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.
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.
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.
Results
Clinical manifestations in PHEV-inoculated BALB/c mice
Six-week-old (6w) and three-week-old (3w) BALB/c mice of both sexes were intranasally challenged with 20 μL of PBS or a 50% tissue culture infective dose (TCID50) of 103.96 PHEV (strain CC14) for behavioral experiments and neuroinvasion experiments. These mice were monitored daily for survival, weight change, and clinical symptoms (Fig 1A). Viral inoculation resulted in 100% mortality at day 5 post-infection (dpi) in PHEV-infected 3w mice and at 6 dpi in PHEV-infected 6w mice (Fig 1B). PHEV-infected mice exhibited a substantial decrease in body weight at 3 dpi in both age groups (Fig 1C) and began to display signs of sickness at 3 dpi in PHEV-infected 3w mice and 4 dpi in PHEV-infected 6w mice, including lethargy, delayed movement, and vocalizations (Fig 1D). Neurological signs such as ruffled fur, hunchback posture, tremors, and ataxic gait were detected at 4 dpi in PHEV-infected 3w mice and 5 dpi in PHEV-infected 6w mice (Fig 1D). No differences in death, weight loss, or clinical symptoms caused by PHEV were observed between sexes but between ages (S1A–S1C Fig). Collectively, these results suggest that BALB/c mice are susceptible to PHEV infection and that infected mice show obvious clinical manifestations.
Fig 1
Intranasal inoculation of PHEV in BALB/c mice results in lethal infection.
Three-week-old (3w) and six-week-old (6w) BALB/c mice were mock-infected (n = 3/sex/age) or intranasally inoculated (n = 5/sex/age) with 103.96 TCID50 PHEV. (A) Schematic diagram of the study design and workflow. Mice were monitored daily for survival (B), relative weight change (C), and clinical signs (D). Statistical analyses were performed using log-rank (Mantel–Cox) tests (B), Wilcoxon matched-pairs rank test (C), and one-way ANOVA, two-tailed Student’s t test (D). Data are representative of three replicate experiments and are shown as the means ± SD.
Intranasal inoculation of PHEV in BALB/c mice results in lethal infection.
Three-week-old (3w) and six-week-old (6w) BALB/c mice were mock-infected (n = 3/sex/age) or intranasally inoculated (n = 5/sex/age) with 103.96 TCID50 PHEV. (A) Schematic diagram of the study design and workflow. Mice were monitored daily for survival (B), relative weight change (C), and clinical signs (D). Statistical analyses were performed using log-rank (Mantel–Cox) tests (B), Wilcoxon matched-pairs rank test (C), and one-way ANOVA, two-tailed Student’s t test (D). Data are representative of three replicate experiments and are shown as the means ± SD.
PHEV infection induces anosmia and ageusia in mice
Given that 6w mice have fully developed organs and have reached sexual maturity, we selected 6w mice for behavioral studies, although PHEV efficiently infects 3w and 6w mice. We performed a sucrose preference test at 1–3 dpi in 6w mice to assess taste function (Fig 2A–2C). No significant difference in the total intake of water overnight was observed between mock and PHEV-infected mice of both sexes (Fig 2B). As expected, mock-infected mice preferred 1% sucrose water to regular water, while infected mice of either sex showed no preference for sucrose-complemented water (Fig 2C). It indicated that PHEV-associated ageusia occurred. Smell function was evaluated by performing a series of behavioral experiments, as previously reported [34]. First, the buried food finding test indicated that infected mice of both sexes took longer time to find the hidden food (Fig 2D and 2E), and a significant proportion of the mice (30% of males vs. 43% of females at 3 dpi) were unable to locate the food at the end of the test (Fig 2F). All infected mice were able to find visible food (S2 Fig), indicating that the delay in locating the buried food was not due to sickness-related behavior, vision impairment, or locomotor deficiency. Second, male mice were exposed to 2-ml Eppendorf tubes filled with either female or male dander (Fig 2G). Male mice were attracted to female dander when olfaction was normal, but we found that the female dander was less attractive to PHEV-infected male mice (Fig 2H). Third, female mice were provided bedding from their home cage (‘familiar scent’) and a different foreign cage (‘novel scent’) (Fig 2J). Mice with normal olfaction preferred the novel bedding, while PHEV-infected female mice spent less time near the foreign bedding (Fig 2K). Preference indices further confirmed anosmia in PHEV-infected mice of both sexes (Fig 2I and 2L). These results suggest that PHEV infection induces obvious smell and taste impairments in mice.
Fig 2
PHEV-associated anosmia and ageusia in BALB/c mice.
The 6w male and female BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV or mock-infected with PBS (M), and a series of behavioral experiments was performed to assess the olfactory and taste functions in mock and PHEV-infected mice. (A) Schematic diagram of the sucrose preference test (n = 6 per sex). (B) Total intake of water overnight. (C) Sucrose preference of mock and PHEV-infected mice at 1–3 dpi. Each circle represents a mouse. (D) Schematic diagram of the buried food finding test (n = 7 per sex). (E) The time it took for male and female mice to find buried food. The dashed line represents the time limit of 3 min. (F) Percentage of mice that successfully found buried food within 3 min. (G) Schematic diagram of the social scent-discrimination test for male mice (n = 10). (H) Time that male mice spent sniffing male or female dander. (I) Preference indices for male mice. (J) Schematic diagram of the social scent-discrimination test for female mice (n = 10). (K) Time that female mice spent exploring familiar or novel scents. (L) Preference indices for female mice. P values were calculated by two-way ANOVA (B, C, E, H and K) and the two-tailed Mann–Whitney U test (I and L). Data are representative of three replicate experiments and are shown as the means ± SD.
PHEV-associated anosmia and ageusia in BALB/c mice.
The 6w male and female BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV or mock-infected with PBS (M), and a series of behavioral experiments was performed to assess the olfactory and taste functions in mock and PHEV-infected mice. (A) Schematic diagram of the sucrose preference test (n = 6 per sex). (B) Total intake of water overnight. (C) Sucrose preference of mock and PHEV-infected mice at 1–3 dpi. Each circle represents a mouse. (D) Schematic diagram of the buried food finding test (n = 7 per sex). (E) The time it took for male and female mice to find buried food. The dashed line represents the time limit of 3 min. (F) Percentage of mice that successfully found buried food within 3 min. (G) Schematic diagram of the social scent-discrimination test for male mice (n = 10). (H) Time that male mice spent sniffing male or female dander. (I) Preference indices for male mice. (J) Schematic diagram of the social scent-discrimination test for female mice (n = 10). (K) Time that female mice spent exploring familiar or novel scents. (L) Preference indices for female mice. P values were calculated by two-way ANOVA (B, C, E, H and K) and the two-tailed Mann–Whitney U test (I and L). Data are representative of three replicate experiments and are shown as the means ± SD.
