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PHEV infection: A promising model of betacoronavirus-associated neurological and olfactory dysfunction [1]

['Junchao Shi', 'State Key Laboratory For Zoonotic Diseases', 'Key Laboratory For Zoonosis Research Of The Ministry Of Education', 'College Of Veterinary Medicine', 'Jilin University', 'Changchun', 'Zi Li', 'Jing Zhang', 'Rongyi Xu', 'Yungang Lan']

Date: 2022-08 

Abstract Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic coronavirus belonging to the genus Betacoronavirus. Similar to pathogenic coronaviruses to which humans are susceptible, such as SARS-CoV-2, PHEV is transmitted primarily through respiratory droplets and close contact, entering the central nervous system (CNS) from the peripheral nerves at the site of initial infection. However, the neuroinvasion route of PHEV are poorly understood. Here, we found that BALB/c mice are susceptible to intranasal PHEV infection and showed distinct neurological manifestations. The behavioral study and histopathological examination revealed that PHEV attacks neurons in the CNS and causes significant smell and taste dysfunction in mice. By tracking neuroinvasion, we identified that PHEV invades the CNS via the olfactory nerve and trigeminal nerve located in the nasal cavity, and olfactory sensory neurons (OSNs) were susceptible to viral infection. Immunofluorescence staining and ultrastructural observations revealed that viral materials traveling along axons, suggesting axonal transport may engage in rapid viral transmission in the CNS. Moreover, viral replication in the olfactory system and CNS is associated with inflammatory and immune responses, tissue disorganization and dysfunction. Overall, we proposed that PHEV may serve as a potential prototype for elucidating the pathogenesis of coronavirus-associated neurological complications and olfactory and taste disorders.

Author summary PHEV, a neurotropic porcine betacoronavirus (β-CoV), primarily infects and replicates in the respiratory tract and CNS in suckling pigs. Neurological complications and anosmia (i.e., inability to perceive odor or loss of olfactory function) are common clinical features in coronavirus-induced diseases, however the underlying mechanisms remain unclear. In this study, we investigated the pathogenesis of neurological and olfactory dysfunction in a murine model of PHEV infection. The data underscore that PHEV invades the CNS via the olfactory and trigeminal nerves, and that anosmia and neurological manifestations are associated with direct OSNs infection and neuroimmune inflammation. The utilization of PHEV prototype will provide a platform for future studies on the neuroinvasion and neuropathogenesis of human pathogenic coronaviruses.

Citation: Shi J, Li Z, Zhang J, Xu R, Lan Y, Guan J, et al. (2022) PHEV infection: A promising model of betacoronavirus-associated neurological and olfactory dysfunction. PLoS Pathog 18(6): e1010667. https://doi.org/10.1371/journal.ppat.1010667 Editor: Debby van Riel, Erasmus Medical Center, NETHERLANDS Received: February 7, 2022; Accepted: June 10, 2022; Published: June 27, 2022 Copyright: © 2022 Shi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (grant numbers 32172805, 32172828, 31902262, and 31872446); the Scientific and Technological Project of Jilin Province (grant numbers 20210202041NC to WH), the Youth Science and Technology Talent Support Project of Jilin Province (grant numbers QT202015 to ZL). The funder had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. Competing interests: The authors have declared that no competing interests exist.

