Abstract
Zika virus (ZIKV) (genus Flavivirus, family Flaviviridae) is an emerging pathogen associated with microcephaly and Guillain-Barré syndrome. The rapid spread of ZIKV disease in over 60 countries and the large numbers of travel-associated cases have caused worldwide concern. Thus, intensified surveillance of cases among immigrants and tourists from ZIKV-endemic areas is important for disease control and prevention. In this study, using Next Generation Sequencing, we reported the first whole-genome sequence of ZIKV strain AFMC-U, amplified from the urine of a traveler returning to Korea from the Philippines. Phylogenetic analysis showed geographic-specific clustering. Our results underscore the importance of examining urine in the diagnosis of ZIKV infection.
Keywords: Zika virus, Next generation sequencing (NGS), Whole-genome sequence, South Korea, Philippines
Zika virus (ZIKV), a single-stranded, positive-sense RNA virus belonging to the Flavivirus genus of the Flaviviridae family, is transmitted by mosquitoes of the Aedes species (Ae. aegypti and Ae. albopictus). ZIKV was first identified in a sentinel rhesus monkey in the Zika Forest in Uganda in 1947 [1–3]. Recently, ZIKV has become one of the most important mosquito-borne viruses, with outbreaks associated with microcephaly [4] and Guillain-Barré syndrome [5] in the Americas, Pacific, and Southeast Asia. As of January 2017, 17 ZIKV infection cases (13 male and 4 female) have been confirmed in Korea, according to the Korea Centers for Disease Control and Prevention (KCDC). All 17 cases have been related to travel to South America and Southeast Asia: one to Brazil (case #1) [6], seven to the Philippines (cases #2, 3, 5, 12, 13, 15, 17), four to Vietnam (cases #4, 9, 11, 16), one to the Dominican Republic (case #6), one to the Republic of Guatemala (case #7), one to Puerto Rico (case #8), and two to Thailand (cases #10, 14) (unpublished data from KCDC).
Here, we report the full-length genome sequence of ZIKV strain AFMC-U, amplified from the urine of a male recruit (case #3) in a Korean Army training center in the Republic of Korea, using next generation sequencing technology. In April 10–14, 2016, two brothers, ages 20 and 21 years (cases #2 and #3), returned to Korea from Boracay, Philippines. The younger brother (case #2) was hospitalized with flu-like symptoms and rash, and ZIKV infection was diagnosed in a urine sample, using Zika Virus Polyprotein gene genesig® Standard Kit (Primerdesign Ltd, United Kingdom) (unpublished data from KCDC). Two weeks after returning, the older brother (case #3) joined the Korean Army, and although he was asymptomatic, serum, saliva and urine samples were collected. Total RNA was extracted from serum, saliva, and urine, using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), and cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen, San Diego, USA) and random hexamers. Oligo-nucleotide primer sequences for nested PCR were ZIKV-1F: 5′–AGTTGTTGATCTGTGTGAATCAGAC–3′ and ZIKV-637R: 5′–CATAGGGCATTCATAGCTCATGGT–3′, ZIKV-1F and ZIKV-397R: 5′–GCATTGATTATTCTCAGCATGGC–3′. Initial denaturation was 94 °C for 5 min, followed by 15 cycles of denaturation at 94 °C for 40 s, annealing at 50 °C for 40 s, elongation at 72 °C 1 min, then 25 cycles of denaturation at 94 °C for 40 s, annealing at 52 °C for 40 s and elongation at 72 °C for 1 min, in a. ProFlex™ PCR system (Applied Biosystems, Foster City, CA, USA). PCR products were purified by the QIAquick PCR purification Kit (Qiagen), and DNA sequencing was performed in both directions, using the Big-Dye terminator v3.1 cycle sequencing kit (Applied Biosystems) on an Applied Biosystems 3500 series Genetic Analyzer (Applied Biosystems). Both urine and saliva were positive, but serum was negative for ZIKV using RT-PCR.
To obtain the whole-genome sequence of ZIKV from urine and saliva of case #3 by next generation sequencing (NGS) technology, a library was prepared using TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA, USA) according to manufacturer’s instruction. The library sizes and molar concentrations were determined by the Bio-analyzer with the Agilent DNA 1000 Kit (Agilent Technologies, Inc., Santa Clara, CA, USA), and the libraries were quantified using the Library Quantification kit for Illumina sequencing platforms (KAPA Biosystems, Wilmington, MA, USA) and a Quantstudio 6 Flex Real-time PCR (Applied Biosystems). Deep sequencing of ZIKV from urine and saliva of case #3 were performed on a MiSeq benchtop sequencer (Illumina) using a MiSeq reagent kit version 2 (Illumina) with 2 × 150 bp paired-end, according to manufacturer’s instructions. The 5′- and 3′-terminal sequences were filled by designing specific primers, using the conventional Sanger sequencing method and SMARTer® RACE 5′/3′ Kit (Takara Bio Inc., Otsu, Japan). Total reads were qualified over Q20 score and trimmed for reference mapping (Reference sequence: NC_012532) and consensus sequences extraction using CLC Genomics Workbench version 7.5.2 (CLC Bio, Cambridge, MA, USA). Depth of coverage was calculated by the number of mapped reads (read length × number of reads matching to the reference/genome size of reference). NGS data from the urine sample (ZIKV strain AFMC-U) generated 1,012,451 reads (depth of coverage; 14,069.6) and saliva sample (ZIKV strain AFMC-S) generated 4791 reads (depth of coverage; 66.6) with a mean read length of 150 bases.
