key: cord-269973-sntnmqqd
authors: To, Kelvin Kai-Wang; Chan, Wan-Mui; Ip, Jonathan Daniel; Chu, Allen Wing-Ho; Tam, Anthony Raymond; Liu, Raymond; Wu, Alan Ka-Lun; Lung, Kwok-Cheung; Tsang, Owen Tak-Yin; Lau, Daphne Pui-Ling; To, Wing-Kin; Kwan, Mike Yat-Wah; Yau, Yat-Sun; Ng, Anthony Chin-Ki; Yip, Cyril Chik-Yan; Chan, Kwok-Hung; Tse, Herman; Hung, Ivan Fan-Ngai; Yuen, Kwok-Yung
title: Unique SARS-CoV-2 clusters causing a large COVID-19 outbreak in Hong Kong
date: 2020-08-05
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1119
sha: 
doc_id: 269973
cord_uid: sntnmqqd

After two months of relative quiescence, a large COVID-19 outbreak occurred in Hong Kong in July 2020 after gradual relaxation of social distancing policy. Two unique SARS-CoV-2 phylogenetic clusters have been identified among locally-acquired cases, with most genomes belonging to cluster HK1 which is phylogenetically related to SARS-CoV-2 reported overseas.

The coronavirus disease 2019 (COVID-19) pandemic has overwhelmed the healthcare system in many parts of the world. To combat the pandemic, social distancing measures have been implemented to reduce community transmission. However, resurgence of COVID-19 cases has been seen in many parts of the world after the relaxation of these social distancing measures.

Hong Kong was one of the first places in the world to report COVID-19 cases [1] .

However, the number of COVID-19 cases remained relatively low due to the early implementation of stringent public health measures, including border control, voluntary community-wide wearing of face masks, hand hygiene and social distancing, prompt isolation of suspected cases, and testing and quarantine of close contacts and travelers from epidemic areas [2, 3] . The first wave, consisting of imported cases from China and limited local transmission, occurred in January and early March, 2020. The second wave, mainly related to imported cases outside Asia and associated local transmission, occurred in mid-March to May, 2020. After stepping up public control measures, only sporadic local cases were reported in May and June. Hence, most social distancing restrictions were lifted on June 18, 2020. However, a third wave started to occur since early July 2020. Locally-acquired cases re-appeared since July 5, 2020. A total of 617 locally-acquired laboratory-confirmed cases have been reported between July 5 and 21 [4] . In this study, we use whole genome sequencing to investigate the genomic epidemiology of this large summer outbreak.

Archived nasopharyngeal swab or posterior oropharyngeal saliva from COVID-19 patients were retrieved. This study was approved by the the Institutional Review Board of the 

Nanopore sequencing was performed as described previously with modifications [1, 5] . RNA was extracted from clinical specimens with Qiagen Viral RNA Mini Kit, and was amplified with sequence-independent single-primer amplification (SISPA) or tiled primers.

Nanopore sequencing was performed with the Oxford Nanopore MinION platform.

Bioinformatic analysis was performed as described previously with modifications [1] . Sanger sequencing was performed on specimens with low coverage at the cluster-defining mutations.

Details on library preparation, bioinformatics analysis, and Sanger sequencing are included in the Supplementary Methods section.

Genomes from Hong Kong and selected genomes from overseas were included for the phylogenetic tree analysis. Nucleotide position was numbered according to the reference genome Wuhan-Hu-1 (GenBank accession number MN908947.3). Please refer to Supplementary Methods section for details.

In this study, a total of 116 high-quality whole genomes of SARS-CoV-2 were Table S1 ). Ten genomes from the first wave were reported previously [1, 5, 6] . For the third wave, we included imported cases up to 2 weeks prior to the first locally-acquired case. Spike protein D614G mutation was not found in any genomes during the first wave, which mainly involved travelers from mainland China or other parts of Asia, or the linked local cases. However, D614G mutation was present in 73.8% (31/42) of the genomes in the second wave, which mainly involved travelers returning from Europe or North America ( Figure 1A ).

In the third wave, 32 specimens collected from locally-acquired cases between July 7 and 14, 2020, and 18 specimens collected from imported cases collected between June 22 and July 14, 2020, were included for analysis. The majority of the locally-acquired cases (29/32) Table S2 ).

Phylogenetically, cluster HK1 is most closely related to 4 imported cases from the Philippines but at least 2 of the HK1-defining mutations were not found in these cases.

Another local cluster during the third wave, consisting of 3 members in a household, also belongs to GISAID clade GR, Nextstrain clade 20B, and Pangolin lineage B.1.1. This cluster is characterized by 3 unique mutations, including A19702G (nsp15 N28D), T22020C

(spike protein M153T) and C28269T (located between ORF8 and NP gene) (referred to as cluster HK2) (Supplementary Table S2 ). Cluster HK2 is most closely related to imported A c c e p t e d M a n u s c r i p t cases from Kazakhstan, but those from Kazakhstan only had A19702G (nsp15 N28D) and T22020C (spike M153T) but not C28269T mutation.

