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.

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.

Multiple types of SARS-CoV-2 vaccines have been used worldwide, but summarizing their immunologic efficacy post-vaccination remains challenging. The BCR and TCR sequencing based on single-cell sorting makes it possible to evaluate the vaccine-induced immune responses of B or T cells. In this study, we compared the repertoire diversities of B cells and T cells between a whole-virus inactivated vaccine and an S1 protein subunit vaccine in rhesus macaques. We found that the inactivated vaccine could induce a large antigen-specific-BCR repertoire with longer VH CDR3 (21 aa), while the CD3+ TCR α chains of the two vaccine groups showed a similar TCRV/J usage frequency. Detailed analysis of the TCR and BCR repertoires might be of interest for further understanding of the mechanisms of vaccine-induced immune responses.

Reinfection risk is a great concern with regard to the COVID-19 pandemic because a large proportion of the population has recovered from an initial infection, and previous reports found that primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques without viral presence and pathological injury; however, a high possibility for reinfection at the current stage of the pandemic has been proven. We found the reinfection of SARS-CoV-2 in Syrian hamsters with continuous viral shedding in the upper respiratory tracts and few injuries in the lung, and nasal mucosa was exploited by SARS-CoV-2 for replication and shedding during reinfection; meanwhile, no viral replication or enhanced damage was observed in the lower respiratory tracts. Consistent with the mild phenotype in the reinfection, increases in mRNA levels in cytokines and chemokines in the nasal mucosa but only slight increases in the lung were found. Notably, the high levels of neutralizing antibodies in serum could not prevent reinfection in hamsters but may play roles in benefitting the lung recovery and symptom relief of COVID-19. In summary, Syrian hamsters could be reinfected by SARS-CoV-2 with mild symptoms but with obvious viral shedding and replication, and both convalescent and vaccinated patients should be wary of the transmission and reinfection of SARS-CoV-2.

The nucleocapsid (N) protein contributes to key steps of the SARS-CoV-2 life cycle, including packaging of the virus genome and modulating interactions with cytoplasmic components. Expanding knowledge of the N protein acting on cellular proteins and interfering with innate immunity is critical for studying the host antiviral strategy. In the study on SARS-CoV-2 infecting human bronchial epithelial cell line s1(16HBE), we identified that the N protein can promote the interaction between GTPase-activating protein SH3 domain–binding protein 2 (G3BP2) and tripartite motif containing 25 (TRIM25), which is involved in formation of the TRIM25-G3BP2-N protein interactome. Our findings suggest that the N protein is enrolled in the inhibition of type I interferon production in the process of infection. Meanwhile, upgraded binding of G3BP2 and TRIM25 interferes with the RIG-I-like receptor signaling pathway, which may contribute to SARS-CoV-2 escaping from cellular innate immune surveillance. The N protein plays a critical role in SARS-CoV-2 replication. Our study suggests that the N protein and its interacting cellular components has potential for use in antiviral therapy, and adding N protein into the vaccine as an antigen may be a good strategy to improve the effectiveness and safety of the vaccine. Its interference with innate immunity should be strongly considered as a target for SARS-CoV-2 infection control and vaccine design.

Respirovirus such as influenza virus infection induces pulmonary anti-viral immune response, orchestration of innate and adaptive immunity restrain viral infection, otherwise causes severe diseases such as pneumonia. Chemokines regulate leukocyte recruitment to the inflammation site. One chemokine CXCL5, plays a scavenging role to regulate pulmonary host defense against bacterial infection, but its role in pulmonary influenza virus infection is underdetermined. Here, using an influenza (H1N1) infected CXCL5-/- mouse model, we found that CXCL5 not only responds to neutrophil infiltration into infected lungs at the innate immunity stage, but also affects B lymphocyte accumulation in the lungs by regulating the expression of the B cell chemokine CXCL13. Inhibition of CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs, and in vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge. CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to an enhanced B cell immune response and facilitating induced bronchus-associated lymphoid tissue formation in the infected lungs during the late infection and recovery stages. These data highlight multiple regulatory roles of CXCL5 in leukocyte chemotaxis during pulmonary influenza infection.

