Emergence of an ancient and pathogenic mammarenavirus

Abstract

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).

Keywords: Mammarenavirus; ancient evolution; evolution history; pathogenicity; plateau pika.

Figure 1.

A novel arenavirus discovered in plateau pikas. (A) A map showing the location of sampling sites in Yushu, Qinghai Province, China. Samples were collected from the GQG, GDC, BTT and JLC counties. (B) The dominant mammalian virus discovered in the intestinal contents of plateau pikas by RNA-seq. (C) A schematic of the genome organization of PPV. (D) The distribution of PPV and the abundance of the dominant mammalian virus from plateau pikas in 2012 and 2015. Darker colour reflects higher virus abundance. (E) Phylogenetic analysis of PPV and other arenaviruses. PPVs are marked with red.

Figure 2.

Co-divergence analyses of arenaviruses and their associated hosts. (A) Distribution of estimated co-divergence and non-codivergence (e.g. duplication, host switching, and extinction) events. (B) Estimated sum of non-codivergence events (red dotted line) in comparison to the associated null distributions under 100 randomizations of virus–host associations. (C) Comparisons of the topologies of virus and host phylogenetic trees. Well-established co-divergence points (black solid dots) are marked on the node. For these nodes, the divergence times of the host groups were used to calibrate the divergence times of the corresponding virus groups.

Figure 3.

Biological features of PPV. (A) Electron microscope images of PPV in cell culture supernatant (arrow, left) and in infected RK-13 cell (IB, right). IB: inclusion body. N: nucleus. Cyt: cytoplasm. (B) The growth of six isolates of PPV in RK-13 cells. RK-13 cells infected with PPV (MOI  = 0.5) for 24, 48, 80 and 96 h. PPV RNA levels were measured by qRT–PCR. Values are the averages of three independent experiments (mean ± SD). (C) The replication of PPV in different cell lines. RK-13, Vero, A549 and SLF-1 cells were infected with PPV (MOI  = 0.5) for 10, 24, 48 and 72 h, and PPV RNA levels were measured by qRT–PCR. Values are the averages of three independent experiments (mean ± SD).

Figure 4.

Histopathological analyses of IFNαβR^-/- mice intraventricularly infected with PPV. (A) Weight loss of IFNαβR-/-mice infected with PPV via intraperitoneal (ip) and intraventricular (icv) infection. (B) The death rate of IFNαβR-/-mice infected with PPV via intraperitoneal (ip) and intraventricular (icv) infection. (C) Viral load in different tissues of IFNαβR-/-mice infected with PPV at 3, 5, and 7 days post-infection (d.p.i.). (D) Histopathological changes in the cerebrum, cerebellum and spinal cord of mice intraventricularly challenged with PPV. Blue arrows indicate the enlargement of normal areas. Red arrows indicate the inflammatory infiltrates. The black arrow indicates cortical edema. Yellow arrows indicate gliosis in the spinal cord. Green arrows indicate hematomyelia. [Cerebrum scale bars: 0.5 mm (left); 20 μm (right); cerebellum scale bars: 0.2 mm (left); 20 μm (right); spinal cord scale bars: 0.2 mm (left); 20 μm (right).]

Figure 5.

Evidence of human exposure by IFA and western blot. (A) Representative immunofluorescence images of a serum sample from a patient (left) and from a nonexposed individual (right). Human serum samples were diluted 1:40. (B) Representative western blot images of a serum sample from a patient (left) from a healthy individual (right). Purified NPs (400 ng) were used as antigens. Nonexposed human serum was used as a negative control. Human serum samples were diluted 1:200. (C) Cross-reactivity between PPV and WENV/LCMV NPs. Mouse antisera against PPV NP, WENV NP and LCMV NP were diluted at 1:2000 and incubated with the PPV, WENV and LCMV NPs, respectively. The loading amount of NP for each lane was 400 ng.