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      Characterization of eight novel full-length genomes of SARS-CoV-2 among imported COVID-19 cases from abroad in Yunnan, China

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          Dear Editor: Recent correspondence in this Journal has highlighted the current threat posed by recently-emerging corona virus disease 2019 (COVID-19) in the world. 1 The COVID-19 is infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is characterized by fever, dry cough, weak, and so on. 2 , 3 SARS-CoV-2 has already caused a global pandemic. By 26 Apr, 2020, the spread of SARS-CoV-2 has led to more than 3.0 million infections and above 200,000 deaths; 4 thus, its outbreak has become a global public health problem. Recently, COVID-19 epidemic in China has been well controlled, whereas the risk of imported COVID-19 cases has increased dramatically. 5 As of April 26, 2020, a total of 1,636 abroad imported patients were reported in China. 6 However, limited studies on full-length genome characterization of SARS-CoV-2 from COVID-19 cases imported from abroad. Here, we characterized the genotype and mutation characteristics of SARS-CoV-2 isolated from eight imported cases from abroad in Yunnan, China. Eight COVID-19 patients imported from overseas were admitted to Yunnan Provincial Infectious Disease Hospital from March 15, 2020 to March 26, 2020. The epidemiological history and respiratory symptoms of the eight patients were summarized in Figure 1 A and 1B. The 8 patients include 4 males and 4 females, with ages ranging from 6 years to 70 years old. No patient has ever been to Wuhan city in China. Two cases YN_Im01 and YN_Im03 were from Spain to Yunnan, YN_Im02 from France, YN_Im04 from Cambodia, YN_Im05 from Sri Lanka, and YN_Im06-08, a family cluster of COVID-19 patients from the United States (Fig. 1A). Six cases showed different degrees of respiratory symptoms before hospitalization. YN_Im06 was severe, YN_Im01, YN_Im05, YN_Im07, and YN_Im08 were moderate, YN_Im02 was mild, and YN_Im03 and YN_Im04 were asymptomatic according to the latest COVID-19 diagnostic criteria (5th edition) published by the National Health Commission of China (Fig. 1B). Figure 1 Maps of the study region, epidemiological characteristics, phylogenetic analysis based on the complete genome sequences of the SARS-CoV-2 from eight COVID-19 cases imported from abroad in Yunnan, China. (A) Eight cases of imported SARS-CoV-2 infection in travelers returning to Yunnan from overseas in 2020. (B) Demographic characteristics and respiratory symptoms. (C) Maximum-likelihood tree based on the complete SARS-CoV-2 genomic sequence obtained from positive serum samples was constructed using IQ-tree software. The genotypes of SARS-CoV-2 were divided into G, S, V and other. Virus clades are shown at right. Figure 1 Fig. 2 . Figure 2 Nucleotide and amino acid substitution differences across the whole genome among clades G, V, S and other strains. Nucleotide and amino acid positions are numbered with a reference to SARS-CoV-2 strain Wuhan-Hu-1 identified earliest in Wuhan seafood market, in Hubei, China. Figure 2 So far, three main clades involving G, V, and S have been identified based on marker mutations in the complete SARS-CoV-2 genome according to the latest genotyping rules recommended by the GISAID databas. 7 G clade containing D614G variant in S protein is predominant in Europe, V clade possessing G251V mutation in ORF3 is more common in Asia and Europe, and S clade having L84S substitution in ORF8 is move prevalent in North America. 