Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.

Susceptibility and Attenuated Transmissibility of SARS-CoV-2 in Domestic Cats.

Domestic cats, an important companion animal, can be infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This has aroused concern regarding the ability of domestic cats to spread the virus that causes coronavirus disease 2019. We systematically demonstrated the pathogenesis and transmissibility of SARS-CoV-2 in cats. Serial passaging of the virus between cats dramatically attenuated the viral transmissibility, likely owing to variations of the amino acids in the receptor-binding domain sites of angiotensin-converting enzyme 2 between humans and cats. These findings provide insight into the transmissibility of SARS-CoV-2 in cats and information for protecting the health of humans and cats.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 via Close Contact and Respiratory Droplets Among Human Angiotensin-Converting Enzyme 2 Mice.

We simulated 3 transmission modes, including close-contact, respiratory droplets and aerosol routes, in the laboratory. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be highly transmitted among naive human angiotensin-converting enzyme 2 (hACE2) mice via close contact because 7 of 13 naive hACE2 mice were SARS-CoV-2 antibody seropositive 14 days after being introduced into the same cage with 3 infected-hACE2 mice. For respiratory droplets, SARS-CoV-2 antibodies from 3 of 10 naive hACE2 mice showed seropositivity 14 days after introduction into the same cage with 3 infected-hACE2 mice, separated by grids. In addition, hACE2 mice cannot be experimentally infected via aerosol inoculation until continued up to 25 minutes with high viral concentrations.

Comparison of media for a human peripheral blood mononuclear cell-based in vitro vaccine evaluation system.

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

Prolonging the delivery of influenza virus vaccine improves the quantity and quality of the induced immune responses in mice.

Introduction: Influenza vaccines play a vital role in protecting individuals from influenza virus infection and severe illness. However, current influenza vaccines have suboptimal efficacy, which is further reduced in cases where the vaccine strains do not match the circulating strains. One strategy to enhance the efficacy of influenza vaccines is by extended antigen delivery, thereby mimicking the antigen kinetics of a natural infection. Prolonging antigen availability was shown to quantitatively enhance influenza virus-specific immune responses but how it affects the quality of the induced immune response is unknown. Therefore, the current study aimed to investigate whether prolongation of the delivery of influenza vaccine improves the quality of the induced immune responses over that induced by prime-boost immunization. Methods: Mice were given daily doses of whole inactivated influenza virus vaccine for periods of 14, 21, or 28 days; the control group received prime-boost immunization with a 28 days interval. Results: Our data show that the highest levels of cellular and humoral immune responses were induced by 28 days of extended antigen delivery, followed by 21, and 14 days of delivery, and prime-boost immunization. Moreover, prolonging vaccine delivery also improved the quality of the induced antibody response, as indicated by higher level of high avidity antibodies, a balanced IgG subclass profile, and a higher level of cross-reactive antibodies. Conclusions: Our findings contribute to a better understanding of the immune response to influenza vaccination and have important implications for the design and development of future slow-release influenza vaccines.

Sequential infection with H1N1 and SARS-CoV-2 aggravated COVID-19 pathogenesis in a mammalian model, and co-vaccination as an effective method of prevention of COVID-19 and influenza.

Influenza A virus may circulate simultaneously with the SARS-CoV-2 virus, leading to more serious respiratory diseases during this winter. However, the influence of these viruses on disease outcome when both influenza A and SARS-CoV-2 are present in the host remains unclear. Using a mammalian model, sequential infection was performed in ferrets and in K18-hACE2 mice, with SARS-CoV-2 infection following H1N1. We found that co-infection with H1N1 and SARS-CoV-2 extended the duration of clinical manifestation of COVID-19, and enhanced pulmonary damage, but reduced viral shedding of throat swabs and viral loads in the lungs of ferrets. Moreover, mortality was increased in sequentially infected mice compared with single-infection mice. Compared with single-vaccine inoculation, co-inoculation of PiCoVacc (a SARS-CoV-2 vaccine) and the flu vaccine showed no significant differences in neutralizing antibody titers or virus-specific immune responses. Combined immunization effectively protected K18-hACE2 mice against both H1N1 and SARS-CoV-2 infection. Our findings indicated the development of systematic models of co-infection of H1N1 and SARS-CoV-2, which together notably enhanced pneumonia in ferrets and mice, as well as demonstrated that simultaneous vaccination against H1N1 and SARS-CoV-2 may be an effective prevention strategy for the coming winter.

