Riding apoptotic bodies for cell-cell transmission by African swine fever virus.

African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.

Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets.

H3N8 avian influenza viruses (AIVs) in China caused two confirmed human infections in 2022, followed by a fatal case reported in 2023. H3N8 viruses are widespread in chicken flocks; however, the zoonotic features of H3N8 viruses are poorly understood. Here, we demonstrate that H3N8 viruses were able to infect and replicate efficiently in organotypic normal human bronchial epithelial (NHBE) cells and lung epithelial (Calu-3) cells. Human isolates of H3N8 virus were more virulent and caused severe pathology in mice and ferrets, relative to chicken isolates. Importantly, H3N8 virus isolated from a patient with severe pneumonia was transmissible between ferrets through respiratory droplets; it had acquired human-receptor-binding preference and amino acid substitution PB2-E627K necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naive to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

Comparative pathology of the nasal epithelium in K18-hACE2 Tg mice, hACE2 Tg mice, and hamsters infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes severe viral pneumonia and is associated with a high fatality rate. A substantial proportion of patients infected by SARS-CoV-2 suffer from mild hyposmia to complete loss of olfactory function, resulting in anosmia. However, the pathogenesis of the olfactory dysfunction and comparative pathology of upper respiratory infections with SARS-CoV-2 are unknown. We describe the histopathological, immunohistochemical, and in situ hybridization findings from rodent models of SARS-CoV-2 infection. The main histopathological findings in the olfactory epithelia of K8-hACE2 Tg mice, hACE2 Tg mice, and hamsters were varying degrees of inflammatory lesions, including disordered arrangement, necrosis, exfoliation, and macrophage infiltration of the olfactory epithelia, and inflammatory exudation. On the basis of these observations, the nasal epithelia of these rodent models appeared to develop moderate, mild, and severe rhinitis, respectively. Correspondingly, SARS-CoV-2 viral RNA and antigen were mainly identified in the olfactory epithelia and lamina propria. Moreover, viral RNA was abundant in the cerebrum of K18-hACE2 Tg mice, including the olfactory bulb. The K8-hACE2 Tg mouse, hACE2 Tg mouse, and hamster models could be used to investigate the pathology of SARS-CoV-2 infection in the upper respiratory tract and central nervous system. These models could help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages.

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

Microarray microRNA profiling of urinary exosomes in a 5XFAD mouse model of Alzheimer's disease.

Background: Alzheimer's disease (AD) is an incurable and irreversible neurodegenerative disease, without a clear pathogenesis. Therefore, identification of candidates before amyloid-ß plaque (Aß) deposition proceeds is of major significance for earlier intervention in AD. Methods: To explore the potential noninvasive earlier biomarkers of AD in a 5XFAD mouse model, microRNAs (miRNAs) from urinary exosomes in 1-month-old pre-Aß accumulation 5XFAD mice models and their littermate controls were profiled by microarray analysis. The differentially expressed miRNAs were further analyzed via droplet digital PCR (ddPCR). Results: Microarray analysis demonstrated that 48 differentially expressed miRNAs (18 upregulated and 30 downregulated), of which six miRNAs - miR-196b-5p, miR-339-3p, miR-34a-5p, miR-376b-3p, miR-677-5p, and miR-721 - were predicted to display gene targets and important signaling pathways closely associated with AD pathogenesis and verified by ddPCR. Conclusions: Urinary exosomal miRNAs showing differences in expression prior to Aß-plaque deposition were identified. These exosomal miRNAs represent potential noninvasive biomarkers that may be used to prevent AD in clinical applications.

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.

Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.

BNT162b2 Vaccine Encoding the SARS-CoV-2 P2 S Protects Transgenic hACE2 Mice against COVID-19.

BNT162b2 is a highly efficacious mRNA vaccine approved to prevent COVID-19. This brief report describes the immunogenicity and anti-viral protective effect of BNT162b2 in hACE2 transgenic mice. Prime-boost immunization with BNT162b2 elicited high titers in neutralizing antibodies against SARS-CoV-2, which correlated with viral clearance and alleviated lung lesions in these mice after viral challenge.

