Apoptosis and pyroptosis in the nasal mucosa of Syrian hamster during SARS-CoV-2 infection and reinfection.

In SARS-CoV-2 infection, it has been observed that viral replication lasts longer in the nasal mucosa than in the lungs, despite the presence of a high viral load at both sites. In hamsters, we found that the nasal mucosa exhibited a mild inflammatory response and minimal pathological injuries, whereas the lungs displayed a significant inflammatory response and severe injuries. The underlying cellular events may be induced by viral infection in three types of cell death: apoptosis, pyroptosis, and necroptosis. Our findings indicate that apoptosis was consistently activated during infection in the nasal mucosa, and the levels of apoptosis were consistent with the viral load. On the other hand, pyroptosis and a few instances of necroptosis were observed only on 7 dpi in the nasal mucosa. In the lungs, however, both pyroptosis and apoptosis were prominently activated on 3 dpi, with lower levels of apoptosis compared to the nasal mucosa. Interestingly, in reinfection, obvious viral load and apoptosis in the nasal mucosa were detected on 3 dpi, while no other forms of cell death were detected. We noted that the inflammatory reactions and pathological injuries in the nasal mucosa were milder, indicating that apoptosis may play a role in promoting lower inflammatory reactions and milder pathological injuries and contribute to the generation of long-term viral replication in the nasal mucosa. Our study provides valuable insights into the differences in cellular mechanisms during SARS-CoV-2 infection and highlights the potential significance of apoptosis regulation in the respiratory mucosa for controlling viral replication.

The Abundant Distribution and Duplication of SARS-CoV-2 in the Cerebrum and Lungs Promote a High Mortality Rate in Transgenic hACE2-C57 Mice.

Patients with COVID-19 have been reported to experience neurological complications, although the main cause of death in these patients was determined to be lung damage. Notably, SARS-CoV-2-induced pathological injuries in brains with a viral presence were also found in all fatal animal cases. Thus, an appropriate animal model that mimics severe infections in the lungs and brain needs to be developed. In this paper, we compared SARS-CoV-2 infection dynamics and pathological injuries between C57BL/6Smoc-Ace2em3(hACE2-flag-Wpre-pA)Smoc transgenic hACE2-C57 mice and Syrian hamsters. Importantly, the greatest viral distribution in mice occurred in the cerebral cortex neuron area, where pathological injuries and cell death were observed. In contrast, in hamsters, viral replication and distribution occurred mainly in the lungs but not in the cerebrum, although obvious ACE2 expression was validated in the cerebrum. Consistent with the spread of the virus, significant increases in IL-1ß and IFN-γ were observed in the lungs of both animals. However, in hACE2-C57 mice, the cerebrum showed noticeable increases in IL-1ß but only mild increases in IFN-γ. Notably, our findings revealed that both the cerebrum and the lungs were prominent infection sites in hACE2 mice infected with SARS-CoV-2 with obvious pathological damage. Furthermore, hamsters exhibited severe interstitial pneumonia from 3 dpi to 5 dpi, followed by gradual recovery. Conversely, all the hACE2-C57 mice experienced severe pathological injuries in the cerebrum and lungs, leading to mortality before 5 dpi. According to these results, transgenic hACE2-C57 mice may be valuable for studying SARS-CoV-2 pathogenesis and clearance in the cerebrum. Additionally, a hamster model could serve as a crucial resource for exploring the mechanisms of recovery from infection at different dosage levels.

Virulence and pathogenesis of SARS-CoV-2 infection in rhesus macaques: A nonhuman primate model of COVID-19 progression.