Tropism of PHEV to neurons in the mouse CNS
The 3w and 6w BALB/c mice were intranasally inoculated with PHEV (strain CC14), and viral genomic RNA and infectious virus particles were detected at 5 dpi to determine the target organs for PHEV infection. PHEV preferentially infects the brain and spinal cord, while no virus antigens or infectious virions were detected in peripheral organs, including heart, liver, spleen, lung, kidney, small intestine, colon, and blood (Fig 3A and 3B). Consistent with these findings, no significant pathological changes in the liver, spleen, kidney, small intestine, or colon were observed in either mock or PHEV-infected mice (S3 Fig).
Fig 3
Brain damage and viral tropism in the CNS of PHEV-infected mice.
(A-B) The 3w and 6w BALB/c mice were inoculated with 103.96 TCID50 PHEV and samples were collected at 5 dpi for qRT–PCR and viral titer determination (n = 6). (A) Viral genome loads were monitored in different organs and blood. The limit of detection (LOD) is shown with a dashed line. (B) Infectious viral titers were detected in different organs (n = 6). (C-L) The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV. Mice were euthanized at 5 dpi, and brain samples were harvested for histopathological examination. (C-H) H&E staining of brain sections from control and PHEV-infected mice. (C-D) Lymphocytic perivascular cuffing (red arrows). (E) Dying neurons undergoing degeneration (black arrows). (F) Microglial nodules (green arrows). (G-H) Brains from mock-infected mice. (I-L) Immunofluorescence images of PHEV-infected neurons in 3w BALB/c mice. MAP-2, GFAP, IBA1, and MBP are markers for neurons, astrocytes, microglia, and oligodendroglia, respectively. Scale bars, 100 μm (C, G), 20 μm (D-F, H), and 50 μm (I-L).
Brain damage and viral tropism in the CNS of PHEV-infected mice.
(A-B) The 3w and 6w BALB/c mice were inoculated with 103.96 TCID50 PHEV and samples were collected at 5 dpi for qRT–PCR and viral titer determination (n = 6). (A) Viral genome loads were monitored in different organs and blood. The limit of detection (LOD) is shown with a dashed line. (B) Infectious viral titers were detected in different organs (n = 6). (C-L) The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV. Mice were euthanized at 5 dpi, and brain samples were harvested for histopathological examination. (C-H) H&E staining of brain sections from control and PHEV-infected mice. (C-D) Lymphocytic perivascular cuffing (red arrows). (E) Dying neurons undergoing degeneration (black arrows). (F) Microglial nodules (green arrows). (G-H) Brains from mock-infected mice. (I-L) Immunofluorescence images of PHEV-infected neurons in 3w BALB/c mice. MAP-2, GFAP, IBA1, and MBP are markers for neurons, astrocytes, microglia, and oligodendroglia, respectively. Scale bars, 100 μm (C, G), 20 μm (D-F, H), and 50 μm (I-L).Because of the higher efficiency of PHEV neuroinvasion in younger mice, we used immature 3w mice to determine the target cells for PHEV infection in the CNS. When neurological symptoms were visible, mice were euthanized and brains were harvested at 5 dpi. Histopathologically, perivascular cuffing was observed in the cerebral parenchyma of PHEV-infected mice due to lymphocyte infiltration (Fig 3C and 3D, red arrow). Degenerated neurons had basophilic, angular, and shrunken cell bodies with contracted and dense nuclei (Fig 3E, black arrows). Activated microglia with large rod-shaped nuclei surrounded degenerating neurons and formed clusters around small foci of degenerate/necrotic neurons (Fig 3F, green arrows). No histological abnormalities were observed in the brain tissue of the mock mice (Fig 3G and 3H). Immunofluorescence staining of the cerebral cortex revealed that the PHEV nucleocapsid (N) protein was predominantly present in neurons, as identified by MAP2 staining (Fig 3I). Astrocytes, microglia, and oligodendroglia identified by GFAP, IBA1 and MBP staining, respectively, rarely showed costaining with the PHEV N protein (Fig 3J, 3K and 3L). These results suggest that PHEV is a neurotropic coronavirus that causes lethal disease resulting from brain infection and targets neurons in the CNS.
PHEV hijacks the olfactory pathway for CNS invasion
Brain and nose tissues from PHEV-infected 3w BALB/c mice were sagittally sectioned, and the viral distribution was examined by performing immunofluorescence staining. In the olfactory epithelium (OE) of the nasal cavity, viral antigens were detected as early as 1 dpi in the cell body of OSNs (Fig 4A), and the number of PHEV-positive cells further increased at 3 and 5 dpi (Fig 4B and 4C). At 1 dpi, PHEV-infected cells were not observed in the brain (Fig 4D and S1 Table). At 3 dpi, the viral antigens were detected mainly in the brainstem and olfactory-associated regions, including the olfactory bulb (OB) and piriform cortex (Fig 4E and S1 Table). PHEV-positive cells were detected in the entire brain at 5 dpi, including the cerebellar regions (Fig 4F and S1 Table). Our findings suggest that PHEV is able to cross the neural-mucosal interface in the OE, and that the site of initial infection is the OB in the CNS.
Fig 4
Viral antigen distribution in the mouse OE and brain during PHEV infection.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV and sacrificed at 1, 3 and 5 dpi, respectively. (A-C) Representative images of immunofluorescence staining for viral antigens in the nasal cavity at 1 dpi (A), 3 dpi (B) and 5 dpi (C). The bottom panels show the magnified images of the dashed rectangles. Scale bars, 500 μm (A-C, top panels), 50 μm (A-C, bottom panels). (D-F) Representative images of immunofluorescence staining for viral antigens in the brains at 1 dpi (D), 3 dpi (E) and 5 dpi (F). Right panels represent the OB (panel 1), cerebral cortex (panel 2), piriform cortex (panel 3), hippocampus (panel 4), brain stem (panel 5), and cerebellum (panel 6). Scale bars, 1,000 μm (D-F, left panels), 30 μm (D-F, right panels). PHEV-N (green), DAPI (blue).