In July, 2020, Hong Kong has experienced the third wave of COVID-19, which represented the largest local COVID-19 outbreak since the beginning of the pandemic. In this study, we have identified two unique clusters causing this COVID-19 summer outbreak in Hong Kong. The majority of genomes from locally-acquired cases (91%) during this third wave belong to a cluster HK1, a unique cluster within the GR clade, which is characterized by 4 non-synonymous mutations (nsp3 A85V, nsp15 A231V, spike protein S12F, NP A12G) and 1 synonymous mutation (NP C29144T). Genomes from a local household cluster with 3 members form another unique cluster (cluster HK2), which is characterized by 2 nonsynonymous mutations (nsp15 N28D, spike protein M153T) and C28269T. Both clusters, especially cluster HK1, were phylogenetically more closely related to imported cases than the strains collected in Hong Kong during the previous waves before June 2020, suggesting that this summer COVID-19 outbreak is unlikely to be related to silent carriers from previous waves. Instead, our results suggest that the current outbreak may be related to imported cases.

Cluster HK1 is most closely related to genomes of patients traveling from the Philippines, while cluster HK2 is most closely related to those from Kazakhstan. However, there are important differences between our locally-acquired clusters and these imported cases. All the genomes from cases imported from the Philippines did not contain nsp3 A85V or NP A12G that define cluster HK1, while those from Kazakhstan lack the mutation C28269T that define cluster HK2. Hence, our results suggest that there is a missing link between these locally-acquired cases in Hong Kong and those cases from the Philippines or Both cluster HK1 and HK2 possess D614G, which is now the predominant clade worldwide. D614G, located on the surface of the spike protein promoter, has become a predominant mutation worldwide [8] . D614G mutation is associated with a higher viral load in patients, and is associated with better viral replication in a pseudovirus assay [8] .

Several factors may explain this explosive summer outbreak in Hong Kong. First, the sudden increase in social gatherings especially at eateries after the stepping down of public health control measures facilitated person-to-person transmission. Second, this outbreak occurred in the summer, when both the temperature and humidity are high. Whether these climate conditions affect virus transmissibility require further investigations. Third, the unique mutations in these clusters, especially in cluster HK1, may have increased the survival or transmissibility of the virus. The mutations specific to cluster HK1 occur in different proteins, including two nonstructural proteins (nsp3 and nsp15) and two structural proteins (spike protein and nucleoprotein). nsp3 is a putative PL-pro domain. nsp15 is a XendoU: poly(U)-specific endoribonuclease, and has been shown to be a potent interferon antagonist [9] . The surface spike protein is responsible for receptor binding, and the nucleoprotein mainly participates in viral genome transcription, replication and virion assembly. Spike protein S12F is located in the signal sequence, while the nucleoprotein A12G is located in the N1a domain, a linker domain [10] . Cluster HK2 contains two non-synonymous mutations, nsp15 N28D and spike protein M153T. M153T is at the N-terminal domain. This residue is located within a region that is newly found in the SARS-CoV-2 strains when compared with SARS-CoV [11] .Whether these mutations affect the function of these proteins remains to be determined.

A c c e p t e d M a n u s c r i p t There are several limitations in this study. First, the current third wave is still ongoing, and there may be other clusters that have not been identified. Second, since whole genome sequencing required samples with relatively high viral load, those with lower viral load could not yield good quality sequence for phylogenetic analysis.

Two unique SARS-CoV-2 clusters have been identified during this large summer outbreak in Hong Kong shortly after the easing of social distancing policies. Further studies are required to determine how environmental, host or viral factors contribute to this outbreak.

Source control at the borders and airport are important to prevent imported cases. Since transmission from asymptomatic or mildly symptomatic patients are common [12, 13] , community-wide wearing of face mask and social distancing measures, especially at eateries, are required for prevent local spread.

A c c e p t e d M a n u s c r i p t M a n u s c r i p t A c c e p t e d M a n u s c r i p t Figure 1B 

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

The epidemiology of COVID-19 cases and the successful containment strategy in Hong Kong

The role of community-wide wearing of face mask for control of coronavirus disease 2019 (COVID-19) epidemic due to SARS-CoV-2

Centre for Health Protection. Latest situation of cases of COVID-19 (as of 15 July

gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing

Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study

Clade and lineage nomenclature aids in genomic epidemiology studies of active hCoV-19 viruses

SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus

SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein

Evolutionary relationships and sequence-structure determinants in human SARS coronavirus-2 spike proteins for host receptor recognition

Asymptomatic transmission during the COVID-19 pandemic and implications for public health strategies

SARS-CoV-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series

We gratefully acknowledge the originating and submitting laboratories who 

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t