The emerging new lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have marked a new phase of coronavirus disease 2019 (COVID-19). Understanding the recognition mechanisms of potent neutralizing monoclonal antibodies (NAbs) against the spike protein is pivotal for developing new vaccines and antibody drugs. Here, we isolated several monoclonal antibodies (MAbs) against the SARS-CoV-2 spike protein receptor-binding domain (S-RBD) from the B cell receptor repertoires of a SARS-CoV-2 convalescent. Among these MAbs, the antibody nCoV617 demonstrates the most potent neutralizing activity against authentic SARS-CoV-2 infection, as well as prophylactic and therapeutic efficacies against the human angiotensin-converting enzyme 2 (ACE2) transgenic mouse model in vivo. The crystal structure of S-RBD in complex with nCoV617 reveals that nCoV617 mainly binds to the back of the "ridge" of RBD and shares limited binding residues with ACE2. Under the background of the S-trimer model, it potentially binds to both "up" and "down" conformations of S-RBD. In vitro mutagenesis assays show that mutant residues found in the emerging new lineage B.1.1.7 of SARS-CoV-2 do not affect nCoV617 binding to the S-RBD. These results provide a new human-sourced neutralizing antibody against the S-RBD and assist vaccine development. IMPORTANCE COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic has posed a serious threat to global health and the economy, so it is necessary to find safe and effective antibody drugs and treatments. The receptor-binding domain (RBD) in the SARS-CoV-2 spike protein is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor. It contains a variety of dominant neutralizing epitopes and is an important antigen for the development of new coronavirus antibodies. The significance of our research lies in the determination of new epitopes, the discovery of antibodies against RBD, and the evaluation of the antibodies' neutralizing effect. The identified antibodies here may be drug candidates for the development of clinical interventions for SARS-CoV-2.

ABSTRACTThe risk of secondary infection with SARS-CoV-2 and influenza A virus is becoming a practical problem that must be addressed as the flu season merges with the COVID-19 pandemic. As SARS-CoV-2 and influenza A virus have been found in patients, understanding the in vivo characteristics of the secondary infection between these two viruses is a high priority. Here, hACE2 transgenic mice were challenged with the H1N1 virus at a nonlethal dose during the convalescent stage on 7 and 14 days post SARS-CoV-2 infection, and importantly, subsequent H1N1 infection showed enhanced viral shedding and virus tissue distribution. Histopathological observation revealed an extensive pathological change in the lungs related to H1N1 infection in mice recovered from SARS-CoV-2 infection, with severe inflammation infiltration and bronchiole disruption. Moreover, upon H1N1 exposure on 7 and 14 dpi of SARS-CoV-2 infection, the lymphocyte population activated at a lower level with T cell suppressed in both PBMC and lung. These findings will be valuable for evaluating antiviral therapeutics and vaccines as well as guiding public health work.

Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.

An ideal vaccine against SARS-CoV-2 is expected to elicit broad immunity to prevent viral infection and disease, with efficient viral clearance in the upper respiratory tract (URT). Here, the N protein and prefusion-full S protein (SFLmut) are combined with flagellin (KF) and cyclic GMP-AMP (cGAMP) to generate a candidate vaccine, and this vaccine elicits stronger systemic and mucosal humoral immunity than vaccines containing other forms of the S protein. Furthermore, the candidate vaccine administered via intranasal route can enhance local immune responses in the respiratory tract. Importantly, human ACE2 transgenic mice given the candidate vaccine are protected against lethal SARS-CoV-2 challenge, with superior protection in the URT compared with that in mice immunized with an inactivated vaccine. In summary, the developed vaccine can elicit a multifaceted immune response and induce robust viral clearance in the URT, which makes it a potential vaccine for preventing disease and infection of SARS-CoV-2.

Glycosylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein mediates viral entry and immune escape. While glycan site is determined by viral genetic code, glycosylation is completely dependent on host cell post-translational modification. Here, by producing SARS-CoV-2 virions from various host cell lines, viruses of different origins with diverse spike protein glycan patterns were revealed. Binding affinities to C-type lectin receptors (CLRs) DC&L-SIGN differed in the different glycan pattern virions. Although none of the CLRs supported viral productive infection, viral trans&cis-infection mediated by the CLRs were substantially changed among the different virions. Specifically, trans&cis-infection of virions with a high-mannose structure (Man5GlcNAc2) at the N1098 glycan site of the spike postfusion trimer were markedly enhanced. Considering L-SIGN co-expression with ACE2 on respiratory tract cells, our work underlines viral epigenetic glycosylation in authentic viral infection and highlights the attachment co-receptor role of DC&L-SIGN in SARS-CoV-2 infection and prevention.