Viral antigen distribution in the mouse OE and brain during PHEV infection.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV and sacrificed at 1, 3 and 5 dpi, respectively. (A-C) Representative images of immunofluorescence staining for viral antigens in the nasal cavity at 1 dpi (A), 3 dpi (B) and 5 dpi (C). The bottom panels show the magnified images of the dashed rectangles. Scale bars, 500 μm (A-C, top panels), 50 μm (A-C, bottom panels). (D-F) Representative images of immunofluorescence staining for viral antigens in the brains at 1 dpi (D), 3 dpi (E) and 5 dpi (F). Right panels represent the OB (panel 1), cerebral cortex (panel 2), piriform cortex (panel 3), hippocampus (panel 4), brain stem (panel 5), and cerebellum (panel 6). Scale bars, 1,000 μm (D-F, left panels), 30 μm (D-F, right panels). PHEV-N (green), DAPI (blue).To further validate this hypothesis, the blood, OE, OB, cerebrum, cerebellum, brainstem, and spinal cord of mock- and PHEV-infected 3w mice were harvested and evaluated by qRT–PCR (Fig 5A–5F). Compared to mock mice (Fig 5A), viral RNA was initially detected in the OE at 1 dpi (Fig 5B), followed by detection in the OB at 2 dpi (Fig 5C), suggesting that the olfactory route may be hijacked by PHEV for rapid neuroinvasion. Comparatively, the cerebrum and brainstem were positive for viral RNA at 3 dpi (Fig 5D). Viral RNA was globally distributed in the whole brain, including the cerebellum, at 4 dpi (Fig 5E) and spread to the spinal cord at 5 dpi (Fig 5F). No viral RNA was detected in blood samples from 1 to 5 dpi (Fig 5A–5F), excluding systemic spread via the blood and access to the CNS via transport across the blood–brain barrier or blood–cerebrospinal fluid barrier. Moreover, the viral antigen was detected in the olfactory nerves that connected the OE and OB (Fig 5G). Therefore, we concluded that PHEV invade the CNS of mice mainly through the olfactory pathway.
Fig 5
PHEV invasion into the CNS via the olfactory nerve.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV and sacrificed at 1–5 dpi. (A-F) Mock and PHEV-infected CNS tissues (including OB, cerebrum, cerebellum, brain stem, and spinal cord), OE, and blood were collected for PHEV N RNA quantification by qRT–PCR (n = 6). The Y axis represents the PHEV N RNA copy number per gram of tissue. (G) Detection of PHEV-positive signals (N, green) in the olfactory nerve (OMP, red) of PHEV-infected 3w mice at 5 dpi. Nuclei stained with DAPI (blue). N, PHEV nucleocapsid protein; OMP, olfactory marker protein; OE, olfactory epithelium; OB, olfactory bulb. Scale bars, 100 μm (left panel), 20 μm (right panel).
PHEV invasion into the CNS via the olfactory nerve.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV and sacrificed at 1–5 dpi. (A-F) Mock and PHEV-infected CNS tissues (including OB, cerebrum, cerebellum, brain stem, and spinal cord), OE, and blood were collected for PHEV N RNA quantification by qRT–PCR (n = 6). The Y axis represents the PHEV N RNA copy number per gram of tissue. (G) Detection of PHEV-positive signals (N, green) in the olfactory nerve (OMP, red) of PHEV-infected 3w mice at 5 dpi. Nuclei stained with DAPI (blue). N, PHEV nucleocapsid protein; OMP, olfactory marker protein; OE, olfactory epithelium; OB, olfactory bulb. Scale bars, 100 μm (left panel), 20 μm (right panel).
The trigeminal nerve is an alternative route for PHEV neuroinvasion
To verify whether there are other neuroinvasion routes other than the olfactory nerve, we chemically destroyed the olfactory nerve endings of the OE by exposing 3w BALB/c mice to zinc sulfate (ZnSO4) or Triton X-100 using previously described methods [35,36]. ZnSO4 destroyed the OE by inducing rapid and selective OSN necrosis [37-39]. Mice in the PBS+PHEV group all died at 6 dpi, but 100% mortality was observed in ZnSO4- and Triton X-100-treated PHEV-infected mice at 11 and 10 dpi, respectively (Fig 6A). This suggested that the chemical treatment delayed the time to death of mice but did not ultimately reduce mortality. We further detected the distribution of the viral antigens in the CNS of 3w mice treated with ZnSO4 for 3 days prior to intranasal PHEV inoculation. Blood, OE, OB, cerebrum, cerebellum, brain stem, and spinal cord were harvested from different mice daily for 7 days and the presence of the viral RNA was detected in these samples by qRT–PCR. Notably, ZnSO4-treated mice were resistant to PHEV neuroinvasion within 4 dpi (Fig 6B). The viral RNA was first detected in the brainstem at 5 dpi (Fig 6C) and throughout the whole brain at 7 dpi (Fig 6D). The data suggested that OE damage impeded PHEV entrance into the CNS via the olfactory nerve, but the olfactory route is not strictly the sole route for PHEV neuroinvasion. We then asked whether PHEV infects the subepithelial trigeminal nerve endings in the respiratory epithelium (RE) of the nose. The detection of viral antigens in the trigeminal nerve and trigeminal ganglion of 3w BALB/c mice provided a direct evidence of trigeminal nerve transmission of PHEV (Fig 7A and 7B). We concluded that in addition to the olfactory route, the subepithelial trigeminal nerve in the RE of the nasal cavity might represent an alternative route for PHEV neuroinvasion.
Fig 6
Chemical treatment delayed the time of animal death.
(A) The 3w BALB/c mice were untreated (n = 10) or intranasally irrigated with 10 μl of PBS (n = 11), ZnSO4 (0.17 M) (n = 16), or a 0.7% Triton X-100 (n = 16) solution in both nostrils daily for 3 days before intranasal inoculation with 103.96 TCID50 PHEV. The survival curves were plotted for these four groups. (B-D) The 3w BALB/c mice were intranasally irrigated with 10 μl of ZnSO4 (0.17 M) in both nostrils 3 days before intranasal inoculation with 103.96 TCID50 PHEV. Mice were euthanized at different time points, and infected CNS tissues, OE, and blood were collected for PHEV N RNA quantification by qRT–PCR. Six mice per time point were analyzed. The Y axis represents the PHEV N RNA copy number per gram of tissue.
Fig 7
The trigeminal nerve is an alternative route for PHEV neuroinvasion.
The 3w BALB/c mice were inoculated with 103.96 TCID50 PHEV by the intranasal route and sacrificed at 5 dpi. (A) Brains from PHEV-infected mice were subjected to immunofluorescence staining using a PHEV-N antibody (red). A magnified image of the trigeminal nerve is shown in the lower panel. (B) Trigeminal ganglions of mock- and PHEV-infected mice were immunofluorescence stained using NSE (green) and PHEV-N (red) antibodies. The right three panels represent magnified images of the area delimited by the dotted box. Arrows indicate cells colocalized with NSE and PHEV-N. DAPI stains nuclei (blue). NSE, neuron-specific enolase.
Chemical treatment delayed the time of animal death.
