Genomic stratification and differential natural selection signatures among human norovirus genogroup II isolates

Abstract

Noroviruses (NoVs), which are members of the family Caliciviridae, are the most common cause of gastroenteritis in humans. Ten NoV genogroups have been reported so far. Of these, genogroup II (GII) is the most prevalent, and it causes serious infections worldwide. The complete genome sequences of NoV GII isolates from different geographical regions were retrieved from the public database. The model-based clustering approach, implemented in the STRUCTURE resource, was employed for assessment of genetic composition. The MEGA X and IQ Tree tools were used for phylogenetic analysis. Genome-wide natural selection analysis was performed using maximum-likelihood-based methods. The demographic features of NoV GII genome sequences were assessed using the BEAST package. All of the NoV GII sequences initially clustered into two main subpopulations at significant K = 2, where the genotype GII.4 samples clearly split from the rest of the genotypes. This indicates a marked genetic distinction between norovirus GII.4 and non-GII.4 samples. Phylogenetic analysis showed the presence of five distinct subclades for genotype GII.2 and seven subclades for GII.4 samples. Several isolates with admixed ancestry were identified that constituted distinct subclusters in the phylogenetic tree. No continental-specific genetic distinctions were observed among the NoV GII samples. Significant genomic signatures of both positive and negative natural selection were identified across the NoV GII genes. A differential pattern of positive selection signals was inferred between the GII.4 and non-GII.4 genotypes. The demographic analysis revealed an increase in the effective population size of NoV GII during 2009-2010, followed by a rapid fall in 2015.

Keywords: genomic diversity; norovirus genogroup II; selection pressure; spatiotemporal.

Fig. 1

[A] Determination of K_opt for NoV GII. The graph shows a plot of K versus delta K, which defines the optimum number of clusters K_opt in the NoV GII population. K represents the number of clusters, while delta K is the rate of change in likelihood posterior probability for the given subcluster K. The plot was executed using large values for simulation burn-ins (100,000) and burn-in length (100,000). The major peak at K = 2 shows that the NoV genetic structure is grouped into two main subpopulations. (B) Estimate of the population genetic structure of NoV at a K_opt of 2 using an admixture model in STRUCTURE software. C-1 comprises genomic entries from genotypes GII.1, GII.2, GII.3, GII.5, GII.6, GII.7, GII.8, GII.12, GII.13, GII.17, and GII.26, and C-2 comprises genotype GII.4 strains [C] The initial clustering pattern of the phylogenetic tree results is consistent with the STRUCTURE result.

Fig. 2

Additional genetic structure analysis of C-1 and C-2 using an admixture model in STRUCTURE. (A) Plot of K versus delta K showing the optimum number of subpopulations in C-1. The plot shows a significant peak at K = 3, followed by two minor peaks at K = 4 and K = 5. [B] Sublevel genetic structure of C-1 (non-GII.4 genotypes) obtained using the STRUCTURE program, applying an admixture model. The analysis suggests the presence of three clusters at K = 3, represented as a color bar plot. At K = 3, C-1.1 contains genotype GII.2, C-1.2 contains genotypes GII.17, and C-1.3 consists of genotype GII.3, GII.5, GII.6, GII.7, GII.8, GII.12, and GII.26. At K = 4, four clusters were observed: C-1.1 (GII.2), C-1.2 (GII.17), C-1.3a (GII.3), and C-1.3b (GII.5, GII.6, GII.7, GII.8, GII.12, and GII.26). At K = 5, possible clustering of genotype GII.2 into two lineages (C-1.1a and C-1.1b) can be seen. (C) Genetic structure of genotype GII.4 (cluster 2). At K = 2, there is clustering of C-2.1 (GII.4 Sydney_2012, New Orleans_2009, and Apeldoorn) and C-2.2 (Den_Haag_2006b), and at K = 5, C-2.1a (Sydney_2012), C-2.1b (Sydney_2012 and New Orleans), C-2.1c (Sydney_2012), and C-2.2a (Den_Haag_2006), C-2.2b (Den_Haag_2006) can be seen. (D) Plot of K versus delta K showing a major peak at K = 2, dividing C-2 (GII.4) into two subpopulations. The second peak was found at K = 5, which shows additional diversification of GII.4 into five lineages.

Fig. 3

The two-dimensional PCA analysis of NoV GII samples

Fig. 4

Phylogenetic analysis of complete genome sequences of NoV GII isolates. (A) NJ-based tree of 822 strains constructed using MEGA X. Ten clades were observed in the phylogenetic tree, which is congruent with the clustering pattern observed using STRUCTURE, i.e., C-1.1a (GII.2), C-1.1b (GII.2), C-1.2(GII.17), C-1.3a (GII.3), C-1.3b (GII.5, 6, 7, 12, 13 and 26), C-2.1a (GII.4- Sydney_2012/P31), C-2.1b (GII.4-Sydney_2012/P4, and New Orleans_2009), C-2.1c (Sydney_2012/p16) C-2.2a (Den Haag_2006b), and C-2.2b (Den Haag_2006b). The branches of recombinant/admixed strains are indicated by an asterisk (*). (B) Maximum-likelihood tree of NoV GII.2. All major clades of NoV are colored and labeled.

Fig. 5

Bayesian skyline plot of Human NoV GII.2. The y-axis represents the effective population size, and the x-axis represents time in years. The solid black line indicates the mean posterior value, and the blue shaded area represents the 95% HPD interval.