Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

Human ACE2 protein is a molecular switch controlling the mode of SARS-CoV-2 transmission.

BACKGROUND: Human angiotensin-converting enzyme 2 (hACE2) is the receptor mediating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. hACE2 expression is low in the lungs and is upregulated after SARS-CoV-2 infection. How such a hACE2-limited pulmonary environment supports efficient virus transmission and how dynamic hACE2 expression affects SARS-CoV-2 infection are unclear. METHODS: We generated stable cell lines with different expression levels of hACE2 to evaluate how the hACE2 expression level can affect SARS-CoV-2 transmission. RESULTS: We demonstrated that the hACE2 expression level controls the mode of SARS-CoV-2 transmission. The hACE2-limited cells have an advantage for SARS-CoV-2 shedding, which leads to cell-free transmission. By contrast, enhanced hACE2 expression facilitates the SARS-CoV-2 cell-to-cell transmission. Furthermore, this cell-to-cell transmission is likely facilitated by hACE2-containing vesicles, which accommodate numerous SARS-CoV-2 virions and transport them to neighboring cells through intercellular extensions. CONCLUSIONS: This hACE2-mediated switch between cell-free and cell-to-cell transmission routes provides SARS-CoV-2 with advantages for either viral spread or evasion of humoral immunity, thereby contributing to the COVID-19 pandemic and pathogenesis.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Natural fucoidans inhibit coronaviruses by targeting viral spike protein and host cell furin.

Fucoidans are a class of long chain sulfated polysaccharides and have multiple biological functions. Herein, four natural fucoidans extracted from Fucus vesiculosus, F. serratus, Laminaria japonica and Undaria pinnatifida, were tested for their HCoV-OC43 inhibition and found to demonstrate EC50 values ranging from 0.15 to 0.61 µg/mL. That from U. pinnatifida exhibited the most potent anti-HCoV-OC43 activity with an EC50 value of 0.15 ± 0.02 µg/mL, a potency largely independent of its sulfate content. Comparison of the gene expression profiles of fucoidan-treated and untreated cells infected with HCoV-OC43 revealed that fucoidan treatment effectively diminished HCoV-OC43 gene expressions associated with induced chemokines, cytokines and viral activities. Further studies using a highly fucoidan-resistant HCoV-OC43 determined that fucoidan inhibited HCoV-OC43 infection via interfering with viral entry and led to the identification of the specific site on the N-terminal region of spike protein, that located adjacent to the host cell receptor binding domain, targeted by the virus. Furthermore, in a SARS-CoV-2 pseudovirus neutralization assay, fucoidan also blocked SARS-CoV-2 entry. In vitro and in vivo, fucoidan decreased SARS-CoV-2 viral loads and inhibited viral infection in Calu-3 or Vero E6 cells and SARS-CoV-2 infected hamsters, respectively. Fucoidan was also found to inhibit furin activity, and reported furin inhibitors were found to inhibit viral infection by wild type HCoV-OC43 or SARS-CoV-2. Accordingly, we conclude that fucoidans inhibit coronaviral infection by targeting viral spike protein and host cell furin to interfere with viral entry.