(A) The 3w BALB/c mice were untreated (n = 10) or intranasally irrigated with 10 μl of PBS (n = 11), ZnSO4 (0.17 M) (n = 16), or a 0.7% Triton X-100 (n = 16) solution in both nostrils daily for 3 days before intranasal inoculation with 103.96 TCID50 PHEV. The survival curves were plotted for these four groups. (B-D) The 3w BALB/c mice were intranasally irrigated with 10 μl of ZnSO4 (0.17 M) in both nostrils 3 days before intranasal inoculation with 103.96 TCID50 PHEV. Mice were euthanized at different time points, and infected CNS tissues, OE, and blood were collected for PHEV N RNA quantification by qRT–PCR. Six mice per time point were analyzed. The Y axis represents the PHEV N RNA copy number per gram of tissue.
The trigeminal nerve is an alternative route for PHEV neuroinvasion.
The 3w BALB/c mice were inoculated with 103.96 TCID50 PHEV by the intranasal route and sacrificed at 5 dpi. (A) Brains from PHEV-infected mice were subjected to immunofluorescence staining using a PHEV-N antibody (red). A magnified image of the trigeminal nerve is shown in the lower panel. (B) Trigeminal ganglions of mock- and PHEV-infected mice were immunofluorescence stained using NSE (green) and PHEV-N (red) antibodies. The right three panels represent magnified images of the area delimited by the dotted box. Arrows indicate cells colocalized with NSE and PHEV-N. DAPI stains nuclei (blue). NSE, neuron-specific enolase.
Axonal transport enables PHEV neural transmission in the CNS
The sequential nature of PHEV distribution in the brain and spinal cord suggests a nonstochastic pathway of neural transmission within the CNS. Three-week BALB/c mice were infected intranasally with PHEV and brains were collected at 5 dpi. Immunofluorescence staining revealed that the N protein of PHEV colocalized with axons, as defined by staining for the marker βIII-tubulin, in several regions of the brain, such as the cerebral cortex and hippocampus (Fig 8A and 8B), suggesting that PHEV may be transported along axons. The localization of PHEV particles and axons was investigated in infected brains using transmission electron microscopy (TEM) to obtain deeper insights into this axonal association. As expected, assembled viral particles were associated with axonal structures (Fig 8C). Collectively, these data suggest that axonal transport may contribute to rapid, transneuronal spread of PHEV from initial sites to connected areas within the CNS.
Fig 8
PHEV N protein and viral particles are associated with axons in vivo.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV. (A-B) Brains were collected at 5 dpi, sagittally sectioned, and subjected to immunofluorescence staining with β III-tubulin (green) and PHEV-N protein (red) antibodies. The nuclei were stained with DAPI (blue). (C) Brains were collected at 5 dpi and analyzed by TEM. Red arrows, microtubules. Yellow arrows, virus particles.
PHEV N protein and viral particles are associated with axons in vivo.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV. (A-B) Brains were collected at 5 dpi, sagittally sectioned, and subjected to immunofluorescence staining with β III-tubulin (green) and PHEV-N protein (red) antibodies. The nuclei were stained with DAPI (blue). (C) Brains were collected at 5 dpi and analyzed by TEM. Red arrows, microtubules. Yellow arrows, virus particles.
PHEV attacks OSNs and causes inflammation in the nasal cavity
In the nasal cavity of infected mice, PHEV-positive cells were scattered in the RE (Fig 9A, panels 1 and 2) and OE (Fig 9A, panels 3 and 4) at 5 dpi. Histopathologically, the PHEV infected RE was largely incomplete, with increased inflammatory cell infiltration and accumulation of luminal cell debris (Fig 9B, panels 1 and 2). Compared with the RE, the integrity of the OE was not significantly damaged, but intraepithelial and submucosal infiltration was also observed in the indicated regions (Fig 9B, panels 3 and 4). These findings indicate that the OE and RE in the nasal mucosa of mice are highly susceptible to PHEV infection.
Fig 9
Viral antigen distribution and histological changes in the nasal cavity.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV, and nose tissue samples were collected at 5 dpi. (A) Immunofluorescence staining of PHEV-N antigens in the RE and OE of the nasal cavity at 5 dpi. Magnified images indicate N-expressing cells in RE (panels 1–2) and OE (panels 3–4). OMP, olfactory marker protein (red); N, PHEV nucleocapsid protein (green). The nuclei were stained with DAPI (blue). Scale bars, 1,000 μm (upper panel), 50 μm (lower panels 1–4). (B) H&E staining of tissue sections showed destruction and inflammatory cell infiltration of the RE (panels 1–2) and OE (panels 3–4) at 5 dpi. The red arrows represent infiltrating inflammatory cells. Scale bars, 1,000 μm (upper panel), 50 μm (lower panels 1–4).
Viral antigen distribution and histological changes in the nasal cavity.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV, and nose tissue samples were collected at 5 dpi. (A) Immunofluorescence staining of PHEV-N antigens in the RE and OE of the nasal cavity at 5 dpi. Magnified images indicate N-expressing cells in RE (panels 1–2) and OE (panels 3–4). OMP, olfactory marker protein (red); N, PHEV nucleocapsid protein (green). The nuclei were stained with DAPI (blue). Scale bars, 1,000 μm (upper panel), 50 μm (lower panels 1–4). (B) H&E staining of tissue sections showed destruction and inflammatory cell infiltration of the RE (panels 1–2) and OE (panels 3–4) at 5 dpi. The red arrows represent infiltrating inflammatory cells. Scale bars, 1,000 μm (upper panel), 50 μm (lower panels 1–4).We subsequently characterized the cells that were attacked in the OE and found that PHEV N protein was localized in OMP-expressing OSNs (Fig 10A), suggesting that PHEV specifically infected olfactory neurons. Furthermore, neural cell adhesion molecule (NCAM), a potential cell interacting partner of PHEV [22,23], was detected in the mouse OE using immunofluorescence staining, and most NCAM-expressing cells were colocalized with OMP-expressing OSNs (Fig 10B). Direct infection and virus-induced inflammatory responses potentially both lead to tissue damage and olfactory dysfunction. We then tested the effect of PHEV infection on the OE, and significant induction of a proinflammatory environment was observed (Fig 10C). Specifically, IL-1β levels were significantly increased as early as 1 dpi, while increases in IL-6 and CXCL10 mRNA levels were observed only at 3 and 5 dpi. CCL5, TNF-α, IFN-α, IFN-β, and IFN-γ were not significantly activated until 5 dpi compared with mock-infected mice (0 dpi). Consistently, immunohistochemical staining revealed a large number of IBA1-positive macrophages in the OE of PHEV-infected mice but few to no IBA1-positive cells in the OE of control mice (S4B and S4E and S4H Fig). Therefore, we demonstrated that PHEV exhibits strong tropism for OE and that CNS infection is associated with the inflammatory response of OE.
