TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection.

Jawahar Raina
TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection.
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ABSTRACT

Transmembrane serine protease TMPRSS2 activates the spike protein of highly pathogenic human coronaviruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV). In vitro, activation induces virus-cell membrane fusion at the cell surface. However, the roles of TMPRSS2 during coronavirus infection in vivo are unclear. Here, we used animal models of SARS-CoV and MERS-CoV infection to investigate the role of TMPRSS2. Th1-prone C57BL/6 mice and TMPRSS2-knockout (KO) mice were used for SARS-CoV infection, and transgenic mice expressing the human MERS-CoV receptor DPP4 (hDPP4-Tg mice) and TMPRSS2-KO hDPP4-Tg mice were used for MERS-CoV infection. After experimental infection, TMPRSS2-deficient mouse strains showed reduced body weight loss and viral kinetics in the lungs. Lack of TMPRSS2 affected the primary sites of infection and virus spread within the airway, accompanied by less severe immunopathology. However, TMPRSS2-KO mice showed weakened inflammatory chemokine and/or cytokine responses to intranasal stimulation with poly(I·C), a Toll-like receptor 3 agonist. In conclusion, TMPRSS2 plays a crucial role in viral spread within the airway of murine models infected by SARS-CoV and MERS-CoV and in the resulting immunopathology.

IMPORTANCE Broad-spectrum antiviral drugs against highly pathogenic coronaviruses and other emerging viruses are desirable to enable a rapid response to pandemic threats. Transmembrane protease serine type 2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the coronavirus spike protein, making it a potential therapeutic target for coronavirus infections. Here, we examined the role of TMPRSS2 using animal models of SARS-CoV and MERS-CoV infection. The results suggest that lack of TMPRSS2 in the airways reduces the severity of lung pathology after infection by SARS-CoV and MERS-CoV. Taken together, the results will facilitate development of novel targets for coronavirus therapy.

KEYWORDS: MERS-CoV, SARS-CoV, TMPRSS2, animal model, immunopathology

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INTRODUCTION

Highly pathogenic human coronaviruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV) (1,–6) and Middle East respiratory syndrome-related coronavirus (MERS-CoV) (7,–9) cause severe infection of the lower respiratory tract in humans. These zoonotic pathogens have mortality rates of >50% in aged and immunosuppressed populations, making them potentially important emerging pathogens (10, 11). Broad-spectrum antiviral drugs against these coronaviruses (and other highly pathogenic viruses) will facilitate rapid responses to pandemic threats. Transmembrane protease serine type 2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the influenza virus hemagglutinin (HA) molecule in human airway epithelial cells (12); however, it can also cleave coronavirus fusion glycoproteins, namely, the spike protein. The protease activates the spike protein to induce virus-cell membrane fusion at the cell surface and facilitate entry of coronaviruses into the host cell (13,–16). Thus, active-site inhibitors of TMPRSS2 are potential therapeutic targets for not only influenza viruses but also coronaviruses (17). Some animal studies show that TMPRSS2-knockout (KO) mice are protected against severe pathology and death after influenza virus infection (18,–21). In addition, a genetic study revealed that those with high expression of certain TMPRSS2 variants are at increased risk of severe outcomes after infection with A (H1N1) pdm09 influenza (22). However, the roles of TMPRSS2 in vivo during coronavirus infection are unclear. Here, we used animal models of coronavirus infection to examine the role of TMPRSS2.

Previously, we established a murine model of SARS based on adult BALB/c mice. The animals were moribund due to severe pulmonary edema caused by skewing the immune response toward a Th2 profile after infection by mouse-adapted SARS-CoV (23, 24). We used adult C57BL/6 mice because the TMPRSS2-KO mice were backcrossed to this strain (20). After infection with mouse-adapted SARS-CoV, Th1-prone C57BL/6 mice developed acute pneumonia, with around 15% body weight loss; however, this was not fatal. In addition, we recently generated an animal model of MERS-CoV using transgenic mice expressing human DPP4 (hDPP4-Tg mice) under the control of an endogenous promoter (25). The hDPP4-Tg mice were susceptible to infection by MERS-CoV and developed acute pneumonia with transient loss of body weight. Next, we generated TMPRSS2-KO hDPP4-Tg (TMPRSS2-KO Tg) mice by crossing male hDPP4-Tg mice with female TMPRSS2-KO mice.

