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Emerging zoonoses of wildlife origin caused by previously unknown agents are one of the most important challenges for human health. The Qinghai-Tibet Plateau represents a unique ecological niche with diverse wildlife that harbours several human pathogens and numerous previously uncharacterized pathogens. In this study, we identified and characterized a novel arenavirus (namely, plateau pika virus, PPV) from plateau pikas (Ochotona curzoniae) on the Qinghai-Tibet Plateau by virome analysis. Isolated PPV strains could replicate in several mammalian cells. We further investigated PPV pathogenesis using animal models. PPV administered via an intraventricular route caused trembling and sudden death in IFNαβR-/- mice, and pathological inflammatory lesions in brain tissue were observed. According to a retrospective serological survey in the geographical region where PPV was isolated, PPV-specific IgG antibodies were detected in 8 (2.4%) of 335 outpatients with available sera. Phylogenetic analyses revealed that this virus was clearly separated from previously reported New and Old World mammarenaviruses. Under the co-speciation framework, the estimated divergence time of PPV was 77–88 million years ago (MYA), earlier than that of OW and NW mammarenaviruses (26-34 MYA).

Feline panleukopenia (FPL), a highly contagious and frequently fatal disease of cats, is caused by Feline parvovirus (FPV) and Canine parvovirus (CPV). We characterized the diversity of these Carnivore protoparvovirus 1 variants in 18 faecal samples collected from domestic cats with FPL during an outbreak, using targeted parvoviral DNA metagenomics to a mean depth of >10,000 X coverage per site. All samples comprised FPV alone. Compared to the reference FPV genome, isolated in 1967, 44 mutations were detected. Ten of these were non-synonymous, including 9 in non-structural genes and one in VP1/VP2 (Val232Ile), which was the only one to exhibit inter-host diversity, being present in five sequences. There were five other polymorphic nucleotide positions, all with synonymous mutations. Intra-host diversity at all polymorphic positions was low with sub-consensus variant frequencies (SVF) of >1% except for two positions (2108 and 3208) in two samples with SVF of 1.1 – 1.3%. Intra-host nucleotide diversity was measured across the whole genome (0.7 - 1.5%) and for each gene, and was highest in the NS2 gene of four samples (1.2 – 1.9%). Overall, intra-host viral genetic diversity was limited and most mutations observed were synonymous, indicative of a low background mutation rate and strong selective constraints.

Additional file 5. Primer and probe sequence information.

