Air travel-related outbreak of multiple SARS-CoV-2 variants (preprint)

Background. A large cluster of 59 cases were linked to a single flight with 146 passengers from New Delhi to Hong Kong in April 2021. This outbreak coincided with early reports of exponential pandemic growth in New Delhi, which reached a peak of >400,000 newly confirmed cases on 7 May 2021. Methods. Epidemiological information including date of symptom onset, date of positive-sample detection, and travel and contact history for individual cases from this flight were collected. Whole genome sequencing was performed, and sequences were classified based on the dynamic Pango nomenclature system. Maximum-likelihood phylogenetic analysis compared sequences from this flight alongside other cases imported from India to Hong Kong on 26 flights between June 2020 and April 2021, as well as sequences from India or associated with India-related travel from February to April 2021, and 1,217 reference sequences. Results. Sequence analysis identified six lineages of SARS-CoV-2 belonging to two variants of concern (Alpha and Delta) and one variant of public health interest (Kappa) involved in this outbreak. Phylogenetic analysis confirmed at least three independent sub-lineages of Alpha with limited onward transmission, a superspreading event comprising 37 cases of Kappa, and transmission of Delta to only one passenger. Additional analysis of another 26 flights from India to Hong Kong confirmed widespread circulation of all three variants in India since early March 2021. Conclusions. The broad spectrum of disease severity and long incubation period of SARS-CoV-2 pose a challenge for surveillance and control. As illustrated by this particular outbreak, opportunistic infections of SARS-CoV-2 can occur irrespective of variant lineage, and requiring a nucleic acid test within 72 hours of departure may be insufficient to prevent importation or in-flight transmission.

Long-Term Persistence of SARS-CoV-2 Neutralizing Antibody Responses After Infection and Estimates of the Duration of Protection (preprint)

The duration of immunity in SARS-CoV-2 infected people remains unclear. Neutralizing antibody responses are the best available correlate of protection against re-infection. Recent studies have estimated that the correlate of 50% protection from re-infection was 20% of the mean convalescent neutralizing antibody titre. We used sera collected from a cohort of 125 individuals with RT-PCR confirmed SARS-CoV-2 infections up to 386 days after symptom onset. In the subset of 65 sera collected from day 151 to 386 after symptom onset, all remained positive in 50% plaque reduction neutralization tests (PRNT50). Because antibody waning follows a bimodal pattern with slower waning beyond day 90 after illness, we fitted lines of decay to 115 sera from 62 patients collected beyond 90 after symptom onset and estimate that PRNT50 antibody will remain detectable for around 1,717 days after symptom onset and that 50% protective antibody titers will be maintained for around 990 days post-symptom onset, in symptomatic patients. Peak PRNT titres in mildly symptomatic children did not differ from those in mildly symptomatic adults but these antibody titres appear to wane faster in children. There was a high level of correlation between PRNT50 antibody titers and the % of inhibition in surrogate virus neutralization tests. We conclude that there will be relatively long-lived protection from re-infection following symptomatic COVID-19 disease.Funding Information: The study was supported by the Health and Medical Research Fund, Commissioned research on Novel Coronavirus Disease (COVID-19) (Reference no COVID190126) from the Food and Health Bureau, Hong Kong SAR Government and the Theme-based Research Scheme project no. T11-712/19-N, the University Grants Committee of the Hong Kong Government.Declaration of Interests: None of the authors have any conflicts of interest to declare.Ethics Approval Statement: Written informed consent was obtained from the participants or their parents (when the participant was a child) and the studies were approved by the institutional review boards of the respective hospitals, viz. Kowloon West Cluster (KW/EX-20-039 (144-27)), Kowloon Central / Kowloon East cluster (KC/KE-20-0154/ER2) and HKU/HA Hong Kong West Cluster (UW 20-273).

