Intraperitoneal versus intranasal administration of lipopolysaccharide in causing sepsis severity in a murine model: a preliminary comparison.

Community-acquired respiratory infection is the commonest cause of sepsis presenting to emergency departments. Yet current experimental animal models simulate peritoneal sepsis with intraperitoneal (I.P.) injection of lipopolysaccharide (LPS) as the predominant route. We aimed to compare the progression of organ injury between I.P. LPS and intranasal (I.N.) LPS in order to establish a better endotoxemia murine model of respiratory sepsis. Eight weeks old male BALB/c mice received LPS-Escherichia coli doses at 0.15, 1, 10, 20, 40 and 100 mg per kg body weight (e.g. LPS-10 is a dose of 10 mg/kg body weight). Disease severity was monitored by a modified Mouse Clinical Assessment Score for Sepsis (M-CASS; range 0-21). A M-CASS score ≥ 10 or a weight reduction of ≥ 20%, was used as a criterion for euthanasia. The primary outcome was the survival rate (either no death or no need for euthanasia). The progression of disease was specified as M-CASS, body weight, blood glucose, histopathological changes to lung, liver, spleen, kidney, brain and heart tissues. Survival rate in I.P. LPS-20 mice was 0% (2/3 died; 1/3 euthanized with M-CASS > 10) at 24 h. Survival rate in all doses of I.N. LPS was 100% (20/20; 3-4 per group) at 96 h. 24 h mean M-CASS post-I.P. LPS-10 was 6.4/21 significantly higher than I.N. LPS-10 of 1.7/21 (Unpaired t test, P < 0.05). Organ injury was present at 96 h in the I.P. LPS-10 group: lung (3/3; 100%), spleen (3/3; 100%) and liver (1/3; 33%). At 24 h in the I.P. LPS-20 group, kidney injury was observed in the euthanized mouse. At 96 h in the post-I.N. LPS-20 group, only lung injury was observed in 2/3 (67%) mice (Kruskal-Wallis test with Dunn's, P < 0.01). At 24 h in the post-I.N. LPS-100 group all (4/4) mice had evidence of lung injury. Variable doses of I.N. LPS in mice produced lung injury but did not produce sepsis. Higher doses of I.P. LPS induced multi-organ injury but not respiratory sepsis. Lethal models of respiratory virus, e.g., influenza A, might provide alternative avenues that can be explored in future research.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Stability of SARS-CoV-2 on Commercial Aircraft Interior Surfaces with Implications for Effective Control Measures.

BACKGROUND: The COVID-19 pandemic from 2019 to 2022 devastated many aspects of life and the economy, with the commercial aviation industry being no exception. One of the major concerns during the pandemic was the degree to which the internal aircraft environment contributed to virus transmission between humans and, in particular, the stability of SARS-CoV-2 on contact surfaces in the aircraft cabin interior. METHOD: In this study, the stability of various major strains of SARS-CoV-2 on interior aircraft surfaces was evaluated using the TCID50 assessment. RESULTS: In contrast to terrestrial materials, SARS-CoV-2 was naturally less stable on common contact points in the aircraft interior, and, over a 4 h time period, there was a 90% reduction in culturable virus. Antiviral and surface coatings were extremely effective at mitigating the persistence of the virus on surfaces; however, their benefit was diminished by regular cleaning and were ineffective after 56 days of regular use and cleaning. Finally, successive strains of SARS-CoV-2 have not evolved to be more resilient to survival on aircraft surfaces. CONCLUSIONS: We conclude that the mitigation strategies for SARS-CoV-2 on interior aircraft surfaces are more than sufficient, and epidemiological evidence over the past three years has not found that surface spread is a major route of transmission.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles.

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.

Mutation nsp6 L232F associated with MERS-CoV zoonotic transmission confers higher viral replication in human respiratory tract cultures ex-vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes zoonotic disease. Dromedary camels are the source of zoonotic infection. We identified a mutation of amino acid leucine to phenylalanine in the codon 232 position of the non-structural protein 6 (nsp6) (nsp6 L232F) that is repeatedly associated with zoonotic transmission. We generated a pair of isogenic recombinant MERS-CoV with nsp6 232L and 232F residues, respectively, and showed that the nsp6 L232F mutation confers higher replication competence in ex-vivo culture of human nasal and bronchial tissues and in lungs of mice experimentally infected in-vivo. Mechanistically, the nsp6 L232F mutation appeared to modulate autophagy and was associated with higher exocytic virus egress, while innate immune responses and zippering activity of the endoplasmic reticulum remained unaffected. Our study suggests that MERS-CoV nsp6 may contribute to viral adaptation to humans. This highlights the importance of continued surveillance of MERS-CoV in both camels and humans.