Optimization and Improvement of qPCR Detection Sensitivity of SARS-CoV-2 in Saliva.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health threat globally, especially during the beginning of the pandemic in 2020. Reverse transcription-quantitative PCR (RT-qPCR) is utilized for viral RNA detection as part of control measures to limit the spread of COVID-19. Collecting nasopharyngeal swabs for RT-qPCR is a routine diagnostic method for COVID-19 in clinical settings, but its large-scale implementation is hindered by a shortage of trained health professionals. Despite concerns over its sensitivity, saliva has been suggested as a practical alternative sampling approach to the nasopharyngeal swab for viral RNA detection. In this study, we spiked saliva from healthy donors with inactivated SARS-CoV-2 from an international standard to evaluate the effect of saliva on viral RNA detection. On average, the saliva increased the cycle threshold (CT) values of the SARS-CoV-2 RNA samples by 2.64 compared to the viral RNA in viral transport medium. Despite substantial variation among different donors in the effect of saliva on RNA quantification, the outcome of the RT-qPCR diagnosis was largely unaffected for viral RNA samples with CT values of <35 (1.55 log10 IU/mL). The saliva-treated viral RNA remained stable for up to 6 h at room temperature and 24 h at 4°C. Further supplementing protease and RNase inhibitors improved the detection of viral RNA in the saliva samples. Our data provide practical information on the storage conditions of saliva samples and suggest optimized sampling procedures for SARS-CoV-2 diagnosis. IMPORTANCE The primary method for detection of SARS-CoV-2 is using nasopharyngeal swabs, but a shortage of trained health professionals has hindered its large-scale implementation. Saliva-based nucleic acid detection is a widely adopted alternative, due to its convenience and minimally invasive nature, but the detection limit and direct impact of saliva on viral RNA remain poorly understood. To address this gap in knowledge, we used a WHO international standard to evaluate the effect of saliva on SARS-CoV-2 RNA detection. We describe the detection profile of saliva-treated SARS-CoV-2 samples under different storage temperatures and incubation periods. We also found that adding protease and RNase inhibitors could improve viral RNA detection in saliva. Our research provides practical recommendations for the optimal storage conditions and sampling procedures for saliva-based testing, which can improve the efficiency of COVID-19 testing and enhance public health responses to the pandemic.

Longitudinal neutralizing antibody responses after SARS-CoV-2 infection: A convalescent cohort study in Taiwan.

BACKGROUND: Understanding the neutralizing antibody (NAb) titer against COVID-19 over time is important to provide information for vaccine implementation. The longitudinal NAb titer over one year after SARS-CoV-2 infection is still unclear. The purposes of this study are to evaluate the duration of the neutralizing NAb titers in COVID-19 convalescents and factors associated with the titer positive duration. METHODS: A cohort study followed COVID-19 individuals diagnosed between 2020 and 2021 May 15th from the COVID-19 database from the Taiwan Centers for Disease Control. We analyzed NAb titers from convalescent SARS-CoV-2 individuals. We used generalized estimating equations (GEE) and a Cox regression model to summarize the factors associated with NAb titers against COVID-19 decaying in the vaccine-free population. RESULTS: A total of 203 convalescent subjects with 297 analytic samples were followed for a period of up to 588 days. Our study suggests that convalescent COVID-19 in individuals after more than a year and four months pertains to only 25% of positive titers. The GEE model indicates that longer follow-up duration was associated with a significantly lower NAb titer. The Cox regression model indicated the disease severity with advanced condition was associated with maintaining NAb titers (adjusted hazard ratio: 2.01, 95% CI: 1.11-3.63) and that smoking was also associated with higher risk of negative NAb titers (adjusted hazard ratio: 0.55, 95% CI: 0.33-0.92). CONCLUSIONS: Neutralizing antibody titers diminished after more than a year. The antibody titer response against SARS-CoV-2 in naturally convalescent individuals provides a reference for vaccinations.

A receptor-binding domain-based nanoparticle vaccine elicits durable neutralizing antibody responses against SARS-CoV-2 and variants of concern.

Numerous vaccines have been developed to address the current COVID-19 pandemic, but safety, cross-neutralizing efficacy, and long-term protectivity of currently approved vaccines are still important issues. In this study, we developed a subunit vaccine, ASD254, by using a nanoparticle vaccine platform to encapsulate the SARS-CoV-2 spike receptor-binding domain (RBD) protein. As compared with the aluminum-adjuvant RBD vaccine, ASD254 induced higher titers of RBD-specific antibodies and generated 10- to 30-fold more neutralizing antibodies. Mice vaccinated with ASD254 showed protective immune responses against SARS-CoV-2 challenge, with undetectable infectious viral loads and reduced typical lesions in lung. Besides, neutralizing antibodies in vaccinated mice lasted for at least one year and were effective against various SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, particle size, polydispersity index, and zeta-potential of ASD254 remained stable after 8-month storage at 4°C. Thus, ASD254 is a promising nanoparticle vaccine with good immunogenicity and stability to be developed as an effective vaccine option in controlling upcoming waves of COVID-19.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Humoral Immunogenicity and Reactogenicity of the Standard ChAdOx1 nCoV-19 Vaccination in Taiwan.

