Complete genome characterization by nanopore sequencing of rotaviruses A, B, and C circulating on large-scale pig farms in Russia

Abstract

Background: Rotaviruses are the major etiological agents of gastroenteritis and diarrheal outbreaks in plenty of mammalian species. The genus Rotavirus is highly diverse and currently comprises nine genetically distinct species, and four of them (A, B, C, and H) are common for humans and pigs. There is a strong necessity to comprehend phylogenetic relationships among rotaviruses from different host species to assess interspecies transmission, specifically between humans and livestock. To reveal the genetic origin of rotaviruses from Russian pig farms, nanopore-based metagenomic sequencing was performed on the PCR-positive specimens.

Methods: Samples were selected among the cases submitted to routine diagnostic or monitoring studies to the Laboratory of Biochemistry and Molecular Biology of "Federal Scientific Center VIEV" (Moscow, Russia). The selected positive samples were genotyped using nanopore sequencing method.

Results: Five porcine RVA isolates were completely sequenced, and genotype analysis revealed various porcine G/P genogroups: G2, G3, G4, G5, G11 and P[6], P[7], P[13], P[23], P[27] with a typical backbone constellation I5-R1-C1-M1-A8-N1-T1/7-E1-H1. The RVB isolate was detected in combination with RVA in a rectal swab from a diseased pig in Krasnoyarsk Krai. It was characterized by the following genogroups: G15-P[X]-I11-R4-C4-M4-A8-N10-T4-E4-H7. The first complete porcine RVC genome from Russia was obtained with genomic constellation G6-P[5]-I14-R1-C1-M1-A7-N9-T6-E1-H1, and the phylogenetic analysis revealed putative novel genotype group for the VP6 gene-I14. Additionally, the first porcine kobuvirus isolate from Russia was phylogenetically characterized.

Conclusions: The applied nanopore sequencing method successfully genotyped the RV isolates and additionally revealed co-circulated species. The study demonstrates high genetic variability of Russian RVA isolates in VP4/VP7 genes and phylogenetically describes local RVB and RVC. Complete characterization of genomic segments is a crucial methodology in tracing the rotavirus's evolution and evaluating interspecies transmissions.

Keywords: Complete genome; Diarrhea; Metagenomics; Nanopore sequencing; Phylogenetics; Porcine rotaviruses.

Fig. 1

Phylogenetic dendrograms constructed with the GTR model for the VP4 and VP7 RVA genes. The RVA isolates identified in this study are colored in bold blue. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified. GenBank accession number, host, country, and collection year of each isolate are also shown. RVA genotypes are specified on the right of the bracket

Fig. 2

Phylogenetic dendrograms constructed with the GTR model for the VP4, VP6, and VP7 RVB genes. The RVB isolate identified in this study is colored in bold blue. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified.. GenBank accession number, host, country, and collection year of each isolate are also shown. RVB genotypes are specified on the right of the bracket

Fig. 3

Phylogenetic dendrograms constructed with the GTR model for the VP4, VP6, and VP7 RVC genes. The RVC isolate identified in this study is colored in bold blue. The putative novel VP6 genotype I14 is additionally highlighted in light green. Scale bars indicate nucleotide substitutions per site. Bootstrap values greater than 80% are specified. GenBank accession number, host, country, and collection year of each isolate are also shown. RVC genotypes are specified on the right of the bracket

Fig. 4

Phylogenetic dendrogram constructed with the GTR model for the partial sequence (2271 nt) of Porcine kobuvirus (PKV). The PKV isolate identified in this study is colored in bold blue. Bootstrap values greater than 80% are specified. GenBank accession number, country and collection year of each isolate are shown in brackets on the right side