Here, we used these animal models to demonstrate a role for TMPRSS2 during infection by SARS-CoV and MERS-CoV. TMPRSS2-deficient mice showed reduced body weight loss and viral replication in the lungs. In addition, histopathological and immunohistochemical analyses revealed that expression of TMPRSS2 influenced both the primary site of infection and virus spread within the airways of both mouse models, which was accompanied by different immunopathologies.

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RESULTS

 

TMPRSS2-KO mice show no body weight loss and weak proinflammatory responses after SARS-CoV infection.

To screen the generated TMPRSS2-KO mice, we confirmed the absence of the TMPRSS2 gene by PCR analysis using a primer set specific for TMPRSS2 (Fig. 1). To examine the effect of TMPRSS2 expression during SARS-CoV infection, we infected C57BL/6 wild-type (WT) and TMPRSS2-KO mice with 105 50% tissue culture infective doses (TCID50) of F-musX, a mouse-adapted SARS-CoV. WT mice showed clear loss of body weight from 2 to 4 days postinfection (p.i.) but recovered later (the exception was a single moribund mouse at day 5 p.i.); these symptoms were not observed in TMPRSS2-KO mice (Fig. 2a). Measurement of the virus titer showed lower viral replication in the lungs of TMPRSS2-KO mice (Fig. 2b). There were no significant differences in the titers of neutralizing antibodies in serum samples from either group (Fig. 2c).

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FIG 1

Genotyping of C57BL/6 (WT), TMPRSS2-KO (KO), hDPP4-Tg (Tg), and TMPRSS2-KO hDPP4 (KO-Tg) mice by PCR analysis. PCR analysis was performed on the genomic DNA from ear punches taken from WT (WT, 4 to 5 weeks old; n = 3 male mice), TMPRSS2-KO (4 to 5 weeks old; n = 3 male mice), hDPP4-Tg (4 to 5 weeks old; n = 3 [male, 1; female, 2]), and TMPRSS2-KO Tg (4 to 5 weeks old; n = 3 [male, 1; female, 2] mice), and the resulting products (391 bp for hDPP4 and 388 bp for TMPRSS2) are shown. Numbers indicate positions of the 500-bp molecular weight marker ladder. M, 100-bp ladder; P, positive controls for hDPP4 or TMPRSS2; N, negative control without the ear punch template.

 

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FIG 8

Immune responses after intranasal inoculation of mice with poly(I·C). Concentrations of inflammatory cytokines and chemokines in the lungs at 24 h postinfection are shown. The assays were done using unicate samples per animal. Numbers of animals per group were as follows: (a) WT, n = 4 per group (male, 2; female, 2); TMPRSS2-KO (KO), n = 4 per group (male, 2; female, 2). (b) hDPP4-Tg (Tg), n = 4 per group (male, 2; female, 2); TMPRSS2-KO Tg (KO-Tg), n = 4 per group [with poly(I·C): male, 2; female, 2; with PBS: male, 1; female, 3]. Mice 14 to 16 weeks old were used. Error bars represent standard errors. P values for the graph were calculated by ANOVA (*, P < 0.05).

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DISCUSSION

Mouse models of SARS-CoV and MERS-CoV infection allow us to investigate disease pathogenesis and vaccine applications and to evaluate antiviral drugs and other therapies. hDPP4-Tg mice are susceptible to infection by a MERS-CoV isolate, resulting in acute pneumonia but no brain disease (25). Here, we generated a TMPRSS2-KO mouse bearing hDPP4. After infection with SARS-CoV or MERS-CoV, TMPRSS2-deficient mice were protected from body weight loss. The results suggest that TMPRSS2 plays an important role in the early phase of disease (lung infection); in particular, SARS-CoV and MERS-CoV replicated in the bronchioles.