Sensitivity of SARS-CoV-2 to different temperatures.

This study was designed to investigate the sensitivity of SARS-CoV-2 to different temperatures, to provide basic data and a scientific basis for the control of COVID-19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 103.2 TCID50/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS-CoV-2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.

Analysis of the molecular mechanism of Pudilan (PDL) treatment for COVID-19 by network pharmacology tools.

BACKGROUND: Pudilan (PDL), a four-herb prescription with the traditional function of heat-clearing and detoxifying, has been clinically used as an anti-SARS-CoV-2 infectory agent in China. PDL might also have therapeutic potentials for COVID-19 while the underlying mechanisms remain to be clarified. METHODS: We used network pharmacology analysis and selected 68 co-targeted genes/proteins as targets of both PDL and COVID-19. These co-targeted genes/proteins were predicted by SwissDock Server for their high-precision docking simulation, and analyzed by STRING for proteins to protein interaction (PPI), pathway and GO (gene ontology) enrichment. The therapeutic effect for PDL treatment on COVID-19 was validated by the TCMATCOV (TCM Anti COVID-19) platform. RESULTS: PDL might prevent the entrance of SARS-CoV-2 entry into cells by blocking the angiotensin-converting enzyme 2 (ACE2). It might inhibit the cytokine storm by affecting C-reactive protein (CRP), interferon-γ (IFN-γ), interleukin- 6 (IL-6), interleukin- 10 (IL-10), tumor necrosis factor (TNF), epidermal growth factor receptor (EGFR), C-C motif chemokine ligand 5 (CCL5), transforming growth factor-ß1 (TGFß1), and other proteins. PDL might moderate the immune system to shorten the course of the disease, delay disease progression, and reduce the mortality rate. CONCLUSION: PDL might have a therapeutic effect on COVID-19 through three aspects, including the moderate immune system, anti-inflammation, and anti-virus entry into cells.

SARS-CoV-2 Causes a Systemically Multiple Organs Damages and Dissemination in Hamsters.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread across the world and impacted global healthcare systems. For clinical patients, COVID-19 not only induces pulmonary lesions but also affects extrapulmonary organs. An ideal animal model that mimics COVID-19 in humans in terms of the induced systematic lesions is urgently needed. Here, we report that Syrian hamster is highly permissive to SARS-CoV-2 and exhibit diffuse alveolar damage and induced extrapulmonary multi-organs damage, including spleen, lymph nodes, different segments of alimentary tract, kidney, adrenal gland, ovary, vesicular gland and prostate damage, at 3-7 days post inoculation (dpi), based on qRT-PCR, in situ hybridization and immunohistochemistry detection. Notably, the adrenal gland is a novel target organ, with abundant viral RNA and antigen expression detected, accompanied by focal to diffuse inflammation. Additionally, viral RNA was also detected in the corpus luteum of the ovary, vesicular gland and prostate. Focal lesions in liver, gallbladder, myocardium, and lymph nodes were still present at 18 dpi, suggesting potential damage after disease. Our findings illustrate systemic histological observations and the viral RNA and antigen distribution in infected hamsters during disease and convalescence to recapitulate those observed in humans with COVID-19, providing helpful data to the pathophysiologic characterization of SARS-CoV-2-induced systemic disease and the development of effective treatment strategies.

Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques.

Coronavirus disease 2019 (COVID-19), which is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. It is unclear whether convalescing patients have a risk of reinfection. We generated a rhesus macaque model of SARS-CoV-2 infection that was characterized by interstitial pneumonia and systemic viral dissemination mainly in the respiratory and gastrointestinal tracts. Rhesus macaques reinfected with the identical SARS-CoV-2 strain during the early recovery phase of the initial SARS-CoV-2 infection did not show detectable viral dissemination, clinical manifestations of viral disease, or histopathological changes. Comparing the humoral and cellular immunity between primary infection and rechallenge revealed notably enhanced neutralizing antibody and immune responses. Our results suggest that primary SARS-CoV-2 exposure protects against subsequent reinfection in rhesus macaques.

Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmitted through the respiratory route, but potential extra-respiratory routes of SARS-CoV-2 transmission remain uncertain. Here we inoculated five rhesus macaques with 1 × 106 TCID50 of SARS-CoV-2 conjunctivally (CJ), intratracheally (IT), and intragastrically (IG). Nasal and throat swabs collected from CJ and IT had detectable viral RNA at 1-7 days post-inoculation (dpi). Viral RNA was detected in anal swabs from only the IT group at 1-7 dpi. Viral RNA was undetectable in tested swabs and tissues after intragastric inoculation. The CJ infected animal had a higher viral load in the nasolacrimal system than the IT infected animal but also showed mild interstitial pneumonia, suggesting distinct virus distributions. This study shows that infection via the conjunctival route is possible in non-human primates; further studies are necessary to compare the relative risk and pathogenesis of infection through these different routes in more detail.

Age-related rhesus macaque models of COVID-19.

BACKGROUND: Since December 2019, an outbreak of the Corona Virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, has become a public health emergency of international concern. The high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models. METHODS: Three 3-5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS-CoV-2, and then analyzed by clinical signs, viral replication, chest X-ray, histopathological changes and immune response. RESULTS: Viral replication of nasopharyngeal swabs, anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge. Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema, notably, old monkeys exhibited diffuse severe interstitial pneumonia. Viral antigens were detected mainly in alveolar epithelial cells and macrophages. CONCLUSION: SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys. Rhesus macaque models infected with SARS-CoV-2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS-CoV-2 infection.

Interleukin-37 Ameliorates Influenza Pneumonia by Attenuating Macrophage Cytokine Production in a MAPK-Dependent Manner.

Viral pneumonitis caused by influenza A (H1N1) virus leads to high levels of morbidity and mortality. Given the limited treatment options for severe influenza pneumonia, it is necessary to explore effective amelioration approaches. Interleukin-37 (IL-37) has been reported to inhibit excessive immune responses and protect against a variety of inflammatory diseases. In this study, by using BALB/c mice intranasally infected with A/California/07/2009 (H1N1), we found that IL-37 treatment increases the survival rate and body weight, and reduces the pulmonary index, impaired the lung injury and decreased production of pro-inflammatory cytokines in the BALF and lung tissue. Moreover, IL-37 administration enhanced not only the percentage of macrophages, but also the percentage of IL-18Rα+ macrophages, suggesting that enhancing the macrophages function may improve outcomes in a murine model of H1N1 infection. Indeed, macrophages depletion reduced the protective effect of IL-37 during H1N1 infection. Furthermore, IL-37 administration inhibited MAPK signaling in RAW264.7 cells infected with H1N1. This study demonstrates that IL-37 treatment can ameliorate influenza pneumonia by attenuating cytokine production, especially by macrophages. Thus, IL-37 might serve as a promising new target for the treatment of influenza A-induced pneumonia.

Human-Derived A/Guangdong/Th005/2017 (H7N9) Exhibits Extremely High Replication in the Lungs of Ferrets and Is Highly Pathogenic in Chickens.

After a series of studies on the pathogenicity of several H7N9 strains from 2013 to 2018, we wanted to dynamically track the pathogenicity of A/Guangdong/Th005/2017 in ferrets and poultry. The pathogenicity and transmissibility of Th005, especially the distribution and replication in tissues, were studied in ferrets. We also aimed to assess the level of Th005 pathogenicity in chickens. The results showed that the pathogenicity of Th005 was significantly increased in ferrets and chickens, especially compared with the Anhui strain. The replication of Th005 in the lung tissues of ferrets was 100-fold higher than that of the Anhui strain. Th005 pathogenicity reached an intravenous pathogenicity index (IVPI) score of 3 in avian models. Continuously high titres of viruses could be detected in the cloacal cavity of chickens infected with Th005. Th005 remained highly pathogenic in mice and chickens after passaging in ferrets. High expression of both the α2,6- and α2,3-sialic acid residues in cells in vitro was beneficial to Th005 replication, which was enhanced compared to the Anhui strain. China needs to strengthen its surveillance of virulent influenza virus strains, such as Th005, which continues to increase in pathogenicity.

The battle against SARS and MERS coronaviruses: Reservoirs and Animal Models.

In humans, infection with the coronavirus, especially the severe acute respiratory syndrome coronavirus (SARS-CoV) and the emerging Middle East respiratory syndrome coronavirus (MERS-CoV), induces acute respiratory failure, resulting in high mortality. Irregular coronavirus related epidemics indicate that the evolutionary origins of these two pathogens need to be identified urgently and there are still questions related to suitable laboratory animal models. Thus, in this review we aim to highlight key discoveries concerning the animal origin of the virus and summarize and compare current animal models.