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

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

Cross-neutralizing antibody against emerging Omicron subvariants of SARS-CoV-2 in infection-naïve individuals with homologous BNT162b2 or BNT162b2(WT + BA.4/5) bivalent booster vaccination.

Since the emergence of SARS-CoV-2, different variants and subvariants successively emerged to dominate global virus circulation as a result of immune evasion, replication fitness or both. COVID-19 vaccines continue to be updated in response to the emergence of antigenically divergent viruses, the first being the bivalent RNA vaccines that encodes for both the Wuhan-like and Omicron BA.5 subvariant spike proteins. Repeated infections and vaccine breakthrough infections have led to complex immune landscapes in populations making it increasingly difficult to assess the intrinsic neutralizing antibody responses elicited by the vaccines. Hong Kong's intensive COVID-19 containment policy through 2020-2021 permitted us to identify sera from a small number of infection-naïve individuals who received 3 doses of the RNA BNT162b2 vaccine encoding the Wuhan-like spike (WT) and were boosted with a fourth dose of the WT vaccine or the bivalent WT and BA.4/5 spike (WT + BA.4/5). While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 (WT + BA.4/BA.5) bivalent vaccine elicited significantly higher plaque neutralizing antibodies to Omicron subvariants BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1, compared to BNT162b2 monovalent vaccine. The single amino acid substitution that differentiates the spike of JN.1 from BA.2.86 resulted in a profound antigenic change.

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

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

Transmission of SARS-CoV-2 delta variant (AY.127) from pet hamsters to humans, leading to onward human-to-human transmission: a case study.

BACKGROUND: Transmission of SARS-CoV-2 from humans to other mammals, including pet animals, has been reported. However, with the exception of farmed mink, there is no previous evidence that these infected animals can infect humans, resulting in sustained human-to-human transmission. Following a confirmed SARS-CoV-2 infection of a pet shop worker, animals in the shop and the warehouse supplying it were tested for evidence of SARS-CoV-2 infection. METHODS: In this case study, viral swabs and blood samples were collected from animals in a pet shop and its corresponding warehouse in Hong Kong. Nasal swab or saliva samples from human COVID-19 patients epidemiologically linked to the pet shop and from subsequent local cases confirmed to be infected by SARS-CoV-2 delta variant were collected. Oral swabs were tested by quantitative RT-PCR (RT-qPCR) for SARS-CoV-2 and blood samples were serologically tested by a surrogate virus neutralisation test and plaque reduction neutralisation test. The SARS-CoV-2 RT-qPCR positive samples were sequenced by next generation viral full genome sequencing using the ISeq sequencing platform (Illumina), and the viral genomes were phylogenetically analysed. FINDINGS: Eight (50%) of 16 individually tested Syrian hamsters in the pet shop and seven (58%) of 12 Syrian hamsters in the corresponding warehouse were positive for SARS-CoV-2 infection in RT-qPCR or serological tests. None of the dwarf hamsters (n=75), rabbits (n=246), guinea pigs (n=66), chinchillas (n=116), and mice (n=2) were confirmed positive for SARS-CoV-2 in RT-qPCR tests. SARS-CoV-2 viral genomes deduced from human and hamster cases in this incident all belong to the delta variant of concern (AY.127) that had not been circulating locally before this outbreak. The viral genomes obtained from hamsters were phylogenetically related with some sequence heterogeneity. Phylogenetic dating suggests infection in these hamsters occurred around Oct 14, 2021 (95% CI Sept 15 to Nov 9, 2021). Multiple zoonotic transmission events to humans were detected, leading to onward human-to-human transmission. INTERPRETATION: Pet hamsters can be naturally infected with SARS-CoV-2. The virus can circulate among hamsters and lead to human infections. Both genetic and epidemiological results strongly suggest that there was more than one hamster-to-human transmission event in this study. This incident also led to onward human transmission. Importation of SARS-CoV-2-infected hamsters was a likely source of this outbreak. FUNDING: US National Institutes of Health, Research Grants Council of Hong Kong, Food and Health Bureau, and InnoHK.

