A total of 135 COVID-19 cases were identified in the present outbreak, 64 cases (47.4%) were employees of Company K and 67 (49.6%) were their contacts or residents living around Company K. The date of onset of the primary case (Case A) was July 9, and the infection was confirmed on July 25 by RT-qPCR testing. The index case was confirmed on July 23. A total of 4 early cases had onset by July 14, and the peak occurred from July 22 to 23 (Figure 1). Serum samples collected from Case A on August 5 tested positive for higher IgM level and lower IgG level (geometric mean titer=1:53.427 and 1:15.139, respectively).
Investigation workflow and the Dalian COVID-19 outbreak. Imported cold-chain seafood products with COVID-19 virus infected wharf workers and then were introduced into the seafood product workshop of Company K before spreading further. The epidemiological investigation strategy was shown bolded in blue. COVID-19 case data were represented by a grey dotted box, and virus introduction from the DK ship was represented by a blue dotted box.
Among 64 Company K employees, 63 were exposed to the environment of the cold-chain seafood processing workspace of Company K. On July 23, we collected 39 environmental smear samples from this location, including samples from the operating table, floor, tools, sinks, sewers, and other environmental areas. COVID-19 virus nucleic acid was tested in these samples, with a positive rate of 35.9% (14/39). In addition, the workers in the same and adjacent working areas to case A were identified as the first infected cases through work contact.
A total of 24 throat swab samples from confirmed cases and 2 environmentally positive samples from Company K were further sequenced, and full-length genomic sequences were acquired. Whole-genome analysis revealed that the sequences of 24 cases and two environmental samples were highly homologous. Compared with the reference sequence of COVID-19 virus, strain Wuhan-1 (GenBank No. NC_045512), all the sequences shared a total of 14 nucleotide mutations (C241T, C2091T, C3037T, A5128G, A8360G, C13860T, C14408T, T19839C, G19999T, A23403G, G28881A, G28882A, G28883C, and C28905T), belonging to PANGO lineage B.1.1.317 (2) (Figure 2).
Phylogenetic tree based on Dalian outbreak-related samples. (A) Phylogenetic tree based on the representative full-length genome sequences of the COVID-19 virus. The genomes of the COVID-19 virus from Dalian case samples were highlighted in yellow, environmental samples from cold-chain seafood processing work area of Company K were highlighted in green and pollock packaging samples were highlighted in blue. The genome of the reference COVID-19 virus from Wuhan in December 2019 was shaded in grey. Strains associated with specific outbreaks in China were indicated by blue squares. The PANGO lineages were marked and colored on the right. The tree was rooted using strain WH04 (EPI_ISL_406801) in accord with the root of PANGO tree. (B) Phylogenetic tree based on the full-length genome sequences of Dalian COVID-19 virus samples, rooted using the Wuhan reference strain.
From June 24 to July 8, which was the 14 days of the COVID-19 incubation period before the onset date of Case A, a total of 8 imported cases were reported in Dalian. Based on the measures to prevent the risk of importation from overseas implemented in China, all the overseas arrivals were swabbed and tested for COVID-19 upon their entry at customs, were then transferred to the centralized quarantine location (usually a designated hotel) for 14 days for medical observation, and were tested again before their release. These 8 cases had no historical contact with early cases of the Dalian outbreak, and the whole-genome sequences from the samples of these cases were distinct from those of the Dalian outbreak (Figure 2). In addition, during this period, smeared swab samples were taken from the inner and outer packaging of a total of 2,119 suspicious packages of cold-chain seafood imported by Company K, and a total of 4,963 samples were collected for COVID-19 virus nucleic acid testing. The results revealed that all samples were negative.
In addition to importing and processing imported cold-chain seafood, Company K temporarily stored bonded cold-chain products for other companies. Imported bonded cold-chain products loaded from two overseas cargo ships were temporarily stored by Company K during that time. We traced the location of these cold-chain seafood products and conducted nucleic acid testing of their inner and outer packaging for COVID-19 virus and detected positive samples from the outer packaging of some cold-chain pollock loaded on one of the cargo ships (cargo ship DK). No positive samples were found from the other cargo ship. We further conducted RT-qPCR testing of the cold-chain pollock packaging that had been stored in four different freezers of DK, and the positive rates were 19.0% (19/100), 36.3% (37/102), 43.9% (29/66), and 49.0% (49/100).
DK was a large ocean-going refrigerated transport ship. From June 5 to 10, the ship DK carried 5,000 tons of cold-chain pollock from four fishing vessels that belonged to the same overseas company H. During the marine operation, the crews of the four overseas fishing vessels participated in processing and loading the cold chain pollock into the freezers of ship DK (temperature minus 25 degrees Celsius), while the crews of ship DK neither participated in cargo handling nor contacted the overseas crew. When the cargo ship arrived at the Dalian terminal on June 27–29, a total of 216 dockworkers from Company K were divided into three shifts; each shift spent 10 hours handling the cold-chain products, while the crews from ship DK did not participate in handling. According to custom inspection records, the ship DK crew’s temperatures were normal, and they presented no symptoms of infection, but they were not tested with RT-qPCR while in China. All of them were quarantined on the ship until the ship left the port, which was confirmed by video from the dock around the ship. We also interviewed the crew of the DK ship, who maintained a distance of at least 10 metres from overseas crews; they had neither talked with the overseas crews nor used the toilet on their vessels. All the dockworkers of Company K wore labour gloves and disposable masks but carried the seafood products close to their faces and drank water and wiped sweat with their face mask off (Figure 1).
We further investigated the COVID-19 virus infection status of the workers who handled the cold-chain pollock. From July 23 to August 6 (14 days since the index case was confirmed), a total of 245 dockworkers and 13 cold storage stevedores of Company K who were exposed to DK cargo were quarantined and screened with RT-qPCR several times until August 5. Among these 258 potential contacts, five dock workers tested positive for COVID-19 virus nucleic acid, and all the others were negative. In addition, on August 11, sera samples of 195 dock workers and 13 cold storage stevedores of Company K were collected for antibody detection, and 50 dock workers were not sampled because they had been released from quarantine and were lost to follow up. Among the 208 potential contacts, 6 dockworkers who tested negative for nucleic acid were positive for COVID-19 IgG and IgM antibodies. Among the 5 confirmed cases and 6 undiscovered cases who had been infected with COVID-19, 2 of them reported physical soreness or cold symptoms at the end of June and early July, and the other nine were asymptomatic. These 11 people were all among the 216 dockworkers who participated in the actual handling of cold-chain pollock carried by the overseas DK cargo ship, and the infection rate was 5.1% (11/216). It is worth noting that 1 of the 6 dockworkers who were positive for COVID-19 IgG and IgM antibodies was the husband of Case A (designated Case B). The geometric mean titer of anti COVID-19 virus IgM and IgG of Case B were 1:3.899 and 1:13.677,respectively (Figure 1).
Most importantly, the COVID-19 whole-genome sequences from 7 nucleic acid-positive samples of the packaging of cold-chain pollock from the ship DK were obtained, all of which had very high homology with the sequences of the Dalian cases, also belonging to PANGO lineage B.1.1.317, only lacking a mutation site (A5128G), which was strongly suggestive of the parental virus of the Dalian cases (Figure 2).
We tried to isolate the virus from COVID-19 virus nucleic acid-positive samples detected in the cold-chain pollock from the DK cargo ship, but the virus was not successfully isolated. However, the result of quantitative fluorescence RT-qPCR detection was 1,090 copies, which was equivalent to 13,080 copies of the gene in the 200-µL environmental swab. Even though the virus was not isolated, such high viral copy numbers indicated that the viral load was large.