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Abstract SARS-CoV-2 JN.1 with an additional L455S mutation on spike when compared with its parental variant BA.2.86 has outcompeted all earlier variants to become the dominant circulating variant. Recent studies investigated the immune resistance of SARS-CoV-2 JN.1 but additional factors are speculated to contribute to its global dominance, which remain elusive until today. Here, we find that SARS-CoV-2 JN.1 has a higher infectivity than BA.2.86 in differentiated primary human nasal epithelial cells (hNECs). Mechanistically, we demonstrate that the gained infectivity of SARS-CoV-2 JN.1 over BA.2.86 associates with increased entry efficiency conferred by L455S and better spike cleavage in hNECs. Structurally, S455 altered the mode of binding of JN.1 spike protein to ACE2 when compared to BA.2.86 spike at ACE2H34, and modified the internal structure of JN.1 spike protein by increasing the number of hydrogen bonds with neighboring residues. These findings indicate that a single mutation (L455S) enhances virus entry in hNECs and increases immune evasiveness, which contribute to the robust transmissibility of SARS-CoV-2 JN.1. We further evaluate the in vitro and in vivo virological characteristics between SARS-CoV-2 BA.2.86/JN.1 and EG.5.1/HK.3, and identify key lineage-specific features of the two Omicron sublineages that contribute to our understanding on Omicron antigenicity, transmissibility, and pathogenicity.

There are only eight approved small molecule antiviral drugs for treating COVID-19. Among them, four are nucleotide analogues (remdesivir, JT001, molnupiravir, and azvudine), while the other four are protease inhibitors (nirmatrelvir, ensitrelvir, leritrelvir, and simnotrelvir-ritonavir). Antiviral resistance, unfavourable drug‒drug interaction, and toxicity have been reported in previous studies. Thus there is a dearth of new treatment options for SARS-CoV-2. In this work, a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity. One compound, designated 172, demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern. Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease (3CLpro) by binding to an allosteric site and reduces 3CLpro dimerization. A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro. In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice. Overall, this study identified an alternative druggable site on the SARS-CoV-2 3CLpro, proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

Abstract Avian influenza A virus H7N9 causes severe human infections with >30% fatality. Currently, there is no H7N9-specific prevention or treatment for humans. Here, from a 2013 H7N9 convalescent case in Hong Kong, we isolate four hemagglutinin (HA)-reactive monoclonal antibodies (mAbs), with three directed to the globular head domain (HA1) and one to the stalk domain (HA2). Two clonally related HA1-directed mAbs, H7.HK1 and H7.HK2, potently neutralize H7N9 and protect female mice from lethal H7N9/AH1 challenge. Cryo-EM structures reveal that H7.HK1 and H7.HK2 bind to a β14-centered surface and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on an adjacent protomer, thereby blocking viral entry. Sequence analysis indicates the lateral patch targeted by H7.HK1 and H7.HK2 to be conserved among influenza subtypes. Both H7.HK1 and H7.HK2 retain HA1 binding and neutralization capacity to later H7N9 isolates from 2016–2017, consistent with structural data showing that the antigenic mutations during this timeframe occur at their epitope peripheries. The HA2-directed mAb H7.HK4 lacks neutralizing activity but when used in combination with H7.HK2 moderately augments female mouse protection. Overall, our data reveal antibodies to a conserved lateral HA1 supersite that confer neutralization, and when combined with a HA2-directed non-neutralizing mAb, augment protection.

Summary: Background: The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. Methods: Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model). Findings: By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. Interpretation: Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Summary: Background: Post-acute sequalae of COVID-19 defines a wide range of ongoing symptoms and conditions long after SARS-CoV-2 infection including respiratory diseases. The histopathological changes in the lung and underlying mechanism remain elusive. Methods: We investigated lung histopathological and transcriptional changes in SARS-CoV-2-infected male hamsters at 7, 14, 42, 84 and 120dpi, and compared with A (H1N1)pdm09 infection. Findings: We demonstrated viral residue, inflammatory and fibrotic changes in lung after SARS-CoV-2 but not H1N1 infection. The most prominent histopathological lesion was multifocal alveolar-bronchiolization observed in every SARS-CoV-2 infected hamster (31/31), from 42dpi to 120dpi. Proliferating (Ki67+) CK14+ basal cells accumulated in alveoli adjacent to bronchioles at 7dpi, where they proliferated and differentiated into SCGB1A+ club cell or Tubulin+ ciliated cells forming alveolar-bronchiolization foci. Molecularly, Notch pathway significantly upregulated with intensive Notch3 and Hes1 protein expression in alveolar-bronchiolization foci at 42 and 120dpi, suggesting Notch signaling involving the persistence of alveolar-bronchiolization. This is further demonstrated by spatial transcriptomic analysis. Intriguingly, significant upregulation of some cell-growth promoting pathways and genes such as Tubb4b, Stxbp4, Grb14 and Mlf1 were spatially overlapping with bronchiolization lesion. Interpretation: Incomplete resolution of SARS-CoV-2 infection in lung with viral residue, chronic inflammatory and fibrotic damage and alveolar-bronchiolization impaired respiratory function. Aberrant activation of CK14+ basal cells during tissue regeneration led to persistent alveolar-bronchiolization due to sustained Notch signaling. This study advances our understanding of respiratory PASC, sheds light on disease management and highlights the necessity for monitoring disease progression in people with respiratory PASC. Funding: Funding is listed in the Acknowledgements section.

Summary: Background: De novo amino acid substitutions (DNS) frequently emerge among immunocompromised patients with chronic SARS-CoV-2 infection. While previous studies have reported these DNS, their significance has not been systematically studied. Methods: We performed a review of DNS that emerged during chronic SARS-CoV-2 infection. We searched PubMed until June 2023 using the keywords “(SARS-CoV-2 or COVID-19) and (mutation or sequencing) and ((prolonged infection) or (chronic infection) or (long term))”. We included patients with chronic SARS-CoV-2 infection who had SARS-CoV-2 sequencing performed for at least 3 time points over at least 60 days. We also included 4 additional SARS-CoV-2 patients with chronic infection of our hospital not reported previously. We determined recurrent DNS that has appeared in multiple patients and determined the significance of these mutations among epidemiologically-significant variants. Findings: A total of 34 cases were analyzed, including 30 that were published previously and 4 from our hospital. Twenty two DNS appeared in ≥3 patients, with 14 (64%) belonging to lineage-defining mutations (LDMs) of epidemiologically-significant variants and 10 (45%) emerging among chronically-infected patients before the appearance of the corresponding variant. Notably, nsp9-T35I substitution (Orf1a T4175I) emerged in all three patients with BA.2.2 infection in 2022 before the appearance of Variants of Interest that carry nsp9-T35I as LDM (EG.5 and BA.2.86/JN.1). Structural analysis suggests that nsp9-T35I substitution may affect nsp9-nsp12 interaction, which could be critical for the function of the replication and transcription complex. Interpretation: DNS that emerges recurrently in different chronically-infected patients may be used as a marker for potential epidemiologically-significant variants. Funding: Theme-Based Research Scheme [T11/709/21-N] of the Research Grants Council (See acknowledgements for full list).