We obtained the complete-genome and partial-genome sequence of ZIKV from urine and saliva sample, respectively. The full-length genome sequence of ZIKV strain AFMC-U was 10,795 nucleotides (GenBank accession no. KY553111) with 51.4% G+C content and 107-(1 to 107) and 428-nucleotide (10,368 to 10,795) 5′- and 3′-untranslated region (UTR), respectively. A 9063 nucleotide of ZIKV strain AFMC-S (GenBank accession no. KY962729) and ZIKV strain AFMC-U were identical (Table 1; Fig. 1). Whole-genome sequence comparison between ZIKV strain AFMC-U and ZIKV/H.sapiens-tc/PHL/2012/CPC-0740 from the Philippines (GenBank accession no. KU681082) showed 98.6 and 99.6% sequence similarity at the nucleotide and amino acid level, respectively (Table 1). Phylogenetic analysis, based on the nucleotide sequences, generated by the neighbor-joining method with 1000 bootstrap replicates using MEGA 6 [7]. The phylogenetic tree showed that ZIKV strain AFMC-U belonged to the Asian lineage and was closely related to a ZIKV strain from the Philippines (Fig. 1) [8, 9].
Table 1.
Nucleotide and amino acid sequence similarity (%) between ZIKV strain AFMC-U and representative flaviviruses
| Virus | Isolate (strain) | Genome (bp) | Nucleotide (%) | Amimo acid (%) |
|---|---|---|---|---|
| Zika | AFMC-S | 9063 | 100.0 | 100.0 |
| Zika | H.sapiens-tc/PHL/2012/CPC-0740 | 10,807 | 98.6 | 99.6 |
| Zika | H.sapiens-tc/KHM/2010/FSS13025 | 10,807 | 97.9 | 99.4 |
| Zika | H.sapiens-tc/THA/2014/SV0127-14 | 10,807 | 97.6 | 99.3 |
| Zika | SZ01/2016/China | 10,272 | 92.7 | 99.4 |
| Zika | SZ-WIV01 | 10,709 | 96.7 | 99.4 |
| Zika | GZ01 | 10,272 | 92.6 | 99.4 |
| Zika | GD01 | 10,574 | 95.4 | 99.4 |
| Zika | PLCal_ZV | 10,141 | 91.7 | 99.4 |
| Zika | TS17-2016 | 10,806 | 97.7 | 99.4 |
| Zika | H/PF/2013 | 10,807 | 97.8 | 99.5 |
| Zika | PRVABC-59 | 10,807 | 97.6 | 99.4 |
| Zika | P6-740 | 10,269 | 90.7 | 99.4 |
| Zika | SSABR1 | 10,648 | 96.2 | 99.5 |
| Zika | Rio-U1 | 10,795 | 97.5 | 99.4 |
| Zika | MEX/InDRE/Sm/2016 | 10,617 | 95.6 | 99.3 |
| Zika | Brazil-ZKV2015 | 10,793 | 97.5 | 99.4 |
| Zika | Brazil/2016/INMI1 | 10,643 | 96.2 | 99.4 |
| Zika | Haiti/1225/2014 | 10,807 | 97.6 | 99.4 |
| Zika | ZikaSPH2015 | 10,676 | 96.4 | 99.3 |
| Zika | ZIKV/H.sapiens/Brazil/PE243/2015 | 10,807 | 97.7 | 99.3 |
| Zika | Paraiba_01 | 10,807 | 97.7 | 99.4 |
| Zika | Natal RGN | 10,808 | 97.6 | 99.4 |
| Zika | Rio-S1 | 10,805 | 97.6 | 99.4 |
| Zika | ArD128000 | 10,272 | 88.7 | 96.5 |
| Zika | ARB13565 | 10,788 | 88.7 | 97.3 |
| Zika | ArD158084 | 10,272 | 84.2 | 97.1 |
| Zika | MR 766 | 10,794 | 89.0 | 96.6 |
| Zika | MR766-NIID | 10,807 | 89.0 | 96.6 |
| Zika | MR 766 | 10,766 | 88.7 | 97.0 |
| Spondweni | SM-6 V-1 | 10,290 | 65.1 | 74.8 |
| West Nile | B956 | 11,038 | 56.5 | 56.9 |
| Dengue 1 | Hawaii | 10,736 | 57.4 | 55.4 |
| Dengue 2 | D2/SG/CT38/2013 | 10,720 | 57.6 | 55.4 |
| Dengue 3 | H87 | 10,696 | 57.4 | 56.1 |
| Dengue 4 | H241 | 10,664 | 57.5 | 55.8 |
Fig. 1.