BACKGROUND: The ChAdOx1 nCoV-19 vaccine has been widely administered against SARS-CoV-2 infection; however, data regarding its immunogenicity, reactogenicity, and potential differences in responses among Asian populations remain scarce. METHODS: 270 participants without prior COVID-19 were enrolled to receive ChAdOx1 nCoV-19 vaccination with a prime-boost interval of 8-9 weeks. Their specific SARS-CoV-2 antibodies, neutralizing antibody titers (NT50), platelet counts, and D-dimer levels were analyzed before and after vaccination. RESULTS: The seroconversion rates of anti-RBD and anti-spike IgG at day 28 after a boost vaccination (BD28) were 100% and 95.19%, respectively. Anti-RBD and anti-spike IgG levels were highly correlated (r = 0.7891), which were 172.9 ± 170.4 and 179.3 ± 76.88 BAU/mL at BD28, respectively. The geometric mean concentrations (GMCs) of NT50 for all participants increased to 132.9 IU/mL (95% CI 120.0-147.1) at BD28 and were highly correlated with anti-RBD and anti-spike IgG levels (r = 0.8248 and 0.7474, respectively). Body weight index was statistically significantly associated with anti-RBD IgG levels (p = 0.035), while female recipients had higher anti-spike IgG levels (p = 0.038). The GMCs of NT50 declined with age (p = 0.0163) and were significantly different across age groups (159.7 IU/mL for 20-29 years, 99.4 IU/mL for ≥50 years, p = 0.0026). Injection-site pain, fever, and fatigue were the major reactogenicity, which were more pronounced after prime vaccination and in younger participants (<50 years). Platelet counts decreased and D-dimer levels increased after vaccination but were not clinically relevant. No serious adverse events or deaths were observed. CONCLUSION: The vaccine is well-tolerated and elicited robust humoral immunity against SARS-CoV-2 after standard prime-boost vaccination in Taiwanese recipients.

Safety and immunogenicity of CpG 1018 and aluminium hydroxide-adjuvanted SARS-CoV-2 S-2P protein vaccine MVC-COV1901: interim results of a large-scale, double-blind, randomised, placebo-controlled phase 2 trial in Taiwan.

BACKGROUND: MVC-COV1901, a recombinant protein vaccine containing pre-fusion-stabilised spike protein S-2P adjuvanted with CpG 1018 and aluminium hydroxide, has been shown to be well tolerated with a good safety profile in healthy adults aged 20-49 years in a phase 1 trial, and provided a good cellular and humoral immune responses. We present the interim safety, tolerability, and immunogenicity results of a phase 2 clinical trial of the MVC-COV1901 vaccine in Taiwan. METHODS: This is a large-scale, double-blind, randomised, placebo-controlled phase 2 trial done at ten medical centres and one regional hospital in Taiwan. Individuals aged 20 years or older who were generally healthy or had stable pre-existing medical conditions were eligible for enrolment. Exclusion criteria included (but were not limited to) travel overseas within 14 days of screening, intention to travel overseas within 6 months of the screening visit, and the absence of prespecified medical conditions, including immunosuppressive illness, a history of autoimmune disease, malignancy with risk to recur, a bleeding disorder, uncontrolled HIV infection, uncontrolled hepatitis B and C virus infections, SARS-CoV-1 or SARS-CoV-2 infections, an allergy to any vaccine, or a serious medical condition that could interfere with the study. Study participants were randomly assigned (6:1) to receive two doses of either MVC-COV1901 or placebo, administered via intramuscular injection on day 1 and day 29. MVC-COV1901 contained 15 µg of S-2P protein adjuvanted with 750 µg CpG 1018 and 375 µg aluminium hydroxide in a 0·5 mL aqueous solution, and the placebo contained the same volume of saline. Randomisation was done centrally by use of an interactive web response system, stratified by age (≥20 to <65 years and ≥65 years). Participants and investigators were masked to group assignment. The primary outcomes were to evaluate the safety, tolerability, and immunogenicity of MVC-COV1901 from day 1 (the day of the first dose) to day 57 (28 days after the second dose). Safety was assessed in all participants who received at least one dose. Immunogenicity was assessed by measuring geometric mean titres (GMTs) and seroconversion rates of neutralising antibody and antigen-specific IgG in the per-protocol population. This study is registered with ClinicalTrials.gov, NCT04695652. FINDINGS: Of 4173 individuals screened between Dec 30, 2020, and April 2, 2021, 3854 were enrolled and randomly assigned: 3304 to the MVC-COV1901 group and 550 to the placebo group. A total of 3844 participants (3295 in the MVC-COV1901 group and 549 in the placebo group) were included in the safety analysis set, and 1053 participants (903 and 150) had received both doses and were included in the per-protocol immunogenicity analysis set. From the start of this phase 2 trial to the time of interim analysis, no vaccine-related serious adverse events were recorded. The most common solicited adverse events in all study participants were pain at the injection site (2346 [71·2%] of 3295 in the MVC-COV1901 group and 128 [23·3%] of 549 in the placebo group), and malaise or fatigue (1186 [36·0%] and 163 [29·7%]). Fever was rarely reported (23 [0·7%] and two [0·4%]). At 28 days after the second dose of MVC-COV1901, the wild-type SARS-CoV-2 neutralising antibody GMT was 662·3 (95% CI 628·7-697·8; 408·5 IU/mL), the GMT ratio (geometric mean fold increase in titres at day 57 vs baseline) was 163·2 (155·0-171·9), and the seroconversion rate was 99·8% (95% CI 99·2-100·0). INTERPRETATION: MVC-COV1901 has a good safety profile and elicits promising immunogenicity responses. These data support MVC-COV1901 to enter phase 3 efficacy trials. FUNDING: Medigen Vaccine Biologics and Taiwan Centres for Disease Control, Ministry of Health and Welfare.