Abstract Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

ABSTRACTHepatitis E virus (HEV) variants infecting humans belong to two species: Paslahepevirus balayani (bHEV) and Rocahepevirus ratti (rat hepatitis E virus; rHEV). R. ratti is a ubiquitous rodent pathogen that has recently been recognized to cause hepatitis in humans. Transmission routes of rHEV from rats to humans are currently unknown. In this study, we examined rHEV exposure in cats and dogs to determine if they are potential reservoirs of this emerging human pathogen. Virus-like particle-based IgG enzymatic immunoassays (EIAs) capable of differentiating rHEV & bHEV antibody profiles and rHEV-specific real-time RT–PCR assays were used for this purpose. The EIAs could detect bHEV and rHEV patient-derived IgG spiked in dog and cat sera. Sera from 751 companion dogs and 130 companion cats in Hong Kong were tested with these IgG enzymatic immunoassays (EIAs). Overall, 13/751 (1.7%) dogs and 5/130 (3.8%) cats were sero-reactive to HEV. 9/751 (1.2%) dogs and 2/130 (1.5%) cats tested positive for rHEV IgG, which was further confirmed by rHEV immunoblots. Most rHEV-seropositive animals were from areas in or adjacent to districts reporting human rHEV infection. Neither 881 companion animals nor 652 stray animals carried rHEV RNA in serum or rectal swabs. Therefore, we could not confirm a role for cats and dogs in transmitting rHEV to humans. Further work is required to understand the reasons for low-level seropositivity in these animals.

Abstract Soluble angiotensin‐converting enzyme 2 (ACE2) can act as a decoy molecule that neutralizes severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by blocking spike (S) proteins on virions from binding ACE2 on host cells. Based on structural insights of ACE2 and S proteins, we designed a “muco‐trapping” ACE2‐Fc conjugate, termed ACE2‐(G4S)6‐Fc, comprised of the extracellular segment of ACE2 (lacking the C‐terminal collectrin domain) that is linked to mucin‐binding IgG1‐Fc via an extended glycine‐serine flexible linker. ACE2‐(G4S)6‐Fc exhibits substantially greater binding affinity and neutralization potency than conventional full length ACE2‐Fc decoys or similar truncated ACE2‐Fc decoys without flexible linkers, possessing picomolar binding affinity and strong neutralization potency against pseudovirus and live virus. ACE2‐(G4S)6‐Fc effectively trapped fluorescent SARS‐CoV‐2 virus like particles in fresh human airway mucus and was stably nebulized using a commercial vibrating mesh nebulizer. Intranasal dosing of ACE2‐(G4S)6‐Fc in hamsters as late as 2 days postinfection provided a 10‐fold reduction in viral load in the nasal turbinate tissues by Day 4. These results strongly support further development of ACE2‐(G4S)6‐Fc as an inhaled immunotherapy for COVID‐19, as well as other emerging viruses that bind ACE2 for cellular entry.

Summary: SARS-CoV-2 Omicron variant has evolved into sublineages. Here, we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However, there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population.

Abstract A pan-sarbecovirus or pan-betacoronavirus vaccine that can prevent current and potential future beta-coronavirus infections is important for fighting possible future pandemics. Here, we report a mucosal vaccine that cross-protects small animal models from sarbecoviruses including SARS-CoV-1, SARS-CoV-2 and its variants. The vaccine comprises a live-but-defective SARS-CoV-2 virus that is envelope deficient and has the orf8 segment replaced by interferon-beta, hence named Interferon Beta Integrated SARS-CoV-2 (IBIS) vaccine. Nasal vaccination with IBIS protected mice from lethal homotypic SARS-CoV-2 infection and hamsters from co-housing-mediated transmission of homotypic virus. Moreover, IBIS provided complete protection against heterotypic sarbecoviruses, including SARS-CoV-2 Delta and Omicron variants, and SARS-CoV-1 in both mice and hamsters. Besides inducing a strong lung CD8 + T cell response, IBIS specifically heightened the activation of mucosal virus-specific CD4 + T cells compared to the interferon-null vaccine. The direct production of interferon by IBIS also suppressed virus co-infection of SARS-CoV-2 in human cells, reducing the risk of genetic recombination when using as live vaccines. Altogether, IBIS is a next-generation pan-sarbecovirus vaccine and warrants further clinical investigations.

Abstract Many of the currently available COVID-19 vaccines and therapeutics are not effective against newly emerged SARS-CoV-2 variants. Here, we developed the metallo-enzyme domain of angiotensin converting enzyme 2 (ACE2)—the cellular receptor of SARS-CoV-2—into an IgM-like inhalable molecule (HH-120). HH-120 binds to the SARS-CoV-2 Spike (S) protein with high avidity and confers potent and broad-spectrum neutralization activity against all known SARS-CoV-2 variants of concern. HH-120 was developed as an inhaled formulation that achieves appropriate aerodynamic properties for rodent and monkey respiratory system delivery, and we found that early administration of HH-120 by aerosol inhalation significantly reduced viral loads and lung pathology scores in male golden Syrian hamsters infected by the SARS-CoV-2 ancestral strain (GDPCC-nCoV27) and the Delta variant. Our study presents a meaningful advancement in the inhalation delivery of large biologics like HH-120 (molecular weight (MW) ~ 1000 kDa) and demonstrates that HH-120 can serve as an efficacious, safe, and convenient agent against SARS-CoV-2 variants. Finally, given the known role of ACE2 in viral reception, it is conceivable that HH-120 has the potential to be efficacious against additional emergent coronaviruses.