Phylogenetic analysis of the complete-genome sequences of Zika virus for a travel-associated case of Zika virus infection in a traveler returning to Korea from Boracay, Philippines, in April 2016. Phylogenetic tree was generated by the neighbor-joining method, using the Kimura 2-parameter model. Scale bar indicates number of base substitutions per site
This is the first report of the whole-genome sequence from a travel-associated case of ZIKV infection in the Republic of Korea. Our results underscore the importance of examining urine for ZIKV RNA. Although no cases of autochthonous transmission of ZIKV have been found in Korea, the presence of Ae. albopictus mosquitoes in rural and urban areas of Korea should heighten awareness of this possibility among physicians, as well as public health and vector control personnel.
Acknowledgement
This work was supported by grants (611665-912388501 and 611665-912543001) from the Agency for Defense Development, Republic of Korea.
Author’s contributions
SHG and DHS conceived and designed the experiments; SHG, DHS, JJ, and MYK performed the experiments; JJ, KIW, MK, WS, HSO, and BSC contributed to the clinical diagnosis and collected samples. SHG, DL, JSA, QP, and STJ contributed to the writing and revision of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
All procedures involving human participant was in accordance with ethical standards.
Footnotes
Se Hun Gu and Dong Hyun Song have contributed equally to this work.
References
- 1.Bonaldo MC, Ribeiro IP, Lima NS, dos Santos AAC, Menezes LSR, da Cruz SOD, de Mello IS, Furtado ND, de Moura EE, Damasceno L, da Silva KAB, de Castro MG, Gerber AL, de Almeida LGP, Lourenco-de-Oliveira R, Vasconcelos ATR, Brasil P. PLoS Negl. Trop. Dis. 2016;10(6):e0004816. doi: 10.1371/journal.pntd.0004816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Ladner JT, Wiley MR, Prieto K, Yasuda CY, Nagle E, Kasper MR, Reyes D, Vasilakis N, Heang V, Weaver SC, Haddow A, Tesh RB, Sovann L, Palacios G. Genome Announc. 2016;4(3):e00377–e00416. doi: 10.1128/genomeA.00377-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Dick GWA, Kitchen SF, Haddow AJ. Trans. R. Soc. Trop. Med. Hyg. 1952;46:509–520. doi: 10.1016/0035-9203(52)90042-4. [DOI] [PubMed] [Google Scholar]
- 4.Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, Kolenc M, Rus KR, Vipotnik TV, Vodušek VF, Vizjak A, Pižem J, Petrovec M, Županc TA. N. Engl. J. Med. 2016;374:951–958. doi: 10.1056/NEJMoa1600651. [DOI] [PubMed] [Google Scholar]
- 5.Oehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F, Baudouin L, Mallet HP, Musso D, Ghawche F. Euro. Surveill. 2014;19:20720. doi: 10.2807/1560-7917.ES2014.19.9.20720. [DOI] [PubMed] [Google Scholar]
- 6.Jang H-C, Park WB, Kim UJ, Chun JY, Choi S-J, Choe PG, Jung S-I, Jee Y, Kim N-J, Choi EH, Oh MD. J. Korean Med. Sci. 2016;31:1173–1177. doi: 10.3346/jkms.2016.31.7.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013;30(12):2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Ellison DW, Ladner JT, Buathong R, Alera MT, Wiley MR, Hermann L, Rutvisuttinunt W, Klungthong C, Chinnawirotpisan P, Manasatienkij W, Melendrez MC, Maljkovic Berry I, Thaisomboonsuk B, Ong-ajchaowlerd P, Kaneechit W, Velasco JM, Tac-An IA, Villa D, Lago CB, Roqu VG, Jr, Plipat T, Nisalak A, Srikiatkhachorn A, Fernandez S, Yoon IK, Haddow AD, Palacios GF, Jarman RG, Macareo LR. Genome Announc. 2016;4(3):16–e00359. doi: 10.1128/genomeA.00359-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Li J, Xiong Y, Wu W, Liu X, Qu J, Zhao X, Zhang S, Li J, Li W, Liao Y, Gong T, Wang L, Shi Y, Xiong Y, Ni D, Li Q, Liang M, Hu G, Li D. Emerg. Infect. Dis. 2016;22(6):1133–1136. doi: 10.3201/eid2206.160273. [DOI] [PMC free article] [PubMed] [Google Scholar]