Gut virome of mammals and birds reveals high genetic diversity of the family Microviridae

Abstract

Nineteen families of phages infecting bacteria or archaea are currently recognized by the International Committee on Taxonomy of Viruses (ICTV). Of these, only two have single-stranded DNA genomes, namely Inoviridae and Microviridae. The distribution, genetic characteristics, and ecological roles of Microviridae remain largely under explored. Here, using viral metagenomics, we investigate the intestinal virome from human and twenty-four species of animals, as well as freshwater samples, containing abundant sequence reads showing similarity to the Microviridae. Eight hundred and sixty complete or near complete Microviridae-related genomes were generated, showing high levels of co-infections and sequence divergence. Sequence comparison and phylogenetic analysis showed that the Microviridae subfamily Gokushovirinae was highly prevalent and that some strains may qualify as new subfamilies. This study significantly augments our knowledge of the genetic diversity, genome evolution, and distribution in animal species of members of the family Microviridae.

Keywords: Microviridae; complete genome; genetic diversity; phylogenetic analysis.

Figure 1.

Identification of viral sequences showing similarity to Microviridae from virome in human, animals, and other samples. (a) Samples size and library numbers. On top of each bar in the upper graph the sample number is shown. On bottom of each bar in the lower graph the library number is shown. The species name of animal is shown on the bottom. Those samples without marked sample types all belong to fecal samples. (b) The abundance of SSM in fecal sample of different animals and water sample. The number of SSM is normalized to reads per million unique reads. (c) The abundance of SSM in intestinal contents from yaks with different health status in three different farms. Health status normal and diarrheic is shown on the bottom of the bars. On top of each bar the farm numbers are shown. The number of SSM is normalized to reads per million unique reads. (d) The abundance of SSM in different types of samples from cattle. On bottom of each bar the sample types are shown. The number of SSM was normalized to reads per million unique reads. (e) The abundance of SSM in different types of samples from wild rats. On bottom of each bar the sample types are shown. The number of SSM is normalized to reads per million unique reads. (f) Sequence length distribution of the 713 complete Microviridae genomes identified in this study. The horizontal axis indicates the sequence numbers and the vertical axis shows the sequence length. (g) Sequence identity distribution based on VP1 amino acid sequence comparison between the 860 Microviridae strains in this study and their best matches in BLASTp search, respectively. The horizontal axis indicates the sequence number and the vertical axis shows the amino acid sequence identity. (h) Pairwise sequence comparison of the 860 amino acid sequences of VP1 identified in this study. The percent identities are shown by heat map, where plot colors and color saturation reflect the identity, ranging from low (blue) to high (red).

Figure 2.

Phylogenetic diversity of Microviridae. (a) Phylogenetic tree based on VP1 amino acid sequence. The sequences in this analysis include VP1 amino acid sequences of the 860 Microviridae strains and 22 representative members of the previously identified subfamilies. Within the tree, the twenty-two representative members of the previously confirmed subfamilies are shown with red dots. The names of the subfamily are shown beside the corresponding clades. The potential new clades are shown with rose lines and marked with numbers 1–10. The scale bar indicates amino acid substitutions per site. (b) Phylogenetic tree based on VP1 amino acid sequences of the representative strain of the potential new clades and the previously identified subfamilies in Microviridae. The previously identified subfamilies and the potential new subfamilies are shaded different colors. GenBank accession numbers, strain names, and isolation sources of each Microviridae identified in this study are shown on tree. (c) Phylogenetic analysis based VP1 amino acid sequences the reflecting the correlation between genetic relationship and isolation hosts and geographic location of the 860 Microviridae sequence identified in this study. Isolation source and geographic information of each sequences are shown with different colors, respectively. (d) Sampling locations included thirteen different provinces which are shown in different colors in map of China.

Figure 3.

Phylogenetic analysis of Microviridae from virome in pigs. (a) Phylogenetic tree based on VP1 amino acid sequence of the 410 Microviridae strains from pigs located in 4 different sampling pig farms and 18 representative members of the previously identified subfamilies. Within the tree, the Microviridae strains from four different farms are shown with four different color dots, respectively. The twenty-two representative members of the previously confirmed subfamilies in Microviridae are marked with black diamonds and the clades in which they are located are shaded with different colors. The subfamily names are shown in text box beside their corresponding shaded colors. The scale bar indicates 0.5 amino acid substitutions per site. Pairwise sequence comparison of the VP1 amino acid sequences of Microviridae identified in this study in each clade within this tree was performed. The percent identities are shown beside each clade by heat map, where plot colors and color saturation reflect the identity, ranging from low (blue) to high (red). (b) Phylogenetic tree based on VP1 amino acid sequence of Microviridae co-existing in four individual pigs. Four pigs which showed about twenty different genomes co-existing in each virome are included in this analysis. The twenty-two representative members of the previously confirmed subfamilies in Microviridae are marked with black diamonds and the clades in which they are located are shaded with different colors. The subfamily names are shown in text box beside their corresponding shaded colors. The scale bar indicates amino acid substitutions per site. As the predominance of Gokushovirinae in the approximate twenty Microviridae in each of the four pigs, pairwise sequence comparison of the VP1 amino acid sequences of Gokushovirinae identified in this study in each clade within these trees was also performed. The percent identities are shown beside each clade by heat map, where plot colors and color saturation reflect the identity, ranging from low (blue) to high (red).

Figure 4.

Phylogenetic analysis of Microviridae from virome in different species or group of animals. Animals species or group names are shown under each tree. Phylogenetic tree based on VP1 amino acid sequence of the Microviridae strains identified in this study and twenty-two representative members of the previously identified subfamilies. Within the tree, the twenty-two representative members of the previously confirmed subfamilies in Microviridae are marked with black dots and the clades in which they are located are shaded with different colors. The subfamily names are shown in text box beside their corresponding shaded colors. The scale bar indicates amino acid substitutions per site. In the phylogenetic analysis based on sequences from cattle (panel a), different sample types are marked with different color dots. In the phylogenetic analysis based on sequences from birds (panel b) or non-human primates (panel e), different species of animals are marked with different color dots.