In humans, TMPRSS2 is expressed widely in epithelial tissues, including those lining the upper airways, bronchi, and lung (30). The protein sequence of human and mouse TMPRSS2 is conserved, with 78% sequence identity between the two species. In situ hybridization analyses of mouse embryos and adult tissues reveal that TMPRSS2 is expressed in the epithelia lining the gastrointestinal, urogenital, and respiratory tracts, including the bronchi and bronchioles, but not in alveolar epithelium (31). Kim et al. showed that depletion of TMPRSS2 (the molecule was inactivated by disrupting the serine protease domain through homologous recombination) from mice did not affect development or survival to adulthood, nor were there abnormalities in organ histology or function (32). While the physiological function of TMPRSS2 is unclear, data suggest that it does regulate sodium currents in lung epithelial cells through proteolytic cleavage of the epithelial sodium channel (33). We also made the interesting observation that TMPRSS2-deficient mice show weaker, or delayed, inflammatory chemokine and cytokine responses mediated by Toll-like receptor 3.

Host cellular proteases such as trypsin, tryptase Clara, miniplasmin, human airway trypsin-like protease, and TMPRSS2 cleave the HA glycoprotein of influenza A viruses. Cleavage of HA is critical for viral entry into cells during fusion between the viral and host cell membranes (34). Serine protease inhibitors such as camostat and aprotinin inhibit both influenza virus replication in human airway epithelial cells and the release of cytokines (IL-6 and TNF-α) into cell supernatants (35). In addition, animal studies using TMPRSS2-KO mice revealed that TMPRSS2 is essential for the spread and pathogenesis of influenza viruses such as emerging H7N9 and seasonal H1N1 and H3N2 (18,–21). TMPRSS2 also cleaves the coronavirus spike protein to generate unlocked, fusion-catalyzing forms at the cell surface and facilitate rapid early entry (14, 15, 36,–40). In addition, SARS-CoV and MERS-CoV enter cells via two distinct pathways, TMPRSS2 via the cell surface and cathepsin L via the endosome (13, 14, 16, 37, 41, 42). A previous antiviral study revealed that a serine protease (TMPRSS2) rather than a cysteine protease (cathepsin L) facilitated the spread of SARS-CoV in the infected mouse (43). Our findings are in agreement with this study; coronavirus replication in the lungs, especially in the bronchioles, was less pronounced in TMPRSS2-deficient mice. However, viral spread and inflammatory infiltration were still detected in the alveoli. Several proteases, including other serine proteases and the cysteine protease cathepsin L, may activate both SARS-CoV and MERS-CoV, allowing the virus to spread to alveolar areas in TMPRSS2-deficient mice. In addition, TLR3 mRNA expression in the lungs at 6 h p.i. of SARS-CoV-inoculated TMPRSS2-deficient mice suggested that TLR3, which recognizes specifically dsRNA and localizes to endosomes (44), recognized viral RNA within the endosomal component. Thus, we speculate that the pathway employing cathepsin L and the endosome mainly contributed to SARS-CoV infection in the TMPRSS2-deficient mice.

MERS-CoV-infected animals had more obvious histopathological differences than SARS-CoV-infected animals in this animal model. In addition, weak-positive SARS-CoV antigens at 1 day p.i. and a few virus antigen-positive cells at 3 days p.i. were detected in the bronchi of TMPRSS2-KO mice but not in MERS-CoV-inoculated TMPRSS2-KO Tg mice. Thus, MERS-CoV may rely more on TMPRSS2 during early infection than mouse-adapted SARS-CoV although differences in viral passage history and in the genetic backgrounds of the animals should also be considered. More work will be required to test the possibility that viral mutations are acquired during virus spread in TMPRSS2-deficient mice.

TMPRSS2-deficient mice, including TMPRSS2-KO and TMPRSS2-KO Tg mice, showed less severe loss of body weight after infection. Peak expression of FGF-basic, also known as FGF2, after infection synchronized with peak body weight loss in WT and hDPP4-Tg mice. FGFs play a role in tissue repair after pneumonia, including bronchiolitis obliterans organizing pneumonia (BOOP) and interstitial pneumonia (both fibrous pulmonary disorders), by promoting proliferation of fibroblasts (45). In fact, formation of granulation tissue was observed in WT mice after SARS-CoV infection. BOOP and pulmonary fibrosis are common in patients with SARS, including those who survive the infection (46). While there is limited evidence for development of fibrosis during end-stage acute respiratory distress syndrome induced by MERS-CoV, clinical data from MERS patients suggest that the situation is similar to that observed for SARS (46, 47). In addition, some metabolic FGFs cause body weight loss (48).