Introduction Coronavirus disease 2019 (COVID-19) is caused by the newly emerged betacoronavirus (β-CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has had global impacts on public healthcare systems and economies [1,2]. In addition to airway and pulmonary symptoms, reduction or sudden loss of smell or taste has been reported in approximately half of all COVID-19 patients [3–5]. Furthermore, a wide range of central and peripheral neurological symptoms have been observed in patients with severe disease [6,7], suggesting that SARS-CoV-2 may target cells within the central nervous system (CNS) [8]. Currently, few animal models of COVID-19-associated anosmia, ageusia, and SARS-CoV-2 neuroinvasion are available [9–11]. The use of other β-CoVs, such as murine hepatitis virus (MHV), has been proposed as an approach for simulating several of the major characteristics of human pathogenic coronaviruses infection, including SARS-CoV-2-induced acute lung injury and systemic symptoms [12,13]. Porcine hemagglutinating encephalomyelitis virus (PHEV), along with SARS-CoV-2 and MHV, is a member of the genus Betacoronavirus within the family Coronaviridae and order Nidovirales [14]. After replicating in the upper respiratory tract, some PHEV strains also cause influenza-like symptoms (ILS) in adult pigs [15]. Notably, PHEV exhibits typical neurotropism and is currently the only known neurotropic coronavirus capable of infecting pigs [14]. Naturally, PHEV infects nasal epithelial cells and the tonsils in the respiratory tract, and then the virus propagates from the peripheral nerves to the CNS [14]. Clinical signs include encephalomyelitis, vomiting and wasting disease (VWD), and ILS. Encephalomyelitis in suckling pigs caused by PHEV infection was first reported in Canada in 1957, and the causative agent was first isolated in 1962 [16,17]. In 1969, another clinical type of PHEV-induced VWD in suckling piglets was observed in England [18]. Both clinical forms were experimentally reproduced in neonatal pigs using PHEV isolates from the same farm [19]. Generally, clinical manifestations of encephalomyelitis and VWD are age-dependent and reported frequently in piglets under 4 weeks old, with mortality rates reaching 100% [14,15,20]. However, an acute outbreak of ILS-like respiratory disease in adult exhibition swine was reported in the USA in 2015, and PHEV was identified as the causative agent [15]. Although only a few reports of PHEV outbreaks have been documented, they are devastating due to the lack of vaccines and effective countermeasures [14]. Furthermore, subclinical circulation of PHEV has been reported in many countries according to serological investigation, further emphasizing the significance of PHEV in pig farming worldwide [14,21]. In recent years, researchers have studied the pathogenesis of PHEV from multiple perspectives using mouse, rat and in vitro nerve cell models. Neural cell adhesion molecule (NCAM) interacts with PHEV, promoting entry into nerve cells [22,23]. In addition, cell-surface glycans, i.e., sialic acid (SA) and heparan sulfate (HS), act as attachment factors for PHEV in nerve cells [24]. Clathrin-mediated endocytosis (CME) and the endosomal system of neurons are hijacked by PHEV for virus intracellular trafficking [25]. Meanwhile, PHEV activates integrin α5β1-FAK-Cofilin signaling to induce rearrangement of the cytoskeleton, which in turn provides energy for the intracellular transport of virions [26]. In PHEV-infected neurons, progeny virions bud and assemble in smooth-surfaced vesicles originating from endoplasmic reticulum–Golgi intermediate compartments and are then released from the cells by the biosynthetic secretory pathway [27]. The vesicle-mediated secretory pathway mediates the transsynaptic transmission of PHEV between neurons. In the CNS, PHEV is mainly located in the neuronal soma and processes in the cerebral cortex, brain stem and spinal cord [28]. Neurodegenerative changes, such as axonal dysplasia, unstable dendritic spine formation, and irregular swelling and disconnection in neurites, are linked to the Ulk1-TrkA-NGF-Rab5 signaling pathway [29,30]. Furthermore, PHEV-induced neurodegeneration is related to lysosome dysfunction and endoplasmic reticulum (ER) stress [31–33], similar to the pathogenesis of human neurodegenerative diseases such as Parkinson’s disease, frontotemporal degeneration and neuronal lipofuscinosis, indicating that PHEV might be a useful model virus to study the mechanisms of human neurodegenerative disease [33]. In this paper, we investigated the neuroinvasiveness of PHEV in BALB/c mice and proposed the potential application of intranasal PHEV infection in BALB/c mice as a model for investigating the pathogenesis of β-CoV-induced anosmia, ageusia, and neurological complications. PHEV-infected mice exhibit significant olfactory and gustatory dysfunction, with effective virus replication, robust inflammation, and functional impairment of the nasal epithelium and CNS. A better understanding of the neuroinvasion route and underlying mechanisms of the neuropathogenesis of PHEV after transport from the upper respiratory tract to and within the CNS will provide important insights into the development of antiviral countermeasures tailored to this specific host compartment for other neurotropic coronaviruses.