The COVID-19 has emerged as an epidemic, causing severe pneumonia with a high infection rate globally. To better understand the pathogenesis caused by SARS-CoV-2, we developed a rhesus macaque model to mimic natural infection via the nasal route, resulting in the SARS-CoV-2 virus shedding in the nose and stool up to 27 days. Importantly, we observed the pathological progression of marked interstitial pneumonia in the infected animals on 5-7 dpi, with virus dissemination widely occurring in the lower respiratory tract and lymph nodes, and viral RNA was consistently detected from 5 to 21 dpi. During the infection period, the kinetics response of T cells was revealed to contribute to COVID-19 progression. Our findings implied that the antiviral response of T cells was suppressed after 3 days post infection, which might be related to increases in the Treg cell population in PBMCs. Moreover, two waves of the enhanced production of cytokines (TGF-α, IL-4, IL-6, GM-CSF, IL-10, IL-15, IL-1ß), chemokines (MCP-1/CCL2, IL-8/CXCL8, and MIP-1ß/CCL4) were detected in lung tissue. Our data collected from this model suggested that T cell response and cytokine/chemokine changes in lung should be considered as evaluation parameters for COVID-19 treatment and vaccine development, besides of observation of virus shedding and pathological analysis.

Hand-Foot-and-Mouth Disease-Associated Enterovirus and the Development of Multivalent HFMD Vaccines.

Hand-foot-and-mouth disease (HFMD) is an infectious disease of children caused by more than 20 types of enteroviruses, with most cases recovering spontaneously within approximately one week. Severe HFMD in individual children develops rapidly, leading to death, and is associated with other complications such as viral myocarditis and type I diabetes mellitus. The approval and marketing of three inactivated EV-A71 vaccines in China in 2016 have provided a powerful tool to curb the HFMD epidemic but are limited in cross-protecting against other HFMD-associated enteroviruses. This review focuses on the epidemiological analysis of HFMD-associated enteroviruses since the inactivated EV-A71 vaccine has been marketed, collates the progress in the development of multivalent enteroviruses vaccines in different technical routes reported in recent studies, and discusses issues that need to be investigated for safe and effective HFMD multivalent vaccines.

Self-Assembling Nanoparticle Vaccines Displaying the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Responses in Rhesus Monkeys.

SARS-CoV-2 caused the COVID-19 pandemic that lasted for more than a year. Globally, there is an urgent need to use safe and effective vaccines for immunization to achieve comprehensive protection against SARS-CoV-2 infection. Focusing on developing a rapid vaccine platform with significant immunogenicity as well as broad and high protection efficiency, we designed a SARS-CoV-2 spike protein receptor-binding domain (RBD) displayed on self-assembled ferritin nanoparticles. In a 293i cells eukaryotic expression system, this candidate vaccine was prepared and purified. After rhesus monkeys are immunized with 20 µg of RBD-ferritin nanoparticles three times, the vaccine can elicit specific humoral immunity and T cell immune response, and the neutralizing antibodies can cross-neutralize four SARS-CoV-2 strains from different sources. In the challenge protection test, after nasal infection with 2 × 105 CCID50 SARS-CoV-2 virus, compared with unimmunized control animals, virus replication in the vaccine-immunized rhesus monkeys was significantly inhibited, and respiratory pathology observations also showed only slight pathological damage. These analyses will benefit the immunization program of the RBD-ferritin nanoparticle vaccine in the clinical trial design and the platform construction to present a specific antigen domain in the self-assembling nanoparticle in a short time to harvest stable, safe, and effective vaccine candidates for new SARS-CoV-2 isolates.

A hSCARB2-transgenic mouse model for Coxsackievirus A16 pathogenesis.