As expected, lower expression of cytokines and chemokines was observed in TMPRSS2-deficient mice than in TMPRSS2-competent mice after coronavirus infection. This result is similar to results reported for TMPRSS2 and TMPRSS4 double-KO mice on day 3 postinfection with H3N2 influenza A virus (49). High levels of virus replication very likely induce severe tissue damage and increased cellular infiltration by immune cells. Viral replication is likely a major cause of the elevated inflammatory chemokine levels observed in WT mice; nevertheless, we assessed the possibility that TMPRSS2, a serine protease, may also contribute to inflammatory reactions after TLR3 stimulation. Intranasal administration of poly(I·C) induced expression of MCP-1, KC, IL-1α, IL-1β, and IL-12 in the lungs of WT mice but not in those of TMPRSS2-KO mice. The genetic backgrounds of WT and TMPRSS2-KO mice were the same because the TMPRSS2-KO mice were produced from TMPRSS2 gene knockout C57BL/6 embryonic stem (ES) cells. The physiological function of TMPRSS2 remains unclear; however, TMPRSS2 may contribute to more severe or rapid immunopathology in WT mice by increasing the levels of inflammatory cytokines and chemokines after TLR3 stimulation.

The immune responses to poly(I·C) treatment in 14- to 16-week-old hDPP4-Tg mice were quite different from those in 14- to 16-week-old WT C57BL/6 mice. The hDPP4-Tg mice were produced from BDF1×C57BL/6 mice; however, the Tg mice were backcrossed with C57BL/6 mice for eight generations. Thus, the genetic backgrounds were almost the same between these strains. On the other hand, hDPP4 expression did not have a marked effect on basal innate immune responses in 10-week-old C57BL/6 and hDPP4-Tg mice; however, hDPP4-Tg mice showed slightly stronger or earlier innate immune responses than C57BL/6 mice (25). In addition, Simeoni et al. reported that hDPP4/CD26 transgene expression induced major phenotypic changes in T-cell populations within the thymus and peripheral blood of their Tg mice and that peripheral blood T-cell reduction was age dependent (50). Thus, the compromised immune responses in our hDPP4-Tg mice were possibly due to hDPP4.

Broad-spectrum antiviral drugs against coronaviruses and other highly pathogenic viruses will enable a rapid response to pandemic threats. Here, we demonstrate a role of TMPRSS2 during infection by SARS-CoV and MERS-CoV. TMPRSS2 played an active role at primary infection sites and influenced the spread of coronaviruses within the airways of both mouse models, modulating the eventual immunopathology. Interestingly, inflammatory chemokine and cytokine levels in TMPRSS2-KO mice were lower even after intranasal stimulation by poly(I·C), suggesting an as-yet-unidentified physiological role for TMPRSS2. In conclusion, we show that TMPRSS2 plays a role in the spread and immunopathology of coronaviruses in the airways.

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MATERIALS AND METHODS

 

Ethics statements.

Experiments using recombinant DNA and pathogens were approved by the Committee for Experiments using Recombinant DNA and Pathogens at the National Institute of Infectious Diseases, Tokyo, Japan. All animal experiments were approved by the Animal Care and Use Committee of the National Institute of Infectious Diseases and were conducted in accordance with institutional Guidelines for the Care and Use of Animals. All animals were housed in a Japan Health Sciences Foundation-certified facility.

 

Cells and viruses.

Vero E6 cells (American Type Culture Collection, Manassas, VA) were cultured in Eagle’s minimal essential medium (MEM) containing 5% fetal bovine serum (FBS), 50 IU/ml penicillin G, and 50 μg/ml streptomycin (5% FBS-MEM). Stocks of a mouse-passaged Frankfurt 1 isolate of SARS-CoV and F-musX-Vero E6 were propagated twice and titrated on Vero E6 cells prior to cryopreservation at −80°C, as previously described (23). MERS-CoV (HCoV-EMC 2012 strain) was kindly provided by Bart Haagmans and Ron Fochier (Erasmus Medical Center, Rotterdam, The Netherlands). Stocks of MERS-CoV were propagated twice, titrated on Vero E6 cells, and cryopreserved at −80°C. Viral infectivity titers are expressed as the TCID50 per milliliter on Vero E6 cells and were calculated according to the Behrens-Kärber method. All work with infectious SARS-CoV and MERS-CoV was performed under biosafety level 3 conditions.