Replication of Novel Zoonotic-Like Influenza A(H3N8) Virus in Ex Vivo Human Bronchus and Lung.

Human infection with avian influenza A(H3N8) virus is uncommon but can lead to acute respiratory distress syndrome. In explant cultures of the human bronchus and lung, novel H3N8 virus showed limited replication efficiency in bronchial and lung tissue but had a higher replication than avian H3N8 virus in lung tissue.

Heterosubtypic immune pressure accelerates emergence of influenza A virus escape phenotypes in mice.

Rapid antigenic evolution of the influenza A virus surface antigen hemagglutinin undermines protection conferred by seasonal vaccines. Protective correlates targeted by universal vaccines such as cytotoxic T cells or HA stem directed broadly neutralizing antibodies have been shown to select for immune escape mutants during infection. We developed an in vivo serial passage mouse model for viral adaptation and used next generation sequencing to evaluate full genome viral evolution in the context of broadly protective immunity. Heterosubtypic immune pressure increased the incidence of genome-wide single nucleotide variants, though mutations found in early adapted populations were predominantly stochastic in nature. Prolonged adaptation under heterosubtypic immune selection resulted in the manifestation of highly virulent phenotypes that ablated vaccine mediated protection from mortality. High frequency mutations unique to escape phenotypes were identified within the polymerase encoding segments. These findings suggest that a suboptimial usage of population-wide universal influenza vaccine may drive formation of escape variants attributed to polygenic changes.

Reduced Pathogenicity and Transmission Potential of Omicron BA.1 and BA.2 Sublineages Compared with the Early Severe Acute Respiratory Syndrome Coronavirus 2 D614G Variant in Syrian Hamsters.

BACKGROUND: The epidemiological advantage of Omicron variant is evidenced by its rapid spread and the ability to outcompete prior variants. Among Omicron sublineages, early outbreaks were dominated by BA.1, while BA.2 has gained dominance since February 2022. The relative pathogenicity and transmissibility of BA.1 and BA.2 have not been fully defined. METHODS: We compared viral loads and clinical signs in Syrian hamsters after infection with BA.1, BA.2, or D614G variant. A competitive transmission model and next-generation sequencing were used to compare the relative transmission potential of BA.1 and BA.2. RESULTS: BA.1 and BA.2 caused no apparent clinical signs, while D614G caused more than 10% weight loss. Higher viral loads were detected in nasal wash samples and nasal turbinate and lung tissues from BA.1-inoculated hamsters compared with BA.2-inoculated hamsters. No aerosol transmission was observed for BA.1 or BA.2 under the experimental condition in which D614G transmitted efficiently. BA.1 and BA.2 were able to transmit among hamsters via direct contact; however, BA.1 transmitted more efficiently than BA.2 under the competitive transmission model. No recombination was detected from direct contacts exposed simultaneously to BA.1 and BA.2. CONCLUSIONS: Omicron BA.1 and BA.2 demonstrated attenuated pathogenicity and reduced transmission potential in hamsters compared with early SARS-CoV-2 strains.

Replication of SARS-CoV-2 Omicron BA.2 variant in ex vivo cultures of the human upper and lower respiratory tract.