BACKGROUND: Coxsackievirus A16 (CA16) is one of the neurotropic pathogen that has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD), but its pathogenesis is not yet clear. The limited host range of CA16 make the establishment of a suitable animal model that can recapitulate the neurological pathology observed in human HFMD more difficult. Because the human scavenger receptor class B, member 2 (hSCARB2) is a cellular receptor for CA16, we used transgenic mice bearing human SCARB2 and nasally infected them with CA16 to study the pathogenicity of the virus. METHODS: Coxsackievirus A16 was administered by intranasal instillation to groups of hSCARB2 transgenic mice and clinical signs were observed. Sampled at different time-points to document and characterize the mode of viral dissemination, pathological change and immune response of CA16 infection. RESULTS: Weight loss and virus replication in lung and brain were observed in hSCARB2 mice infected with CA16, indicating that these animals could model the neural infection process. Viral antigens were observed in the alveolar epithelia and brainstem cells. The typical histopathology was interstitial pneumonia with infiltration of significant lymphocytes into the alveolar interstitial in lung and diffuse punctate hemorrhages in the capillaries of the brainstem. In addition, we detected the expression levels of inflammatory cytokines and detected high levels of interleukin IL-1ß, IL-6, IL-18, and IFN-γ in nasal mucosa, lungs and brain tissues. CONCLUSIONS: The hSCARB2-transgenic mice can be productively infected with CA16 via respiratory route and exhibited a clear tropism to lung and brain tissues, which can serve as a model to investigate the pathogenesis of CA16 associated respiratory and neurological disease.

Distinct infection process of SARS-CoV-2 in human bronchial epithelial cell lines.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leads to a series of clinical symptoms of respiratory and pulmonary inflammatory reactions via unknown pathologic mechanisms related to the viral infection process in tracheal or bronchial epithelial cells. Investigation of this viral infection in the human bronchial epithelial cell line (16HBE) suggests that SARS-CoV-2 can enter these cells through interaction between its membrane-localized S protein with the angiotensin-converting enzyme 2 molecule on the host cell membrane. Further observation indicates distinct viral replication with a dynamic and moderate increase, whereby viral replication does not lead to a specific cytopathic effect but maintains a continuous release of progeny virions from infected cells. Although messenger RNA expression of various innate immune signaling molecules is altered in the cells, transcription of interferons-α (IFN-α), IFN-ß, and IFN-γ is unchanged. Furthermore, expression of some interleukins (IL) related to inflammatory reactions, such as IL-6, IL-2, and IL-8, is maintained at low levels, whereas that of ILs involved in immune regulation is upregulated. Interestingly, IL-22, an IL that functions mainly in tissue repair, shows very high expression. Collectively, these data suggest a distinct infection process for this virus in respiratory epithelial cells, which may be linked to its clinicopathological mechanism.

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

Single B cells reveal the antibody responses of rhesus macaques immunized with an inactivated enterovirus D68 vaccine.

Enterovirus D68 (EV-D68) infection may cause severe respiratory system manifestations in pediatric populations. Because of the lack of an effective preventive vaccine or specific therapeutic drug for this infection, the development of EV-D68-specific vaccines and antibodies has become increasingly important. In this study, we prepared an experimental EV-D68 vaccine inactivated by formaldehyde and found that the serum of rhesus macaques immunized with the inactivated EV-D68 vaccine exhibited potent neutralizing activity against EV-D68 virus in vitro. Subsequently, the antibody-mediated immune response of B cells elicited by the inactivated vaccine was evaluated in a rhesus monkey model. The binding activity, in vitro neutralization activity, and sequence properties of 28 paired antibodies from the rhesus macaques' EV-D68-specific single memory B cells were analyzed, and the EV-D68 VP1-specific antibody group was found to be the main constituent in vivo. Intriguingly, we also found a synergistic effect among the E15, E18 and E20 monoclonal antibodies from the rhesus macaques. Furthermore, we demonstrated the protective efficacy of maternal antibodies in suckling C57BL/6 mice. This study provides valuable information for the future development of EV-D68 vaccines.

A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice.