8 In this study, eight complete genome sequences of SARS-CoV-2 isolated from sputum samples were successfully amplified and sequenced with 38 overlapping fragments. Dataset comprise SARS-CoV-2 full-length sequences of representative clade G, V, and S as previously reported, and reference sequences with the highest similarity (12 sequences) based on BLAST in Genbank using the eight sequences obtained in this study as the query set. Further, phylogenetic trees for SARS-CoV-2 full-length nucleotide sequences were constructed based on the obtained datasets with the maximum-likelihood method using IQ-tree. Phylogenetic analyses revealed that the six isolates, including one from France (YN_Im02), two from Spain (YN_Im01 and YN_Im03), and three from the United States (YN_Im06-08) were clustered as G clade with a high bootstrap value of 99%, one strain from Cambodia (YN_Im04) was grouped into S clade with a bootstrap value of 80%, and the remaining one from Sri Lanka was classified within other clade, a large unclassified sequences because lack the signature variants (Fig. 1C). Of note, the three sequences YN_Im06-08 isolated from a family cluster of SARS-CoV-2 infection formed a close monophyletic subclade supported by a bootstrap value of 100% and had 99.99% nucleotide identity, indicating the three sequences originate from the same strain. To further characterize the characteristics of virus variation, the sequence analyses based on SARS-CoV-2 full-length nucleotide and amino acid sequences was performed using the strain Wuhan-Hu-1 (Genbank no. NC_045512) identified earliest in Wuhan seafood market, in Hubei, China as the reference strain for nucleotide and amino acid positions. 9 The results revealed that 15, 12 and 10 nucleotide mutations to clades G, S, and other, respectively, were mapped across the SARS-CoV-2 full-length genome. Corresponding to these nucleotide substitutions, 8, 6, and 5 non-synonymous amino acid substitutions were detected in clades G, S, and other, respectively. Of note, all clade G strains possessed another P4715L marker substitution in nsp12 besides the signature mutation D614G variant in S protein. YN_Im05 strain belonging to other clade have a unique 3-nucleotide deletion between 518 and 520 nt that was not found in G and S clades, leading to a methionine deletion at position 58 in leader protein. Moreover, three novel mutations, including D1962V in nsp3 from the strain YN_Im03, L1375F in nsp3 and A829T in S protein from the isolate YN_Im04 were first identified in this study according to the comparison with 11,231 genomic sequences available at GISAID on 4/26/2020.10 Interestingly, S23T and R203W mutations located in N protein were identified in the three isolates from a family cluster of SARS-CoV-2 infection. Given that the three COVID-19 patients were diagnosed on the same day and the viruses originated from the same strain, indicated that the strain is replicating and mutating rapidly in different individuals. In summary, we characterized the full-length genomes of SARS-CoV-2 strains from eight COVID-19 cases imported from abroad in Yunnan, China. Our results showed that the predominant SARS-CoV-2 clade was G (6 cases), followed by S clade (one case) and unclassified clade (one case). Further, comparative genomic analyses revealed that a novel signature amino acid substitution P4715L in nsp12 was found in the G clade strains. Moreover, three novel mutations, including D1962V in nsp3, L1375F in nsp3, and A829T in S protein were first identified in this study. The present study highlights the urgent need for continuous molecular screening and epidemic surveillance for SARS-CoV-2 among COVID-19 individuals imported from abroad to prevent future outbreaks of SARS-CoV-2 infection in China. Declaration of Competing Interest The authors declare no competing financial interests.