SARS-CoV-2 infection aggravates chronic comorbidities of cardiovascular diseases and diabetes in mice.

Background: Cardiovascular diseases (CVDs) and diabetes mellitus (DM) are top two chronic comorbidities that increase the severity and mortality of COVID-19. However, how SARS-CoV-2 alters the progression of chronic diseases remain unclear. Methods: We used adenovirus to deliver h-ACE2 to lung to enable SARS-CoV-2 infection in mice. SARS-CoV-2's impacts on pathogenesis of chronic diseases were studied through histopathological, virologic and molecular biology analysis. Results: Pre-existing CVDs resulted in viral invasion, ROS elevation and activation of apoptosis pathways contribute myocardial injury during SARS-CoV-2 infection. Viral infection increased fasting blood glucose and reduced insulin response in DM model. Bone mineral density decreased shortly after infection, which associated with impaired PI3K/AKT/mTOR signaling. Conclusion: We established mouse models mimicked the complex pathological symptoms of COVID-19 patients with chronic diseases. Pre-existing diseases could impair the inflammatory responses to SARS-CoV-2 infection, which further aggravated the pre-existing diseases. This work provided valuable information to better understand the interplay between the primary diseases and SARS-CoV-2 infection.

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.

Comprehensive Proteomic Profiling of Urinary Exosomes and Identification of Potential Non-invasive Early Biomarkers of Alzheimer's Disease in 5XFAD Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by irreversible progressive cognitive deficits. Identification of candidate biomarkers, before amyloid-ß-plaque deposition occurs, is therefore of great importance for early intervention of AD. OBJECTIVE: To investigate the potential non-invasive early biomarkers of AD in 5XFAD mouse model, we investigate the proteome of urinary exosomes present in 1-month-old (before amyloid-ß accumulation) 5XFAD mouse models and their littermate controls. Another two groups of 2 and 6 months-old urinary samples were collected for monitoring the dynamic change of target proteins during AD progression. METHODS: Proteomic, bioinformatics analysis, multiple reaction monitoring (MRM), western blotting (WB) or ELISA were performed for analyzing these urinary exosomes. RESULTS: A total of 316 proteins including 44 brain cell markers were identified using liquid chromatography tandem mass spectrometry. Importantly, 18 proteins were unique to the 5XFAD group. Eighty-eight proteins including 11 brain cell markers were differentially expressed. Twenty-two proteins were selected to be verified by WB. Furthermore, based on an independent set of 12 urinary exosomes samples, five in these proteins were further confirmed significant difference. Notably, Annexin 2 and Clusterin displayed significant decreased in AD model during the course detected by ELISA. AOAH, Clusterin, and Ly86 are also brain cell markers that were first reported differential expression in urinary exosomes of AD model. CONCLUSION: Our data demonstrated that some urinary exosome proteins, especially Annexin 2 and Clusterin, as nanometer-sized particles, enable detection of differences before amyloid-ß-plaque deposition in 5XFAD mouse model, which may present an ideal non-invasive source of biomarkers for prevention of AD.

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.

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.

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.

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.

Brain Derived Exosomes Are a Double-Edged Sword in Alzheimer's Disease.

Brain derived exosomes (BDEs) are extracellular nanovesicles that are collectively released by all cell lineages of the central nervous system and contain cargo from their original cells. They are emerging as key mediators of communication and waste management among neurons, glial cells and connective tissue during both physiological and pathological conditions in the brain. We review the rapidly growing frontier of BDEs biology in recent years including the involvement of exosomes in neuronal development, maintenance and communication through their multiple signaling functions. Particularly, we highlight the important role of exosomes in Alzheimer's disease (AD), both as a pathogenic agent and as a disease biomarker. Our understanding of such unique nanovesicles may offer not only answers about the (patho) physiological course in AD and associated neurodegenerative diseases but also ideal methods to develop these vesicles as vehicles for drug delivery or as tools to monitor brain diseases in a non-invasive manner because crossing the blood brain barrier is an inherent capability of exosomes. BDEs have potential as biomarkers and as therapeutic tools for AD and related brain disorders in the near future.

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.

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.