1 Possibility of Gene Rearrangement between Porcine Rotavirus H and Porcine Rotavirus C in Nonstructural Protein 3

Rotavirus species H (RVH) has been detected in pigs, humans and bats. Moreover, porcine RVHs have been recently identified in several swine-producing countries. Despite their zoonotic impact, genome information of RVHs is still limited. This study aimed to establish a tentative complete genome-based genotyping system for RVHs, by appending genomic sequences from 12 porcine RVHs identified in Japan between 2013 and 2015 to those from human and other porcine RVHs reported in previous studies. Phylogenetic analysis of 11 RNA segments indicated that porcine RVHs could be classified into multiple genotypes. Consequently, the genotype classification for RVHs revealed the existence of genotypes 10G, 6P, 6I, 3R, 4C, 7M, 6A, 2N, 4T, 6E, 3H for the genes VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Surprisingly, two distinctive types in NSP1 and NSP3 genes were identified from among the twelve porcine RVHs. Our data suggest a potentially novel gene rearrangement event between porcine RVH and rotavirus species C in the NSP3 gene. These findings would provide a new insight in understanding for evolution of RV.


Introduction
Rotaviruses (RVs), member of the family Reoviridae, are major causative agents of gastroenteritis in humans and animals worldwide [1].RVs are currently classified into nine species, rotavirus species A to I (RVA-RVI), based on sequence diversity of the inner capsid protein, VP6 [2, 3].The genome comprises 11 segments of double-stranded RNA (dsRNA), which encode six structural proteins (VP1-VP4, VP6, and VP7), and five non-structural proteins (NSP1-NSP5).The structural proteins form infectious triplelayered particles surrounding the dsRNA.NSPs are primarily involved in dsRNA replication and transcription, cellular pathogenesis, and maturation of virions [4].
RVH was first isolated from fecal samples of pigs less than 30 days of age from Japan during 1991-1995 as a causative agent of diarrhea; this porcine RVH strain was designated as SKA-1 [5].Subsequently, human RVHs have were detected in cases of epidemic and sporadic diarrhea in adults in China and Bangladesh, in 1997 and 2002, respectively [6][7][8].During 2007 to 2012, porcine RVH infections have been reported in many countries, including the United States, Brazil, South Africa, and Vietnam [9][10][11][12].
Currently, RVH has been also identified in bats in Cameroon [13].However, in Japan, the prevalence and epidemiology of porcine RVHs are unclear since the discovery of the SKA-1.
Recently, we identified twelve porcine RVH strains from fecal samples of pigs of various ages, in six farms in Nagasaki prefecture, Japan, between 2013 and 2015, most of which co-infected with other porcine rotavirus species, porcine rotaviruses C (RVCs).
In this study, we attempted to establish a provisional full genome-based classification system for RVHs by appending further complete genomic sequences from the twelve by the Rotavirus Classification Working Group.Simultaneously, we performed detailed analysis for unique genetic characteristics found among the NSP3 gene from the twelve porcine RVHs.The data presented in this study would facilitate the molecular epidemiology and evolutionary study of RVHs.

Samples
Twelve porcine RVH strains was obtained from fecal samples of pigs from six farms in Nagasaki prefecture, Japan, from 2013 to 2015 (Table 1).Viral RNA was extracted from a 10% fecal suspension in minimum essential medium, using a QIAamp viral RNA mini kit (Qiagen, Venlo, Limburg, Netherlands), in accordance with the manufacturer's instructions.Genomic sequences of individual RNA segments were amplified via reverse transcription polymerase chain reaction (RT-PCR), using a set of primers (Table S1) designed in reference to terminal sequences in the 5'-and 3'-ends of all 11 segments from MRC-DPRU1575 (GenBank accession nos.KT962027-

Comparative sequence analysis
The amplification by RT-PCR using a set of primers designed in reference to complete genome of porcine RVH strain, MRC-DPRU1575 detected in South Africa succeeded to determine the nearly full-length nucleotide sequences of all genes from the twelve porcine RVH strains (Table 2).The NSP1 open reading frame (ORF) nucleotide sequences from four porcine RVH strains (NGS-3, NGS-5, NGS-6, and NGS-7) were 1203 nt in length, which different from those (1206 nt) from the eight remaining strains, the same as the reference MRC-DPRU1575 strain (Table 1).The ORF nucleotide sequences of the NSP3 products from seven porcine RVH strains (NGS-8, NGS-9, NGS-10, NGS-12, NGS-14, NGS-17 and NGS-18) were 801 nt in length, identical to that of the reference strain; however, the sequences of three RVH strains (NGS-3, NGS-5, and NGS-7) were 1218 nt in length.Beside the two genes, the products from the remaining genes were similar to those of the reference porcine RVH strain.These facts show that each terminal sequence in the 5'-and 3'-ends was also highly conserved among RVHs as well as other RVs, and all products were originated from porcine RVHs [19].
Aside from NSP2 gene, the remaining genes displayed highly variable identity among the twelve porcine RVHs, higher than that observed among human RVHs.
Furthermore, comparisons of ORFs of all genes between human and porcine RVHs, including the twelve porcine RVHs analyzed herein, revealed that porcine RVHs exhibit high diversity and are clearly distinct from human RVHs.