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          Imported COVID-19 Cases Pose New Challenges for China

          Dear editor, Recently, a letter in your journal predicted the trend of the spread of the novel coronavirus disease 2019 (COVID-19) in China, an illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1 , 2 would end after March 20, 2020. 3 Currently, the COVID-19 has spread around the globe, with the center of the epidemic shifting from China to Europe and the United States. 4 As of March 24, 2020, a total of 372,757 cases have been confirmed worldwide, with a death toll of 16,231 (WHO, Coronavirus disease 2019 Situation Report 64, March 24, 2020). Italy, in particular, had thus far diagnosed 63,927 patients, 6077 of whom had lost their lives. That translates to a mortality rate of 9.51%, which is more than twice as high as that of China's 4.02% (3283/81,747). The greater share of elderly patients with confirmed COVID-19 infection in Italy along with the population's significantly higher median age may partly explain the differences in cases and case-fatality rates between the two nations. 5 Countries such as the United States (42,164 cases), Spain (33,089 cases), Germany (29,212 cases), and France (19,615 cases) have seen an explosive increase in confirmed cases, with the rate of growth showing no hint of slowing down. For China—the initial epicenter of the outbreak—two stages of the epidemic have passed (Figure 1 A). The first stage is the outbreak period (December 31, 2019 to February 29, 2020), which entailed the period from the first detection of cases to the peak of the epidemic which saw a rapid increase in the number of confirmed cases, and to the time when the growth rate slowed down to less than 200 new confirmed cases per day. In the second stage, which lasted from March 1, 2020 to March 21, 2020, the number of existing cases in most Chinese provinces was reduced to less than 10, respectively, whilst the number of newly confirmed cases in Wuhan, Hubei province, the worst-hit city, was slowly approaching zero. It was during this stage—more specifically on the March 4—that foreign imported cases to appear. During these two stages, the Chinese government, its populous, and its medical professionals had managed to stabilized the deadly epidemic with great deliberation and sacrifices. 6 Currently, however, the situation in China has entered its third stage—recontamination through close contact with foreign infection, as demonstrated by the emergence of second-generation case originated from imported cases first reported in Guangzhou, Guangdong province on March 22, 2020 (Figure 1B). As of March 24, 2020, there were 427 imported cases and 3 second-generation cases originated from imported cases, one each in Beijing, Shanghai and Guangzhou (National Health Commission of the People's Republic of China). It shows that China needs to pay more attention to the control of imported cases and reflect on the measures previously taken against imported cases. Fig. 1 Three stages of China's COVID-19 epidemic (updated on March 24, 2020). (A) The first and second stages of China's COVID-19 epidemic. (B) The third stage of China's COVID-19 epidemic. Data for all cases are from World Health Organization (https://www.who.int/emergencies/diseases/novel-coronavirus-2019) and National Health Commission of the People's Republic of China (http://www.nhc.gov.cn). Fig 1 Due to the outbreak of COVID-19 in Europe and America, many overseas Chinese or ethnic Chinese had chosen to return to China where the epidemic had remained under control, many of whom were themselves COVID-19 patients without realizing that they had been infected. As a result, imported cases from outside of China appeared in early March and began to increase sharply in mid-March (Figure 1B). China is now facing increasing pressure in the face of import cases from overseas, especially in cities that a are international travel hubs such as Beijing, Shanghai and Guangzhou. On the onset of this surge in the number of overseas patients, the operational capacity of airports with high volume of international flights was not capable of efficient screening for every arrival, resulting in a large number of passenger flow jams in the airport lobby, and subsequently causing high risk of infection. Therefore, the conundrum regarding the control over overseas imported cases as well as the prevention of a second epidemic outbreak that is fast approaching is a problem that China needs to pay special attention to, especially after the first second-generation case imported from abroad had appeared. It shows that the current prevention and control measures have yet proven to be consummate. As the global epidemic continues in the outbreak period, more and more overseas Chinese citizens or ethnic Chinese, even non-Chinese, will choose to come to China to escape from the epidemic. 7 The government has shortened the processing time for immigration procedures and required all arrivals from other countries to be quarantined. However, the Chinese government needs to ensure that every overseas arrival would have passed a quarantine period of at least 14 days, and tested negative to COVID-19 before they can conduct social activities in the country, and such measures are not easy to implement. China has managed to control the outbreak of the domestic epidemic, but there are still new issues waiting for them to deal with. In addition, China's outstanding performance in the first stage of the epidemic has provided the world with valuable insights and earned two months of breathing room for the world to respond the novel virus. However, due to the lack of attention and the spread of misinformation regarding COVID-19 in many countries, the disease has seriously threatened the whole world, 4 bringing China's epidemic to the third stage. Therefore, based on the One Health model, 8 the outbreak of COVID-19 should concern all humankind, for no country can survive alone. The real victory over COVID-19 will require concerted efforts from around the globe. Declaration of Competing Interest None.
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            Global COVID-19 fatality analysis reveals Hubei-like countries potentially with severe outbreaks

            Highlights • CFR in Iran in the early stage of the outbreak is highest among all the countries. • CFRs in the USA and Italy are similar to Hubei Province in the early stage. • CFRs in South Korea are similar to outside Hubei, indicating less severity. • Our findings highlight the severity of outbreaks globally, particular in the USA.

              Author and article information

              J Infect
              J. Infect
              The Journal of Infection
              Published by Elsevier Ltd on behalf of The British Infection Association.
              15 May 2020
              15 May 2020
              [a ]Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China
              [b ]Clinical Laboratory, Yunnan Provincial Infectious Disease Hospital, Kunming, China
              [c ]Yunnan SCISpark Medical Laboratory, Yunnan SCISpark Biotech Co.,Ltd, Kunming, China
              [d ]Infectious Diseases Division II, Yunnan Provincial Infectious Disease Hospital, Kunming, China
              [e ]Faculty of Life Science and Technology, Kunming University of Science and Technology & Yunnan SCISpark Medical Laboratory, Yunnan SCISpark Biotech Co.,Ltd, Kunming, China
              [f ]Yunnan Provincial Infectious Disease Hospital, Kunming, China
              Author notes
              [* ]Corresponding author: Tel.: +86 65920756; fax: +86 65920562. fyky2005@ 123456kust.com.cn dongxq8001@ 123456126.com oliverxia2000@ 123456aliyun.com

              These first authors contributed equally in this work

              © 2020 Published by Elsevier Ltd on behalf of The British Infection Association.

              Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.


              Infectious disease & Microbiology


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