Enterovirus D68 (EV-D68) belongs to the picornavirus family and was first isolated in CA, USA, in 1962. EV-D68 can cause severe cranial nerve system damage such as flaccid paralysis and acute respiratory diseases such as pneumonia. There are currently no efficient therapeutic methods or effective prophylactics. In this study, we isolated the mAb A6-1 from an EV-D68-infected rhesus macaque (Macaca mulatta) and found that the Ab provided effective protection in EV-D68 intranasally infected suckling mice. We observed that A6-1 bound to the DE loop of EV-D68 VP1 and interfered with the interaction between the EV-D68 virus and α2,6-linked sialic acids of the host cell. The production of A6-1 and its Ab properties present a bridging study for EV-D68 vaccine design and provide a tool for analyzing the process by which Abs can inhibit EV-D68 infection.

Immunogenicity and Safety of an F-Genotype Attenuated Mumps Vaccine in Healthy 8- to 24-Month-Old Children.

Background: Mumps vaccine immunizations have reduced the incidence of this disease. With the variation of mumps circulating strain, novel vaccine strains are always important. Methods: A 2-center parallel, randomized, double-blind noninferiority trial was performed to compare an F-genotype attenuated mumps vaccine (SP strain) to the A-genotype vaccine (S-79, Jeryl-Lynn strain) in 1080 healthy children aged 8-24 months in Hubei, China. Results: Participants were randomly assigned to receive a high or low dose of the SP or S79 vaccine and then assessed clinically at 30 minutes and 1-28 days postinoculation. No differences in local or systemic reactivity were observed. A similar incidence of severe adverse events associated with the vaccine was observed in the high-dose group and the positive control group. Based on throat swab collections, no viral shedding was present at the 4th and 10th days in any group. Neutralizing and hemagglutination-inhibiting antibody assays with the F- or A-genotype strains showed similar trends in geometric mean titers in the high-dose SP and S79 groups. Increased cytotoxic T lymphocyte responses were observed in all groups. Conclusions: The F-genotype attenuated mumps vaccine is safe, offers immunogenicity against a homologous virus, and is noninferior to the A-genotype vaccine in 8- to 24-month-old children.

The altered gut virome community in rhesus monkeys is correlated with the gut bacterial microbiome and associated metabolites.

BACKGROUND: The gut microbiome is closely associated with the health of the host; although the interaction between the bacterial microbiome and the whole virome has rarely been studied, it is likely of medical importance. Examination of the interactions between the gut bacterial microbiome and virome of rhesus monkey would significantly contribute to revealing the gut microbiome composition. METHODS: Here, we conducted a metagenomic analysis of the gut microbiome of rhesus monkeys in a longitudinal cohort treated with an antibiotic cocktail, and we documented the interactions between the bacterial microbiome and virome. The depletion of viral populations was confirmed at the species level by real-time PCR. We also detected changes in the gut metabolome by GC-MS and LC-MS. RESULTS: A majority of bacteria were depleted after treatment with antibiotics, and the Shannon diversity index decreased from 2.95 to 0.22. Furthermore, the abundance-based coverage estimator (ACE) decreased from 104.47 to 33.84, and the abundance of eukaryotic viruses also changed substantially. In the annotation, 6 families of DNA viruses and 1 bacteriophage family were present in the normal monkeys but absent after gut bacterial microbiome depletion. Intriguingly, we discovered that changes in the gut bacterial microbiome composition may promote changes in the gut virome composition, and tryptophan, arginine, and quinone may play roles in the interaction between the bacterial microbiome and virome. CONCLUSION: Our results indicated that the clearly altered composition of the virome was correlated with depletion in the bacterial community and that metabolites produced by bacteria possibly play important roles in the interaction.

Suppression of the toll-like receptor 7-dependent type I interferon production pathway by autophagy resulting from enterovirus 71 and coxsackievirus A16 infections facilitates their replication.

