SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Sequential Delivery of Live Attenuated Influenza Vaccine and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the Ferret Model Can Reduce SARS-CoV-2 Shedding and Does Not Result in Enhanced Lung Pathology.

Cocirculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in coinfected patients. The initial lack of a readily available coronavirus disease 2019 (COVID-19) vaccine has reinforced the importance of influenza vaccine programs during the COVID-19 pandemic. Live attenuated influenza vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration influences the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV was administered to ferrets 3 days before or after SARS-CoV-2 infection. LAIV administration did not exacerbate the SARS-CoV-2 disease course or lung pathology with either regimen. In addition, LAIV administered before SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract. This study demonstrated that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

Infants Younger Than 6 Months Infected With SARS-CoV-2 Show the Highest Respiratory Viral Loads.

There is a paucity of reports on the characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in infants, because most studies have grouped infants with older children. We analyzed the viral loads of 45318 SARS-CoV-2-positive nasopharyngeal swab samples obtained in Buenos Aires, Argentina. Infants younger than 6 months presented higher viral loads than any other age group. Children older than 6 months showed significantly lower viral loads, similar to those founds in adults. This observation raises new questions regarding the role of infants in the spreading of SARS-CoV-2 infection.

The Impact of Cocirculating Pathogens on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/Coronavirus Disease 2019 Surveillance: How Concurrent Epidemics May Introduce Bias and Decrease the Observed SARS-CoV-2 Percentage Positivity.

BACKGROUND: Circulation of seasonal non-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) respiratory viruses with syndromic overlap during the coronavirus disease 2019 (COVID-19) pandemic may alter the quality of COVID-19 surveillance, with possible consequences for real-time analysis and delay in implementation of control measures. METHODS: Using a multipathogen susceptible-exposed-infectious-recovered (SEIR) transmission model formalizing cocirculation of SARS-CoV-2 and another respiratory virus, we assessed how an outbreak of secondary virus may affect 2 COVID-19 surveillance indicators: testing demand and positivity. Using simulation, we assessed to what extent the use of multiplex polymerase chain reaction tests on a subsample of symptomatic individuals can help correct the observed SARS-CoV-2 percentage positivity and improve surveillance quality. RESULTS: We find that a non-SARS-CoV-2 epidemic strongly increases SARS-CoV-2 daily testing demand and artificially reduces the observed SARS-CoV-2 percentage positivity for the duration of the outbreak. We estimate that performing 1 multiplex test for every 1000 COVID-19 tests on symptomatic individuals could be sufficient to maintain surveillance of other respiratory viruses in the population and correct the observed SARS-CoV-2 percentage positivity. CONCLUSIONS: This study showed that cocirculating respiratory viruses can distort SARS-CoV-2 surveillance. Correction of the positivity rate can be achieved by using multiplex polymerase chain reaction tests, and a low number of samples is sufficient to avoid bias in SARS-CoV-2 surveillance.

Mutations that adapt SARS-CoV-2 to mink or ferret do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs, and farmed mink. Since the start of the 2019 pandemic, several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all three mink adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

Systematic Investigation of SARS-CoV-2 Receptor Protein Distribution along Viral Entry Routes in Humans.

BACKGROUND: The novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enters the human body via mucosal surfaces of the upper and/or lower respiratory tract. Viral entry into epithelial cells is mediated via angiotensin-converting enzyme 2 (ACE2) and auxiliary molecules, but the precise anatomic site of infection still remains unclear. METHODS: Here, we systematically investigated the main SARS-CoV-2 receptor proteins ACE2 and transmembrane serine protease 2 (TMPRSS2), as well as 2 molecules potentially involved in viral entry, furin and CD147, in formalin-fixed, paraffin-embedded human tissues. Tissue microarrays incorporating a total of 879 tissue cores from conjunctival (n = 84), sinonasal (n = 95), and lung (bronchiolar/alveolar; n = 96) specimens were investigated for protein expression by immunohistochemistry. RESULTS: ACE2 and TMPRSS2 were expressed in ciliated epithelial cells of the conjunctivae and sinonasal tissues, with highest expression levels observed in the apical cilia. In contrast, in the lung, the expression of those molecules in bronchiolar and alveolar epithelial cells was much rarer and only very focal when present. Furin and CD147 were more uniformly expressed in all tissues analyzed, including the lung. Interestingly, alveolar macrophages consistently expressed high levels of all 4 molecules investigated. CONCLUSIONS: Our study confirms and extends previous findings and contributes to a better understanding of potential SARS-CoV-2 infection sites along the human respiratory tract.

Age-associated SARS-CoV-2 breakthrough infection and changes in immune response in a mouse model.