Abstract Background Whipple’s disease is a chronic infection due to Tropheryma whipplei, commonly reported in the Caucasian but not in the Chinese population. Case presentation A 52-year-old female with good past health, was diagnosed with Whipple’s disease, presenting with constipation, unintentional weight gain, and fleeting polyarthralgia. Investigations prior to admission showed raised CA125 and computed tomography of the abdomen showed multiple retroperitoneal mesenteric lymphadenopathies. Extensive investigations performed on secondary causes of weight gain were unrevealing. Subsequent PET-CT scan revealed generalized lymphadenopathy involving the left deep cervical, supraclavicular, and retroperitoneal mesenteric area. Excisional biopsy of the left supraclavicular lymph node was performed, with histology showing infiltrations of Periodic acid-Schiff positive foamy macrophages. T. whipplei DNA was detected in her serum, saliva, stool, and lymph node by PCR targeting the 16S ribosomal RNA gene. She was started on intravenous ceftriaxone, and then stepped down to oral antibiotics for a total of 44 months. The recurrence of fever after 12 days of ceftriaxone raised the suspicion of Immune Reconstitution Inflammatory Syndrome (IRIS). Serial imaging showed a gradual reduction in the size of retroperitoneal lymphadenopathies. Literature review on Whipple’s disease in the Chinese population identified 13 reports of detectable T. whipplei DNA in clinical specimens. The majority of the cases were pneumonia, followed by culture-negative endocarditis, encephalitis, and skin and soft tissue infection. However, most patients with pneumonia were diagnosed based on next generation sequencing alone, with the resolution of pulmonary infiltrates without adequate duration of antibiotics, suggesting the possibility of colonization instead of infection. The recommendation of long-term doxycycline suppression after treatment may be supported by the slow response of retroperitoneal lymphadenopathies to antibiotics in our patient. Conclusions Unintentional weight gain and constipation could be atypical presentations of Whipple’s disease. It is a rare disease in the Chinese population despite the advancement of molecular techniques in the diagnosis of infections. A prolonged course of antibiotics may be required due to slow clinical response as documented by serial imaging in our case. The possibility of IRIS should be considered in patients with breakthrough fever during treatment of Whipple’s disease.

Summary: Background: Earlier Omicron subvariants including BA.1, BA.2, and BA.5 emerged in waves, with a subvariant replacing the previous one every few months. More recently, the post-BA.2/5 subvariants have acquired convergent substitutions in spike that facilitated their escape from humoral immunity and gained ACE2 binding capacity. However, the intrinsic pathogenicity and replication fitness of the evaluated post-BA.2/5 subvariants are not fully understood. Methods: We systemically investigated the replication fitness and intrinsic pathogenicity of representative post-BA.2/5 subvariants (BL.1, BQ.1, BQ.1.1, XBB.1, CH.1.1, and XBB.1.5) in weanling (3–4 weeks), adult (8–10 weeks), and aged (10–12 months) mice. In addition, to better model Omicron replication in the human nasal epithelium, we further investigated the replication capacity of the post-BA.2/5 subvariants in human primary nasal epithelial cells. Findings: We found that the evaluated post-BA.2/5 subvariants are consistently attenuated in mouse lungs but not in nasal turbinates when compared with their ancestral subvariants BA.2/5. Further investigations in primary human nasal epithelial cells revealed a gained replication fitness of XBB.1 and XBB.1.5 when compared to BA.2 and BA.5.2. Interpretation: Our study revealed that the post-BA.2/5 subvariants are attenuated in lungs while increased in replication fitness in the nasal epithelium, indicating rapid adaptation of the circulating Omicron subvariants in the human populations. Funding: The full list of funding can be found at the Acknowledgements section.

ABSTRACT The availability of simple, inexpensive assays for coronavirus (CoV) detection is critical for conducting animal surveillance studies to prevent new zoonotic epidemics. We have previously developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosing coronavirus disease 2019 (COVID-19) in humans using respiratory samples. In this study, we improved and extended the application of this assay to detect other sarbecoviruses in bats. Twenty-four and twenty-one out of 838 oral and alimentary samples collected during 2017–2019 were sarbecovirus-positive by quantitative reverse transcription PCR (qRT-PCR) and our colorimetric RT-LAMP assay, respectively. PCR and Sanger sequencing of the partial spike (S) gene in the S1 subunit region and RNA-dependent RNA polymerase gene of the 21 sarbecovirus-positive samples showed that they were all closely related to bat SARS-related coronavirus HKU3. A green fluorescent nucleic acid stain, SYTO9, was also added for real-time quantification and evaluated in the colorimetric RT-LAMP assay. We observed a positive correlation (Spearman’s rank correlation coefficient of 0.77, P < 0.0001) between the time to positivity in the colorimetric RT-LAMP assay and cycle threshold (Ct) values in qRT-PCR assay, suggesting that our assay allows quantitative analysis of viral load in samples. This easily performed, highly sensitive, and specific colorimetric RT-LAMP assay could facilitate mass screening for sarbecoviruses in bats and other animal populations. It will be particularly useful for field studies without sophisticated laboratory equipment and expertise. IMPORTANCE We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.

Prevention of robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) requires in vivo evaluation of IgA neutralizing antibodies. Here, we report the efficacy of receptor binding domain (RBD)-specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1, B8-dIgA2 and TH335-dIgA1 against intranasal SARS-CoV-2 challenge in Syrian hamsters. These antibodies exhibited comparable neutralization potency against authentic virus by competing with human angiotensin converting enzyme-2 (ACE2) receptor for RBD binding. While reducing viral loads in lungs significantly, prophylactic intranasal B8-dIgA unexpectedly led to high amount of infectious viruses and extended damage in NT compared to controls. Mechanistically, B8-dIgA failed to inhibit SARS-CoV-2 cell-to-cell transmission, but was hijacked by the virus through dendritic cell-mediated trans-infection of NT epithelia leading to robust nasal infection. Cryo-EM further revealed B8 as a class II antibody binding trimeric RBDs in 3-up or 2-up/1-down conformation. Neutralizing dIgA, therefore, may engage an unexpected mode of SARS-CoV-2 nasal infection and injury.