Discussion Olfactory impairment and/or neurological manifestation are common symptoms of coronavirus diseases, but the exact mechanisms of neurological and olfactory dysfunction have not yet been clarified [40–43]. PHEV, a typical neurotropic coronavirus, is the causative agent of CNS disease in suckling pigs. Here, we performed virological, behavioral, and molecular studies in a PHEV-infected BALB/c mouse model that replicates olfactory, taste, and neurological dysfunction in coronavirus-related disease, providing a potential in vivo platform for investigating viral pathogenesis. The nasal epithelium is the primary site for the neuroinvasiveness of most neurotropic respiratory viruses [8,44–48]. It consists of the RE and OE, which are located in the inferior-anterior and superior-posterior regions of the nasal cavity, respectively. Many OSNs in the OE comprise the olfactory nerves, and olfactory nerves coalesce to form larger nerve bundles that traverse the bony cribriform plate and terminate in the OB [49]. The olfactory nerve thus provides a direct pathway from the nasal cavity to the CNS [50–53]. In our model, we found that OE and RE are two areas infected by PHEV in the nasal cavity, with OSNs representing the major target cells in OE. The initial site of PHEV colonization in the brain is the OB and brain stem, followed by global transmission through the CNS, indicating that the olfactory nerve may be hijacked by PHEV for rapid CNS invasion. The essential function of OE during PHEV neuroinvasion was also confirmed, as chemically mediated degeneration of OSNs blocked PHEV access to the CNS and significantly improved the survival rate in mice in the early stage of infection. In addition, the ophthalmic branch of the trigeminal nerve, a sensory nerve that senses tactile stimuli, pain, and temperature, also innervates the nasal mucosa in the RE [54]. As shown in Fig 7, detection of the viral antigen in the trigeminal nerve and trigeminal ganglion provides direct evidence of trigeminal nerve transmission of PHEV. Thus, we conclude PHEV invades the CNS via the trigeminal nerve by infecting subepithelial nerve endings in the RE of the nasal cavity. Three main cell types and some glands are present in the RE, including ciliated cells, goblet cells, basal cells, serous glands, seromucous glands, and intraepithelial glands [55,56]. Nasal secretions and mucus produced by seromucous glands and goblet cells provide a physical barrier for the host against invading pathogens. This barrier may lead to less efficient PHEV infection of the subepithelial trigeminal nerve compared to the olfactory nerve. Interestingly, olfactory transmucosal invasion is also a port of CNS entry for SARS-CoV-2 and other neurotropic viruses [45]. SARS-CoV-2 was found in Neuropilin-1 (NRP-1) -positive OSNs of the OE, OB and olfactory tracts in COVID-19 patients, indicating the critical roles of NPR1 in virus entry of the OSNs and anosmia [57–59]. Further research is needed to clarify if NRP1 serves as a host factor for PHEV infection. Intranasal inoculation of PHEV resulted in anosmia and ageusia and induced an inflammatory response in the nasal cavity of mice, which characterized by the presence of IBA1-positive macrophages in the nasal epithelium and elevated levels of inflammatory cytokines. We presume that the olfactory dysfunction may be caused by direct OSN infection and virus-induced inflammation. This hypothesis was supported by a report showing that SARS-CoV-2 infects OSNs in the OE of COVID-19 patients presenting with acute loss of smell, and viral replication in the OE is related to local inflammation [10]. SARS-CoV-2 also causes acute anosmia in golden Syrian hamsters, which lasted as long as the virus remained in the OE and OB with an enhanced immune response [10,60]. In the S2 Table, we showed how the PHEV-infected model and the SARS-CoV-2-infected models relate to findings identified in humans vis-a-vis anosmia and infection of the OE, RE, and OB. Furthermore, disruption of the cilial architecture in the OE or loss of cilia in the OSNs may also contribute to olfactory dysfunction [61]. Further studies should be performed to examine any correlations between the deciliation of the OE and olfactory dysfunction in PHEV-infected mice. In this paper, we developed a novel neurological and olfactory dysfunction model based on intranasal inoculation of BALB/c mice with PHEV, and demonstrated its rapid CNS entry via the olfactory nerve in OE and/or the trigeminal nerve in