Yang Yang, Gary Wong, Baoguo Ye, Shihua Li, Shanqin Li, Haixia Zheng, Qiang Wang, Mifang Liang, George F Gao, Lei Liu, Yingxia Liu and Yuhai Bi. Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages[J]. Virologica Sinica, 2017, 32(3): 199-206. doi: 10.1007/s12250-017-3958-y
Citation: Yang Yang, Gary Wong, Baoguo Ye, Shihua Li, Shanqin Li, Haixia Zheng, Qiang Wang, Mifang Liang, George F Gao, Lei Liu, Yingxia Liu, Yuhai Bi. Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages .VIROLOGICA SINICA, 2017, 32(3) : 199-206.  http://dx.doi.org/10.1007/s12250-017-3958-y

亚洲和非洲型寨卡病毒通用型实时荧光定量RT-PCR检测方法的建立

  • 寨卡病毒是一种虫媒传播病毒,在世界范围内广泛流行。越来越多的研究表明寨卡病毒感染与小头畸形和格林-巴利综合征的发生密切相关。因此建立一种适用于不同流行地区的快速、灵敏和特异的检测方法非常必要。由于寨卡病毒地域分布广泛、遗传多样性复杂,目前已有的病原检测的引物和探针不能覆盖所有寨卡病毒谱系。针对这些问题,本研究建立了一种针对寨卡病毒E蛋白的荧光定量RT-PCR方法。该方法能够特异性检测亚洲型和非洲型寨卡病毒,与其它黄病毒属成员没有交叉反应;检测灵敏度能够达到每个反应5个拷贝RNA标准品以及2.94×10-3 TCID50的病毒RNA;通过临床样本检测,进一步验证了该方法的可靠性。该方法的建立和应用将对不同寨卡疫情流行区域的病原学检测和诊断发挥重要作用,具有实际应用价值。

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages

Novel RT-PCR assay to detect African and Asian Zika lineages

  • The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times.There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans.The ZIKV is genetically diverse and can be separated into Asian and African lineages.A rapid,sensitive,and specific assay is needed for the detection of ZIKV across various pandemic regions.So far,the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains.To this end,we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene.The detection limit of the assay was determined to be five RNA transcript copies and 2.94×10-3 50% tissue culture infectious doses (TCID50) of live ZIKV per reaction.The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification,when tested against other flaviviruses.The assay was also successful in testing for ZIKV in clinical samples.Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

  • 加载中
    1. Aubry M, Richard V, Green J, Broult J, Musso D. 2016. Inactivation of Zika virus in plasma with amotosalen and ultraviolet A illumination. Transfusion, 56: 33–40.
        doi: 10.1111/trf.13271

    2. Basarab M, Bowman C, Aarons EJ, Cropley I. 2016. Zika virus. BMJ, 352: i1049.

    3. Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. 2014. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill, 19. pii: 20751.

    4. Deng C, Liu S, Zhang Q, Xu M, Zhang H, Gu D, Shi L, He J, Xiao G, Zhang B. 2016a. Isolation and characterization of Zika virus imported to China using C6/36 mosquito cells. Virol Sin, 31: 176–179.
        doi: 10.1007/s12250-016-3778-5

    5. Deng YQ, Zhao H, Li XF, Zhang NN, Liu ZY, Jiang T, Gu DY, Shi L, He JA, Wang HJ, Sun ZZ, Ye Q, Xie DY, Cao WC, Qin CF. 2016b. Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China. Sci China Life Sci, 59: 428–430.