The devastating Coronavirus Disease 2019 (COVID-19) pandemic indicates that early detection of candidates with pandemic potential is vital. However, comprehensive metagenomic sequencing of the total microbiome is not practical due to the astronomical and rapidly evolving numbers and species of micro-organisms. Analysis of previous pandemics suggests that an increase in human–animal interactions, changes in animal and arthropod distribution due to climate change and deforestation, continuous mutations and interspecies jumping of RNA viruses, and frequent travels are important factors driving pandemic emergence. Besides measures mitigating these factors, surveillance at human–animal interfaces targeting animals with unusual tolerance to viral infections, sick heathcare workers, and workers at high biosafety level laboratories is crucial. Surveillance of sick travellers is important when alerted by an early warning system of a suspected outbreak due to unknown agents. These samples should be screened by multiplex nucleic acid amplification and subsequent unbiased next-generation sequencing. Novel viruses should be isolated in routine cell cultures, complemented by organoid cultures, and then tested in animal models for interspecies transmission potential. Potential agents are candidates for designing rapid diagnostics, therapeutics, and vaccines. For early detection of outbreaks, there are advantages in using event-based surveillance and artificial intelligence (AI), but high background noise and censorship are possible drawbacks. These systems are likely useful if they channel reliable information from frontline healthcare or veterinary workers and large international gatherings. Furthermore, sufficient regulation of high biosafety level laboratories, and stockpiling of broad spectrum antiviral drugs, vaccines, and personal protective equipment are indicated for pandemic preparedness.

ABSTRACTChildren infected by SARS-CoV-2 Omicron variant may develop neurological complications. To study the pathogenesis in the growing brain, we intranasally challenged newly-weaned or mature hamsters with SARS-CoV-2 Omicron BA.2, BA.5, or Delta variant. Omicron BA.2 and Delta infection produced a significantly lower viral load in the lung tissues of newly-weaned than mature hamsters despite comparable histopathological damages. Newly-weaned hamsters had higher brain viral load, significantly increased cerebrospinal fluid concentration of TNF-α and CXCL10 and inflammatory damages including mild meningitis and parenchymal vascular congestion, despite sparse expression of nucleocapsid antigen in brain cells. Furthermore, 63.6% (28/44) of all SARS-CoV-2 infected newly-weaned hamsters showed microgliosis in olfactory bulb (OB), cerebral cortex, and hippocampus. In infected mature hamsters, microgliosis was observed mainly in OB and olfactory cortex of 35.3% (12/34) of their brains. Neuronal degeneration was found in 75% (33/44) of newly-weaned hamsters affecting multiple regions including OB, olfactory cortex, midbrain cortex, and hippocampus, while such changes were mainly observed in the hippocampus of mature hamsters. Importantly, similar brain histopathology was also observed in Omicron BA.5-infected newly-weaned hamsters. Our study suggested that SARS-CoV-2 may affect the brain at a young age. This kind of brain involvement and histological changes are not virus variant or subvariant specific. Incidentally, a moderate amount of eosinophilic infiltration was observed in the mucosa of nasal turbinate and trachea of newly-weaned hamsters infected by Omicron BA.2 and BA.5 but not Delta variant. This histological finding is consistent with the higher incidence of laryngotracheobronchitis in young children infected by the Omicron variant.Summary Intranasal infection of newly-weaned Syrian hamsters by SARS-CoV-2 Omicron variants can lead to brain inflammation and neuron degeneration with detectable low level of viral load and sparse expression of viral nucleoprotein.

ABSTRACTCytokine dynamics in patients with coronavirus disease 2019 (COVID-19) have been studied in blood but seldomly in respiratory specimens. We studied different cell markers and cytokines in fresh nasopharyngeal swab specimens for the diagnosis and for stratifying the severity of COVID-19. This was a retrospective case-control study comparing Myeloperoxidase (MPO), Adenosine deaminase (ADA), C–C motif chemokine ligand 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in 490 (327 patients and 163 control) nasopharyngeal specimens from 317 (154 COVID-19 and 163 control) hospitalized patients. Of the 154 COVID-19 cases, 46 died. Both total and normalized MPO, ADA, CCL22, TNFα, and IL-6 mRNA expression levels were significantly higher in the nasopharyngeal specimens of infected patients when compared with controls, with ADA showing better performance (OR 5.703, 95% CI 3.424–9.500, p

ABSTRACTIncreasing spread by SARS-CoV-2 Omicron variants challenges existing vaccines and broadly reactive neutralizing antibodies (bNAbs) against COVID-19. Here we determine the diversity, potency, breadth and structural insights of bNAbs derived from memory B cells of BNT162b2-vaccinee after homogeneous Omicron BA.1 breakthrough infection. The infection activates diverse memory B cell clonotypes for generating potent class I/II and III bNAbs with new epitopes mapped to the receptor-binding domain (RBD). The top eight bNAbs neutralize wildtype and BA.1 potently but display divergent IgH/IgL sequences and neuralization profiles against other variants of concern (VOCs). Two of them (P2D9 and P3E6) belonging to class III NAbs display comparable potency against BA.4/BA.5, although structural analysis reveals distinct modes of action. P3E6 neutralizes all variants tested through a unique bivalent interaction with two RBDs. Our findings provide new insights into hybrid immunity on BNT162b2-induced diverse memory B cells in response to Omicron breakthrough infection for generating diverse bNAbs with distinct structural basis.

Abstract Background Opportunistic infection is an under-recognized complication of Cushing’s syndrome, with infection due to atypical mycobacterium rarely reported. Mycobacterium szulgai commonly presents as pulmonary infection, with cutaneous infection seldom reported in the literature. Case Presentation 48-year-old man with a newly-diagnosed Cushing’s syndrome secondary to adrenal adenoma presented with a subcutaneous mass on the dorsum of his right hand, was diagnosed with cutaneous Mycobacterium szulgai infection. The most likely source of the infection was through minor unnoticed trauma and inoculation from a foreign body. The patient’s Cushing’s syndrome, high serum cortisol levels and secondary immune suppression facilitated mycobacterial replication and infection. The patient was successfully treated with adrenalectomy, surgical debridement of cutaneous lesion, and a combination of rifampicin, levofloxacin, clarithromycin, and ethambutol for 6 months. There were no signs of relapse one year after cessation of anti-mycobacterial treatment. A literature review on cutaneous M. szulgai infection to further characterize the clinical characteristics of this condition, identified 17 cases of cutaneous M. szulgai infection in the English literature. Cutaneous M. szulgai infections with subsequent disease dissemination are commonly reported in immunocompromised hosts (10/17, 58.8%), as well as in immunocompetent patients with a history of breached skin integrity, such as invasive medical procedures or trauma. The right upper extremity is the most commonly involved site. Cutaneous M. szulgai infection is well controlled with a combination of anti-mycobacterial therapy and surgical debridement. Disseminated infections required a longer duration of therapy than localized cutaneous infections. Surgical debridement may shorten the duration of antibiotics. Conclusions Cutaneous M. szulgai infection is a rare complication of adrenal Cushing’s syndrome. Further studies are needed to provide evidence-based guidelines on the best combination of anti-mycobacterial and surgical therapy for managing this rare infective complication.