 

Generation of mice.

TMPRSS2−/− mice were established from TMPRSS2 gene knockout C57BL/6 embryonic stem (ES) cells (product number VG13341), which were obtained from the Knockout Mouse Project (KOMP) Repository (University of California Davis). The ES cells were injected into C57BL/6 mouse blastocysts, and chimeric mice with a complete C57BL/6 genetic background were generated. TMPRSS2−/− mice with a homologous genotype were obtained by crossing male and female TMPRSS2+/− C57BL/6 mice (20).

The transgenic mice expressing the human DPP4 gene (hDPP4-Tg mice) were generated by microinjection of purified bacterial artificial chromosome (BAC) clones carrying the hDPP4 gene into the pronuclei of fertilized eggs from BDF1×C57BL/6NCr mice (25). The transgenic mice were then backcrossed with C57BL/6NCr mice for eight generations and subsequently crossed with homozygous TMPRSS2 knockout (TMPRSS2−/−) mice.

Genomic DNA isolated from ear punch tissues was subjected to genotyping by PCR analysis using hDPP4-specific primers (forward, 5′-ACACACACACTCTCACACACT-3′; reverse, 5′-TCTCAGTGCCATAAAAGCCCA-3′) (25) or TMPRSS2-specific primers P11 (5′-ACCTGGAGTATACGGGAACGTGA-3′) and P12 (5′-GTGAGTGGGTGAAGGTTGGGTAG-3′) (32).

 

Animal experiments.

Mice were anesthetized by intraperitoneal injection of a mixture of 1.0 mg of ketamine and 0.02 mg of xylazine (0.08 ml/10 g of body weight). TMPRSS2-KO mice, C57BL/6 mice lacking a homologous genotype of the TMPRSS2 gene (TMPRSS2−/−) (20), and C57BL/6 mice (WT mice; TMPRSS2+/+) were inoculated intranasally with SARS-CoV (105 TCID50 in 30 μl of F-musX). Human DPP4-expressing transgenic mice (hDPP4-Tg mice; C57BL/6 mice heterozygous for the human DPP4 gene [hDPP4+/− TMPRSS2+/+]) (25) and hDPP4+/− TMPRSS2−/− C57BL/6 mice (TMPRSS2-KO Tg mice) were obtained by crossing hDPP4-Tg mice with TMPRSS2-KO mice. These mice were inoculated intranasally with MERS-CoV (106 TCID50 in 30 μl of HCoV-EMC 2012). Infected mice were observed for clinical signs of infection, and body weight was measured daily for 10 days or 14 days (n = 6 to 14 mice, all aged 12 to 28 weeks). For analysis of virus replication, cytokine expression, and pathology, animals were sacrificed at various time points after inoculation (n = 3 to 4 mice per group, all aged 13 to 30 weeks).

WT, TMPRSS2-KO, hDPP4-Tg, and TMPRSS2-KO Tg mice (n = 4 mice per group, all aged 14 to 16 weeks) were anesthetized by intraperitoneal injection of a mixture of 1.0 mg of ketamine and 0.02 mg of xylazine (0.08 ml/10 g of body weight). Mice then received 20 μg of poly(I·C) (Invitrogen, San Diego, CA) in 20 μl of phosphate-buffered saline (PBS) (intranasally) (26). All mice were sacrificed 24 h after administration for analysis of cytokine expression.

 

Virus titration.

Lung tissue homogenates (10% [wt/vol]) were prepared in MEM containing 2% FBS, 50 IU/ml penicillin G, 50 μg/ml streptomycin, and 2.5 μg/ml amphotericin B. Samples were clarified by centrifugation at 740 × g for 20 min, and the supernatant was inoculated onto Vero E6 cell cultures for virus titration.