Phylogenetic analysis
All 11 genes of the twelve porcine RVH strains analyzed herein, the classical porcine RVH strain from Japan, numerous porcine RVH strains from the United States, Brazil, South Africa, and Vietnam, and 2-3 human RVH strains were differentiated (Fig. 1 and Table 3).Cut-off values for differentiating among VP4 and VP7 genotypes were calculated from the frequency distribution of pairwise sequence identities and were set to 86% and 86% at the nucleotide level, respectively (Table S2).Based on these cut-off values, we observed six (P1-P6) and ten (G1-G10) genotypes for VP4 and VP7 genes, respectively (Fig. 1).The genotype classification for the VP6 gene, based on a cut-off value of 87%, revealed the presence of six genotypes (I1-I6).Analysis of VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4, and NSP5 genes, based on cut-off values of 85%, 87%, 86%, 84%, 67%, 87%, 83%, and 89% revealed the presence of three (R1-R3), four (C1-C4), seven (M1-M7), six (A1-A6), two (N1-N2), four (T1-T4), six (E1-E6), and three (H1-H3) genotypes, respectively (Table 3).Thus, for the ten remaining genes other than NSP2 gene analyzed herein, porcine RVHs were classified into multiple genotypes, in contrast to human RVHs being grouped into one genotype.Moreover, our analysis indicates that porcine RVHs from Japan might belong to multiple genotypes; however, porcine RVHs from the United States, Brazil, South Africa, and Vietnam might be classified into 1-2 different genotypes in accordance with the country of collection.between porcine RVH and porcine RVC.Furthermore, we establish a tentative complete genome-based genotyping system for RVHs from pigs and humans.In future, a better complete genome-based genotyping system for RVHs, the same as RVAs, would be constructed, by adding complete genomic sequence data from new host species to these sequence data.The finding presented in this study would further proceed the current understanding of the evolution of RV.
Supplementary Materials: Funding: The authors received no specific grant from any funding agency.
Japanese porcine RVHs to classical porcine RVH, multiple porcine RVHs from other countries, and human RVHs available in GenBank, and by calculating the cut-off values for genotype classification on each RNA segment based on the definition recommended Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 9 August 2018 doi:10.20944/preprints201808.0187.v1

Figure 1 .
Figure 1.Phylogenetic tree constructed from the open reading frames of individual genes

Figure 2 .
Figure 2. Multiple-sequence alignment of the full-length nucleotide sequences of the

Figure 3 .
Figure 3. Phylogenetic tree constructed from entire (A) and partial (B) NSP3 nucleotide

Figure 4 .
Figure 4. Schema of gene rearrangement between porcine RVH and porcine RVC in

Figure S1 :
Multiple alignment between RVH NSP3nucleotide sequence (positions 820-1240) from three strains grouped into long-type and RVC NSP3 nucleotide sequences (positions 550-970) from two strains grouped into T6 genotype; TableS1: A set of specific primers used for reverse transcription amplification of complete genomes from twelve porcine rotaviruses H strains; TableS2: Identities of individual genes at the nucleotide level among RVHs based on genotype classification as shown in Fig. 1 Author contributions: Conceptualization, S.T.; Methodology, S.T.; Validation, S.T.; Investigation, I.D.; Resources, I.D.; Data Curation, I.D.; Writing -Original Draft Preparation, S.T.; Writing -Review & Editing, S.T.All authors read and approved the manuscript.

Table 1 .
Origin of twelve porcine rotavirus H strains detected in Nagasaki prefecture, Japan from 2013 to 2015.Table2).The sequence data were aligned using the ClustalW method in the MEGA6 software[15].Genetic distances were calculated using the Kimura two-parameter correction at the nucleotide level[16].Phylogenetic analyses KT962037)[11].RVC NSP3 gene was also amplified via RT-PCR with specific primers reported in our previous study[14].RT-PCR was carried out using a PrimeScript II High Fidelity One Step RT-PCR Kit (Takara Bio, Inc., Shiga, Japan) with the following cycling were conducted using the maximum-likelihood method with the general time reversible nucleotide substitution model and 1,000 bootstrap replicates.Genotype classification of individual RVH genes was conducted using cut-off values calculated per the definition recommended by the Rotavirus Classification Working Group[16, 17].In addition, genotype classification of RVC NSP3 gene was performed in accordance with our previous report[18].

Table 2 .
Full-length (open reading frame) and GenBank accession number of individual genes from the twelve porcine rotavirus H strains.

Table 3 .
Genotypes for individual genes of representative rotavirus H strains used in this study.
*Each genotype shown in Fig.1.ND: not determined.