Abstract Current available vaccines for COVID-19 are effective in reducing severe diseases and deaths caused by SARS-CoV-2 infection but less optimal in preventing infection. Next-generation vaccines which are able to induce mucosal immunity in the upper respiratory to prevent or reduce infections caused by highly transmissible variants of SARS-CoV-2 are urgently needed. We have developed an intranasal vaccine candidate based on a live attenuated influenza virus (LAIV) with a deleted NS1 gene that encodes cell surface expression of the receptor-binding-domain (RBD) of the SARS-CoV-2 spike protein, designated DelNS1-RBD4N-DAF. Immune responses and protection against virus challenge following intranasal administration of DelNS1-RBD4N-DAF vaccines were analyzed in mice and compared with intramuscular injection of the BioNTech BNT162b2 mRNA vaccine in hamsters. DelNS1-RBD4N-DAF LAIVs induced high levels of neutralizing antibodies against various SARS-CoV-2 variants in mice and hamsters and stimulated robust T cell responses in mice. Notably, vaccination with DelNS1-RBD4N-DAF LAIVs, but not BNT162b2 mRNA, prevented replication of SARS-CoV-2 variants, including Delta and Omicron BA.2, in the respiratory tissues of animals. The DelNS1-RBD4N-DAF LAIV system warrants further evaluation in humans for the control of SARS-CoV-2 transmission and, more significantly, for creating dual function vaccines against both influenza and COVID-19 for use in annual vaccination strategies.

The rebound characteristics of respiratory infections after lifting pandemic control measures were uncertain. From January to November 2023, patients presenting at a teaching hospital were tested for common respiratory viruses and Mycoplasma pneumoniae using a combination of antigen, nucleic acid amplification, and targeted next-generation sequencing (tNGS) tests. The number and rate of positive tests per month, clinical and microbiological characteristics were analyzed. A rapid rebound of SARS-CoV-2 was followed by a slower rebound of M. pneumoniae, with an interval of 5 months between their peaks. The hospitalization rate was higher, with infections caused by respiratory viruses compared to M. pneumoniae. Though the pediatric hospitalization rate of respiratory viruses (66.1%) was higher than that of M. pneumoniae (34.0%), the 4094 cases of M. pneumoniae within 6 months posed a huge burden on healthcare services. Multivariate analysis revealed that M. pneumoniae-infected adults had more fatigue, comorbidities, and higher serum C-reactive protein, whereas children had a higher incidence of other respiratory pathogens detected by tNGS or pathogen-specific PCR, fever, and were more likely to be female. A total of 85% of M. pneumoniae-positive specimens had mutations detected at the 23rRNA gene, with 99.7% showing A2063G mutation. Days to defervescence were longer in those not treated by effective antibiotics and those requiring a change in antibiotic treatment. A delayed but significant rebound of M. pneumoniae was observed after the complete relaxation of pandemic control measures. No unusual, unexplained, or unresponsive cases of respiratory infections which warrant further investigation were identified.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48–299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95–36.60) and 25.94 (range 16.37–36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman’s ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

Summary: Background: Among the Omicron sublineages that have emerged, BA.1, BA.2, BA.5, and their related sublineages have resulted in the largest number of infections. While recent studies demonstrated that all Omicron sublineages robustly escape neutralizing antibody response, it remains unclear on whether these Omicron sublineages share any pattern of evolutionary trajectory on their replication efficiency and intrinsic pathogenicity along the respiratory tract. Methods: We compared the virological features, replication capacity of dominant Omicron sublineages BA.1, BA.2 and BA.5 in the human nasal epithelium, and characterized their pathogenicity in K18-hACE2, A129, young C57BL/6, and aged C57BL/6 mice. Findings: We found that BA.5 replicated most robustly, followed by BA.2 and BA.1, in the differentiated human nasal epithelium. Consistently, BA.5 infection resulted in higher viral gene copies, infectious viral titres and more abundant viral antigen expression in the nasal turbinates of the infected K18-hACE2 transgenic mice. In contrast, the Omicron sublineages are continuously attenuated in lungs of infected K18-hACE2 and C57BL/6 mice, leading to decreased pathogenicity. Nevertheless, lung manifestations remain severe in Omicron sublineages-infected A129 and aged C57BL/6 mice. Interpretation: Our results suggested that the Omicron sublineages might be gaining intrinsic replication fitness in the upper respiratory tract, therefore highlighting the importance of global surveillance of the emergence of hyper-transmissive Omicron sublineages. On the contrary, replication and intrinsic pathogenicity of Omicron is suggested to be further attenuated in the lower respiratory tract. Effective vaccination and other precautions should be in place to prevent severe infections in the immunocompromised populations at risk. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Background & Aims: Rat hepatitis E virus (Rocahepevirus ratti; HEV-C1) is an emerging cause of hepatitis E that is divergent from conventional human-infecting HEV variants (Paslahepevirus balayani; HEV-A). Validated serological assays for HEV-C1 are lacking. We aimed to develop a parallel enzymatic immunoassay (EIA) system that identifies individuals with HEV-C1 exposure. We also aimed to conduct the first HEV-C1 seroprevalence study in humans using this validated EIA system. Methods: Expressed HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) peptides were characterised. Blood samples were simultaneously tested in HEV-A4 p239 and HEV-C1 p241 IgG EIAs. An optical density (OD) cut-off-based interpretation algorithm for identifying samples seropositive for HEV-A or HEV-C1 was validated using RT-PCR-positive infection sera. This algorithm was used to measure HEV-C1 seroprevalence in 599 solid organ transplant recipients and 599 age-matched immunocompetent individuals. Results: Both peptides formed virus-like particles. When run in HEV-A4 p239 and HEV-C1 p241 EIAs, HEV-A and HEV-C1 RT-PCR-positive samples formed distinct clusters with minimal overlap in a two-dimensional plot of optical density values. The final EIA interpretation algorithm showed high agreement with RT-PCR results (Cohen’s κ = 0.959) and was able to differentiate HEV-A and HEV-C1 infection sera with an accuracy of 94.2% (95% CI: 85.8–98.4%). HEV-C1 IgG seroprevalence was 7/599 (1.2%) among solid organ transplant recipients and 4/599 (0.7%) among immunocompetent individuals. Five of 11 (45.5%) of these patients had history of transient hepatitis of unknown cause. Conclusions: HEV-C1 exposure was identified in 11/1198 (0.92%) individuals in Hong Kong indicating endemic exposure. This is the first estimate of HEV-C1 seroprevalence in humans. The parallel IgG EIA algorithm is a valuable tool for investigating epidemiology and risk factors for HEV-C1 infection. Impact and Implications: Rat hepatitis E virus has recently been discovered to infect humans, but antibody tests for this infection are lacking, making it difficult to gauge how common this infection is. We developed an antibody test algorithm that can identify individuals with past rat hepatitis E virus exposure. We used this algorithm to estimate rat hepatitis E exposure rates in humans in Hong Kong and found that approximately 1% of all tested people had been exposed to this virus previously.

