Extended analyses of rotavirus C (RVC) G-types and P-types reveal new cut-off value for the G-types and reclassification of strains

Abstract

Rotavirus C (RVC) is an important cause of gastroenteritis in humans and pigs and has also been detected in cattle, ferrets, minks, and dogs. Incidental zoonotic transmissions have been described. In contrast to rotavirus A (RVA), a complete genotyping system for RVC has not yet been established due to limited or incomplete sequence data. In this study, 138 complete nucleotide sequences for VP7 (G-type) and 97 complete nucleotide sequences for VP4 (P-type) of porcine RVC-positive samples have successfully been generated and genotyped. Together with available sequences from the NCBI database, phylogenetic analyses were conducted, cut-off values were re-evaluated, and the current classification system was adapted. Pairwise identity frequency analyses revealed a new cut-off value of 82% instead of the previous 85% for the G-type and confirmed the current cut-off value of 85% for the P-type. This resulted in the identification of 21 G-types and 39 P-types, including 4 new G-types and 10 new P-types. The results of the investigations expand the existing knowledge about the genetics of RVC and demonstrate the enormous diversity of porcine RVC sequences in particular.IMPORTANCEThis article provides a new sequence data set of porcine rotavirus C (RVC) strains. The extended full-length analysis of RVC G-types and P-types enabled us to review the current classification system. According to the guidelines of the rotavirus classification working group (RCWG), the results led to a new cut-off value of RVC G-types and required the reclassification of numerous RVC G-types. In addition, several new genotypes have been found. The present work closes the aforementioned knowledge gap and provides important, comprehensive data for RVC genetic diversity.

Keywords: genetic diversity; genotyping; rotavirus; rotavirus group C; sequence analysis.

Fig 1

Phylogenetic analyses and pairwise identity frequency distribution graph for the G-type (VP7). (A) Phylogenetic tree based on the ORF nucleotide sequences of the VP7 gene (G-type) of RVC strains. The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. Genotypes are represented by compressed clusters and designated on the right; the accession number of the respective reference strain is shown in brackets. The determined cut-off value of 82% is shown by the vertical line. (B) Nucleotide pairwise identity frequency graph of 295 complete VP7 ORF sequences. The proposed 82% nucleotide cut-off value is shown by the vertical line. (C) Phylogenetic tree based on the ORF nucleotide sequences of the VP7 gene (G-type) of RVC strains. The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. The visualization of the tree was conducted using iTOL (58). Symbols (filled dots) indicate the sequences (n = 138) obtained in this study. Genotypes are specified with colored branches and associated designation outside of the tree.

Fig 2

Phylogenetic analyses and pairwise identity frequency distribution graph for the P-type (VP4). (A) Phylogenetic tree based on the ORF nucleotide sequences of the VP4 gene (P-type) of RVC strains. The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. Genotypes are represented by compressed clusters and designated on the right; the accession number of the respective reference strain is shown in brackets. The current cut-off value of 85% is shown by the vertical line. (B) Nucleotide pairwise identity frequency graph of 165 complete VP4 ORF sequences. The current 85% nucleotide cut-off value is shown by the vertical line. (C) Phylogenetic tree based on the ORF nucleotide sequences of the VP4 gene (P-type) of RVC strains. The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. The visualization of the tree was conducted using iTOL (58). Symbols (filled dots) indicate the sequences (n = 97) obtained in this study. Genotypes are specified with colored branches and associated designation outside of the tree.

Fig 3

Subtree of cluster G1 displayed separately from the detailed phylogenetic tree of VP7 (Fig. S1). The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. Symbols (filled dots) indicate the sequences obtained in this study. Subtypes are specified with brackets and associated designation on the right.

Fig 4

Subtree of cluster G5 displayed separately from the detailed phylogenetic tree of VP7 (Fig. S1). The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. Symbols (filled dots) indicate the sequences obtained in this study. Subtypes are specified with brackets and associated designation on the right.

Fig 5

Subtree with compressed subclusters of cluster G6 displayed separately from the detailed phylogenetic tree of VP7 (Fig. S1). The dendrogram was constructed in MegaX using Maximum Likelihood with General Time Reversible Model + G + I and 1,000 bootstrap replicates. Subtypes are designated on the right.

Fig 6

Nucleotide pairwise identity frequency graph of 102 complete VP4 ORF sequences without the genotype P21. The current 85% nucleotide cut-off value is shown by the vertical line.