Toll-like receptors (TLRs) act as molecular sentinels, detecting invading viral pathogens and triggering host innate immune responses, including autophagy. However, many viruses have evolved a series of strategies to manipulate autophagy for their own benefit. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16), as the primary agents causing hand, foot and mouth disease (HFMD), can induce autophagy leading to their replication. Therefore, the objective of this study was to investigate whether enhanced viral replication caused by autophagy in EV71 and CA16 infections was associated with a TLR-related signaling pathway. Our results demonstrate that complete autophagy and incomplete autophagy were observed in human bronchial epithelial (16HBE) cells infected with EV71 and CA16. Moreover, suppression of autophagy by the pharmacological modulator 3-MA significantly and clearly decreased the survival rates and viral replication of EV71 and CA16 in 16HBE cells. Inhibition of autophagy also enhanced the expression of molecules related to the TLR7-dependent type I interferon (IFN-I) production pathway, such as TLR7, MyD88, IRF7 and IFN-α/ß. Finally, immunofluorescence staining demonstrated that TLR7 endosome marker M6PR levels were clearly reduced in EV71- and CA16-infected cells, while they were markedly elevated in infected cells treated with 3-MA. These findings suggest that increased EV71 and CA16 replication meditated by autophagy in 16HBE cells might promote degradation of the endosome, leading to suppression of the TLR7-mediated IFN-I signaling pathway.

An inactivated enterovirus 71 vaccine in healthy children.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease in children and may be fatal. A vaccine against EV71 is needed. METHODS: We conducted a randomized, double-blind, placebo-controlled phase 3 trial involving healthy children 6 to 71 months of age in Guangxi Zhuang Autonomous Region, China. Two doses of an inactivated EV71 vaccine or placebo were administered intramuscularly, with a 4-week interval between doses, and children were monitored for up to 11 months. The primary end point was protection against hand, foot, and mouth disease caused by EV71. RESULTS: A total of 12,000 children were randomly assigned to receive vaccine or placebo. Serum neutralizing antibodies were assessed in 549 children who received the vaccine. The seroconversion rate was 100% 4 weeks after the two vaccinations, with a geometric mean titer of 170.6. Over the course of two epidemic seasons, the vaccine efficacy was 97.4% (95% confidence interval [CI], 92.9 to 99.0) according to the intention-to-treat analysis and 97.3% (95% CI, 92.6 to 99.0) according to the per-protocol analysis. Adverse events, such as fever (which occurred in 41.6% of the participants who received vaccine vs. 35.2% of those who received placebo), were significantly more common in the week after vaccination among children who received the vaccine than among those who received placebo. CONCLUSIONS: The inactivated EV71 vaccine elicited EV71-specific immune responses and protection against EV71-associated hand, foot, and mouth disease. (Funded by the National Basic Research Program and others; ClinicalTrials.gov number, NCT01569581.).

Functional effector memory T cells contribute to protection from superinfection with heterologous simian immunodeficiency virus or simian-human immunodeficiency virus isolates in Chinese rhesus macaques.

Many studies have revealed a protective effect of infection of an individual with an immunodeficiency virus against subsequent infection with a heterologous strain. However, the extent of protection against superinfection conferred by the first infection and the biological consequences of superinfection are not well understood. Here, we report that a rhesus monkey model of mucosal superinfection was established to investigate the protective immune response. Protection against superinfection was shown to correlate with the extent of the polyfunctionality of CD4+ effector memory T cells, whereas neutralizing antibody responses did not protect against superinfection in this model. Notably, immunodeficiency-virus-associated effector memory T-cell responses might significantly contribute to the suppression of virus superinfection. This provides a potential theoretical basis for the development of an HIV/AIDS vaccine.

The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription.

