in green

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Genomic comparison revealed SJZ217 had almost the same genome sequence with strain Chungnam but 21 SNPs. However, the segments of SJZ217 presented different branch structures from the concatenated sequence. All 11 fragments were clustered within human origin sequences. Segments VP4, NSP1 and NSP2 of SJZ217 were closely related to strain Chungnam, while VP1, VP2 and VP3 clustered with strain Chungnam and CAU14-1-242 from Korean. The VP7 gene is closely related to strain CAU14-1-242, CAU14-1-246 and Chungnam from Korean. The segments NSP3, NSP4 and NSP5 had the highest homology with HO-65 [39] isolated from Hokkaido, Japan, while the VP6 gene fell into the clade with strains ERN6210, ERN6216 and ERN6233 [40] from Hungarian (Supplementary Figure S1). These 11 segments all had > 99% homology to these above closely related strains (Supplementary Table S4).

Recombination analysis

In general, the 11 fragments of SJZ217 were clustered in Asian branches such as China, Japan and South Korea. And the VP1, VP2, VP3, VP4, VP6, NSP1, NSP2, NSP3 and NSP5 genes of SJZ217 were also mixed with European Hungarian strains in Asian branches such as China, Japan and South Korea, while the NSP4 gene has been found to be mixed with strains of Italy and Brazil in the Asian branches.

The recombination analysis showed that the recombinant locus was at 1082 ~ 3322 bp, which was on the VP4 gene, and the parental strains were OS-270 of Japan and CAU13-177 of Korean, respectively (Fig. 2a). Inconsistent genetic evolutionary relationships were observed at the recombination breakpoints (Fig. 2b), which further confirming the occurrence of gene recombination in the VP4 gene of SJZ217 strain.

Fig. 2
figure 2

Detection of possible recombination events in the SJ217 strain. (a) The recombination detection software SimPlot software v.3.5.1 and Recombination Detection Program v.4.39 was used to recognize recombination events in the CAU13-1-77 (blue) and OS-270 (red) genomes. The Y-axis represents the pairwise identity between the recombinant and its putative parents. The X-axis represents the position in alignment with a 30-nt sliding window. The comparison of recombinant-major parent and recombinant-minor parent was indicated as blue and red, respectively. (b) Phylogenetic analysis of the recombination loci in VP4. The SJ217 strain is highlighted in red and non-human origin RVCs are colored in blue

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Discussion

Rotavirus is an important pathogen causing severe diarrhea in humans and animals. In this study, the whole genome sequence of SJZ217 isolated from an outbreak of acute diarrhea in China was analyzed and characterized. By comparing human and animal RVC sequences available in the limited NCBI database, it was found that all fragments in this study were of human origin and no relevant molecular and epidemiological evidence of zoonotic transmission of RVC strains was observed (Supplementary Figure S1). However, G and P types of some human RVC can be found in animals, indicating that these genotypes may originate from animal strains through direct interspecific transmission or through reclassification of homologous genes between heterologous and homologous strains [41]. Both virological and serological investigations suggest that animal RVC may have zoonotic potential [42, 43]. RVC interspecific transmission and its zoonotic potential have been reported [19]. And gene transfer was observed between porcine RVC and human RVC [44]. Therefore, the increasing detection frequency of human RVC strains, the possibility of recombination of zoonotic human RVC strains, and the lack of adequate molecular studies of RVC strains worldwide require further research on human RVC strains that may have important genetic characteristics.

Phylogenetic analysis indicated that strain SJZ217 showed the highest similarity with strain Chungnam and 10 genes were closely related to strains of Korean-Japan, except for the VP6 gene, which shared the highest homology with strain ERN6210, ERN6216 and ERN6233 from Hungarian. These results suggest that dissemination of strain SJZ217 may have a global scope. The previous study indicated that the transmission and intraspecific recombination of RVC may occur [45]. Researches have suggested that RVCs may have the potential for interspecies transmission between humans and animals in Brazil and Japan [46,47,48]. The recombination analysis proves that the VP4 gene of strain SJZ217 may be recombined from RVC strains from Japanese and Korean, indicating a possible interspecies transmission. The phylogenetic analysis suggested that genetic information of SJZ217 is unrelated to non-human origin sequences. Further epidemiological and molecular biological analyses are required to provide additional support for this conclusion.

Conclusion

In this study, we performed genome sequencing to investigate a RVC outbreak of acute gastroenteritis in China. Phylogenetic analysis showed that the outbreak strain SJZ217 belongs to the G4P[2] genotype and shares almost identical genomic sequences with Chungnam isolated in Korea and reassortment in the VP4 fragment was observed. Our results helped to understand the genetic diversity and possible spread of RVC strains. These results suggest the importance of focusing on the work related to entry-exit inspection and quarantine. And enhancing epidemiological and genetic surveillance of rotavirus is beneficial in providing more advantageous information for the development of specific drugs and vaccines against.