 

Neutralizing antibody test.

Serum was collected from mice sacrificed on day 10 or 14 p.i. After inactivation at 56°C for 30 min, Vero E6 cells were infected with virus (100 TCID50 per well) in the presence of plasma (serially diluted 2-fold), incubated for 3 or 5 days, and then examined for cytopathic effects. Plasma titers of neutralizing antibodies were calculated as the reciprocal of the highest dilution at which no cytopathic effect was observed. The lowest and highest dilutions tested were 4 and 256 or 64, respectively.

 

Histopathology and immunohistochemistry.

Mice were anesthetized and perfused with 2 ml of 10% phosphate-buffered formalin. The lungs were harvested, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Masson’s trichrome staining was also conducted to detect fibrosis in the lungs. Immunohistochemical analysis was performed using a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO; Nichirei Biosciences, Inc., Tokyo, Japan). Antigen retrieval from formalin-fixed mouse tissue sections was performed by autoclaving in retrieval solution (pH 6.0) (Nichirei Biosciences) at 121°C for 10 min. Hyperimmune rabbit serum raised against SARS-CoV (23) or an anti-MERS-CoV nucleocapsid antibody (Sino Biological Inc., Beijing, China) was used as the primary antibody to detect viral antigens. Peroxidase activity was detected with 3,3′-diaminobenzidine (Sigma-Aldrich). Hematoxylin was used for counterstaining.

 

Detection of inflammatory cytokines and chemokines.

Cytokines and chemokines in mouse lung homogenates (10%, wt/vol) were measured using a commercial Mouse Cytokine 20-Plex antibody bead kit (Thermo Fisher Scientific) and a Luminex 100 apparatus (Luminex Co., Austin, TX), as described previously (23). A panel of inflammatory cytokines and chemokines (FGF-basic, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 [p40/p70], IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α, TNF-α, and vascular endothelial growth factor [VEGF]) was detected according to the manufacturer’s protocols.

 

Quantitative real-time RT-PCR.

To measure the levels of type I IFN and TLR3 mRNA expression, RNA was extracted from 20% (wt/vol) lung homogenates of mice infected with viruses using RNeasy minikits (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. mRNAs encoding IFN-α, IFN-β, and TLR3 were examined by real-time RT-PCR using an ABI Prism 7900HT Fast real-time PCR system (Applied Biosystems, Foster City, CA). The TaqMan probes and primers and the reaction conditions have been described previously (24, 26). Expression of each gene was normalized to that of β-actin.

 

Statistical analysis.

Data are expressed as the means and standard errors of the means. Statistical analyses were performed using GraphPad Prism, version 7, software (GraphPad Software, Inc., La Jolla, CA). Body weight curves, virus titers, and multiplex assay results were analyzed using one-way or two-way analysis of variance (ANOVA). Significant effects of viral titers in different animal strains at different time points were assessed by two-way ANOVA, and P values were calculated using Bonferroni’s multiple-comparison test. The results of the neutralizing antibody titer assays were analyzed using a Mann-Whitney test. A P value of <0.05 was considered statistically significant.

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ACKNOWLEDGMENTS

We thank Ron A. M. Fouchier and Bart L. Haagmans (Erasmus Medical Center, The Netherlands) for providing MERS-CoV (isolate HCoV-EMC/2012) and Kazuya Shirato, Shutoku Matsuyama, Masaki Anraku, and Kohji Sakai (National Institute of Infectious Diseases) for helpful discussions. We also thank our colleagues at the Institute, especially Midori Ozaki and Hitoshi Kujirai, for technical assistance.

This work was supported by the following: a grant-in-aid for research (H25-Shinko-Wakate-004) from the Ministry of Health, Labor, and Welfare, Japan; the Research Program on Emerging and Re-emerging Infectious Diseases (grants JP17fk0108313 and JP18fk0108058) from the Japan Agency for Medical Research and Development; grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (16K09951 and 18H02665); and a grant from the National Center for Global Health and Medicine (27A1102).

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FOOTNOTES

 

For a companion article on this topic, see https://doi.org/10.1128/JVI.01818-18.

 

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