Older individuals are at higher risk of SARS-CoV-2 infection and severe outcomes, but the underlying mechanisms are incompletely understood. In addition, how age modulates SARS-CoV-2 re-infection and vaccine breakthrough infections remain largely unexplored. Here, we investigated age-associated SARS-CoV-2 pathogenesis, immune responses, and the occurrence of re-infection and vaccine breakthrough infection utilizing a wild-type C57BL/6N mouse model. We demonstrated that interferon and adaptive antibody response upon SARS-CoV-2 challenge are significantly impaired in aged mice compared to young mice, which results in more effective virus replications and severe disease manifestations in the respiratory tract. Aged mice also showed increased susceptibility to re-infection due to insufficient immune protection acquired during the primary infection. Importantly, two-dose COVID-19 mRNA vaccination conferred limited adaptive immune response among the aged mice, making them susceptible to SARS-CoV-2 infection. Collectively, our findings call for tailored and optimized treatments and prevention strategies against SARS-CoV-2 among older individuals.

Sequential infections with rhinovirus and influenza modulate the replicative capacity of SARS-CoV-2 in the upper respiratory tract.

Although frequently reported since the beginning of the pandemic, questions remain regarding the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interaction with circulating respiratory viruses in coinfected patients. We here investigated dual infections involving early-pandemic SARS-CoV-2 and the Alpha variant and three of the most prevalent respiratory viruses, rhinovirus (RV) and Influenza A and B viruses (IAV and IBV), in reconstituted respiratory airway epithelial cells cultured at air-liquid interface. We found that SARS-CoV-2 replication was impaired by primary, but not secondary, rhino- and influenza virus infection. In contrast, SARS-CoV-2 had no effect on the replication of these seasonal respiratory viruses. Inhibition of SARS-CoV-2 correlated better with immune response triggered by RV, IAV and IBV than the virus entry. Using neutralizing antibody against type I and III interferons, SARS-CoV-2 blockade in dual infections could be partly prevented. Altogether, these data suggested that SARS-CoV-2 interaction with seasonal respiratory viruses would be modulated by interferon induction and could impact SARS-CoV-2 epidemiology when circulation of other respiratory viruses is restored.

Analysis of Severe Acute Respiratory Syndrome 2 Replication in Explant Cultures of the Human Upper Respiratory Tract Reveals Broad Tissue Tropism of Wild-Type and B.1.1.7 Variant Viruses.

BACKGROUND: The upper respiratory tract (URT) is the primary entry site for severe acute respiratory syndrome 2 (SARS-CoV-2) and other respiratory viruses, but its involvement in viral amplification and pathogenesis remains incompletely understood. METHODS: In this study, we investigated primary nasal epithelial cultures, as well as vital explanted tissues, to scrutinize the tropism of wild-type SARS-CoV-2 and the recently emerged B.1.1.7 variant. RESULTS: Our analyses revealed a widespread replication competence of SARS-CoV-2 in polarized nasal epithelium as well as in the examined URT and salivary gland tissues, which was also shared by the B.1.1.7 virus. CONCLUSIONS: In our analyses, we highlighted the active role of these anatomic sites in coronavirus disease 2019.

SARS-CoV-2 pseudovirus infectivity and expression of viral entry-related factors ACE2, TMPRSS2, Kim-1, and NRP-1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems.

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.

Efficient Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Exhaled Breath.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease, coronavirus disease-2019 (COVID-19), in some patients. Samples for SARS-CoV-2 testing are typically collected by nasopharyngeal swab, with the virus detected by PCR; however, patients can test positive for 3 months after infection. Without the capacity to assay SARS-CoV-2 in breath, it is not possible to understand the risk for transmission from infected individuals. To detect virus in breath, the Bubbler-a breathalyzer that reverse-transcribes RNA from SARS-CoV-2 particles into a sample-specific barcoded cDNA-was developed. In a study of 70 hospitalized patients, the Bubbler was both more predictive of lower respiratory tract involvement (abnormal chest X-ray) and less invasive than alternatives. Samples tested using the Bubbler were threefold more enriched for SARS-CoV-2 RNA than were samples from tongue swabs, implying that virus particles were being directly sampled. The barcode-enabled Bubbler was used for simultaneous diagnosis in large batches of pooled samples at a lower limit of detection of 334 genomic copies per sample. Diagnosis by sequencing can provide additional information, such as viral load and strain identity. The Bubbler was configured to sample nucleic acids in water droplets circulating in air, demonstrating its potential in environmental monitoring and the protective effect of adequate ventilation.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population.

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus' reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV's horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group.