Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak

Tsuyoshi Sekizuka; Kentaro Itokawa; Tsutomu Kageyama; Shinji Saito; Ikuyo Takayama; Hideki Asanuma; Nao Naganori; Rina Tanaka; Masanori Hashino; Takuri Takahashi; Hajime Kamiya; Takuya Yamagishi; Kensaku Kakimoto; Motoi Suzuki; Hideki Hasegawa; Takaji Wakita; Makoto Kuroda

Haplotype networks from genomic SNVs (HN-GSNVs) were used to map the genotypes of the SARS-CoV-2 isolates that disseminated in the DP cruise ship after isolation of the passengers on 5 Feb. 5. In addition to identifying the genetic alterations, we generated HN-GSNVs to highlight the clonality of DP isolates compared with other SARS-CoV-2 genome sequences which are publicly available on GISAID (update as of March 10, 2020). Analysis clearly suggested that the DP cluster had been generated from the potential haplotype ancestor of the global pandemic, Wuhan-Hu-1 haplotype, by G11083T transversion before initial dissemination in the DP ( Fig. 2A , 10 and see Supplementary Table S2 for SNVs). Thus far, all DP isolates identified have been rooted on a single brunch of the HN-GSNVs map ( Fig. 2A) , showing different genealogy to other isolates in China, Europe, North America, and an outbreak on another cruise ship reported from the USA. The finding that all isolates in the DP cluster exhibit G11083T transversion strongly suggest that SARS-CoV-2 dissemination in the DP could have originated from a single 15 introduction event before the quarantine started on Feb. 3. Further focusing of the HN-GSNVs map on only the DP isolates in the cluster revealed that the 29 isolates of the DP-A cluster (indicated A in Fig. 2B ) predominated among the DP isolates, indicating that the DP-A cluster is the ancestral haplotype for subsequent transmission. Based on the estimated speed of SARS-CoV-2 evolution (temporal resolution assumes a nucleotide substitution rate of 8 × 10 -4 subs per 20 site per year among the 528 SARS-CoV-2 WGSs, described at the Nextstrain web site for realtime tracking of pathogen evolution [update as of March 16, 2020] (8), one mutation per genome occurs in every 15 days in average, suggesting that predominant isolates in the DP-A cluster could be a very early generation of progeny from Wuhan-Hu-1 isolated on Dec. 26, 2020. This predomination of the DP-A cluster might explain the large epidemics within the cruise ship; it is 25 possible that super-spreading occurred immediately after introduction of the virus, well before the quarantine starting on Feb. 3. This super-spreading may have originated from the COVID-19 patient who had disembarked in Hong Kong on Jan. 25. Although further explosive spreading might have been prevented after the quarantine, some of the subsequent progeny clusters, including DP-B (5 isolates) and DP-C (6 isolates) (Fig. 2B) , may have formed via transmission 30 through hidden links, such as eating at the same dinner table. Additionally, 33 patients (45%) not included in the DP-A, -B and -C clusters were revealed to have unique SARS-CoV-2 haplotypes with patient-specific unique SNVs and/or deletions. The finding of such unique progenies might explain how further subsequent minor spreading occurred after the quarantine. Indeed, the haplotype network suggests the existence of direct transmissions among passengers who shared 35 same cabins during the quarantine; three cabin mates had been infected by the virus with same haplotype (two pairs in DP-A cluster, and one pair in other cluster), and 5 pairs shared haplotypes linked by a single SNV (A to DP0290, A to DP0481, A to DP0645, A to DP0803, and DP0764 to DP0765).