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
• evolution history
• ancient evolution
• pathogenicity
• plateau pika

Data availability

The genome sequences of PPV are available from GenBank (accession numbers MN443982-MN444000 and MN444002-MN444020).

Results

Identification of a novel arenavirus in plateau pikas

A total of 113 fecal samples were collected and grouped into two pools for meta-transcriptomic sequencing. Data analyses revealed that arenavirus-associated contigs had the highest abundance levels, although other viruses such as astroviruses, picobirnaviruses, and rotaviruses were also discovered in both pools (Figure 1(A) and (B)). For each pool, both ambisense L and S segments were identified, which encoded zinc-binding RING protein (ZP) (89 aa), RNA-dependent RNA polymerase (RdRp) (2,206 aa), glycoprotein (GP) (536 aa), and nucleoprotein (NP) (559 aa). The final lengths for the L and S segments were determined to be 7106 and 3540 bases, respectively, by 5’ and 3’ RACE. There were 15 bp conserved reverse complementary sequences, namely, GGATTCCCAGGTTTT and GGATTCCAGGGTTT(G)T, at both the 5’ and 3’ termini of the L and S segments, respectively (Figure 1(C)). We designated this newly identified arenavirus plateau pika arenavirus (PPV).

Distribution and tissue tropism of PPV arenaviruses

We surveyed the distribution of new PPV arenaviruses in the plateau pikas from BTT, GDC and JLC in 2012 and from GDC and GQG in 2015. The data showed that PPV RNA was detected in 34 (6.3%) of 541 intestinal content samples from these counties (Figure 1(D)). Nineteen positive samples (9 samples collected in 2012, and 10 samples collected in 2015) were randomly selected for RNA-seq sequencing (Table S2). Evolutionary divergence analysis between these sequences by MEGA7.0 showed 27∼39 (RdRp), 30∼33 (NP), 31∼37 (GP) and 26∼39 (ZP) amino acid identity (%) to known mammarenavirus (Table S3). This relationship among the PPV sequences was supported by the highest identities between each other, with 82∼100 nucleotide identity (%) to the L and S segments (Table S4). The data showed that PPV was the dominant virus among the identified mammalian viruses (Figure 1(D)). PPV RNA was detected in infected plateau pikas in the lungs (100%), kidney (97%), heart (96%), liver (94%), and spleen (96%) (Table S5); brain samples were not collected. Viral loads in the tested organs of plateau pikas ranged from 7 × 10² to 6 × 10⁷ copies/g, and the lung showed the highest concentration of viral RNA (Figure S1). These results indicated that plateau pikas were a natural host of PPV.

Evolutionary history of PPV arenaviruses

Previous reports showed that the family Arenaviridae contained 4 clades, including two mammarenaviruses (Old World and New World), one associated with reptiles and one with fish [23]. We further investigated the evolutionary history of PPV arenaviruses within the context of the entire Arenaviridae family (Figure 1(E)). Interestingly, the newly identified PPV arenavirus formed a divergent sister clade to both Old World and New World mammarenaviruses. Nevertheless, it shared much closer relationship with members of mammarenavirueses (33∼37% amino acid identity at RdRp) than reptile (27%) or fish arenaviruses (30%). This phylogenetic position, as well as the fact that the virus was identified from plateau pika, a mammalian species, suggests that it is more likely to be a divergent member of mammarenavirus. Furthermore, this topology was also supported by phylogenies constructed based on nucleoprotein and glycoprotein (Figure S2), although for glycoproteins the fish and reptile counterparts are too divergent for sequence alignment and therefore are not included in the tree (Figure S2).

To estimate the divergence time of PPV arenaviruses from the other arenaviruses, we first reconciled the phylogenetic history of viruses with that of their hosts, with the assumption that if the virus co-speciate with the host, the host divergence time could be used on the virus tree. Our reconciliation analyses with the programme Jane 4 revealed 16 co-speciation events and 26 noncospeciation event (duplications = 7; extinction = 8; host-jumping = 11, Figure 2(A)). The total cost estimated was 37, which was significantly less than that expected by chance (p < 0.01) and therefore strongly favoured the co-speciation hypothesis (Figure 2(B)). Under the co-speciation framework, the estimated divergence time of the ancient group was 77–88 million years ago (MYA), earlier than that of Old World and New World mammarenaviruses (26-34 MYA) (Figure 2(A-C)). The ancient group was proposed (Figure 1(E), Figure 2(C)).

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.

Isolation and growth properties of PPV arenaviruses

We successfully isolated PPV strain #4 from the lung tissue of plateau pikas. PPV strain #4 was stably passaged in RK-13 cells but caused no visible CPE. Negative-stain electron microscopy showed an 80-nm round particle with an outer layer formed by surface projections, indicating that PPV had morphologic similarities to viruses in the genus mammarenavirus (Figure 3(A)). Inclusion bodies of approximately 1 μm in diameter were observed in the infected RK-13 cells by transmission electron microscopy (Figure 3(A)). Subsequently, we isolated five additional strains from lung and liver tissues of plateau pikas. After sequence alignment, these isolates shared 85∼98% (for the L segment) and 82%∼99% (for the S segment) nucleotide identity with each other (Table S6). All isolates replicated in RK-13 cells, of which strain #4 seemed slightly more efficient than the other isolates (Figure 3(B)). Hence, we focused on strain #4 for further study including the growth properties and pathogenicity of PPV.