RE. Similar to SARS-CoV-2, this prototype shows anosmia, ageusia, and neurological disorders along with productive replication of virus, robust inflammation, and tissue disorganization in the OE and CNS. Given that the high pathogenicity of human coronaviruses, relevant experiments must be conducted in a BSL-3 laboratory, which has undoubtedly hindered research progress. Therefore, the PHEV-infected BALB/c mice represent an excellent animal model for studying the viral pathogenesis, neuroinvasiveness, and neurovirulence of human pathogenic coronaviruses, albeit with limitations. For example, there is a large difference in the ability of neuroinvasiveness and neurotropism between SARS-CoV-2 and PHEV. SARS-CoV-2 does not replicate efficiently in the neurons and spread throughout the CNS causing a lethal encephalitis [62–64], although SARS-CoV-2 antigen is occasionally detected in the CNS of humans and relevant animal models [45,65]. In contrast, PHEV is strongly neurotropic but has little or no lung damage, leading to the restriction of using this model to evaluate pathogenic coronavirus-induced lung injury. Other limitation of experimental PHEV infection in mice as a model for SARS-CoV-2 associated CNS complications is the difference in host cell receptor usage between SARS-CoV-2 and PHEV. Angiotensin receptor 2 (ACE2) is a critical entry receptor for SARS-CoV-2 and it expresses diffusely on the mucous membrane of the entire oral cavity [66], but PHEV does not appear to use ACE2 as an entry receptor. Notably, both SARS-CoV-2 and PHEV could bind to the sialic acid (SA) of host cells [24,67,68]. Sialic acid is an essential element in the salivary mucin and may protect glycoproteins that transduce gustatory signals inside taste pores from enzymatic destruction [69,70]. PHEV may occupy sialic acid binding sites on taste buds, leading to gustatory particle destruction, but this remains to be elucidated in the future.

Materials and methods Ethics statement All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the College of Veterinary Medicine, Jilin University, China (permission number KT202108025). All applicable institutional and/or national guidelines for the care and use of animals were followed. Study design The main objective of this study was to investigate PHEV neuroinvasion in BALB/c mice and its potential use as a promising model to clarify coronavirus-associated neurological and olfactory and taste dysfunction. For assessments olfactory and taste functions, 6w BALB/c mice were used in behavioral experiments according to previously published protocols [34,71]. Intranasal PHEV inoculation has been successfully used in mice to mimic aerosol droplet infection in pigs [30,72]. We used 3w BALB/c mice to test neuroinvasion, which resulted in improved efficiency of intranasal inoculation [73]. Cells and virus Mouse neuroblastoma cells (Neuro-2a; ATCC CCL-131, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Meilunbio, Dalian, CN) supplemented with 6% fetal bovine serum (FBS; BI, Kibbutz Beit Haemek, Israel), penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively) and incubated at 37°C with 5% CO 2 . The PHEV CC14 strain (GenBank accession number: MF083115) was isolated from naturally infected piglets with neurological symptoms, vomiting, diarrhea, and wasting, and was propagated in Neuro-2a cells [74,75]. Antibodies Rabbit polyclonal anti-olfactory marker protein antibody (ab183947), rabbit polyclonal anti-MAP2 antibody (ab21693), rabbit polyclonal anti-GFAP antibody (ab207165), rabbit polyclonal anti-IBA1 antibody (ab178846) and rabbit polyclonal anti-MBP antibody (ab218011) were obtained from Abcam (Cambridge, MA). The rabbit polyclonal anti-NSE antibody was obtained from Servicebio (Wuhan, CN). The mouse monoclonal anti-β3-tubulin (TU-20) antibody (#4466), Alexa Fluor 488-conjugated goat anti-rabbit antibody (#4412), Alexa Fluor 488-conjugated goat anti-mouse antibody (#4408), Alexa Fluor 594-conjugated goat anti-rabbit antibody (#8889) and Alexa Fluor 594-conjugated goat anti-mouse antibody (#8890) were purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal anti-PHEV-nucleoprotein antibodies were prepared and stored in our laboratory. Briefly, the recombinant full-length PHEV-N protein was generated based on the sequence of the PHEV CC14 strain. First, the PHEV-N coding region was amplified using RT–PCR and cloned into the pET-32a(+) vector. Second, the recombinant protein was expressed and purified with His-tag Purification Resin (P2233, Beyotime). Third, the polyclonal antibody was prepared in New Zealand white rabbits by subcutaneously injecting 100 μg of recombinant protein combined with an equal volume of Freund’s incomplete adjuvant at eight different sites. Rabbits were boosted three times at 2-week intervals. Animal experiments Three-week-old (3w) and six-week-old (6w) BALB/c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Shengyang, CN), provided ad libitum access to water and a standard chow diet, and maintained in the animal facility in the College of Veterinary Medicine, Jilin University. Mice were subjected to intranasal inoculation with 103.96 TCID 50 of PHEV (strain CC14) diluted in 20 μl of phosphate-buffered saline (PBS, 0.01 M, pH 7.4) (10 μl were instilled dropwise in each nostril) or a sham inoculation with the same dose of sterile PBS under 1–3% isoflurane anesthesia. Following inoculation, mice were monitored twice daily and a clinical score was recorded based on an Institutional Animal Care and Use Committee (IACUC)-approved scoring system for a maximum score of 5, and the evaluation indicators included body weight, respiration, general appearance, responsiveness and neurological signs (Table 1) [76]. Humane endpoints were established based on the IACUC-approved clinical scoring system. Mice were considered moribund and humanely euthanized when the cumulative clinical score of 4 or weight loss greater than 20% was observed. Mice were euthanized when they reached humane endpoints. Briefly, mice were exposed to 5% isoflurane for 5 min in a plexiglass chamber before being decapitated when fully sedated, as determined by the absence of an active paw reflex. PPT PowerPoint slide

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TIFF original image Download: Table 1. Clinical scoring system used for PHEV-infected mice. https://doi.org/10.1371/journal.ppat.1010667.t001 For survival curve experiments, any mice that reached the humane endpoints were humanely euthanized immediately, regardless of the day. For time course experiments, a subset of mice were humanely euthanized and tissue samples were collected at 1, 2, 3, 4, and 5 dpi. Blood was collected through eyeball extraction under deep anesthesia with isoflurane, and serum was obtained by centrifugation at 4°C, 4,000 xg for 10 minutes. Samples of heart, liver, spleen, lung, kidney, small intestine, colon, brain and spinal cord were collected after transcardial perfusion with 4% (wt/vol) paraformaldehyde, and then stored at -80°C for qRT-PCR analysis or fixed in 10% neutral buffered formalin solution for histology analysis. Sucrose preference test Taste function in mice was assessed with a sucrose preference test as previously described [71]. Briefly, the 6w BALB/c mice (male and female) were subjected to 48 h of continuous exposure to both 1% sucrose water and regular water for adaptation. All mice had ad libitum access to laboratory chow. During the preference test after intranasal PHEV and PBS inoculation, mice were deprived of water for 6 h, and individual overnight (12 h) testing, which corresponds to the circadian rhythms of the drinking of mice, was performed. Abnormalities in taste function were indicated by the reduction in the sucrose preference ratio (preference = sucrose intake/total intake × 100%) in PHEV-infected and control mice. Buried food finding test The buried food finding test was performed as previously described with a few modifications [34]. Six-week BALB/c mice (male and female) were used only once per day for each test, and the food position was changed daily. Mice were fasted for 12 h and sensitized to food for 5 min before testing and then individually placed into a fresh cage with food pellets hidden below the bedding. The latency to locate and dig the buried food was recorded using a stopwatch. The experiment was carried out for a 3 min period, and if the mice could not find the food, the time was recorded as 3 min. Social scent discrimination tests The social scent discrimination test was performed to evaluate the ability of mice to detect and differentiate different odors using previously described method, with a few modifications [9]. Briefly, two identical 2-ml Eppendorf tubes or 3-cm dishes were separately sealed with bedding from different mouse cages overnight and placed at two different corners in a fresh cage (28 cm × 24 cm × 17 cm). For male 6w mice, the bedding was collected from female mouse cages and male cages. For female 6w mice, the bedding was collected