BACKGROUND: UL7, a tegument protein of Herpes Simplex Virus type I (HSV-1), is highly conserved in viral infection and proliferation and has an unknown mechanism of action. METHODS: A HSV-1 UL7 mutant (UL7-MU) was constructed using the CRISPR-cas9 system. The replication rate and plaque morphology were used to analyze the biological characteristics of the wild-type (WT), UL7-MU and MU-complemented P1 viruses. The virulence of the viruses was evaluated in mice. Real-time RT-qPCR and ChIP assays were used to determine the expression levels of relevant genes. RESULTS: The replication capacity of a recombinant virus (UL7-MU strain) was 10-fold lower than that of the WT strain. The neurovirulence and pathologic effect of the UL7-MU strain were attenuated in infected mice compared with the WT strain. In the latency model, the expression of latency-associated transcript (LAT) in the central nervous system (CNS) and trigeminal nerve was lower in UL7-MU-infected mice than in WT strain-infected mice. The transcription level of the immediate-early gene α-4 in UL7-MU-infected cells was reduced by approximately 2-fold compared with the clear transcriptional peak identified in WT strain-infected Vero cells within 7 h post-infection (p.i.). CONCLUSION: By modulating the transcription of the α-4 gene, UL7 may be involved in transcriptional regulation through its interaction with the transcript complex structure of the viral genome during HSV-1 infection.

Immunity and clinical efficacy of an inactivated enterovirus 71 vaccine in healthy Chinese children: a report of further observations.

BACKGROUND: To investigate the long-term effects on immunity of an inactivated enterovirus 71 (EV71) vaccine and its protective efficacy. METHODS: A sub-cohort of 1,100 volunteers from Guangxi Province in China was eligible for enrolment and randomly administered either the EV71 vaccine or a placebo on days 0 and 28 in a phase III clinical trial and then observed for the following 2 years with approval by an independent ethics committee of Guangxi Zhuang Autonomous Region, China. Serum samples from the 350 participants who provided a full series of blood samples (at all the sampling points) within the 2-year period were collected. Vaccine-induced immune effects, including the neutralizing antibody titres and cross-protection against different genotypes of EV71, were examined. This study also evaluated the protective efficacy of this vaccine based upon clinical diagnosis. RESULTS: This sub-cohort showed a >60% drop-out rate over 2 years. The seroconversion rates among the 161 immunized subjects remained >95% at the end of study. The geometric mean titres of neutralizing antibodies (anti-genotype C4) 360 days after vaccination in 350 subjects were 81.0 (subjects aged 6-11 months), 98.4 (12-23 months), 95.0 (24-35 months), and 81.8 (36-71 months). These titres subsequently increased to 423.1, 659.0, 545.0, and 321.9, respectively, at 540 days post-immunization (d.p.i.), and similar levels were maintained at 720 d.p.i. Higher IFN-γ/IL-4-specific responses to the C4 genotype of EV71 and cross-neutralization reactivity against major EV71 genotype strains were observed in the vaccine group compared to those in the placebo group. Five EV71-infected subjects were observed in the placebo-treated control group and none in the vaccine-immunized group in per-protocol analysis. CONCLUSION: These results are consistent with the induction of dynamic immune responses and protective efficacy of the vaccine against most circulating EV71 strains. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT01569581, Trial registration date: March 2012.

The Structure, Pathogenicity and Immunogenicity of Two Virion Fractions Harvested from Cell Cultures Infected with the CA16 Virus.

OBJECTIVES: To investigate the biological characteristics of the two types of virion fractions of Coxsackievirus A 16 (CA16), which include the real virion fraction and pseudo-virion fraction in their structure, pathogenicity and immunogenicity. METHODS: We obtained the two CA16 virion fractions by density gradient centrifugation. The morphology of virion fractions was analyzed by electron microscopy, while the antigenic characteristics and immunogenicity of two virion fractions were determined by ELISA, SDS-PAGE, Western blot, qRT-PCR, and the mouse model of immune response. RESULTS: The two virion fractions contained the major viral antigen components in their structures, showed similar pathogenicity in a neonatal murine model and were capable of inducing an effective primary immune response in adult mice, regardless of the essential distinction between the two virion fractions, which was the cleavage of VP0 to VP2 and VP4. CONCLUSIONS: The two CA16 virion fractions showed antigenicity and immunogenicity with inducing a specific immune response in animals.