Fig 10
Cell target and inflammatory responses in the OE of PHEV-infected mice.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV, and nose samples were collected at indicated times. (A) Immunofluorescence staining of PHEV-N (green) and olfactory neuron marker OMP (red) in OE at 5 dpi. Magnified images (panels 1–4) show the colocalization of N with OMP-expressing OSNs. (B) Immunofluorescence staining of NCAM (green) and olfactory neuron marker OMP (red) in the mice OE at 5 dpi. NCAM, neural cell adhesion molecule. (C) Expression of IL-1β, IL-6, CXCL10, CCL5, TNF-α, IFN-α, IFN-β, and IFN-γ mRNA in homogenized nasal turbinate tissues was determined by qRT–PCR. Three mice per time point were analyzed. Data are normalized to GAPDH and presented as fold changes in expression relative to mock (0 dpi) and shown as the means ± SD. P values were determined by one-way ANOVA.
Cell target and inflammatory responses in the OE of PHEV-infected mice.
The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV, and nose samples were collected at indicated times. (A) Immunofluorescence staining of PHEV-N (green) and olfactory neuron marker OMP (red) in OE at 5 dpi. Magnified images (panels 1–4) show the colocalization of N with OMP-expressing OSNs. (B) Immunofluorescence staining of NCAM (green) and olfactory neuron marker OMP (red) in the mice OE at 5 dpi. NCAM, neural cell adhesion molecule. (C) Expression of IL-1β, IL-6, CXCL10, CCL5, TNF-α, IFN-α, IFN-β, and IFN-γ mRNA in homogenized nasal turbinate tissues was determined by qRT–PCR. Three mice per time point were analyzed. Data are normalized to GAPDH and presented as fold changes in expression relative to mock (0 dpi) and shown as the means ± SD. P values were determined by one-way ANOVA.
Dissemination of PHEV to the brain drives neuroinflammation
Because PHEV-infected mice exhibit olfactory dysfunction and rapid anterograde PHEV neuroinvasion via the olfactory route, we examined the viral distribution and the inflammatory response in the OB at 5 dpi. PHEV invades many regions in the OB, including the glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), internal plexiform layer (IPL), and granular cell layer (GCL) (Fig 11A). Furthermore, the number of IBA1-positive macrophages/microglial cells increased significantly in the OB of PHEV-infected mice (S4C and S4F and S4I Fig). The high viral loads in the OB, together with macrophage/microglial cell infiltration, suggest that PHEV infection induces a bulbar inflammatory response.
Fig 11
Differentially expressed genes screened in the OB of PHEV-infected mice.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV. (A) At 5 dpi, mice were euthanized and OB samples were collected for immunofluorescence staining with antibodies against OMP (green) and PHEV-N (red). PHEV-positive cells were widely distributed in the OB, especially in the MCL and GCL. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, granular cell layer. (B-C) Differentially expressed genes in the OB of PHEV-infected mice at 5 dpi were analyzed by RNA-seq. Scatter plot of biological processes identified in the GO enrichment analysis. The vertical axis represents the functional annotation information, and the horizontal axis represents the Rich factor corresponding to the function. (Only the 30 most enriched KEGG and GO terms are plotted for the significantly differentially expressed gene set).
Differentially expressed genes screened in the OB of PHEV-infected mice.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 PHEV. (A) At 5 dpi, mice were euthanized and OB samples were collected for immunofluorescence staining with antibodies against OMP (green) and PHEV-N (red). PHEV-positive cells were widely distributed in the OB, especially in the MCL and GCL. GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GCL, granular cell layer. (B-C) Differentially expressed genes in the OB of PHEV-infected mice at 5 dpi were analyzed by RNA-seq. Scatter plot of biological processes identified in the GO enrichment analysis. The vertical axis represents the functional annotation information, and the horizontal axis represents the Rich factor corresponding to the function. (Only the 30 most enriched KEGG and GO terms are plotted for the significantly differentially expressed gene set).Next, an RNA sequencing (RNA-seq) analysis was conducted to elucidate the characteristics of differentially expressed genes in the OB of PHEV-infected mice. An analysis of enriched Gene Ontology (GO) terms indicated that the 3542 upregulated genes were mainly associated with immune and inflammatory responses, including innate immunity (Toll-like and NOD-like receptor signaling pathway, NF-κB and Jak-STAT signaling pathway, cytokine–cytokine interaction, complement, and coagulation cascades) and adaptive immunity (antigen processing and presentation, T and B cell receptor signaling pathway, and natural killer cell-mediated cytotoxicity) (Figs 11B and S5). The 1174 downregulated genes were mainly involved in nervous system functions, including neuronal development and differentiation, synaptic signaling, neurotransmitter transport, and anterograde transsynaptic signaling (Figs 11C and S5). These data indicate that PHEV infection induces a neuroinflammatory response and neuronal dysfunction in the OB, which may be responsible for anosmia.
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% CO2. 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 TCID50 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.
Table 1
Clinical scoring system used for PHEV-infected mice.
Category
Score = Criteria
Body weight
1 = 10–19% of body weight loss
Respiration
1 = rapid, shallow, polypnea
Appearance
1 = ruffled fur, hunched posture
Responsiveness
1 = low to moderate movement, paralysis
Neurologic signs
1 = tremors, ataxia, circling, and seizures
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 ZnSO4 (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 ΔΔCT 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% CO2 for 1 h. After removing the inocula, DMEM containing 2% FBS was added to the plates for 3 days. The TCID50 was calculated by the method of Reed-Muench and presented as TCID50/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.
Sex-independent clinical symptoms of mice after PHEV infection.
Male and female 3w and 6w BALB/c mice were intranasally inoculated with 103.96 TCID50 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.(TIF)Click here for additional data file.
Visible food finding test.
The 6w male and female BALB/c mice were intranasally inoculated with 103.96 TCID50 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.(TIF)Click here for additional data file.
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 TCID50 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.(TIF)Click here for additional data file.
Inflammatory cell accumulation in the OE and OB of PHEV-infected mice.
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 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.(TIF)Click here for additional data file.
Differentially expressed genes (DEGs) in the OB identified by transcriptomic sequencing (RNA-seq).
The 3w BALB/c mice were inoculated intranasally with 103.96 TCID50 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.(TIF)Click here for additional data file.
Distribution of viral antigen in the brains of PHEV-infected mice.
(DOCX)Click here for additional data file.
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.