from the home cage (‘familiar’) and another cage (‘novel’). Next, PHEV-infected mice or mock-infected mice were released into fresh cages. Time spent sniffing within a 3-min period was measured. Each mouse was performed one trial each day with the position of tubes or dishes changed daily. To avoid the interference of decreased mobility or malaise, the preference index for both sexes was also calculated as previously reported [9]. Chemical deafferentation of olfactory nerve To destroy the OE and block olfactory viral neuroinvasion, the 3w BALB/c mice were intranasally irrigated with 20 μL of ZnSO 4 (0.17 M) or 0.7% Triton X-100 solution in both nostrils (10 μl were instilled dropwise in each nostril) daily for 3 days before intranasal PHEV inoculation. We investigated the effect of OE destruction on mouse survival by plotting survival curves using the aforementioned humane endpoint criteria. Mice were euthanized, and whole infected CNS tissues (including OB, cerebrum, cerebellum, brain stem, and spinal cord) and blood were collected from different mice daily until the fifth day post-infection for the qRT–PCR analysis. qRT–PCR Total RNA was purified from 100 mg of homogenized tissues using a TransZol Up Plus RNA Kit (ER501-01, Transgen, Beijing, CN) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed to first-strand cDNAs using EasyScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (AE341-02, Transgen). The primers used to measure the abundance of mouse cytokines and chemokines were previously reported [9]. The comparative ΔΔC T method was used to calculate the relative abundance of transcripts using the average values of each gene and normalized to GAPDH. For the detection of viral RNA, cDNAs from mouse tissues were subjected to the amplification of genomic RNA for the PHEV N protein by qRT–PCR using the following primers: PHEV-N-F, 5´-TCTGGGAATCCTGACGAG-3´; PHEV-N-R, 5´-AGGCGCTGCAACACTTAC-3´. A standard curve was constructed for each PCR using 101−108 copies of a PHEV-N plasmid to calculate copy numbers for each reaction. Histopathology, immunofluorescence (IF), and immunohistochemistry (IHC) Mice were anesthetized and transcardially perfused with 4% (wt/vol) paraformaldehyde. Then, tissues were removed and fixed with a 10% neutral buffered formalin solution. For histopathology, paraffin-embedded tissues were sectioned routinely (3–5 μm thickness) and stained with hematoxylin and eosin (H&E). For IF, sections were deparaffinized, rehydrated, and antigen retrieval was performed using pH 6.0 citrate buffer (Servicebio, CN). Sections were incubated with blocking reagent (5% skim milk) followed by primary antibodies overnight at 4°C and then incubated with the appropriate fluorescently labeled secondary antibody for 1 h at room temperature. All sections were then mounted using antifade mounting medium with DAPI (P0131, Beyotime) and scanned with a PANNORAMIC MIDI II automatic digital slide scanner (3DHISTECH, Budapest, Hungary). For IHC, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval according to the instructions of the UltraSensitive SP (Mouse/Rabbit) IHC Kit. Viral titration Briefly, tissue samples were weighed and homogenized in 1 ml of 2% DMEM, and tissue homogenate supernatants were serially diluted ten-fold in DMEM. Neuro-2a cells seeded in ninety-six-well plates were inoculated with serial dilutions of tissue homogenates at 37°C with 5% CO 2 for 1 h. After removing the inocula, DMEM containing 2% FBS was added to the plates for 3 days. The TCID 50 was calculated by the method of Reed-Muench and presented as TCID 50 /g of tissue weight. Transmission electron microscopy (TEM) The TEM analysis was performed as previously described [32]. Briefly, PHEV-infected mouse brain samples were collected, fixed with 2.5% glutaraldehyde for 24 h, and then postfixed with 1% osmium tetroxide for 2 h. After dehydration using a graded ethanol series, the specimens were embedded in Renlam resin. The ultrathin sections were cut using a diamond knife, transferred onto slot grids and then stained with 2% uranyl acetate and 0.4% lead citrate. The ultrathin sections were visualized using a HITACHI HT7800 microscope. Transcriptome sequencing Transcriptome sequencing and data analysis were performed at Sangon Biotech (Shanghai) Co., Ltd. Briefly, cDNA libraries were constructed according to the manufacturers’ instructions (Hieff NGS MaxUp Dual-mode mRNA Library Prep Kit for Illumina) and sequenced by an Illumina HiSeq 2,500 sequencer. After quality control