IntroductionTherapeutic monoclonal antibodies (mAbs) against the SARS-CoV-2 spike protein have been shown to improve the outcome of severe COVID-19 patients in clinical trials. However, novel variants with spike protein mutations can render many currently available mAbs ineffective.MethodsWe produced mAbs by using hybridoma cells that generated from mice immunized with spike protein trimer and receptor binding domain (RBD). The panel of mAbs were screened for binding and neutralizing activity against different SARS-CoV-2 variants. The in vivo effectiveness of WKS13 was evaluated in a hamster model.ResultsOut of 960 clones, we identified 18 mAbs that could bind spike protein. Ten of the mAbs could attach to RBD, among which five had neutralizing activity against the ancestral strain and could block the binding between the spike protein and human ACE2. One of these mAbs, WKS13, had broad neutralizing activity against all Variants of Concern (VOCs), including the Omicron variant. Both murine or humanized versions of WKS13 could reduce the lung viral load in hamsters infected with the Delta variant.ConclusionsOur data showed that broad-spectrum high potency mAbs can be produced from immunized mice, which can be used in humans after humanization of the Fc region. Our method represents a versatile and rapid strategy for generating therapeutic mAbs for upcoming novel variants.

Objective: Infectious diseases are global health challenge, impacted the communities worldwide particularly in the midst of COVID-19 pandemic. The need of rapid and accurate automated systems for detecting pathogens of concern has always been critical. Ideally, such systems shall detect a large panel of pathogens simultaneously regardless of well-equipped facilities and highly trained operators, thus realizing on-site diagnosis for frontline healthcare providers and in critical locations such as borders and airports. Methods & Results: Avalon Automated Multiplex System, AAMST, is developed to automate a series of biochemistry protocols to detect nucleic acid sequences from multiple pathogens in one test. Automated processes include isolation of nucleic acids from unprocessed samples, reverse transcription and two rounds of amplifications. All procedures are carried out in a microfluidic cartridge performed by a desktop analyzer. The system was validated with reference controls and showed good agreement with their laboratory counterparts. In total 63 clinical samples, 13 positives including those from COVID-19 patients and 50 negative cases were detected, consistent with clinical diagnosis using conventional laboratory methods. Conclusions: The proposed system has demonstrated promising utility. It would benefit the screening and diagnosis of COVID-19 and other infectious diseases in a simple, rapid and accurate fashion. Clinical and Translational Impact Statement— A rapid and multiplex diagnostic system proposed in this work can clinically help to control spread of COVID-19 and other infectious agents as it can provide timely diagnosis, isolation and treatment to patients. Using the system at remoted clinical sites can facilitate early clinical management and surveillance.

Abstract Horseshoe bats host numerous SARS-related coronaviruses without overt disease signs. Bat intestinal organoids, a unique model of bat intestinal epithelium, allow direct comparison with human intestinal organoids. We sought to unravel the cellular mechanism(s) underlying bat tolerance of coronaviruses by comparing the innate immunity in bat and human organoids. We optimized the culture medium, which enabled a consecutive passage of bat intestinal organoids for over one year. Basal expression levels of IFNs and IFN-stimulated genes were higher in bat organoids than in their human counterparts. Notably, bat organoids mounted a more rapid, robust and prolonged antiviral defense than human organoids upon Poly(I:C) stimulation. TLR3 and RLR might be the conserved pathways mediating antiviral response in bat and human intestinal organoids. The susceptibility of bat organoids to a bat coronavirus CoV-HKU4, but resistance to EV-71, an enterovirus of exclusive human origin, indicated that bat organoids adequately recapitulated the authentic susceptibility of bats to certain viruses. Importantly, TLR3/RLR inhibition in bat organoids significantly boosted viral growth in the early phase after SARS-CoV-2 or CoV-HKU4 infection. Collectively, the higher basal expression of antiviral genes, especially more rapid and robust induction of innate immune response, empowered bat cells to curtail virus propagation in the early phase of infection.

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell–cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2–36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2–36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378 T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2–36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

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.

Host circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA–mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P

The novel SARS-CoV-2 Omicron variant may increase the risk of re-infection and vaccine breakthrough infections as it possesses key mutations in the spike protein that affect neutralizing antibody response. Most studies on neutralization susceptibility were conducted using specimens from adult COVID-19 patients or vaccine recipients. However, since the paediatric population has an antibody response to SARS-CoV-2 infection that is distinct from the adult population, it is critical to assess the neutralization susceptibility of pediatric serum specimens. This study compared the neutralization susceptibility of serum specimens collected from 49 individuals of

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

The SARS-CoV-2 Omicron variant has led to a major wave of COVID-19 in Hong Kong between January and May 2022. Here, we used seroprevalence to estimate the combined incidence of vaccination and SARS-CoV-2 infection, including subclinical infection which were not diagnosed at the acute stage. The overall seropositive rate of IgG against receptor binding domain (anti-RBD IgG) increased from 52.2% in December 2021 to 89.3% in May 2022. The level of anti-RBD IgG was lowest in the 0–9 and ≥80 year-old age groups in May 2022. The seropositive rate of antibody against ORF8, which reflects the rate of prior infection, was 23.4% in May 2022. Our data suggest that although most individuals were either vaccinated or infected after the fifth wave, children and older adults remain most vulnerable. Public health measures should target these age groups in order to ameliorate the healthcare consequences of upcoming waves.

