Highly diverse ribonucleic acid viruses in the viromes of eukaryotic host species in Yunnan province, China

Abstract

Background: The diversity in currently documented viruses and their morphological characteristics indicates the need for understanding the evolutionary characteristics of viruses. Notably, further studies are needed to obtain a comprehensive landscape of virome, the virome of host species in Yunnan province, China.

Materials and methods: We implemented the metagenomic next-generation sequencing strategy to investigate the viral diversity, which involved in 465 specimens collected from bats, pangolins, monkeys, and other species. The diverse RNA viruses were analyzed, especially focusing on the genome organization, genetic divergence and phylogenetic relationships.

Results: In this study, we investigated the viral composition of eight libraries from bats, pangolins, monkeys, and other species, and found several diverse RNA viruses, including the Alphacoronavirus from bat specimens. By characterizing the genome organization, genetic divergence, and phylogenetic relationships, we identified five Alphacoronavirus strains, which shared phylogenetic association with Bat-CoV-HKU8-related strains. The pestivirus-like virus related to recently identified Dongyang pangolin virus (DYPV) strains from dead pangolin specimens, suggesting that these viruses are evolving. Some genomes showed higher divergence from known species (e.g., calicivirus CS9-Cali-YN-CHN-2020), and many showed evidence of recombination events with unknown or known strains (e.g., mamastroviruses BF2-astro-YN-CHN-2020 and EV-A122 AKM5-YN-CHN-2020). The newly identified viruses showed extensive changes and could be assigned as new species, or even genus (e.g., calicivirus CS9-Cali-YN-CHN-2020 and iflavirus Ifla-YN-CHN-2020). Moreover, we identified several highly divergent RNA viruses and estimated their evolutionary characteristics among different hosts, providing data for further examination of their evolutionary dynamics.

Conclusion: Overall, our study emphasizes the close association between emerging viruses and infectious diseases, and the need for more comprehensive surveys.

Keywords: RNA viruses; coronavirus; metagenomic next-generation sequencing (mNGS); picornavirus; viral evolution; virome.

FIGURE 1

Characteristics of virome data. (A) Pie chart of the viral species classifications based on the output data in the eight libraries. (B) The assembly statistics and viral taxonomic identifications in each library.

FIGURE 2

Virome differences among the eight libraries based on read counts. (A) Rarefaction curves for each library, with the x-axis showing the sample size of each library and y-axis showing the viral species identified. (B) Heatmap showing the normalized abundance of the eight libraries at the family level. The red columns represent high abundance, and the blue columns represent low abundance. The taxonomical information was list in the right panel. (C) Virome composition at the family level. The top 10 virus families are shown. (D) Principal component analysis (PCA) showing multivariate variation (host information) and the major contributions of different factors (viral families) to PC1 and PC2. The first two PCA were used, and the groups are shown in different colors.

FIGURE 3

Analysis of coronavirus genomes. (A) The top two hits obtained using BLASTN for two representative coronavirus strains. (B) Maximum likelihood tree of representative genomes of coronaviruses, including alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. The new strains identified in this study are shown in blue. The scale bars show the substitutions per site per year. The values at each node indicate the bootstrap and SH-like approximate likelihood ratio test (SH-aLRT) values, with 1,000 bootstrap replicates.

FIGURE 4

Characterization of the pestivirus genome contigs. (A) Graphics of mapped sequences. (B) The top two hits in the nucleotide (NT) and protein (NR) databases using the BLASTN and BLASTP algorithms. (C) Maximum likelihood tree of the newly identified pestivirus species, neighboring genomes, and newly identified flavivirus, based on the amino acid sequences. The scale bars show the substitutions per site per year, and the values at each node indicate the bootstrap and SH-like approximate likelihood ratio tests (SH-aLRT), with 1,000 bootstrap replicates. The black arrows represent the newly identified strains.

FIGURE 5

Characterization of iflavirus genome contigs. (A) Genomic organization of the detected iflaviruses and annotation of conserved domains. (B) Maximum likelihood tree of the complete amino acid sequences of the detected iflaviruses along with reference genomes of known species of Iflaviridae and neighboring strains. The scale bars show the substitutions per site per year, and the values at each node indicate the bootstrap and SH-like approximate likelihood ratio tests (SH-aLRT), with 1,000 bootstrap replicates. The black arrows represent the newly identified strains. Picornaviridae was used as an outgroup. (C) The top two hits in the nucleotide (NT) and protein (NR) databases obtained using BLASTN and BLASTP.