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. We report herein a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

Understanding the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clarifying antiviral immunity in hosts are critical aspects for the development of vaccines and antivirals. Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation. However, normal adult mice are not susceptible to SARS-CoV-2. Here, we developed a viral receptor (human angiotensin-converting enzyme 2, hACE2) pulmonary transfection mouse model to establish SARS-CoV-2 infection rapidly in the mouse lung. Based on the model, the virus successfully infected the mouse lung 2 days after transfection. Viral RNA/protein, innate immune cell infiltration, inflammatory cytokine expression, and pathological changes in the infected lungs were observed after infection. Further studies indicated that neutrophils were the first and most abundant leukocytes to infiltrate the infected lungs after viral infection. In addition, using infected CXCL5-knockout mice, chemokine CXCL5 was responsible for neutrophil recruitment. CXCL5 knockout decreased lung inflammation without diminishing viral clearance, suggesting a potential target for controlling pneumonia.

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-1β), chemokines (MCP-1/CCL2, IL-8/CXCL8, and MIP-1β/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.

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Two imported family cases (mother and daughter) of fatal H7N9 infection in Kunming, China were reported in 2017. Epidemiological investigation showed that the two family members had both been exposed to sick chickens in a poultry market. The onset of illness and death of the mother was 7 days later than her daughter, raising concerns about human-to-human transmission of H7N9 in the locality. Sequence alignment and phylogenetic analysis of the virus strains isolated from the two patients revealed high sequence similarity (≥ 99%) and homology to each other. The two virus strains shared a PEIPKGR/G cleavage motif and the same key amino acid mutations across 8 viral genes except for a R292K mutation in the neuraminidase (NA) gene isolated from the mother who had been treated with oseltamivir in the clinic. Moreover, the isolated H7N9 virus possesses avian and human dual-receptor specificity and is able to efficiently proliferate in human cell lines in vitro. Further epidemiological study demonstrated that five family members who had close contacted with the patients were free of illness and negative for the H7N9 genomic test. Collectively, the H7N9 virus described here is still limited to transmit efficiently from human-to-human.

High-level biosafety laboratories (BSL), such as BSL-3 and BSL-4, which deal with high infectivity and virulence pathogens, have become indispensable. Mice are frequently used in animal BSL (ABSL) to establish animal models for infection and to evaluate in vivo immune responses. A project of monitoring and evaluation on physiology and immune status of mice housed in different ABSL labs was performed in the ABSL-2/3/4 labs of Kunming National High-level Biosafety Research Center, China. Female Kunming mice were housed in the ABSL-2/3/4 labs for one month, and mouse behaviors, body physiology/immune status, pulmonary immune status and respiratory bacteria composition were evaluated and compared among mice from the different labs. Mice settled in their new housing environment of the different labs after transfer and gained weight steadily. Blood hematology testing, serum cytokine/chemokine profiles, and blood/spleen lymphocyte constitutions were comparable between the ABSL-2/3/4 labs. The numbers of different pulmonary leukocytes in the bronchoalveolar lavage fluid were at baseline levels in mice from the ABSL-2/3/4 labs. Diversity and dominance of mice respiratory bacteria were semblable among the ABSL-2/3/4 labs. Our results confirm the stability of physiology and immune status of Kunming mice maintained in different ABSL-2/3/4 labs for at least one month.

The aim of this nested case-control study was to assess the combined use of urinary kidney injury molecule (KIM)-1 and interleukin (IL)-18 for acute kidney injury (AKI) after cardiopulmonary bypass surgery (CPB). From a cohort of 122 subjects who underwent CPB, serial urinary KIM-1 and IL-18 concentrations were determined in 30 AKI and 92 non-AKI patients. An increased level of urinary KIM-1 was associated with the occurrence of AKI, whereas an increased level of IL-18 was related to progressive AKI. The combination of these two biomarkers facilitates the early diagnosis and assessment of the likely progression of AKI after CPB.