(DOCX)Click here for additional data file.17 Mar 2022Dear Dr. He,Thank you very much for submitting your manuscript "PHEV infection: a promising model of betacoronavirus-associated neurological and olfactory dysfunction" (PPATHOGENS-D-22-00231) for review by PLOS PATHOGENS. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time. We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.We are returning your manuscript with three reviews. All Reviewers acknowledged that the manuscript contains a comprehensive set of new and interesting data, but raised concerns towards the context of the study as it is currently presented and the imprecise description of the experimental design. Currently, the experimental design and the rationale behind this design is not always clear. This includes f.e. the rationale to initially perform experiments in 3 and 6 week old mice, and in the latter experiments only 3 week old mice (Reviewer 2) as well as the methodological choices and descriptions (reviewer 1 & 3). In addition, the number of infected mice should be clear for each set of data, as well as the time post inoculation for the presented data (this is unclear in f.e. Figure 2).In addition, the current manuscript is written in the context of SARS-CoV-2, and how studying the pathogenesis of PHEV could provide insights into the pathogenesis of SARS-CoV-2 infection. However, the reviewers agree that extrapolation of the acquired data to SARS-CoV-2 infection is too far-fetched. There is no side-by-side comparison between PHEV and SARS-CoV-2 in the experimental design, nor in the discussion based on previously published papers. Despite some overlap in the route that both PHEV and SARS-CoV-2 can use to enter the CNS, the cell tropism and ability to spread throughout the CNS differs largely.We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Debby van RielGuest EditorPLOS PathogensRon FouchierSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens
orcid.org/0000-0002-7699-2064
***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: In this manuscript by Shi et al, authors utilize the BALB/c mouse model to investigate the infectivity and tropism of a porcine betacoronavirus (PHEV) in neurological and olfactory-associated tissues. Authors conduct several behavioral studies to measure changes in CNS-related functions in infected mice, and extensively employ immunohistochemistry-based assessments to identify specific tissue structures post-inoculation that likely contribute to these in vivo readouts. They identify specific nerves targeted by PHEV infection and link viral replication at these sites with pathogenic responses. The study appears generally well-controlled, though there are places in the text that make it difficult to follow what exactly is being shown. The manuscript is generally well-referenced and logically organized, with a few exceptions. However, the authors heavily interpret their results in the context of SARS-CoV-2 infection, at the expense of focusing on what their findings mean in the context of PHEV infection. Overall there are several areas throughout the manuscript that warrant improvement.Reviewer #2: In the manuscript by Shi et al the authors investigate the neuroinvasion routes for the betacornavirus called Porcine hemagglutingating encephalomyelitis virus (PHEV) using a BALB/c murine system. Their work provides mechanistic understanding regarding PHEV neuroinvasion and neurotropism. The main findings are that the virus can invade the CNS via the olfactory epithelium/olfactory nerve, in addition to via the trigeminal nerve. The findings are convincing and the studies are well executed. This work adds to our understanding of PHEV and its ability to invade the CNS. However, the authors do not discuss how this work relates to PHEV pathogenesis, despite this virus being an important agricultural pathogen and a major source of economic loss on many farms (Mora-Diaz, et al; Front Vet Sci. 2019 doi: 10.3389/fvets.2019.00053). Rather, these data are postulated to represent a low containment model to study SARS-CoV-2 neuropathogenesis. Here the authors also fall short because they do not adequately related their findings to relevant SARS-CoV-2 models, most importantly the hamster models that have provided insight into anosmia and brain infection that is observed in humans. The manuscript also needs a clear discussion pertaining to the limitations of the surrogate PHEV model as it relates to SARS-CoV-2.Reviewer #3: The manuscript „PHEV infection: a promising model of betacoronavirus-associated neurological and olfactory disfunction” by Shi et al. is describing at mouse model to study the neuroinvasion and neurotropism about PHEV. The authors give a detailed description about pathogenicity and time course of infection and give a great overview of the mechanism of CNS infection of PHEV. Although this manuscript presents a comprehensive set of new data, the description of experiments and the interpretation of results is making it hard for the reader to follow the thoughts of the authors.**********Part II – Major Issues: Key Experiments Required for AcceptancePlease use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: -The majority of the introduction and discussion sections pertain to SARS-CoV-2 infection, not PHEV infection. It is understandable that this study with PHEV was undertaken to provide contextual information to SARS-CoV-2 infection, but without side-by-side data showing that SARS-CoV-2 and PHEV infection target similar tissues or elicit comparable responses, it’s difficult to use the PHEV model established here as a surrogate virus to directly inform SARS-CoV-2/COVID-19 studies. What receptor(s) does PHEV employ to infect CNS-related cells, what is their relative distribution on the specific CNS-associated tissues employed in this study, and are SARS-CoV-2 receptors present on the same tissues in permissive species? This is all critical information that appears to be missing or underemphasized within the text. While SARS-CoV-2 cannot infect BALB/c mice (limiting side-by-side assessments of both viruses in the same model), providing some scope of data regarding which mammalian models would be appropriate for employing any subsequent results obtained in the PHEV mouse model discussed here seems appropriate; without this context and additional information, the authors should temper their applicability of PHEV results to SARS-CoV-2 infection studies.-The methods section is lacking the humane endpoints employed in this study (were mice euthanized once they reached specific endpoint cutoffs)? The survival curves presented are difficult to interpret without understanding which specific criteria (weight loss, lethargy, specific neurological symptoms, etc) necessitated this euthanasia. It is also unclear as currently written if the mice that succumbed to infection in Fig 4A (with delays in death relative to Figure 1B) were due to the same manifestation of endpoint criteria or if the prolonged course of infection resulted in additional sequalae to develop. The method of humane euthanasia also needs to be disclosed (chemical, physical, etc).