Additional file 1: Table S1. Detailed information on the 96 tick libraries examined here. Figure S1. Gene arrangements in tick mitochondrial genomes across eight genera. There are 23 linear maps in 19 species of Haemaphysalis, nine in Amblyomma species, two in Bothriocroton species, three in two Hyalomma species, 13 in 10 Rhipicephalus species, 18 in 16 Ixodes species, one of Nuttalliellidae species and 26 linear maps of Argasid ticks. Each mitochondrial genome has 13 protein-coding genes, two ribosomal genes, 22 tRNA genes and misc-features varying with tick species. Mitochondrial gene arrangement and direction in Haemaphysalis, Dermacentor, Amblyomma, Hyalomma, Rhipicephalus and Bothriocroton are almost identical and those in Ixodes, Nuttalliellidae and Argasidae are almost identical. Protein-coding genes are denoted by yellow arrows, rRNA are denoted by red arrows, tRNA are denoted by pink arrows, and control regions are denoted by gray arrows. The direction of arrows indicated the direction of protein translation. Abbreviations are as follows: ND1 = NADH dehydrogenase subunit 1, ND2 = NADH dehydrogenase subunit 2, ND3 = NADH dehydrogenase subunit 3, ND4 = NADH dehydrogenase subunit 4, ND4L = NADH dehydrogenase subunit 4L, ND5 = NADH dehydrogenase subunit 5, ND6 = NADH dehydrogenase subunit 6, COX1 = cytochrome oxidase I, COX2 = cytochrome oxidase II, COX3 = cytochrome oxidase III, ATP8 = ATPase8, ATP6 = ATPase8, 16 s rRNA = large ribosomal subunit, 12 s rRNA = small ribosomal subunit, CYTB = cytochrome b. Figure S2. Sequence alignment of special tandem repeat regions in misc-features of Dermacentor mt genome. The first hypervariable region located between tRNA-Gln and NAD1 (A), and the second hypervariable region located tRNA-Gln between tRNA-Phe (B), and its cloning of sequencing of PCR products spanning the control region implied various copy numbers of short repeat sequences within the same D. marginatus E1 sample (C). Table S2. Special tandem repeat regions in misc-features of Dermacentor mt genome. Figure S3. Detailed phylogenetic tree of ticks based on all 13 protein-coding genes and two rRNA genes inferred using both ML and Bayesian methods with two mites as the outgroup. Highly similar tree topologies were obtained. The trees are midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. For clarity, the bootstrap values are shown on the left with posterior probability on the right for each node. Mt genomes identified in this study are marked colored font according to different tick genera; the name of each tick genus is shown beside. Figure S4. Detailed phylogenetic tree of ticks based on only 13 protein-coding genes estimated using both ML and Bayesian methods with two mites as the outgroup. Highly similar tree topologies were obtained. The trees are midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. For each node, the bootstrap values are shown on the left with posterior probability on the right. Mt genomes identified in this study are marked colored font according to different tick genera; the name of each tick genus is shown beside. Figure S5. Detailed phylogenetic tree of ticks based on mt 12S rRNA gene estimated using ML methods. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. Mt 12S rRNA gene identified in this study are marked colored font according to different tick genera, the name of each tick genus is shown beside. Figure S6. Detailed phylogenetic tree of ticks based on mt 16S rRNA gene estimated using ML methods. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. Mt 16S rRNA gene identified in this study are marked colored font according to different tick genera, the name of each tick genus is shown beside. Figure S7. Differential diagnosis for D. nuttalli and D. silvarum. Adult trochanter l dorsal spur of D. nuttalli (A) is short, broad and blunt apically, while that of D. silvarum (B) is slightly long, pointed at the apex. Figure S8. Relative abundance of bacteria and fungi at the level of genus based on metaphlan2 results. Table S3. Prevalence of tick-borne bacteria in ticks at the level of genus. A total of 43 tick associated bacterial species were identified from 54 libraries and 7 tick genera (with the exception of Hyalomma). Bracketed numbers denote numbers of bacteria identified in each bacterial group at the level of genera. Table S4. The genetic similarity of bacterial strains identified in this study with its closest reference sequence. Figure S9. ML phylogenetic tree of the order Rickettsiales based on the groEL gene. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The strains identified here are marked by colored font according to different tick genera, and reference sequences are represented by black font with corresponding accession number nearby. Each bacterial group at genus level is denoted by gray font and a vertical line, while bacterial group at subfamily level is denoted by black font and a square bracket. Figure S10. ML phylogenetic tree of the order Rickettsiales based on the gltA gene. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The strains identified here are marked by colored font according to different tick genera, and reference sequences are represented by black font with corresponding accession number nearby. Each bacterial group at genus level is denoted by gray font and vertical line, while bacterial group at subfamily level is denoted by black font and square bracket. Figure S11. ML phylogenetic tree of the genus Rickettsia based on the six conserved housekeeping genes, including the atpD, coxB, ftsZ, gltA, groEL and sucA genes. Individual genes were first aligned and then concatenated to form super-alignment for phylogenetic analysis. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The strains identified here are marked by colored font according to different tick genera, and reference sequences are represented by black font with corresponding accession number nearby. The Rickettsiae collected here could further assigned into three groups: Spotted fever group (SFG), Typhus group (TG) and Belli group (BG). Figure S12. ML phylogenetic tree of the genus Coxiella based on the groEL gene. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The strains identified here are marked by colored font according to different tick genera, and reference sequences are represented by black font with corresponding accession number nearby. A significant clustering of Coxiella genetic diversity at the host general level was observed, and the position of C. burnetii was highlighted by an arrow. Figure S13. ML phylogenetic tree of the genus Borrelia based on the flaB gene. The tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The trees are midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. The B. henanensis strain X1 identified here is marked in pink font according to its tick genus (Carios), and reference sequences are represented by black font with corresponding accession number nearby. The Borrelia collected here could be assigned into three groups: The Relapsing fever group (RFG), Reptile and echidna-associated Borrelia (REB) and Lyme borreliosis group (LB). Table S5. Multiple regression of bacteria genetic distance against tick genetic distance and geographic distance shows that bacterial genetic diversity was mainly structured by tick genetic distance rather than geographical distribution.