Cross-reactive antibody responses to coronaviruses elicited by SARS-CoV-2 infection or vaccination (preprint)

Background: The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. Methods Pre- and post-vaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. Results Among SARS-CoV-2 infection naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The pre-vaccination antibody response to the common cold human coronaviruses did not negatively impact the post-vaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralisation tests. Conclusions Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2 (preprint)

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins ([~]18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

Role of 1'-Ribose Cyano Substitution for Remdesivir to Effectively Inhibit both Nucleotide Addition and Proofreading in SARS-CoV-2 Viral RNA Replication (preprint)

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) provides a promising but challenging drug target due to its intrinsic proofreading exoribonuclease (ExoN) function. Nucleoside triphosphate (NTP) analogues added to the growing RNA chain should supposedly terminate viral RNA replication, but ExoN can cleave the incorporated compounds and counteract their efficacy. Remdesivir targeting SARS-CoV-2 RdRp exerts high drug efficacy in vitro and in vivo. However, its underlying inhibitory mechanisms remain elusive. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 12.6 microseconds to elucidate the molecular mechanisms underlying the inhibitory effects of remdesivir in nucleotide addition (RdRp complex: nsp12-nsp7-nsp8) and proofreading (ExoN complex: nsp14-nsp10). We found that the 1-cyano group of remdesivir possesses the dual role of inhibiting both nucleotide addition and proofreading. For nucleotide addition, we showed that incorporation of one remdesivir is not sufficient to terminate RNA synthesis. Instead, the presence of the polar 1-cyano group of remdesivir at an upstream site causes instability via its electrostatic interactions with a salt bridge formed by Asp865 and Lys593, rendering translocation unfavourable. This may eventually lead to a delayed chain termination of RNA extension by three nucleotides. For proofreading, remdesivir can inhibit cleavage via the steric clash between the 1-cyano group and Asn104. To further examine the role of 1-cyano group in remdesivirs inhibitory effects, we studied three additional NTP analogues with other types of modifications: favipiravir, vidarabine, and fludarabine. Our simulations suggest that all three of them are prone to ExoN cleavage. Our computational findings were further supported by an in vitro assay in Vero E6 cells using live SARS-CoV-2. The dose-response curves suggest that among tested NTP analogues, only remdesivir exerts significant inhibitory effects on viral replication. Our work provides plausible mechanisms at molecular level on how remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.

Transmission routes of Covid-19 virus in the Diamond Princess Cruise ship (preprint)

Background: An outbreak of COVID-19 occurred on the Diamond Princess cruise ship in January and February 2020. We analysed information about cases to infer transmission dynamics and potential modes of transmission. Methods: We collected the daily number of 197 symptomatic cases, and that of the 146 passenger cases in two categories, i.e. those who stayed and did not stay in the same stateroom. We retrieved the quarantine details and the ship's 14-day itinerary. We searched the websites of national/local health authority along the cruise routes and local news using Google for locally confirmed cases associated with the ship. We obtained the design of air conditioning and sewage treatment of the ship from literature. We back-calculated the dates of infection from the epidemic curve and compared with the start of on-board quarantine. Results: Major infections started on Jan 28 and completed by Feb 6 for passengers except those who stayed in the same stateroom with infected individual(s). No other confirmed cases were identified among the disembarked people in Hong Kong except an 80 years old passenger. No confirmed cases were reported in three other stopovers between Jan 27-31 associated with disembarked passengers or visitors from the ship, however two Okinawa taxi drivers became confirmed cases in association with driving the ship passengers. Infection among passengers after Feb 6 was limited to those who stayed in the same stateroom with an infected passenger. Infections in crew members peaked on Feb 7, suggesting significant transmission among crew members after quarantine on Feb 5. Conclusions: We infer that the ship central air conditioning system did not play a role, i.e. the long-range airborne route was absent in the outbreak. Most transmission appears to have occurred through close contact and fomites.