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).

We further investigated cell susceptibility to PPV using cell lines originating from nonhuman primates (Vero) and humans (A549, and SLF-1). No visible CPEs were observed in these three cell lines, which was consistent with observations in RK-13 cells. IFA showed that PPV could replicate in Vero, A549, and SLF-1 cells (Figure 3(C)). Furthermore, qRT–PCR showed that PPV replication in Vero and SLF-1 cells was more efficient than that in A549 cells, with viral loads increasing by 2.0 × 10⁶ copies/mL in the supernatant (Figure 3(C)).

The histopathologic lesions in brain tissues of mice

We then conducted animal experiments to investigate the potential pathogenesis of PPV using multiple mouse genotypes, and New Zealand white rabbits. The body weights of SPF BALB/c mice and New Zealand white rabbits decreased transiently but recovered quickly (Figure S3). We also found that the body weights of suckling mice and IFNαβR^-/- mice continually increased throughout the observation period after intraperitoneal infection (Figure S3). However, the body weights of IFNαβR^-/- mice infected intraventricularly did not change until 5 d.p.i. but showed a steep drop at 7 d.p.i. (Figure 4(A)). At this time, clinical observation included limb weakness, trembling of the whole body and sudden death (Figure 4(B)).

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).]

Therefore, IFNαβR^-/- mice were chosen as the model for further analysis. The mice were infected by intraventricular PPV injection. The animals were then euthanized. The brain, spinal cord, heart, liver, spleen, lung and kidney of intraventricularly inoculated mice were collected for viral load and pathological analysis at 0, 3, 5, and 7 d.p.i. (n = 3) respectively (Figure 4(C) and (D)). All organs tested were positive for PPV, and the brain had the highest viral load. The viral loads in the brain, lung, spleen and heart increased at the initial 5 d.p.i and maintained a high level at 7 d.p.i (Figure 4(C)). H&E staining of the tissue sections showed that infection with PPV led to inflammatory infiltrates in the cerebral cortex at 5 d.p.i, and further progressed to cortical edema by 7 d.p.i. (Figure 4(D)). The cerebellar cortex showed inflammatory infiltrates and cortical edema on 7 d.p.i. (Figure 4(D)). IFNαβR^-/- mice infected with PPV displayed a small amount of inflammatory cell infiltration around the spinal meningeal vessels and the nerve root of the spinal cord, with proliferation of glial cells and hemorrhage in the spinal cord at 7 d.p.i (Figure 4(D)). No obvious lesions were observed in the heart, liver, spleen, lung, or kidney at 7 d.p.i (Figure S4).

Serological evidence of PPV infection in humans

We further investigated potential PPV infection cases in Yushu, Qinghai Province where PPV was isolated. Serological screening of human sera was initially performed by IFA using PPV-infected cells. A total of 335 sera from outpatients in Yushu were screened for anti-PPV IgG antibodies, with 8 (2.4%) samples testing positive (Figure 5(A), Figure S5(A)). In addition, we conducted specific detection of anti-PPV IgG antibodies by western blot assay using purified nucleoprotein of PPV. Consistent with the IFA results, WB showed that the same 8 (2.4%) samples, out of the 335 tested were positive (Figure 5(B), Figure S5(B)). The PPV IgG WB assay was used with LCMV- and Wenzhou virus-positive mouse sera (Figure 5(C)). There was obvious cross-reactivity between LCMV and WENV, but no serological cross-reactivity was observed between PPV and LCMV/WENV (Figure 5(C)). Thus, PPV-specific IgG antibodies were detected in 8 (2.4%) of 335 outpatients with available sera (Table 1).

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.

Table 1. Details of sera screening for PPV.

Discussion

Our virome study of plateau pikas in the Qinghai-Tibet Plateau identified a new mammarenavirus, named plateau pika virus (PPV). PPV has the same genome structure and protein-encoding form as existing members of mammarenavirus. Nevertheless, it shared less than 40% amino acid identity at RdRp with other known mammarenaviruses (Table S3). A phylogenetic analysis based on the amino acid sequences of the RdRp protein encoded by PPV using reptarenaviruses and hartmaniviruses as outgroups suggested that PPV is distinct from the known NW and OW mammarenaviruses and is classified as an ancient group of the genus Mammarenavirus (Figure 1(E)). Following LCMV and WENV, PPV was the third arenavirus reported and isolated in Asia. Nevertheless, despite this discovery, the diversity of non-rodent mammarenaviruses is still limited, and substantial gaps remain between mammarenaviruses and reptarenaviruses, which need to be filled by further study of arenaviruses from diverse mammalian host groups.