of the original sequencing data, reads were mapped to the reference genome sequence. Reads mapped to the genes were counted, and gene expression was calculated. Statistical analysis Graphics and statistical tests were performed using GraphPad Prism v8.0 software (GraphPad Software, San Diego, CA). Data are presented as the means ± SD. Statistical significance was considered at *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Supporting information S1 Fig. Sex-independent clinical symptoms of mice after PHEV infection. Male and female 3w and 6w BALB/c mice were intranasally inoculated with 103.96 TCID 50 PHEV per mouse (n = 5 mice/age/sex). (A) Mortality of PHEV-infected female and male mice. (B) Percentage of initial body weight. (C) Clinical scores. P values were calculated by log-rank (Mantel–Cox) tests (survival) (A), Wilcoxon matched-pairs rank test (B), and one-way ANOVA (C). Data are presented as the means ± SD. https://doi.org/10.1371/journal.ppat.1010667.s001 (TIF) S2 Fig. Visible food finding test. The 6w male and female BALB/c mice were intranasally inoculated with 103.96 TCID 50 PHEV or mock-infected with PBS (M) before the visible food finding test. (A) Time spent by mock or infected mice (1–3 dpi) finding the visible food. The dashed line represents the time limit of 3 min. Each circle represents a mouse. (B) Percentage of mice that successfully found visible food within 3 min. For male mice (mock: n = 13, 1–3 dpi: n = 6), female mice (mock: n = 13, 1–3 dpi: n = 7). P values were calculated by one-way ANOVA (A). Data are presented as the means ± SD. https://doi.org/10.1371/journal.ppat.1010667.s002 (TIF) S3 Fig. Histopathological analysis of the liver, spleen, kidney, small intestine and colon in PHEV-infected mice. The 3w BALB/c mice were euthanized at 0, 3 and 5 dpi after 103.96 TCID 50 PHEV inoculation, and tissues were collected for the histological examination. The liver (A), spleen (B), kidney (C), small intestine (D), and colon (E) were analyzed. There were no substantial histopathological changes in the organs of PHEV-infected mice. Scale bars, 50 μm (A), 100 μm (B, C and E), 200 μm (D), H&E staining. Two sections of each organ from 3 mice per group were analyzed. https://doi.org/10.1371/journal.ppat.1010667.s003 (TIF) S4 Fig. Inflammatory cell accumulation in the OE and OB of PHEV-infected mice. The 3w BALB/c mice were inoculated intranasally with 103.96 TCID 50 PHEV. OB and nose tissues were collected at 0, 3 and 5 dpi for IHC with an antibody against IBA1. Compared with control tissues (A-C), incremental accumulation of IBA1-positive macrophages/microglia cells was observed in the OE and OB at 3 (D-F) and 5 (G-I) dpi. Scale bars, 500 μm (A, D, G), 50 μm (B, C, E, F, H, I). Two sections of each tissue from 3 mice per group were analyzed, and representative images are shown. https://doi.org/10.1371/journal.ppat.1010667.s004 (TIF) S5 Fig. Differentially expressed genes (DEGs) in the OB identified by transcriptomic sequencing (RNA-seq). The 3w BALB/c mice were inoculated intranasally with 103.96 TCID 50 PHEV and OB samples were collected at 5 dpi for RNA-seq. (A) Volcano plot. The horizontal axis represents the fold change in DEGs, and the vertical axis represents the Benjamini–Hochberg corrected p value on a logarithmic scale (-log10). Each dot represents a gene, where red dots represent up-regulated genes, green dots represent down-regulated genes, and black dots represent non-differentially expressed genes. b, Cluster heatmap of DEGs. Each row represents a gene, and each column represents a sample. The color represents the expression level of the gene, the red color represents a high expression level, and the green color represents a low expression level. c, Scatter plot of the KEGG pathway enrichment analysis. P, PHEV-infected sample; M, mock sample. https://doi.org/10.1371/journal.ppat.1010667.s005 (TIF) S1 Table. Distribution of viral antigen in the brains of PHEV-infected mice. https://doi.org/10.1371/journal.ppat.1010667.s006 (DOCX) S2 Table. Comparison of anosmia and brain infection among the PHEV-infected mouse model, SARS-CoV-2-infected hamster model, SARS-CoV-2-infected humanized ACE2 mouse model and deceased COVID-19 patients. https://doi.org/10.1371/journal.ppat.1010667.s007 (DOCX)

Acknowledgments The authors thank Dr. Chen-Hsuan Liu from the School of Veterinary Medicine, National Taiwan University for his assistance with the histopathological analysis.

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