Natural Reassortment of Eurasian Avian-Like Swine H1N1 and Avian H9N2 Influenza Viruses in Pigs, China.

Several zoonotic influenza A viruses detected in humans contain genes derived from avian H9N2 subtypes. We uncovered a Eurasian avian-like H1N1 swine influenza virus with polymerase basic 1 and matrix gene segments derived from the H9N2 subtype, suggesting that H9N2 viruses are infecting pigs and reassorting with swine influenza viruses in China.

Recombinant BA.1/BA.2 SARS-CoV-2 Virus in Arriving Travelers, Hong Kong, February 2022.

We studied SARS-CoV-2 genomes from travelers arriving in Hong Kong during November 2021-February 2022. In addition to Omicron and Delta variants, we detected a BA.1/BA.2 recombinant with a breakpoint near the 5' end of the spike gene in 2 epidemiologically linked case-patients. Continued surveillance for SARS-CoV-2 recombinants is needed.

Probable Transmission of SARS-CoV-2 Omicron Variant in Quarantine Hotel, Hong Kong, China, November 2021.

We report detection of severe acute respiratory syndrome coronavirus 2 Omicron variant (B.1.1.529) in an asymptomatic, fully vaccinated traveler in a quarantine hotel in Hong Kong, China. The Omicron variant was also detected in a fully vaccinated traveler staying in a room across the corridor from the index patient, suggesting transmission despite strict quarantine precautions.

Monitoring International Travelers Arriving in Hong Kong for Genomic Surveillance of SARS-CoV-2.

We sequenced ≈50% of coronavirus disease cases imported to Hong Kong during March-July 2021 and identified 70 cases caused by Delta variants of severe acute respiratory syndrome coronavirus 2. The genomic diversity detected in Hong Kong was similar to global diversity, suggesting travel hubs can play a substantial role in surveillance.

SARS-CoV-2 Superspread in Fitness Center, Hong Kong, China, March 2021.

To investigate a superspreading event at a fitness center in Hong Kong, China, we used genomic sequencing to analyze 102 reverse transcription PCR-confirmed cases of severe acute respiratory syndrome coronavirus 2 infection. Our finding highlights the risk for virus transmission in confined spaces with poor ventilation and limited public health interventions.

Genetic Diversity of SARS-CoV-2 among Travelers Arriving in Hong Kong.

We sequenced 10% of imported severe acute respiratory syndrome coronavirus 2 infections detected in travelers to Hong Kong and revealed the genomic diversity of regions of origin, including lineages not previously reported from those countries. Our results suggest that international or regional travel hubs might be useful surveillance sites to monitor sequence diversity.

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

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

Circulation of Influenza A(H5N8) Virus, Saudi Arabia.

Highly pathogenic avian influenza A(H5N8) viruses have been detected in several continents. However, limited viral sequence data are available from countries in the Middle East. We report full-genome analyses of highly pathogenic H5N8 viruses recently detected in different provinces in Saudi Arabia.

Evidence-Based interventions of Norovirus outbreaks in China.

BACKGROUND: In resource-limited settings where laboratory capacity is limited and response strategy is non-specific, delayed or inappropriate intervention against outbreaks of Norovirus (NoV) are common. Here we report interventions of two norovirus outbreaks, which highlight the importance of evidence-based modeling and assessment to identify infection sources and formulate effective response strategies. METHODS: Spatiotemporal scanning, mathematical and random walk modeling predicted the modes of transmission in the two incidents, which were supported by laboratory results and intervention outcomes. RESULTS: Simulation results indicated that contaminated water was 14 to 500 fold more infectious than infected individuals. Asymptomatic individuals were not effective transmitters. School closure for up to a week still could not contain the outbreak unless the duration was extended to 10 or more days. The total attack rates (TARs) for waterborne NoV outbreaks reported in China (n = 3, median = 4.37) were significantly (p < 0.05) lower than worldwide (n = 14, median = 41.34). The low TARs are likely due to the high number of the affected population. CONCLUSIONS: We found that school closure alone could not contain Norovirus outbreaks. Overlooked personal hygiene may serve as a hotbed for infectious disease transmission. Our results reveal that evidence-based investigations can facilitate timely interventions of Norovirus transmission.

Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children.

Cross-reactive antibodies with Fc receptor (FcR) effector functions may mitigate pandemic virus impact in the absence of neutralizing antibodies. In this exploratory study, we use serum from a randomized placebo-controlled trial of seasonal trivalent influenza vaccination in children (NCT00792051) conducted at the onset of the 2009 H1N1 pandemic (pH1N1) and monitored for infection. We found that seasonal vaccination increases pH1N1 specific antibodies and FcR effector functions. Furthermore, prospective baseline antibody profiles after seasonal vaccination, prior to pH1N1 infection, show that unvaccinated uninfected children have elevated ADCC effector function, FcγR3a and FcγR2a binding antibodies to multiple pH1N1 proteins, past seasonal and avian (H5, H7 and H9) strains. Whereas, children that became pH1N1 infected after seasonal vaccination have antibodies focussed to seasonal strains without FcR functions, and greater aggregated HA-specific profiles for IgM and IgG3. Modeling to predict infection susceptibility, ranked baseline hemagglutination antibody inhibition as the highest contributor to lack of pH1N1 infection, in combination with features that include pH1-IgG1, H1-stem responses and FcR binding to seasonal vaccine and pH1 proteins. Thus, seasonal vaccination can have benefits against pandemic influenza viruses, and some children already have broadly reactive antibodies with Fc potential without vaccination and may be considered 'elite influenza controllers'.

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

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

Wastewater genomic sequencing for SARS-CoV-2 variants surveillance in wastewater-based epidemiology applications.

Wastewater-based epidemiology (WBE) has been widely used as a complementary approach to SARS-CoV-2 clinical surveillance. Wastewater genomic sequencing could provide valuable information on the genomic diversity of SARS-CoV-2 in the surveyed population. However, reliable detection and quantification of variants or mutations remain challenging. In this study, we used mock wastewater samples created by spiking SARS-CoV-2 variant standard RNA into wastewater RNA to evaluate the impacts of sequencing throughput on various aspects such as genome coverage, mutation detection, and SARS-CoV-2 variant deconvolution. We found that wastewater datasets with sequencing throughput greater than 0.5 Gb yielded reliable results in genomic analysis. In addition, using in silico mock datasets, we evaluated the performance of the adopted pipeline for variant deconvolution. By sequencing 86 wastewater samples covering more than 6 million people over 7 months, we presented two use cases of wastewater genomic sequencing for surveying COVID-19 in Hong Kong in WBE applications, including the replacement of Delta variants by Omicron variants, and the prevalence and development trends of three Omicron sublineages. Importantly, the wastewater genomic sequencing data were able to reveal the variant trends 16 days before the clinical data did. By investigating mutations of the spike (S) gene of the SARS-CoV-2 virus, we also showed the potential of wastewater genomic sequencing in identifying novel mutations and unique alleles. Overall, our study demonstrated the crucial role of wastewater genomic surveillance in providing valuable insights into the emergence and monitoring of new SARS-CoV-2 variants and laid a solid foundation for the development of genomic analysis methodologies for WBE of other novel emerging viruses in the future.

Refining detection methods for emerging SARS-CoV-2 mutants in wastewater: A case study on the Omicron variants.

