The importation of the Phylogenetic-transition state of Zika virus to China in 2014.

We read with interest about the article published by Sun J. et al. in your journal.1Sun J. Wu Zhong H. Guan D. Zhang H. Tan Q. et al.Returning ex-patriot Chinese to Guangdong, China, increase the risk for local transmission of Zika virus.J Infect. 2017; (PII: S0163-4453(17)30231-1)Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar They found 19 Zika virus (abbreviated to ‘ZIKV’) infected cases in Guangdong, China were imported from America (From February 12 to September 16, 2016), and inferred it may increase the risk of ZIKV local transmission. That is an exactly alarming Issue. However, we suspected ZIKV has been introduced into China before this time because the Asian-lineage virus had been reported to cause sporadic outbreaks and low-scaled epidemics in Asia before 2010.2Pettersson J.H. Eldholm V. Seligman S.J. Lundkvist Å. Falconar A.K. Gaunt M.W. et al.How did Zika virus emerge in the Pacific Islands and Latin America?.MBio. 2016 Oct 11; 7https://doi.org/10.1128/mBio.01239-16Crossref PubMed Scopus (105) Google Scholar, 3Faria N.R. Azevedo Rdo S. Kraemer M.U. Souza R. Cunha M.S. Hill S.C. et al.Zika virus in the Americas: early epidemiological and genetic findings.Science. 2016; 352: 345-349Crossref PubMed Scopus (716) Google Scholar Here, we report a ZIKV imported case from Bangladesh, a South Asian country. This may be the first recorded ZIKV imported case in China. Our finding may complement the considered transmission profile of ZIKV in China. Since the first discovery of ZIKV in Uganda,4Dick G.W. Kitchen S.F. Haddow A.J. Zika virus. Isolations and serological specificity.Trans R Soc Trop Med Hyg. 1952; 46: 509-520Abstract Full Text PDF PubMed Scopus (1841) Google Scholar only limited ZIKV circulation had been observed in Africa and South/Southeast Asia (abbreviated to ‘SSEA’). It came as a surprise that large-scaled outbreaks of the Asian-lineage viruses had been reported in the French Polynesian islands,5Kindhauser M.K. Allen T. Frank V. Santhana R.S. Dye C. Zika: the origin and spread of a mosquito-borne virus.Bull World Health Organ. 2016; 94 (675–86C)Crossref PubMed Scopus (341) Google Scholar and more surprisingly, they had taken place as well in the Americas where it was first detected in Brazil in 2015.6Hennessey M. Fischer M. Staples J.E. Zika virus spreads to new areas – region of the Americas, May 2015–January 2016.Morb Mortal Wkly Rep. 2016; 65: 55-58Crossref PubMed Scopus (308) Google Scholar Since then, ZIKV had rapidly spread to more than 32 countries and territories7Zhang Y. Chen W. Wong G. Bi Y. Yan J. Sun Y. et al.Highly diversified Zika viruses imported to China, 2016.Protein Cell. 2016; 7: 461-464https://doi.org/10.1007/s13238-016-0274-5Crossref PubMed Scopus (45) Google Scholar, 8Deng Y.Q. Zhao H. Li X.F. Zhang N.N. Liu Z.Y. Jiang T. et al.Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China.Sci China Life Sci. 2016; 59: 428-430Crossref PubMed Scopus (85) Google Scholar, 9Li J. Xiong Y. Wu W. Liu X. Qu J. Zhao X. et al.Zika virus in a traveler returning to China from Caracas, Venezuela, February.Emerg Infect Dis. 2016; 22: 1133-1136Crossref PubMed Scopus (30) Google Scholar and has become an emerging global public health problem. Imported infection was one of the main channels for the rapid spread of ZIKV from epidemic areas to other countries, especially to China.1Sun J. Wu Zhong H. Guan D. Zhang H. Tan Q. et al.Returning ex-patriot Chinese to Guangdong, China, increase the risk for local transmission of Zika virus.J Infect. 2017; (PII: S0163-4453(17)30231-1)Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Zhang Y. Chen W. Wong G. Bi Y. Yan J. Sun Y. et al.Highly diversified Zika viruses imported to China, 2016.Protein Cell. 2016; 7: 461-464https://doi.org/10.1007/s13238-016-0274-5Crossref PubMed Scopus (45) Google Scholar, 8Deng Y.Q. Zhao H. Li X.F. Zhang N.N. Liu Z.Y. Jiang T. et al.Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China.Sci China Life Sci. 2016; 59: 428-430Crossref PubMed Scopus (85) Google Scholar The Asian-lineage virus had been reported to cause sporadic outbreaks and low-scaled epidemics in Asia before 2010,2Pettersson J.H. Eldholm V. Seligman S.J. Lundkvist Å. Falconar A.K. Gaunt M.W. et al.How did Zika virus emerge in the Pacific Islands and Latin America?.MBio. 2016 Oct 11; 7https://doi.org/10.1128/mBio.01239-16Crossref PubMed Scopus (105) Google Scholar, 3Faria N.R. Azevedo Rdo S. Kraemer M.U. Souza R. Cunha M.S. Hill S.C. et al.Zika virus in the Americas: early epidemiological and genetic findings.Science. 2016; 352: 345-349Crossref PubMed Scopus (716) Google Scholar while it had been found out that all the recent ZIKV cases reported in Asia were imported from South America or Oceania.9Li J. Xiong Y. Wu W. Liu X. Qu J. Zhao X. et al.Zika virus in a traveler returning to China from Caracas, Venezuela, February.Emerg Infect Dis. 2016; 22: 1133-1136Crossref PubMed Scopus (30) Google Scholar We analyzed 133 ice-stored serum samples with retrospect, as they were collected from travelers coming to Yunnan Province from January 2014 to June 2016, and we discovered that one of them was determined as ZIKV-positive. The carrier of this ZIKV strain, named ZK-YN001, was a 56-year-old male citizen of Bangladesh, arriving to China from Dhaka for a business trip at the Kunming Airport on November 3rd, 2014. Apart from the fever with a recorded axillary temperature of 37.8 °C, the patient had no other obvious symptoms. Although there had never been reported an infection casein Bangladesh so far until 2014, this finding suggests that ZIKV epidemic was occurred and even caused the outcome of infection cases in the next couple of years. A complete polyprotein-coding sequence of the ZK-YN001 strain was obtained and assembled using the Clustal program and has been submitted to GenBank (accession number KY328290). The complete coding DNA sequence (abbreviated to ‘CDS’) is 10,272-nucleotide (nt) long, and the only open reading frame is predicted to encode a large polyprotein of 3424 amino acids. Phylogenetic analysis of the ZK-YN001 polyprotein CDS was performed by the alignment with corresponding sequences of 49 reference strains, eight of the African and 41 of the Asian lineage, using the MEGA 6 software. A phylogenetic tree had been constructed using the neighbor-joining method, and has revealed that strain ZK-YN001 belonged to the Asian lineage. It also had a closer genetic relationship with SSEA strains, but they did not cluster together with imported strains from Latin America (S1 Fig). Furthermore, a total of eight specific amino acid variations were found in the core, envelope (E), and NS3, NS4, and NS5 proteins. The biological effect of these mutations requires further investigation. Additional analysis was performed by downloading 127 reference CDS sequences from NCBI GenBank for the estimation of Asian-lineage evolutionary rates and the construction of the Maximum Clade Credibility (abbreviated to ‘MCC’) tree using BEAST 1.8.3. In different evolutionary models of Bayesian phylogenetic analysis, the estimated ZIKV rates varied from 5.929 × 10−4 to 7.709 × 10−4 nt substitutions per site per year, and the time to the most recent common ancestor (TMRCA) varied from 66.00 to 70.87 years (Table 1). The MCC tree showed that the currently-circulating ZIKV strains branched together with the Malaysia strains reported earlier. A large tight independent cluster, which was included in all recent outbreak strains in Latin America was formed in the MCC tree. Our ZK-YN001 strain and strains from SSEA and French Polynesia were located between the outbreak cluster and the Malaysia cluster. Thus, the history of the epidemic caused by ZIKV of the Asian lineage can be divided into three stages: Origin Stage before the 1990s, Low-Prevalence stage between 1990 and 2013, and Explosive Pandemic stage after 2013 (Fig. 1). The Asian-lineage ZIKV was first identified in Malaysia in 1966, and then spread to neighboring countries