Whole Genome Analysis of Selected Human Group A Rotavirus Strains Revealed Evolution of DS-1-Like Single- and Double-Gene Reassortant Rotavirus Strains in Pakistan During 2015-2016

Abstract

Acute gastroenteritis due to group A rotaviruses (RVAs) is the leading cause of infant and childhood morbidity and mortality particularly in developing countries including Pakistan. In this study we have characterized the whole genomes of five RVA strains (PAK56, PAK419, PAK585, PAK622, and PAK663) using the Illumina HiSeq platform. The strains PAK56 and PAK622 exhibited a typical Wa-like genotype constellation (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, respectively), whereas PAK419, PAK585, and PAK663 exhibited distinct DS-1-like genotype constellations (G3P[4]-I2-R2-C2-M2-A2-N2-T1-E2-H2, G1P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, and G3P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, respectively). Despite their DS-1-like genotype constellation, strain PAK585 possessed the typical Wa-like G1P[8] genotypes, whereas both PAK419 and PAK663 possessed the G3 genotype. In addition, PAK419 also possessed the Wa-like NSP3 genotype T1, suggesting that multiple reassortments have occurred. On Phylogenetic analysis, all of the gene segments of the five strains examined in this study were genetically related to globally circulating human G1, G2, G3, G6, G8, G9, and G12 strains. Interestingly, the NSP2 gene of strain PAK419 showed closest relationship with Indian bovine strain (India/HR/B91), suggesting the occurrence of reassortment between human and bovine RVA strains. Furthermore, strains PAK419, PAK585, and PAK663 were closely related to one another in most of their gene segments, indicating that these strains might have been derived from a common ancestor. To our knowledge this is the first whole genome-based molecular characterization of human rotavirus strains in Pakistan. The results of our study will enhance our existing knowledge on the diversity and evolutionary dynamics of novel RVA strains including DS-1-like intergenogroup reassortant strains spreading in Asian countries including Pakistan, in the pre-vaccine era. Therefore, continuous surveillance is recommended to monitor the evolution, spread and genetic stability of novel reassortant rotavirus strains derived from such events.

Keywords: genotype constellation; morbidity; mortality; reassortant; surveillance.

FIGURE 1

Comparison of the genotype constellation of 5 Pakistani strains (PAK56, PAK622, PAK585, PAK419 and PAK663) having the G9P[8], G3P[8], G1P[8], G3P[4], and G3P[4] genotypes, respectively, compared with those of selected human and animal strains. The green and red shading represent human Wa-like, and DS-1-like genotypes, respectively. Different shades of green are used for VP7 G genotypes mostly found in combination with the Wa-like genotype constellation. Yellow is used to indicate gene segments of bovine origin and purple gene segments of caprine origin.

FIGURE 2

Phylogenetic tree constructed from the nucleotide sequences of the VP7 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 3

Phylogenetic tree constructed from the nucleotide sequences of the VP4 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.1 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 4

Phylogenetic tree constructed from the nucleotide sequences of the NSP2 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.02 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 5

Phylogenetic tree constructed from the nucleotide sequences of the NSP3 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 6

Phylogenetic tree constructed from the nucleotide sequences of the VP1 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 7

Phylogenetic tree constructed from the nucleotide sequences of the VP3 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 8

Phylogenetic tree constructed from the nucleotide sequences of the VP6 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.1 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 9

Phylogenetic tree constructed from the nucleotide sequences of the NSP1 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.1 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 10

Phylogenetic tree constructed from the nucleotide sequences of the NSP4 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.5 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 11

Phylogenetic tree constructed from the nucleotide sequences of the NSP5 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.

FIGURE 12

Phylogenetic tree constructed from the nucleotide sequences of the VP2 genes of the study strains and representative RVA strains. RVA strains sequenced in this study are represented by the red color. The vaccine strains and an out group strain are represented by Blue and green color, respectively while black shading represent strains isolated all over the world. The RVA strains sequenced in this study and reference strains obtained from GenBank database are represented by Accession number, Strain Name, Country and year of Isolation. Scale bar: 0.05 substitutions per nucleotide. Bootstrap values <60 are not shown.