Novel Naturally Occurring Mutations of Enterovirus 71 Associated With Disease Severity.

Infection with the re-emerging enterovirus 71 (EV-A71) is associated with a wide range of disease severity, including herpangina, encephalitis, and cardiopulmonary failure. At present, there is no FDA-approved therapeutics for EV-A71. Early diagnosis for the high-risk children is the key to successful patient care. We examined viral genome sequences at the 5' untranslated region (UTR) and the capsid protein VP1 from 36 mild and 27 severe cases. We identified five EV-A71 mutations associated with severe diseases, including (1) the 5' UTR mutations C580U, A707G, C709U; (2) a VP1 alanine-to-threonine mutation at position 280 (280T), and (3) a VP1 glutamic acid-to-(non-glutamic acid) at position 145 [145(non-E)]. SCARB2 is a known entry receptor for EV-A71. Based on a recent cryoEM structure of the EV-A71-SCARB2 binding complex, VP1-280T is near the binding interface between the VP1-VP2 complex and its entry receptor SCARB2. A de novo created hydrogen bonding between the mutant VP1-280T and the VP2-139T, could help strengthen a web-like interaction structure of the VP1-VP2 complex. A stabilized loop turn of VP2, once in contact with SCARB2, can enhance interaction with the host SCARB2 receptor for viral entry. Our findings here could facilitate early detection of severe cases infected with EV-A71 in clinical medicine. In addition, it opens up the opportunity of functional studies via infectious cDNA cloning, site-directed mutagenesis, and animal models in the future.

Hypoxia and therapeutic treatment of EV-A71 with an immune modulator TLR7 agonist in a new immunocompetent mouse model.