SARS-CoV-2 specific T cell responses are lower in children and increase with age and time after infection (preprint)

SARS-CoV-2 infection of children leads to a mild illness and the immunological differences with adults remains unclear. We quantified the SARS-CoV-2 specific T cell responses in adults and children (<13 years of age) with RT-PCR confirmed asymptomatic and symptomatic infection for long-term memory, phenotype and polyfunctional cytokines. Acute and memory CD4 + T cell responses to structural SARS-CoV-2 proteins significantly increased with age, whilst CD8 + T cell responses increased with time post infection. Infected children had significantly lower CD4 + and CD8 + T cell responses to SARS-CoV-2 structural and ORF1ab proteins compared to infected adults. SARS-CoV-2-specific CD8 + T cell responses were comparable in magnitude to uninfected negative adult controls. In infected adults CD4 + T cell specificity was skewed towards structural peptides, whilst children had increased contribution of ORF1ab responses. This may reflect differing T cell compartmentalisation for antigen processing during antigen exposure or lower recruitment of memory populations. T cell polyfunctional cytokine production was comparable between children and adults, but children had a lower proportion of SARS-CoV-2 CD4 + T cell effector memory. Compared to adults, children had significantly lower levels of antibodies to β-coronaviruses, indicating differing baseline immunity. Total T follicular helper responses was increased in children during acute infection indicating rapid co-ordination of the T and B cell responses. However total monocyte responses were reduced in children which may be reflective of differing levels of inflammation between children and adults. Therefore, reduced prior β-coronavirus immunity and reduced activation and recruitment of de novo responses in children may drive milder COVID-19 pathogenesis.

The SARS-CoV-2 antibody landscape is lower in magnitude for structural proteins, diversified for accessory proteins and stable long-term in children (preprint)

BackgroundChildren are less clinically affected by SARS-CoV-2 infection than adults with the majority of cases being mild or asymptomatic and the differences in infection outcomes are poorly understood. The kinetics, magnitude and landscape of the antibody response may impact the clinical severity and serological diagnosis of COVID-19. Thus, a comprehensive investigation of the antibody landscape in children and adults is needed. MethodsWe tested 254 plasma from 122 children with symptomatic and asymptomatic SARS-CoV-2 infections in Hong Kong up to 206 days post symptom onset, including 146 longitudinal samples from 58 children. Adult COVID-19 patients and pre-pandemic controls were included for comparison. We assessed antibodies to a 14-wide panel of SARS-CoV-2 structural and accessory proteins by Luciferase Immunoprecipitation System (LIPS). FindingsChildren have lower levels of Spike and Nucleocapsid antibodies than adults, and their cumulative humoral response is more expanded to accessory proteins (NSP1 and Open Reading Frames (ORFs)). Sensitive serology using the three N, ORF3b, ORF8 antibodies can discriminate COVID-19 in children. Principal component analysis revealed distinct serological signatures in children and the highest contribution to variance were responses to non-structural proteins ORF3b, NSP1, ORF7a and ORF8. Longitudinal sampling revealed maintenance or increase of antibodies for at least 6 months, except for ORF7b antibodies which showed decline. It was interesting to note that children have higher antibody responses towards known IFN antagonists: ORF3b, ORF6 and ORF7a. The diversified SARS-CoV-2 antibody response in children may be an important factor in driving control of SARS-CoV-2 infection.

SARS-CoV-2 virus culture from the upper respiratory tract: Correlation with viral load, subgenomic viral RNA and duration of illness. (preprint)

In 68 respiratory specimens from a cohort of 35 COVID-19 patients, 32 of them with mild disease, we found SARS coronavirus-2 virus culture and sub-genomic RNA was rarely detectable beyond 8 days after onset of illness although virus RNA by RT-PCR remained detectable for many weeks.

Beyond the Spike: identification of viral targets of the antibody response to SARS-CoV-2 in COVID-19 patients (preprint)