Our study provides key evidence that demonstrates the ancient history of the family Arenaviridae, which is mainly reflected by the high resemblance of virus and host phylogenies (Figure 2). Such co-divergence theory was initially proposed for arenaviruses because of the observation that each arenavirus species is associated with a single rodent host species and that the virus and host share matching phylogeographic history [6,24]. However, a later co-phylogenetic analysis based on 49 mammarenavirus and their associated host suggested that the hypothesis of virus–host co-speciation is unlikely to hold. The divergence of Old World and New World arenaviruses is estimated at a timescale of ∼45,000 ya [25], although such estimation was mainly based on contemporary sampled sequences. Nevertheless, a recent survey of arenaviruses from non-mammalian hosts revealed large diversity of arenaviruses in reptiles (golden reptarenavirus and California reptarenavirus) [26,27] and ray-fined fish (Wenling frogfish arenavirus 1 and Wenling frogfish arenavirus 1) [27]. Evolutionary analyses including these viruses from divergent hosts revealed a striking similarity between the virus and host trees such that a co-divergence theory is strongly favoured for the entire family Arenaviridae [27]. In this study, the phylogenetic position of the newly identified arenavirus from Lagomorpha was sister to those identified from Rodentia, which was consistent with the evolutionary relationship of the hosts. Therefore, our findings further support the idea that arenaviruses co-diverge with their vertebrate hosts for billions of years, as the estimated divergence time of PPV was far earlier than that of existing mammarenaviruses.

Unlike the NW and OW mammarenaviruses, which infect rodents of the Sigmodontinae, Neotominae, and Murinae subfamilies, PPV can persistently infect plateau pikas of the Lagomorpha, which expands the host range of mammarenaviruses. Our results also provide direct evidence of extensive PPV host tropism in mammalian cell lines, including humans, primates, and rodents. PPV replication was similarly efficient in leporidae-originated RK-13 cells and human originated SLF-1 cells, a kind of embryo lung fibroblast diploid cell. Compared to RK-13 and SLF-1 cells, PPV showed reduced replication efficiency in Vero cells and A549 cells. The broad cell tropism may not indicate that PPV has a broad host range but will provide effective tools to further investigate the characteristics of PPV.

Previous studies have demonstrated that IFNαβR^-/- mice are susceptible to infection by mammarenaviruses (e.g. LASV and LCMV) and are commonly used in arenavirus pathogenicity [28–30]. We found that PPV could infect these mice, and the inflammatory infiltrates, edema in the cerebral cortex, and hemorrhage in the spinal cord upon intraventricular PPV challenge were observed, suggesting that PPV can apparently cause central nervous system-like lesions. However, similar nervous disease symptoms or lesions were not observed in animal models of SPF BALB/c mice, suckling mice and rabbits. Thus, our results indicate that PPV has potential pathogenicity in immunocompromised hosts.

Expanding human populations have a major impact on wildlife habitats, especially viral pathogens, accelerating the emergence of new infectious diseases through increased animal/human interactions [31]. Plateau pika is a keystone species of the Qinghai-Tibetan Plateau, with a high density of approximately 300 animals per hectare [32]. It has been reported that plateau pikas harbour zoonotic or related viruses, such as H5N1, H9N2, H7N2 and coronavirus [33–36]. Our retrospective serological survey showed that PPV antibodies were present in 2.4% of the serum samples from outpatients who lived in Yushu, Qinghai Province. We tried to detect viral RNA in the sera using specific primers against PPV and RNA-seq transcriptome analysis. No trace of PPV RNA was detected in any of the patient sera. However, this might be however inconclusive, as RNA degradation likely occurs in retrospective sera due to the long-term preservation. Serological surveillance in nearby inhabitants nearby is therefore needed. To strengthen the case for PPV as an agent of human disease, future surveys would ideally include a comparison of the seroprevalence in exposed and unexposed populations. LASV and LCMV cause more severe disease in immunocompromised individuals and pregnant women [9,37]. Therefore, these particular individuals should also be examined for PPV infection in the future.

In conclusion, we isolated a new mammarenavirus PPV from plateau pikaS in the Qinghai-Tibet Plateau. Our study elaborated on the characteristics, divergence history and potential infection of PPV. Its ability to replicate in IFNαβR^-/- mice and multiple cell lines revealed its potential infectivity. Antibodies against PPV present in human serum provide preliminary clues to the possibility of cross-species transmission to humans; thus, PPV may pose a risk to human health. Due to limited available samples, more wildlife and livestock samples (e.g. plateau pika, Himalayan marmot and yak) should be screened to assess how prevalent this virus is in the ecosystem. Furthermore, more microbiological and epidemiological studies are needed explore the prevalence of PPV in nature and evaluate its potential risk to human health.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This study was supported by Science & Technology Fundamental Resources Investigation Program [grant no 2022FY100902], the National Natural Science Foundation of China [grant no 81802017], National Key R&D Program of China [grant no 2019YFC1200500, 2019YFC1200505], Research Units of Discovery of Unknown Bacteria and Function [grant no 2018RU010], Shenzhen Science and Technology Program [grant no KQTD20200820145822023], National Mega projects of China for Major Infectious Diseases [grant no 2017ZX10304402].