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Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively. As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.

Influenza is a highly contagious respiratory illness that commonly causes outbreaks among human communities. Details about the exact nature of the droplets produced by human respiratory activities such as breathing, and their potential to carry and transmit influenza A and B viruses is still not fully understood. The objective of our study was to characterize and quantify influenza viral shedding in exhaled aerosols from natural patient breath, and to determine their viral infectivity among participants in a university cohort in tropical Singapore. Using the Gesundheit-II exhaled breath sampling apparatus, samples of exhaled breath of two aerosol size fractions (“coarse” > 5 µm and “fine” ≤ 5 µm) were collected and analyzed from 31 study participants, i.e., 24 with influenza A (including H1N1 and H3N2 subtypes) and 7 with influenza B (including Victoria and Yamagata lineages). Influenza viral copy number was quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Infectivity of influenza virus in the fine particle fraction was determined by culturing in Madin–Darby canine kidney cells. Exhaled influenza virus RNA generation rates ranged from 9 to 1.67 × 105 and 10 to 1.24 × 104 influenza virus RNA copies per minute for the fine and coarse aerosol fractions, respectively. Compared to the coarse aerosol fractions, influenza A and B viruses were detected more frequently in the fine aerosol fractions that harbored 12-fold higher viral loads. Culturable virus was recovered from the fine aerosol fractions from 9 of the 31 subjects (29%). These findings constitute additional evidence to reiterate the important role of fine aerosols in influenza transmission and provide a baseline range of influenza virus RNA generation rates.

Bats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host-pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air-liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

Genome-wide associative studies can potentially uncover novel pathways which modulate anti-viral immune responses against SARS-CoV-2 or identify drivers of severe disease. To date, these studies have yielded loci mostly in non-functional domains of unknown biological significance and invariably require large sample sizes, potentially missing lower frequency variants, especially in under-represented or minority populations. To identify unique genetic traits predisposing to severe COVID-19 in Asians, we employed an alternative strategy using whole exome sequencing of representative cohort of severe versus mild COVID-19 patients. Candidate gene variants were identified by performing logistic regression against top genetic principal components, prioritised for missense variants with likely causal impact. Then, functional sequelae of variants were replicated and re-validated in patients ex vivo to demonstrate causality between genotype and clinical phenotype. Of 136 COVID-19 patients in Singapore (of whom 25% had severe disease), a single nucleotide polymorphism rs2980619 (p.L252F substitution) belonging to NudC-Domain-Containing-1 (NUDCD1) was highly-placed. Homozygous bearers of variant p.L252F had higher (3.97x) odds of severe disease. Age >50 years and male sex were significant covariates which increased the odds of severe disease by 3.38x and 3.16x, respectively. We showed that variant p.L252F reduced NUDCD1 activity, leading to reduced antiviral signalling through RNA helicase DHX15 and antiviral signalling adaptor MAVS, reduced activation of NFκB components RelB and p65, and resultant 1-log higher SARS-CoV-2 viral load compared to wild type (L252) cells. Patients bearing p.L252F had lower NUDCD1, MAVS, and RelB expressions, affirming the above findings. A gene variant of NUDCD1 influences COVID-19 severity in Asians through interacting with DHX15 and MAVS, affecting effective response against SARS-CoV-2.

Highly pathogenic avian influenza (HPAI) virus (e.g. H5N1) infects the lower airway to cause severe infections, and constitute a prime candidate for the emergence of disease X. The nasal epithelium is the primary portal of entry for respiratory pathogens, serving as the airway's physical and immune barrier. While HPAI virus predominantly infects the lower airway, not much is known about its interactions with the nasal epithelium. Hence, we sought to elucidate and compare the differential responses of the nasal epithelium against HPAI infection that may contribute to its pathology, and to identify critical response markers. We infected human nasal epithelial cells (hNECs) cultured at the air-liquid interface from multiple healthy donors with clinical isolates of major human seasonal influenza viruses (H1N1, H3N2, influenza B) and HPAI H5N1. The infected cells were subjected to virologic, transcriptomic and secretory protein analyses. While less adapted to infecting the nasal epithelium, HPAI H5N1 elicited unique host responses unlike seasonal influenza. Interestingly, H5N1 infection of hNECs induced responses indicative of subdued antiviral activity (e.g. reduced expression of IFNβ, and inflammasome mediators, IL-1α and IL-1β); decreased wound healing; suppressed re-epithelialization; compromised epithelial barrier integrity; diminished responses to oxidative stress; and increased transmembrane solute and ion carrier gene expression. These unique molecular changes in response to H5N1 infection may represent potential targets for enhancing diagnostic and therapeutic strategies for better surveillance and management of HPAI infection in humans.

During the SARS-CoV-2 pandemic, angiotensin-converting enzyme 2 (ACE2) was identified as the major entry receptor for the virus and transmembrane serine protease 2 (TMPRSS2) as an important SARS-CoV-2 entry factor. Previous studies investigating the impact of ACE2 and TMPRSS2 gene expression on SARS-CoV-2 susceptibility in adults have yielded inconsistent results, thereby underscoring the need for further research in this domain. We obtained nasopharyngeal swabs from infected adults during the acute and late convalescent phase of SARS-CoV-2 infection and compared the expression of both genes with non-infected household member contacts. We found that ACE2 and TMPRSS2 gene expression is temporarily reduced during the acute phase of SARS-CoV-2 infection presumably due to viral disruption of transcription. Post-recovery, however, the expression of ACE2 and TMPRSS2 was comparable to non-infected household contacts. The lack of significant differences in ACE2 and TMPRSS2 gene expression between SARS-CoV-2-positive adults and uninfected household controls suggests that factors influencing susceptibility to SARS-CoV-2 infection in adults may extend beyond ACE2 and TMPRSS2 expression. However, the findings should be interpreted with caution due to the study’s limited sample size and the heterogeneity of COVID-19 cases. Other non-physiological factors, such as enhanced hygiene practices following the infection of a household member, may also contribute to absence of infection among healthy controls.

Extensive testing is essential to break the transmission of the new coronavirus SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Recently, CRISPR-based diagnostics have emerged as attractive alternatives to quantitative real-time PCR due to their faster turnaround time and their potential to be used in point-of-care testing scenarios. However, existing CRISPR-based assays for COVID-19 have not considered viral genome mutations and RNA editing in human cells. Here, we present the VaNGuard (Variant Nucleotide Guard) test that is not only specific and sensitive for SARS-CoV-2, but can also detect the virus when its genome or transcriptome has evolved or has been edited by deaminases in infected human cells. We show that an engineered AsCas12a enzyme is more tolerant of mismatches than wildtype LbCas12a and that multiplexed Cas12a targeting can overcome the presence of single nucleotide variations. Our assay can be completed in 30 minutes with a dipstick for a rapid point-of-care test. Competing Interest Statement The authors have declared no competing interest.

Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+ cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+ T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.Competing Interest StatementThe authors have declared no competing interest.