COVID-19 is an ongoing public health threat worldwide driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Wastewater surveillance has emerged as a complementary tool to clinical surveillance to control the COVID-19 pandemic. With the emergence of new variants of SARS-CoV-2, accumulated mutations that occurred in the SARS-CoV-2 genome raise new challenges for RT-qPCR diagnosis used in wastewater surveillance. There is a pressing need to develop refined methods for modifying primer/probes to better detect these emerging variants in wastewater. Here, we exemplified this process by focusing on the Omicron variants, for which we have developed and validated a modified detection method. We first modified the primers/probe mismatches of three assays commonly used in wastewater surveillance according to in silico analysis results for the mutations of 882 sequences collected during the fifth-wave outbreak in Hong Kong, and then evaluated them alongside the seven original assays. The results showed that five of seven original assays had better sensitivity for detecting Omicron variants, with the limits of detection (LoDs) ranging from 1.53 to 2.76 copies/µL. UCDC-N1 and Charité-E sets had poor performances, having LoDs higher than 10 copies/µL and false-positive/false-negative results in wastewater testing, probably due to the mismatch and demonstrating the need for modification of primer/probe sequences. The modified assays exhibited higher sensitivity and specificity, along with better reproducibility in detecting 81 wastewater samples. In addition, the sequencing results of six wastewater samples by Illumina also validated the presence of mismatches in the primer/probe binding sites of the three assays. This study highlights the importance of re-configuration of the primer-probe sets and refinements for the sequences to ensure the diagnostic effectiveness of RT-qPCR detection.

Risk of within-hotel transmission of SARS-CoV-2 during on-arrival quarantine in Hong Kong: an epidemiological and phylogenomic investigation.

Background: On-arrival quarantine has been one of the primary measures to prevent the introduction of SARS-CoV-2 into Hong Kong since the start of the pandemic. Most on-arrival quarantines have been done in hotels, with the duration of quarantine and testing frequency during quarantine modified over time along with other pandemic control measures. However, hotels are not designed with infection control in mind. We aimed to systematically study the potential risk of acquisition of SARS-CoV-2 infection among individuals undergoing hotel quarantine. Methods: We examined data on each laboratory-confirmed COVID-19 case identified in on-arrival quarantine in a hotel in Hong Kong between 1 May 2020 and 31 January 2022. We sequenced the whole genomes of viruses from cases that overlapped with other confirmed cases in terms of the hotel of stay, date of arrival and date of testing positive. By combining multiple sources of evidence, we identify probable and plausible transmission events and calculate the overall risk of transmission. Findings: Among 221 imported cases that overlapped with other cases detected during hotel quarantine with available sequence data, phylogenomic analyses identified five probable and two plausible clusters of within-hotel transmission. Only two of these clusters were recognised at the time. Including other clusters reported in Hong Kong, we estimate that 8-11 per 1000 cases identified in hotel quarantine may be infected by another unlinked case during quarantine, or 2-3 per 100,000 overseas arrivals. Interpretation: We have identified additional undetected occurrences of COVID-19 transmission within hotel quarantine in Hong Kong. Although hotels provide suboptimal infection control as improvised quarantine facilities, the risk of contracting infection whilst in quarantine is low. However, these unlikely events could have high consequences by allowing the virus to spread into immunologically naïve communities. Additional vigilance should be taken in the absence of improved controls to identify such events. If on-arrival quarantine is expected to be used for a long time, quarantine facilities could be purpose-built to minimise the risk of transmission. Funding: Health and Medical Research Fund, Hong Kong.

Development of multiplex RT-ddPCR assays for detection of SARS-CoV-2 and other common respiratory virus infections.

BACKGROUND: Measures for mitigation of Coronavirus Disease 2019 (COVID-19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co-circulation of SARS-CoV-2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses. METHODS: A panel of three reverse transcription droplet digital PCR (RT-ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID-19 cases in Hong Kong were screened for co-infection with other common respiratory viruses. RESULTS: All detected viral targets showed distinct signal clusters using the multiplex RT-ddPCR assays. These assays have a broad range of linearity and good intra-/inter-assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID-19 were found to be co-infected with other respiratory viruses, whereas no local case of co-infection was observed. CONCLUSIONS: The multiplex RT-ddPCR assays were demonstrated to be useful for screening of respiratory virus co-infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.