-As described in the text, Figure 1 purports to show differences between mock-infected and PHEV-infected mice, but many of the display images (1D-O) only appear to show breakdowns between male and female mice, and not infected vs mock. Figure legend for Fig 1 also states that bars represent mean + SD, but SD does not appear to be included in panels E-F, making it difficult for the reader to understand if the bars represent a different parameter or not.Reviewer #2: 1. While the authors cite the hamster model papers that examined SARS-CoV-2 in the olfactory epithelium and brain infection (de Melo at al), there is virtually no discussion about the hamster SARS-CoV-2 model or these cited studies. The hamster system is extensively used for SARS-COV-2 research, including infection in the olfactory cavity. I think this should be at least a paragraph in the discussion addressing the hamster models (and other models) and a comparison to their results. Ideally, the authors should consider a table showing how the PHEV BALB/c model and the hamster models relate to what has been identified in humans vis-a-vis anosmia and infection of the olfactory bulb.2. There appears to be no lung involvement in the PHEV/BALB/c system. Some evidence suggests that SARS-COV-2 can usurp the vagus nerve subsequent to lung infection to invade the CNS (Bulfamante, G et al J. Neurol; 10.1007/s00415-021-10604-8). Does a lack of respiratory involvement impair the in the PHEV system as a surrogate for SARS-CoV-2 neuroinvasion? This should be considered in a limitations paragraph along with other pros and cons of the PHEV surrogate model.3. The introduction and discussion should clearly and conspicuously address the agricultural disease caused by PHEV, including the fact that it is an important pathogen. How the author’s data relates to what is known about this disease should also be added to the discussion in its own paragraph. The authors may want to consider reformatting the manuscript as a PHEV pathogenesis paper, then mention its potential use as a surrogate SARS-COV-2 system in the dissuasion.4. The discussion mentions the potential involvement of ACE2 binding as being important for loss of taste, etc. However, I don’t believe that PHEV binds ACE2, rather it binds sialic acid and potentially the Neural Cell Adhesion Molecule. If binding to ACE2 were critical for SARS-CoV-2 neuroinvasion, but PHEV didn’t use this receptor, it would make this a poor model to mimic COVID-19. The authors need to explicitly state the receptor that PHEV uses and then modify the discussion as to how this may be a limitation to this model.5. It isn’t clear why the authors used 3 and 6 week old mice initially, then changed over to just 3 week old mice for subsequent studies. 6 week old mice would be preferred as these are adult mice. Is there a reason to only have used 3 week old mice?Reviewer #3: - I am missing a consistency of methodological workup of samples in terms of titration and PCR. For some samples, titrations are performed to quantify infectious virus, for some not. Either explain your workflow or keep it consistent throughout all experiments- It is very hard to follow which animals (gender, age) and how many (n=?) were used for the different experiments. It seems as if a lot of different sub-experiments were performed with partially different conditions. It would be ideal for reader to follow the workflow and thoughts of the authors if a graphical experimental setup or a table would give an overview of those aspects.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: -Abstract and author summary require editing for English prose (furthermore, use of “biosafety” and “biosafe” in the abstract do not make logical sense as current written). There are also some passages within the main text where phrasing is too colloquial (e.g. line 100, “attractive”, line 152 “suffering mice”).-anosmia and ageusia should be better defined in the author summary as this is intended for a more general audience.-lines 127-8, what additional “neurological complications” are being alluded to outside of anosmia and ageusia?-Authors should be commended for employing both male and female mice of two ages in this study, however, it is unclear why authors elected to use the 3 week vs the 6 week old mice in different studies. Was there a research purposes for choosing one age over another in selected studies (e.g. studies shown in Figure 3, line 147) or were findings interchangeable? If there was a reason, the authors should better disclose this for clarity. There are also places the age of mice employed is not clearly stated (e.g. Figure 5 legend)-Figure 4, it is unclear if the data presented is from ZnSO4-treated mice or not (e.g. Figure 4B, the text implies that the treated mice are being shown, but this is not specified in the legend text, and then panels 4C-F seem to imply that what is being shown is from non-treated mice). If ZnSO4-treated mice are being shown, showing both treated- and untreated- mice here would seem appropriate so that the reader can better appreciate differences that are specifically attributed to the destroyed olfactory nerve endings induced by ZnSO4.-Figure 5D, what is fold induction relative to? Uninfected mice? The methods, figure legend, or text do not specify this. It would be beneficial if baseline quantities of each analyte examined were disclosed in the legend or methods so that the reader could better understand the relative expression (and magnitude of change when appropriate) for each.Reviewer #2: 1. line 26, “a biosafety betaconronavirus” does not make sense. This should be reworded. Maybe “non-human pathogenic..”?2. Line 41-43. “...utilized as a protoype to recapitulate neurological and olfactory dysfunction in a subset of COVID-19 involvement under...” should be reworded. Perhaps “....olfactory dysfunction observed in human SARS-COV-2 infections?3. Line 106. The strain of virus used should be added to be consistent (CC14?).4. The method to disrupt the olfactory epithelium via TritonX and ZnSO4 needs to be explicitly in the methods section, not merely a reference. One of the papers referenced, also neglected to put the method in the paper and only referenced another paper.5. Line 249. “absence of a decreased viral load”. It is unclear what they are trying to convey with this statement. Please clarify.6. The paper should reference Reyna, RA et al “Recovery of anosmia in hamsters infected with SARS-CoV-2 is correlated with repair of the olfactory epithelium” Scientific reports doi.org.10/1038/S41598-021-04622-9. The findings should incorporated into the discussion.7. The authors should look at a recent Trends in Neuroscience paper “The neuroinvasiveness, neurotropism and neurovirulence of SARS-CoV-2”. This may help as they revamp the discussion to talk about the hamster models and how these rodent systems relate to neuroinvasiveness of SARS-CoV-2. doi.org/10.1016/j.tins.2022.02.006.Reviewer #3: - Revise English language! Especially the abstract and author summary need to be revised by a native speaker. Those two paragraphs are nearly impossible to understand due discrepancies in English language. It is getting better within the main text. However, I recommend a complete revision by a native English speaker- Throughout all figure captions be consistent in the description of your animal groups. Sometimes the age of mice is specified, sometimes not.- The authors are generally describing a set of very interesting and valuable data for this field of research! However, the lack of a general overview and clear experimental strategy is partially making it hard to follow.Specific comments:l.26: “biosafety betacoronavirus” is no scientifically correct term. It is a BSL-2 betacoronavirusl.28: what is meant with “pathological test”? Please find a specific term. This leads to missunderstandingsl.34: I do not see the aspect of “low cost” being of such importance that it needs to be stated in the abstract. Money shouldn’t be a justification for the value and applicability of an animal modell.61: OE is susceptible to SARS-CoV-2 infection and not the other way aroundl.83: It is the order of Nidovirales (delete order in Nidoviralesorder)ll.90-101: the whole paragraph describes your results. This is an extensive description of results for an introduction. Please keep it shorter and only give an overview of key findings.l.106: and throughout the whole manuscript: Don’t write 20µl of 10^4.66 TCID50/0.1ml but give a precise titer per animal! Since you know the volume and titer/volume and both are consistent for all animals, calculate the titer/animal.l.107: Day of 100% mortality does vary between the two age groups. Should be mentioned here.ll.111: No differences between sexes but between ages. This finding should be mentioned here or above.ll.112: In the figure caption it is described that only 6 week old mice were used for this test. Also for the description of the animal experiments, is there a reason to do some experiments with both age groups, some with 6 week old, some with 3 week old mice? Rational should be explained somewhere.l.132: Use another word than “propagated”. Which part of the brain?ll.140: Figure S2 is mentioned here, but not that it also shows that you observed no changes in peripheral tissues of infected mice. This observation with referring to Figure S2 should be included