Background: The SARS-CoV-2 virus emerged in December 2019 and caused a pandemic associated with a spectrum of COVID-19 disease ranging from asymptomatic to lethal infection. Serology testing is important for diagnosis of infection, determining infection attack rates and immunity in the population. It also informs vaccine development. Although several serology tests are in use, improving their specificity and sensitivity for early diagnosis on the one hand and for detecting past infection for population-based studies, are priorities. Methods: We evaluated the anti-SARS-CoV-2 antibody profiles to 15 SARS-CoV-2 antigens by cloning and expressing 15 open reading frames (ORFs) in mammalian cells and screened antibody responses to them in COVID-19 patients using the Luciferase Immunoprecipitation System (LIPS). Results: The LIPS technique allowed us to detect antibody responses in COVID-19 patients to 11 of the 15 SARS-CoV-2 antigens tested, identifying novel immunogenic targets. This technique shows that antigens ORF3b and ORF8 allow detection of antibody early in infection in a specific manner and reveals the immuno-dominance of the N antigen in COVID-19 patients. Conclusion: Our report provides an unbiased characterization of antibody responses to a range of SARS-CoV-2 antigens. The combination of 3 SARS-CoV-2 antibody LIPS assays, i.e. N, ORF3b, and ORF8, is sufficient to identify all COVID-19 patients of our cohort even at early time-points of illness, whilst Spike alone fails to do so. Furthermore, our study highlights the importance of investigating new immunogens NSP1, ORF3b, ORF7a and ORF8 which may mediate immune functions other than neutralization which may be beneficial or harmful to the patient.

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.

Tropism of the Novel Coronavirus SARS-CoV-2 in Human Respiratory Tract: An Analysis in Ex Vivo and In Vitro Cultures (preprint)

Background: A novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, to cause a respiratory disease (COVID-19) of varying severity in Wuhan China, subsequently spreading to other parts of China and beyond. Methods: We infected ex vivo explant cultures of the human conjunctiva, bronchus and lung, and in vitro cultures of primary human alveolar epithelial cells and macrophages with SARS-CoV-2, and assessed viral tropism, replication competence and innate immune responses, in comparison with SARS-CoV, MERS-CoV, and the 2009 pandemic influenza H1N1 (pdmH1N1) virus.Findings: SARS-CoV-2 infected ciliated, mucus secreting and club cells of bronchial epithelium, spindled morphologically type I pneumocytes in the lung, and the conjunctival mucosa. Virus replication competence of SARS-CoV-2 in the bronchus was higher than that of SARS-CoV but lower than pdmH1N1. SARS-CoV-2 replication was comparable with SARS-CoV and pdmH1N1 in the lung but was lower than MERS-CoV. SARS-CoV-2 virus was a less potent inducer of pro-inflammatory cytokines compared with H5N1 and MERS-CoV. Influenza virus infection of alveolar epithelial cells increased ACE2 expression.Interpretation: The conjunctival epithelium and the conducting airways appear to be potential portals of infection of SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated comparably in the alveolar epithelium explaining the progression of infection to a primary viral pneumonia.Funding Statement: US National Institute of Allergy and Infectious Diseases (NIAID) under Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract no. HHSN272201400006C and the Theme Based Research Scheme (Ref: T11-705/14N), Hong Kong Special Administrative Region.Declaration of Interests: There is no conflict of interest for all authors.Ethics Approval Statement: All experiments were carried out in a Bio-safety level 3 (BSL-3) facility. Informed consent was obtained from all subjects and approval was granted by the Institutional Review Board (IRB) of the University of Hong Kong and the Hospital Authority (Hong Kong West) (approval no: UW 20-167).

Canine SARS-CoV-2 infection (preprint)

SARS-CoV-2 emerged in Wuhan in December 2019 and has caused the pandemic respiratory disease, COVID-19. Following what is presumed to be an initial zoonotic transmission event, the virus is now spreading efficient in humans. Very little is known about the susceptibility of domestic mammals kept as pets to this virus. Samples were collected over a 13-day period from a 17 year-old neutered male Pomeranian in Hong Kong SA that was taken into isolation after two members of the household tested positive for the virus. Nasal swabs were consistently positive on the five occasions the dog was tested using quantitative RT- PCR with viral loads between 7.5xE2 to 2.6 x10E4 RNA copies per mL of sample. The dog remained asymptomatic. Cultures attempted on three RT-PCR positive nasal samples were negative. Gene sequences from samples from two household members were identical. The viral sequence from the dog differed at three nucleotide positions; two of these resulted in amino acid changes but their significance is yet to be determined. Seroconversion was not detected but this was expected given the asymptomatic infection and low virus load. The evidence suggests that this is an instance of human-to-animal transmission of SARS-COV-2. It is likely that we could see similar events in other infected households. We do not have information yet on whether this virus can cause illness in dogs but no specific signs were seen in this dog. Whether infected dogs could transmit the virus to other animals or back to humans remains unknown. In this case it did not appear to have occurred.