Marmota himalayana in the Qinghai-Tibetan plateau as a special host for bi-segmented and unsegmented picobirnaviruses

Abstract

Wildlife has been considered the main source of novel viruses causing emerging infectious diseases. Marmota himalayana is endemic to the Qinghai-Tibetan Plateau, China. Here, based on a high-throughput method using Illumina RNA sequencing, we studied the RNA virome of M. himalayana and discovered multiple novel viruses, especially picobirnaviruses (PBVs), which have a bi-segmented genome and belong to the family Picobirnaviridae. A total of 63% of the viral contigs corresponded to PBVs, comprising 274 segment 1 and 56 segment 2 sequences. Unexpectedly, four unsegmented PBV genomes were also detected and confirmed by PCR and resequencing. According to the phylogenetic analysis, the following nine PBV assortment types are proposed: C1:GI, C2:GIV, C4:GI, C4:GV, C5:GI, C7:GI, C8:GIV, C8:GV and C8:GII. We hypothesize a model of segmentation for the PBV genome, mediated by a 6-bp direct repeat sequence, GAAAGG. The model is supported by detection of the segmentation-associated sequence GAAAGG not only in the 5' untranslated regions of segment 1 (221 in 289) and segment 2 (57 in 80) of bi-segmented PBVs but also in the 5' untranslated regions and junction sequences between the capsid and RdRp genes of unsegmented PBVs. Therefore, with RNA sequencing, we found an unexpected biodiversity of PBVs in M. himalayana, indicating that M. himalayana is a special host for PBVs. We also proposed a putative model of how bi-segmented PBVs could be converted into unsegmented PBVs, which sheds new light on the processes of RNA virus genome evolution.

Fig. 1. Map showing the location of the collection sites in Yushu, Qinghai province, China.

Red dots are the collection sites. Positive rates in three locations are indicated

Fig. 2. Virus families detected in the intestine of Marmota himalayana.

Numbers of virus contigs are shown in parentheses

Fig. 3. Genome organization of picobirnaviruses.

(a) Schematic representation of the two segments of human picobirnavirus strain Hy005102 (GenBank accession no. NC_007026 and NC_007027). (B) The unsegmented genomes of picobirnaviruses detected in marmot. Arrows indicate the position and direction of primers for nested PCR. Stop codon of the capsid gene and the initiation codon of the RdRp are underlined. Junction sequences between segments 1 and 2 of bi-segmented picobirnaviruses are shown below the triangles. Segmentation-associated motifs are shown in red

Fig. 4. Maximum-likelihood phylogenetic trees and picobirnavirus assortment types.

a The phylogenetic trees of the full-sequence capsid (left) and RdRp (right) proteins. Sequences of picobirnaviruses obtained from marmot are shown in red. Sequences of picobirnaviruses reported previously are shown in black. Unsegmented picobirnaviruses from marmot are indicated by black triangles. Capsid and RdRp sequences of picobirnaviruses that underwent assortment are linked by lines. b Proposed assortment types of bi-segmented and unsegmented picobirnaviruses

Fig. 5. Model of segmentation of unsegmented picobirnaviruses.

Relative positions of the 6-bp direct repeat sequence GAAAGG in the 5′ UTR and junction between the capsid and RdRp genes of unsegmented Marmot picobirnaviruses are indicated by rectangles. A schematic of the three types of segment 1 harboring GAAAGG (S1-1-3) and two types of segment 2 harboring GAAAGG (S2-1-2) from bi-segmented picobirnaviruses. The proportions of S1-1-3 and S2-1-2 among the total sequences analyzed, together with their genotypes, are shown in the following tables. *The published segment 1 sequences (GenBank accession no. KJ495689; KF861768; JQ776551; AB186897; KR902506; KC692367; KR902502; KR902504; KF861770; KF861771; KF861772). The published segment 2 sequences (GenBank accession no. JQ776552; AAG53583; AHX00960; ACT64131; AFK81927; BAJ53294; AHZ46150; KR902507; KR902503)