BACKGROUND: Enterovirus 71 (EV71 or EV-A71) was first identified in California about half a century ago. In recent years, outbreaks of EV-A71 were prevalent worldwide, including Taiwan, Malaysia, Singapore, Japan, and China. Between 2008 and 2011, China alone reported 1894 deaths associated with EV-A71 infection. In mild cases, EV-A71 can cause herpangina and hand-foot-and-mouth disease (HFMD). However, in severe cases, it could cause neurological disorders, including meningitis and encephalitis. Cardiopulmonary failure is common among hospitalized children with EV-A71 infection. No effective FDA-approved therapeutics against EV-A71 are clinically available. METHODS: We report the establishment of an immunocompetent wild type strain 129 (wt-129) mouse model, which can be cross-species infected with human EV-A71 clinical isolates via an intraperitoneal route. RESULTS: One intriguing disease phenotype of this new model is the development of characteristic "White-Jade" patches in the muscle, which lost sporadically the normal pink color of uninfected muscle. Viral VP1 protein and massive leukocyte infiltration were detected in muscles with or without white-jades. We demonstrated further that hypoxia is a general phenomenon associated with white-jades in both immunocompetent and immunodeficient mouse models. Therefore, hypoxia appears to be a feature intrinsic to EV-A71 infection, irrespective of its host's immunogenetic background. To date, no effective treatment for EV-A71 is available. Here, using this new wt-129 mouse model, we showed that timely treatment with compound R837 (a TLR7 immune modulator) via oral or intraperitoneal routes, rescued the hypoxia, limb paralysis, and death at a high therapeutic efficacy. CONCLUSIONS: In this new immunocompetent mouse 129 model, we observed an unexpected white-jade phenotype and its associated hypoxia. The successful treatment with TLR7 immune modulators via an oral route, provide us a new research direction for EV-A71 basic science and translational research. It remains an open issue whether R837 or its related compounds, will be a promising drug candidate in clinical trials in EV-A71 endemic or epidemic areas in the future.

Enterovirus 71 targets the cardiopulmonary system in a robust oral infection mouse model.

Severe infection with the re-emerging enterovirus 71 (EV71 or EV-A71) can cause cardiopulmonary failure. However, in patients' heart and lung, viral protein has not been detected. In mouse models, heart disease has not been reported. EV71-infected brainstem is generally believed to be responsible for the cardiopulmonary collapse. One major limitation in EV71 research is the lack of an efficient oral infection system using non-mouse-adapted clinical isolates. In a robust oral infection NOD/SCID mouse model, we detected EV71 protein at multiple organs, including heart and lung, in 100% of moribund mice with limb paralysis. Infiltrating leukocytes were always detected in heart and muscle, and VP1-positive M2 macrophages were abundant in the lung. Functional dissection on the pathogenesis mechanism revealed severe apoptosis, inflammatory cytokines, and abnormal electrocardiogram (EKG) in orally infected hearts. Therefore, cardiopulmonary disease could be one plausible cause of death in this mouse model. Inoculation of EV71 through an oral route resulted in viral infection in the intestine, viremia, and EV71 appeared to spread to peripheral tissues via blood circulation. Infectious virus was no longer detected in the blood on day 5 post-infection by the plaque formation assay. We demonstrated that both EV71 clinical isolate and cloned virus can target the cardiopulmonary system via a natural infection-like oral route.

Immunocompetent and Immunodeficient Mouse Models for Enterovirus 71 Pathogenesis and Therapy.

Enterovirus 71 (EV71) is a global health threat. Children infected with EV71 could develop hand-foot-and-mouth disease (HFMD), encephalitis, paralysis, pulmonary edema, and death. At present, no effective treatment for EV71 is available. We reviewed here various mouse models for EV71 pathogenesis and therapy. Earlier studies relied on the use of mouse-adapted EV71 strains. To avoid artificial mutations arising de novo during the serial passages, recent studies used EV71 clinical isolates without adaptation. Several human receptors for EV71 were shown to facilitate viral entry in cell culture. However, in vivo infection with human SCARB2 receptor transgenic mice appeared to be more limited to certain strains and genotypes of EV71. Efficacy of oral infection in these transgenic models is extremely low. Intriguingly, despite the lack of human receptors, immunodeficient neonatal mouse models can still be infected with EV71 clinical isolates via oral or intraperitoneal routes. Crossbreeding between SCARB2 transgenic and stat1 knockout mice generated a more sensitive and user-friendly hybrid mouse model. Infected hybrid mice developed a higher incidence and earlier onset of CNS disease and death. Different pathogenesis profiles were observed in models deficient in various arms of innate or humoral immunity. These models are being actively used for antiviral research.

A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71.

Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis.

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

UNLABELLED: Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE: In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.