accordingly.l.147: Again, why now 3 week old mice?l.151: Was that the nasal cavity also screened in this time course after infection (as described in table S1)? I think this table and the Figure 3A,B are highly interesting and can elucidate mechanisms of neuroinvasion. To have a complete picture, it would be perfect if the nasal turbinates would be included in the time course after infection.l.152: Use another word than “suffering”l.165: You observe reduced mortality but still the same neurological symptoms? Were functional assays (burying etc.) also performed with these groups?l.166: You describe RNA data, which says nothing about infectious virus. Please re-phrase.ll.222: This statement is not true. Syrian golden hamsters are currently the most widely used animal model for SARS-CoV-2 and also show neuroinvasion to a certain extent. Please also discuss the advantage of your model compared to the SARS-CoV-2 hamster modelll.227: To me this hypothesis mostly seems to be based on extrapolation. How can a model with another pathogen be used to study efficacy of therapeutic interventions? No direct comparions were performed that can proof the applicabilty of the model in this respect. Consider toning down the use of your model in this respect.ll.291: What is the specific use for this model in the future?ll.308: How were the polyclonal antibodies prepared? Either refer to previous publications or describe more details here. Were animals immunized? Which animals, which antigen, including adjuvants etc.?ll.318: Please be more precise about the use of animals and different sub-experiments. It is very confusing to get an overview which and how many animals were used to answer the different questions, addressed by different experiments. See also general commentl.634: Please change “viral loads” to “viral genome loads”l.635: Why is it n=4 for titrations and n=6 for PCR?l.644: Sacrificed at what day post infection?l.654: What is the added value of the PBS group compared to the untreated animals? Why do all groups have different group sizes?l.656: Which of the above mentioned groups is subset (B) describing? And How can it be 6 mice per time point (3 in total) if one treatment group consists of maximum 16 animals?ll.657: Which animals are described in (C) and (D)?l.691: n=7 is described here, whereas in caption of Figure 1 it is n=5. Or are these separate experiments?Figure 1 D-L: I am missing the mock-controls or are they reflected in the 0 dpi data? Don’t you assume a behavioral adaptation of animals over time and wouldn’t it be ideal to include measurements for mock-controls at all time points?Figure 2 A,B: Use a Log10 y-axis for both graphsFigure 3 A,B: Very impressive images. Was this also done for the 1 dpi time point and for the nasal cavity?**********PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: NoReviewer #3: NoFigure Files:While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .Data Requirements:Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Reproducibility:To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols11 May 2022Submitted filename: Response to reviewers-20220510lz.docxClick here for additional data file.2 Jun 2022Dear Dr. He,Thank you very much for submitting your manuscript "PHEV infection: a promising model of betacoronavirus-associated neurological and olfactory dysfunction" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by an independent reviewer. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the editors recommendations below.The authors have addressed the reviewers' comments concerning the experimental design and the presentation of the data accurately. However, the comments concerning the interpretation of the results in the context of SARS-CoV-2 infection are only partly addressed. Although the introduction and abstract have been adjusted, the authors summary and discussion are still largely written in the context of SARS-CoV-2. In a new version of the manuscript this should be adjusted.1. In the authors summary the focus is the usefulness of PHEV infections in mice as a model to study the pathogenesis neurological complications of SARS-CoV-2. Please modify this according to the previous comments of the reviewers.2. The discussion is too long and too much focused on SARS-CoV-2. In addition, it lacks a thorough discussion on the limitations of experimental PHEV infection in mice as a model for SARS-CoV-2 associated CNS complications. It is for example not included that there is a large difference in the ability of SARS-CoV-2 and PHEV to spread throughout the CNS. Although SARS-CoV-2 virus antigen/RNA is occasionally detected in the CNS of humans (or relevant animal models), it does NOT replicate/spread efficiently throughout the CNS causing a lethal encephalitis. This is an crucial difference, and for this reason the mechanism of SARS-CoV-2 associated neurological disease (including anosmia and ageusia) are likely very different.Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all r comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).mportant additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Debby van RielGuest EditorPLOS PathogensRon FouchierSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064*********************************While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.Data Requirements:Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Reproducibility:To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsReferences:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.5 Jun 2022Submitted filename: Response letter-R2-PLOS Pathogens-20220605.docxClick here for additional data file.10 Jun 2022Dear Dr. He,We are pleased to inform you that your manuscript 'PHEV infection: a promising model of betacoronavirus-associated neurological and olfactory dysfunction' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Debby van RielGuest EditorPLOS PathogensRon FouchierSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens
orcid.org/0000-0002-7699-2064
***********************************************************22 Jun 2022Dear Dr. He,We are delighted to inform you that your manuscript, "PHEV infection: a promising model of betacoronavirus-associated neurological and olfactory dysfunction," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064
Authors: Mathieu Dubé; Alain Le Coupanec; Alan H M Wong; James M Rini; Marc Desforges; Pierre J Talbot Journal: J Virol Date: 2018-08-16 Impact factor: 5.103
Authors: Carly G K Ziegler; Samuel J Allon; Sarah K Nyquist; Ian M Mbano; Vincent N Miao; Constantine N Tzouanas; Yuming Cao; Ashraf S Yousif; Julia Bals; Blake M Hauser; Jared Feldman; Christoph Muus; Marc H Wadsworth; Samuel W Kazer; Travis K Hughes; Benjamin Doran; G James Gatter; Marko Vukovic; Faith Taliaferro; Benjamin E Mead; Zhiru Guo; Jennifer P Wang; Delphine Gras; Magali Plaisant; Meshal Ansari; Ilias Angelidis; Heiko Adler; Jennifer M S Sucre; Chase J Taylor; Brian Lin; Avinash Waghray; Vanessa Mitsialis; Daniel F Dwyer; Kathleen M Buchheit; Joshua A Boyce; Nora A Barrett; Tanya M Laidlaw; Shaina L Carroll; Lucrezia Colonna; Victor Tkachev; Christopher W Peterson; Alison Yu; Hengqi Betty Zheng; Hannah P Gideon; Caylin G Winchell; Philana Ling Lin; Colin D Bingle; Scott B Snapper; Jonathan A Kropski; Fabian J Theis; Herbert B Schiller; Laure-Emmanuelle Zaragosi; Pascal Barbry; Alasdair Leslie; Hans-Peter Kiem; JoAnne L Flynn; Sarah M Fortune; Bonnie Berger; Robert W Finberg; Leslie S Kean; Manuel Garber; Aaron G Schmidt; Daniel Lingwood; Alex K Shalek; Jose Ordovas-Montanes Journal: Cell Date: 2020-04-27 Impact factor: 41.582
Authors: Ludovico Cantuti-Castelvetri; Ravi Ojha; Liliana D Pedro; Minou Djannatian; Jonas Franz; Suvi Kuivanen; Franziska van der Meer; Katri Kallio; Tuğberk Kaya; Maria Anastasina; Teemu Smura; Lev Levanov; Leonora Szirovicza; Allan Tobi; Hannimari Kallio-Kokko; Pamela Österlund; Merja Joensuu; Frédéric A Meunier; Sarah J Butcher; Martin Sebastian Winkler; Brit Mollenhauer; Ari Helenius; Ozgun Gokce; Tambet Teesalu; Jussi Hepojoki; Olli Vapalahti; Christine Stadelmann; Giuseppe Balistreri; Mikael Simons Journal: Science Date: 2020-10-20 Impact factor: 47.728
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6
Fig 7
Fig 8
Fig 9
Fig 10
Fig 11