with the low prevalence in SSEA, including Thailand, Cambodia, the Philippines and French Polynesia (red dotted line in Fig. 1); when a large-scaled outbreak occurred in America, these viruses were re-imported to Asian countries (purple dotted line in Fig. 1). The position of our ZK-YN001 strain was the intermediate between low-epidemic SSEA lineages and explosive pandemic lineages, which might indicate the existence of a transition-state ZIKV with characteristics between those of Low-Prevalence and Explosive Pandemic strains.Table 1Mean evolutionary rates and the time to the most recent common ancestor (TMRCA) of Asian-lineage ZIKV in different model.Clock modelSubstitution modelSite heterogeneity modelMean rates [95%] (sub/s/y)TMRCA [95%] (years)Strict clockHKYGama5.971 × 10−4 [5.04–6.94]70.87 [63.34–78.89]Invariant sites5.997 × 10−4 [5.11–6.90]70.59 [63.46–78.15]Gama + Invariant sites6.007 × 10−4 [5.09–6.93]70.77 [63.45–78.53]GTRGama5.983 × 10−4 [5.02–6.93]70.47 [62.89–78.20]Invariant sites5.925 × 10−4 [4.94–6.86]70.59 [62.92–78.68]Gama + Invariant sites5.967 × 10−4 [5.04–6.90]70.60 [63.54–78.48]TN93Gama5.962 × 10−4 [4.10–6.87]70.63 [63.43–78.62]Invariant sites5.929 × 10−4 [5.39–8.78]70.62 [62.68–79.30]Gama + Invariant sites5.954 × 10−4 [4.98–6.90]70.72 [63.23–78.82]Random clockHKYGama7.107 × 10−4 [5.39–8.78]66.04 [50.19–87.74]Invariant sites7.036 × 10−4 [5.47–8.61]64.64 [50.15–86.03]Gama + Invariant sites7.152 × 10−4 [5.46–8.81]65.43 [50.17–86.73]GTRGama7.039 × 10−4 [5.43–8.65]65.74 [50.14–87.40]Invariant sites7.025 × 10−4 [5.31–8.67]65.25 [50.16–86.48]Gama + Invariant sites6.901 × 10−4 [5.24–8.45]66.00 [50.18–88.07]TN93Gama6.844 × 10−4 [5.14–8.46]66.38 [50.18–88.40]Invariant sites6.738 × 10−4 [5.12–8.44]67.39 [50.19–90.81]Gama + Invariant sites6.978 × 10−4 [5.39–8.51]66.43 [50.17–88.44] Open table in a new tab The presence of the transition-state ZIKV may change the transmission profile of the virus from Latin America and Oceania to China where the first cases were reported on February 2nd, 2016. This finding indicates that ZIKV infection might have been occurred in Bangladesh several years ago, even though no cases had been detected. It also suggests that China is still under the threat of imported ZIKV infection, not only from overseas but also from our neighboring SSEA countries. There had been low prevalence and infection rates of ZIKV in SSEA for nearly half a century, as well as the failure to timely identify the imported case in 2014. This emphasized the urgency of updating the detection methods for emerging infectious diseases, which is particularly important for monitoring infectious pathogens in ports of entry. Phylogenetic and evolutionary analysis of the ZK-YN001 strain should contribute to a better understanding of the Asian-lineage ZIKV evolution and genetic diversity, as well as the mechanism of its transformation from Low-Prevalence strains to Explosive Pandemic strains. To sum up, we reported a case of ZIKV infection imported in 2014, which was, to the best of our knowledge, the first ZIKV infection case in Yunnan Province and in China. The phylogenetic and evolutionary characteristics of ZK-YN001 confirmed its uniqueness as an Asian-lineage transition-state intermediate between Low-Prevalence and Explosive Pandemic strains. The origin and transmission pattern of Asian-lineage ZIKV in Asia indicates that the transition-state intermediates, including ZK-YN001 and other SSEA strains, played as a crucial part in the transformation of ZIKV from Low-prevalence to Explosive pandemic strains. This work was supported by the National Natural Science Foundation of China (NFSC) (81460509), Yunnan provincial innovation team project (2015HC030) and Science and technology project (2016RA014). The authors declare no competing financial interests. We thank Prof Kok Keng Tee (Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia) for helpful discussions. The following is the supplementary data related to this article: Download .zip (.14 MB) Help with zip files Figure S1