SARS-CoV-2 B.1.1.529.1 (Omicron BA.1) emerged in November 2021 and quickly became the predominant circulating SARS-CoV-2 variant globally. Omicron BA.1 contains more than 30 mutations in the spike protein, which contribute to its altered virological features when compared to the ancestral SARS-CoV-2 or previous SARS-CoV-2 variants. Recent studies by us and others demonstrated that Omicron BA.1 is less dependent on transmembrane serine protease 2 (TMPRSS2), less efficient in spike cleavage, less fusogenic, and adopts an altered propensity to utilize the plasma membrane and endosomal pathways for virus entry. Ongoing studies suggest that these virological features of Omicron BA.1 are in part retained by the subsequent Omicron sublineages. However, the exact spike determinants that contribute to these altered features of Omicron remain incompletely understood. In this study, we investigated the spike determinants for the observed virological characteristics of Omicron. By screening for the individual changes on Omicron BA.1 and BA.2 spike, we identify that 69–70 deletion, E484A, and H655Y contribute to the reduced TMPRSS2 usage while 25–27 deletion, S375F, and T376A result in less efficient spike cleavage. Among the shared spike mutations of BA.1 and BA.2, S375F and H655Y reduce spike-mediated fusogenicity. Interestingly, the H655Y change consistently reduces serine protease usage while increases the use of endosomal proteases. In keeping with these findings, the H655Y substitution alone reduces plasma membrane entry and facilitates endosomal entry when compared to SARS-CoV-2 WT. Overall, our study identifies key changes in Omicron spike that contributes to our understanding on the virological determinant and pathogenicity of Omicron.

SARS-CoV-2 has caused the COVID-19 pandemic since early 2020. As of January 2022, the worldwide spreading of SARS-CoV-2 leads to approximately 0.35 billion of human infections and five millions of deaths. Current vaccination is one of the effective ways to control SARS-CoV-2 transmission and reduce the disease severity. However, the antibody level against the immunogen significantly drops several months after the standard two-dose vaccination, and hence a third or fourth dose booster (the same immunogen) has been suggested to boost the antibody response. Here, we described an ultra-effective nasal vaccine booster that potently induced the extraordinary high-level of neutralizing antibody in pre-vaccinated mice. The vaccine booster is composed of a recombinant receptor binding domain of SARS-CoV-2 spike (either wild-type or omicron) fused with a domain of SARS-CoV-2 nucleoprotein. In the absence of adjuvants, a single intranasal administration of the booster in pre-vaccinated mice significantly induced systemic and mucosal antibody responses as evidenced by the elevation of the cross-variant neutralizing antibody and induction of IgA in bronchoalveolar lavage respectively. Most importantly, the single dose nasal vaccine booster (omicron version) potently enhanced the neutralizing activity against authentic SARS-CoV-2 omicron virus infection. Taken together, the induction of respiratory mucosal immunity and the enhancement of cross-variant neutralizing activity by the nasal vaccine booster warrants further clinical trials in humans.

Pandemic influenza virus and SARS-CoV-2 vaiants have posed major global threats to public health. Broad-spectrum antivirals blocking viral entry can be an effective strategy for combating these viruses. Here, we demonstrate a frog-defensin-derived basic peptide (FBP), which broadly inhibits the influenza virus by binding to haemagglutinin so as to block low pH-induced HA-mediated fusion and antagonizes endosomal acidification to inhibit the influenza virus. Moreover, FBP can bind to the SARS-CoV-2 spike to block spike-mediated cell–cell fusion in 293T/ACE2 cells endocytosis. Omicron spike shows a weak cell–cell fusion mediated by TMPRSS2 in Calu3 cells, making the Omicron variant sensitive to endosomal inhibitors. In vivo studies show that FBP broadly inhibits the A(H1N1)pdm09 virus in mice and SARS-CoV-2 (HKU001a and Delta)in hamsters. Notably, FBP shows significant inhibition of Omicron variant replication even though it has a high number of mutations in spike. In conclusion, these results suggest that virus-targeting FBP with a high barrier to drug resistance can be an effective entry-fusion inhibitor against influenza virus and SARS-CoV-2 in vivo.

Abstract Defective interfering genes (DIGs) are short viral genomes and interfere with wild-type viral replication. Here, we demonstrate that the new designed SARS-CoV-2 DIG (CD3600) can significantly inhibit the replication of SARS-CoV-2 including Alpha, Delta, Kappa and Omicron variants in human HK-2 cells and influenza DIG (PAD4) can significantly inhibit influenza virus replication in human A549 cells. One dose of influenza DIGs prophylactically protects 90% mice from lethal challenge of A(H1N1)pdm09 virus and CD3600 inhibits SARS-CoV-2 replication in hamster lungs when DIGs are administrated to lungs one day before viral challenge. To further investigate the gene delivery vector in the respiratory tract, a peptidic TAT2-P1&LAH4, which can package genes to form small spherical nanoparticles with high endosomal escape ability, is demonstrated to dramatically increase gene expression in the lung airway. TAT2-P1&LAH4, with the dual-functional TAT2-P1 (gene-delivery and antiviral), can deliver CD3600 to significantly inhibit the replication of Delta and Omicron SARS-CoV-2 in hamster lungs. This peptide-based nanoparticle system can effectively transfect genes in lungs and deliver DIGs to inhibit SARS-CoV-2 variants and influenza virus in vivo, which provides the new insight into the drug delivery system for gene therapy against respiratory viruses.

Abstract Background Orientia tsutsugamushi (O. tsutsugamushi), an obligate intracellular bacterium, is transmitted to humans through infected larval trombiculid mite bites, causing scrub typhus. Mixed genotypes of O. tsutsugamushi in canonical conserved genes were reported in 8–25% of blood samples from patients. Yet, there are few clinical descriptions of these mixed O. tsutsugamushi-infected patients. Case presentation We report a patient with scrub typhus complicated with pulmonary involvement and hepatic dysfunction, who carried mixed genotypes of the conserved genes but had a single immune-dominant 56-kDa type-specific antigen (tsa56) genotype. The patient was successfully recovered by doxycycline treatment. Conclusions In this reported case, both patient’s eschar and blood samples have repeatedly shown the same results, i.e., no variants were discovered in tsa56 gene that bears multiple hypervariable regions. Whereas the selected highly conserved genes were identified with up to 32 variants in a 2700 base-pair concatenated sequence. The prevalence, disease severity and mechanism of these single-tsa56-genotype mixed infections remain to be investigated on a large scale with more cases.