Toilets dominate environmental detection of SARS-CoV-2 virus in a hospital (preprint)

Background: Respiratory and faecal aerosols play a suspected role in transmitting the SARS-CoV-2 virus. We performed extensive environmental sampling in a dedicated hospital building for Covid-19 patients in both toilet and non-toilet environments, and analysed the associated environmental factors. Methods: We collected data of the Covid-19 patients. 107 surface samples, 46 air samples, two exhaled condensate samples, and two expired air samples were collected were collected within and beyond the four three-bed isolation rooms. We reviewed the environmental design of the building and the cleaning routines. We conducted field measurement of airflow and CO2 concentrations. Findings: The 107 surface samples comprised 37 from toilets, 34 from other surfaces in isolation rooms (ventilated at 30-60 L/s), and 36 from other surfaces outside isolation rooms in the hospital. Four of these samples were positive, namely two ward door-handles, one bathroom toilet-seat cover and one bathroom door-handle; and three were weakly positive, namely one bathroom toilet seat, one bathroom washbasin tap lever and one bathroom ceiling-exhaust louvre. One of the 46 air samples was weakly positive, and this was a corridor air sample. The two exhaled condensate samples and the two expired air samples were negative. Interpretation: The faecal-derived aerosols in patients' toilets contained most of the detected SARS-CoV-2 virus in the hospital, highlighting the importance of surface and hand hygiene for intervention.

Pathogenesis and transmission of SARS-CoV-2 virus in golden Syrian hamsters (preprint)

A pandemic caused by the novel SARS-CoV-2 virus with high nucleotide identity to SARS-CoV and SARS-related coronaviruses detected in horseshoe bats is spreading across the world and impacting the healthcare systems and global economy1,2. A suitable small animal model is urgently needed to support the development of vaccines and antiviral treatments against the SARS-CoV-2 virus. We report the pathogenesis and transmissibility of the SARS-CoV-2 in the golden Syrian hamster model. The SARS-CoV-2 virus replicated in the epithelial cells of respiratory and gastrointestinal tracts. Immunohistochemistry demonstrated viral antigens in the areas of lung consolidation on day 2 and 5 post- inoculation, followed by rapid viral clearance and tissue repairing on day 7. Viral antigen was also detected in the epithelial cells of duodenum without apparent inflammatory response on day 2. Notably, we observed that the SARS-CoV-2 virus can be transmitted efficiently from the inoculated hamsters to co-housed naïve contact hamsters. The inoculated hamsters and naturally-infected hamsters lost greater than 10% of the body weight, and all animals recovered with the detection of neutralizing antibodies within 14 days. Our results suggest that SARS-CoV-2 infection in golden Syrian hamsters resemble features found in human patients with mild infections.Authors Sin Fun Sia, Li-Meng Yan, and Alex WH Chin contributed equally to this work.

Short-range airborne route dominates exposure of respiratory infection during close contact (preprint)

A susceptible person experiences the highest exposure risk of respiratory infection when he or she is in close proximity with an infected person. The large droplet route has been commonly believed to be dominant for most respiratory infections since the early 20th century, and the associated droplet precaution is widely known and practiced in hospitals and in the community. The mechanism of exposure to droplets expired at close contact, however, remains surprisingly unexplored. In this study, the exposure to exhaled droplets during close contact (< 2 m) via both the short-range airborne and large droplet sub-routes is studied using a simple mathematical model of expired flows and droplet dispersion/deposition/inhalation, which enables the calculation of exposure due to both deposition and inhalation. The short-range airborne route is found to dominate at most distances studied during both talking and coughing. The large droplet route only dominates when the droplets are larger than 100 m and when the subjects are within 0.2 m while talking or 0.5 m while coughing. The smaller the exhaled droplets, the more important the short-range airborne route. The large droplet route contributes less than 10% of exposure when the droplets are smaller than 50 m and when the subjects are more than 0.3 m apart, even while coughing.

Stability of SARS-CoV-2 in different environmental conditions (preprint)

Stability of SARS-CoV-2 in different environmental conditions.

Respiratory Virus Shedding in Exhaled Breath and Efficacy of Face Masks (preprint)

There are few studies describing the presence of respiratory viruses in respiratory droplets and aerosols in the exhaled breath of infected persons, and the efficacy of facemasks as a source control to prevent respiratory virus transmission. Here, we recruited children and adults with acute respiratory illness and collected respiratory droplets and aerosols, with and without surgical facemasks. We identified human coronaviruses, influenza virus and rhinovirus from both respiratory droplets and aerosols. Surgical face masks reduced detection of coronavirus RNA in both respiratory droplets and aerosols, but only respiratory droplets and not aerosols for influenza virus RNA. Our results provide mechanistic evidence that surgical facemasks could prevent transmission of human coronavirus and influenza virus infections if worn by symptomatic individuals.Authors Donald K Milton and Benjamin J Cowling are joint senior authors.