BACKGROUND: The Omicron BA.2 sublineage has replaced BA.1 worldwide and has comparable levels of immune evasion to BA.1. These observations suggest that the increased transmissibility of BA.2 cannot be explained by the antibody evasion. METHODS: Here, we characterized the replication competence and respiratory tissue tropism of three Omicron variants (BA.1, BA.1.1, BA.2), and compared these with the wild-type virus and Delta variant, in human nasal, bronchial and lung tissues cultured ex vivo. FINDINGS: BA.2 replicated more efficiently in nasal and bronchial tissues at 33°C than wild-type, Delta and BA.1. Both BA.2 and BA.1 had higher replication competence than wild-type and Delta viruses in bronchial tissues at 37°C. BA.1, BA.1.1 and BA.2 replicated at a lower level in lung parenchymal tissues compared to wild-type and Delta viruses. INTERPRETATION: Higher replication competence of Omicron BA.2 in the human upper airway at 33°C than BA.1 may be one of the reasons to explain the current advantage of BA.2 over BA.1. A lower replication level of the tested Omicron variants in human lung tissues is in line with the clinical manifestations of decreased disease severity of patients infected with the Omicron strains compared with other ancestral strains. FUNDING: This work was supported by US National Institute of Allergy and Infectious Diseases and the Theme-Based Research Scheme under University Grants Committee of Hong Kong Special Administrative Region, China.

SARS-CoV-2 Omicron variant replication in human bronchus and lung ex vivo.

The emergence of SARS-CoV-2 variants of concern with progressively increased transmissibility between humans is a threat to global public health. The Omicron variant of SARS-CoV-2 also evades immunity from natural infection or vaccines1, but it is unclear whether its exceptional transmissibility is due to immune evasion or intrinsic virological properties. Here we compared the replication competence and cellular tropism of the wild-type virus and the D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants in ex vivo explant cultures of human bronchi and lungs. We also evaluated the dependence on TMPRSS2 and cathepsins for infection. We show that Omicron replicates faster than all other SARS-CoV-2 variants studied in the bronchi but less efficiently in the lung parenchyma. All variants of concern have similar cellular tropism compared to the wild type. Omicron is more dependent on cathepsins than the other variants of concern tested, suggesting that the Omicron variant enters cells through a different route compared with the other variants. The lower replication competence of Omicron in the human lungs may explain the reduced severity of Omicron that is now being reported in epidemiological studies, although determinants of severity are multifactorial. These findings provide important biological correlates to previous epidemiological observations.

Cellular tropism of SARS-CoV-2 in the respiratory tract of Syrian hamsters and B6.Cg-Tg(K18-ACE2)2Prlmn/J transgenic mice.

Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

Neutralizing Monoclonal Antibodies That Target the Spike Receptor Binding Domain Confer Fc Receptor-Independent Protection against SARS-CoV-2 Infection in Syrian Hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main target for neutralizing antibodies. These antibodies can be elicited through immunization or passively transferred as therapeutics in the form of convalescent-phase sera or monoclonal antibodies (MAbs). Potently neutralizing antibodies are expected to confer protection; however, it is unclear whether weakly neutralizing antibodies contribute to protection. Also, their mechanism of action in vivo is incompletely understood. Here, we demonstrate that 2B04, an antibody with an ultrapotent neutralizing activity (50% inhibitory concentration [IC50] of 0.04 µg/ml), protects hamsters against SARS-CoV-2 in a prophylactic and therapeutic infection model. Protection is associated with reduced weight loss and viral loads in nasal turbinates and lungs after challenge. MAb 2B04 also blocked aerosol transmission of the virus to naive contacts. We next examined three additional MAbs (2C02, 2C03, and 2E06), recognizing distinct epitopes within the receptor binding domain of spike protein that possess either minimal (2C02 and 2E06, IC50 > 20 µg/ml) or weak (2C03, IC50 of 5 µg/ml) virus neutralization capacity in vitro. Only 2C03 protected Syrian hamsters from weight loss and reduced lung viral load after SARS-CoV-2 infection. Finally, we demonstrated that Fc-Fc receptor interactions were not required for protection when 2B04 and 2C03 were administered prophylactically. These findings inform the mechanism of protection and support the rational development of antibody-mediated protection against SARS-CoV-2 infections. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has resulted in the loss of millions of lives. Safe and effective vaccines are considered the ultimate remedy for the global social and economic disruption caused by the pandemic. However, a thorough understanding of the immune correlates of protection against this virus is lacking. Here, we characterized four different monoclonal antibodies and evaluated their ability to prevent or treat SARS-CoV-2 infection in Syrian hamsters. These antibodies varied in their ability to neutralize the virus in vitro. Prophylactic administration of potent and weakly neutralizing antibodies protected against SARS-CoV-2 infection, and this effect was Fc receptor independent. The potent neutralizing antibody also had therapeutic efficacy and eliminated onward aerosol transmission. In contrast, minimally neutralizing antibodies provided no protection against infection with SARS-CoV-2 in Syrian hamsters. Combined, these studies highlight the significance of weakly neutralizing antibodies in the protection against SARS-CoV-2 infection and associated disease.

Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism.

BACKGROUND: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined. METHODS: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients' liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. FINDINGS: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15-6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. INTERPRETATION: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. FUNDING: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Introduction of ORF3a-Q57H SARS-CoV-2 Variant Causing Fourth Epidemic Wave of COVID-19, Hong Kong, China.

We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells.

Tropism of SARS-CoV-2, SARS-CoV, and Influenza Virus in Canine Tissue Explants.

BACKGROUND: Human spillovers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns on the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses, which might result in further reassortment. METHODS: We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7, and H9 subtypes, and influenza B viruses of Yamagata-like and Victoria-like lineages in ex vivo canine nasal cavity, soft palate, trachea, and lung tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues. RESULTS: There was limited productive replication of SARS-CoV-2 in canine nasal cavity and SARS-CoV in canine nasal cavity, soft palate, and lung, with unexpectedly high ACE2 levels in canine nasal cavity and soft palate. Canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors. CONCLUSIONS: Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the many chances for spillover during outbreaks.

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses.

BACKGROUND: Neutrophil is of the most abundant number in human immune system. During acute influenza virus infection, neutrophils are already active in the early phase of inflammation - a time in which clinical biopsy or autopsy material is not readily available. However, the role of neutrophil in virus infection is not well understood. Here, we studied the role of neutrophil in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. METHODS: Neutrophils were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro. The ability of the naïve neutrophils to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of neutrophils upon influenza virus infection were evaluated. RESULTS: Our results demonstrated that influenza virus infected alveolar epithelium allowed neutrophil transmigration. Significantly more neutrophils migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Neutrophils were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected neutrophils. H5N1 induced higher cytokine and chemokine gene transcription than H1N1 infected neutrophils, including TNF-α, IFN-ß, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was only observed in H1N1 infected neutrophils at 6 hpi while no NET formation was observed upon H5N1 infection. CONCLUSION: Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection and might contribute to the severity of H5N1 disease.

Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, causing a respiratory disease (coronavirus disease 2019, COVID-19) of varying severity in Wuhan, China, and subsequently leading to a pandemic. The transmissibility and pathogenesis of SARS-CoV-2 remain poorly understood. We evaluate its tissue and cellular tropism in human respiratory tract, conjunctiva, and innate immune responses in comparison with other coronavirus and influenza virus to provide insights into COVID-19 pathogenesis. METHODS: We isolated SARS-CoV-2 from a patient with confirmed COVID-19, and compared virus tropism and replication competence with SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and 2009 pandemic influenza H1N1 (H1N1pdm) in ex-vivo cultures of human bronchus (n=5) and lung (n=4). We assessed extrapulmonary infection using ex-vivo cultures of human conjunctiva (n=3) and in-vitro cultures of human colorectal adenocarcinoma cell lines. Innate immune responses and angiotensin-converting enzyme 2 expression were investigated in human alveolar epithelial cells and macrophages. In-vitro studies included the highly pathogenic avian influenza H5N1 virus (H5N1) and mock-infected cells as controls. FINDINGS: SARS-CoV-2 infected ciliated, mucus-secreting, and club cells of bronchial epithelium, type 1 pneumocytes in the lung, and the conjunctival mucosa. In the bronchus, SARS-CoV-2 replication competence was similar to MERS-CoV, and higher than SARS-CoV, but lower than H1N1pdm. In the lung, SARS-CoV-2 replication was similar to SARS-CoV and H1N1pdm, but was lower than MERS-CoV. In conjunctiva, SARS-CoV-2 replication was greater than SARS-CoV. SARS-CoV-2 was a less potent inducer of proinflammatory cytokines than H5N1, H1N1pdm, or MERS-CoV. INTERPRETATION: The conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens. FUNDING: US National Institute of Allergy and Infectious Diseases, University Grants Committee of Hong Kong Special Administrative Region, China; Health and Medical Research Fund, Food and Health Bureau, Government of Hong Kong Special Administrative Region, China.