SUMMARY: Background: The coronavirus disease 2019 (COVID-19) has influenced antimicrobial consumption and incidence of multidrug-resistant organisms (MDROs). We aimed to study the epidemiology of MDROs before and during the COVID-19 pandemic in Hong Kong. Methods: With the maintenance of infection control measures, we described the trend of MDRO infections, including methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter species (CRA), and extended-spectrum-beta-lactamase-(ESBL)-producing Enterobacterales, in a healthcare region with 3100-bed before (1 January 2016 to 31 December 2019, period 1) and during COVID-19 (1 January 2020 to 30 September 2022, period 2), together with the antimicrobial consumption using piecewise Poisson regression. The epidemiological characteristics of newly diagnosed COVID-19 patients with or without MDRO infections were analyzed. Results: Between period 1 and 2, we observed a significant increase in the trend of CRA infections (P

Hong Kong experienced a severe wave of SARS-CoV-2 in early 2022. Here, the authors use genomic and serosurveillance data and show that this wave was dominated by the Omicron BA.2 sublineage, and that low protective immunity, particularly in older age groups, contributed to its severity.

Abstract The emergence of highly transmissible SARS-CoV-2 variants has led to the waves of the resurgence of COVID-19 cases. Effective antivirals against variants are required. Here we demonstrate that a human-derived peptide 4H30 has broad antiviral activity against the ancestral virus and four Variants of Concern (VOCs) in vitro. Mechanistically, 4H30 can inhibit three distinct steps of the SARS-CoV-2 life cycle. Specifically, 4H30 blocks viral entry by clustering SARS-CoV-2 virions; prevents membrane fusion by inhibiting endosomal acidification; and inhibits the release of virions by cross-linking SARS-CoV-2 with cellular glycosaminoglycans. In vivo studies show that 4H30 significantly reduces the lung viral titers in hamsters, with a more potent reduction for the Omicron variant than the Delta variant. This is likely because the entry of the Omicron variant mainly relies on the endocytic pathway which is targeted by 4H30. Moreover, 4H30 reduces syncytia formation in infected hamster lungs. These findings provide a proof of concept that a single antiviral can inhibit viral entry, fusion, and release.

SARS-CoV-2 variants of concern such as the Omicron variant pose a challenge for vaccination and antibody immunotherapy. Here, Zhou et al. isolate a broadly neutralizing antibody (bNAb), named ZCB11, that protects Golden Syrian hamsters against Omicron. Applying CryoEM the authors show that ZCB11 heavy chain predominantly interacts with RBD in up confirmation, which interferes with ACE2 receptor binding.

Scrub typhus, caused by mite-borne Orientia tsutsugamushi (O. tsutsugamushi), is a major febrile disease in the Asia-Pacific region. The DNA load of O. tsutsugamushi in the blood was previously found to be significantly higher in patients with fatal disease than those with non-fatal disease and correlated with the duration of illness, presence of eschar, and hepatic enzyme levels. In this prospective observation study, we analyzed the association of bacterial DNA load with clinical features, disease severity, and genotype using real-time PCR targeting the 56 kDa TSA gene of O. tsutsugamushi in the blood samples of 117 surviving patients with scrub typhus who had not received appropriate antibiotic treatment. The median O. tsutsugamushi DNA load was 3.11×103 copies/mL (range, 44 to 3.3×106 copies/mL). The severity of patients was categorized as mild, moderate, and severe based on the number of dysfunctional organs, and no significant difference in O. tsutsugamushi DNA load was found among these groups. Patients infected with the Karp group showed a significantly higher O. tsutsugamushi DNA load than those in the Gilliam (P

Summary: Background: Obesity is a worldwide epidemic and is considered a risk factor of severe manifestation of Coronavirus Disease 2019 (COVID-19). The pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses to infection, re-infection, and vaccination in individuals with obesity remain incompletely understood. Methods: Using the diet-induced obese (DIO) mouse model, we studied SARS-CoV-2 Alpha- and Omicron BA.1-induced disease manifestations and host immune responses to infection, re-infection, and COVID-19 mRNA vaccination. Findings: Unlike in lean mice, Omicron BA.1 and Alpha replicated to comparable levels in the lungs of DIO mice and resulted in similar degree of tissue damages. Importantly, both T cell and B cell mediated adaptive immune responses to SARS-CoV-2 infection or COVID-19 mRNA vaccination are impaired in DIO mice, leading to higher propensity of re-infection and lower vaccine efficacy. However, despite the absence of neutralizing antibody, vaccinated DIO mice are protected from lung damage upon Omicron challenge, accompanied with significantly more IFN-α and IFN-β production in the lung tissue. Lung RNAseq and subsequent experiments indicated that COVID-19 mRNA vaccination in DIO mice boosted antiviral innate immune response, including the expression of IFN-α, when compared to the nonvaccinated controls. Interpretation: Our findings suggested that COVID-19 mRNA vaccination enhances host innate antiviral responses in obesity which protect the DIO mice to a certain degree when adaptive immunity is suboptimal. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Summary: Background: The ongoing outbreak of SARS-CoV-2 Omicron BA.2 infections in Hong Kong, the model city of universal masking of the world, has resulted in a major public health crisis. Although the third vaccination resulted in strong boosting of neutralization antibody, vaccine efficacy and correlate of immune protection against the major circulating Omicron BA.2 remain to be investigated. Methods: We investigated the vaccine efficacy against the Omicron BA.2 breakthrough infection among 470 public servants who had received different SARS-CoV-2 vaccine regimens including two-dose BNT162b2 (2 × BNT, n = 169), three-dose BNT162b2 (3 × BNT, n = 168), two-dose CoronaVac (2 × CorV, n = 34), three-dose CoronaVac (3 × CorV, n = 67) and third-dose BNT162b2 following 2 × CorV (2 × CorV+1BNT, n = 32). Humoral and cellular immune responses after three-dose vaccination were further characterized and correlated with clinical characteristics of BA.2 infection. Findings: During the BA.2 outbreak, 27.7% vaccinees were infected. The timely third-dose vaccination provided significant protection with lower incidence rates of breakthrough infections (2 × BNT 46.2% vs 3 × BNT 13.1%, p