    6. Dick GW, Kitchen SF, Haddow AJ. 1952. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg, 46: 509–520.
        doi: 10.1016/0035-9203(52)90042-4

    7. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB. 2009. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med, 360: 2536–2543.
        doi: 10.1056/NEJMoa0805715

    8. Faria NR, Azevedo Rdo S, Kraemer MU, Souza R, Cunha MS, Hill SC, Theze J, Bonsall MB, Bowden TA, Rissanen I, et al. 2016. Zika virus in the Americas: Early epidemiological and genetic findings. Science, 352: 345–349.
        doi: 10.1126/science.aaf5036

    9. Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA. 2013. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Virol J, 10: 311.
        doi: 10.1186/1743-422X-10-311

    10. Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, Zanotto PM, Sall AA. 2014. Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Negl Trop Dis, 8: e2636.
        doi: 10.1371/journal.pntd.0002636

    11. Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. 2015. Detection of Zika virus in urine. Emerg Infect Dis, 21: 84–86.
        doi: 10.3201/eid2101.140894

    12. Govero J, Esakky P, Scheaffer SM, Fernandez E, Drury A, Platt DJ, Gorman MJ, Richner JM, Caine EA, Salazar V, Moley KH, Diamond MS. 2016. Zika virus infection damages the testes in mice. Nature, 540: 438–442.
        doi: 10.1038/nature20556

    13. Grard G, Moureau G, Charrel RN, Holmes EC, Gould EA, de Lamballerie X. 2010. Genomics and evolution of Aedes-borne flaviviruses. J Gen Virol, 91: 87–94.
        doi: 10.1099/vir.0.014506-0

    14. Hamel R, Liegeois F, Wichit S, Pompon J, Diop F, Talignani L, Thomas F, Despres P, Yssel H, Misse D. 2016. Zika virus: epidemiology, clinical features and host-virus interactions. Microbes Infect, 18: 441–449.
        doi: 10.1016/j.micinf.2016.03.009

    15. Kuno G, Chang GJ. 2007. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Arch Virol, 152: 687–696.
        doi: 10.1007/s00705-006-0903-z

    16. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR. 2008. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis, 14: 1232–1239.
        doi: 10.3201/eid1408.080287

    17. Liu L, Wu W, Zhao X, Xiong Y, Zhang S, Liu X, Qu J, Li J, Nei K, Liang M. 2016. Complete Genome Sequence of Zika Virus from the First Imported Case in Mainland China. Genome Announc, 4. pii: e00291-16.

    18. Lucchese G, Kanduc D. 2016. Zika virus and autoimmunity: From microcephaly to Guillain-Barre syndrome, and beyond. Autoimmun Rev, 15: 801–808.
        doi: 10.1016/j.autrev.2016.03.020

    19. Ma W, Li S, Ma S, Jia L, Zhang F, Zhang Y, Zhang J, Wong G, Zhang S, Lu X, Liu M, Yan J, Li W, Qin C, Han D, Qin C, Wang N, Li X, Gao GF. 2016. Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell, 167: 1511–1524.
        doi: 10.1016/j.cell.2016.11.016

    20. Macnamara FN. 1954. Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg, 48: 139–145.
        doi: 10.1016/0035-9203(54)90006-1

    21. Malkki H. 2016. CNS infections: Zika virus infection could trigger Guillain-Barre syndrome. Nat Rev Neurol, 12: 187.
        doi: 10.1038/nrneurol.2016.30

    22. Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodusek V, Vizjak A, Pizem J, Petrovec M, Avsic Zupanc T. 2016. Zika Virus Associated with Microcephaly. N Engl J Med, 374: 951–958.
        doi: 10.1056/NEJMoa1600651

    23. Musso D, Gubler DJ. 2016. Zika Virus. Clin Microbiol Rev, 29: 487–524.
        doi: 10.1128/CMR.00072-15

    24. Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. 2015. Potential sexual transmission of Zika virus. Emerg Infect Dis, 21: 359–361.
        doi: 10.3201/eid2102.141363

    25. Parra B, Lizarazo J, Jimenez-Arango JA, Zea-Vera AF, Gonzalez-Manrique G, Vargas J, Angarita JA, Zuniga G, Lopez-Gonzalez R, Beltran CL, et al. 2016. Guillain-Barre Syndrome Associated with Zika Virus Infection in Colombia. N Engl J Med. 375:1513–1523
        doi: 10.1056/NEJMoa1605564

    26. Reed LJ, Muench H. 1938. A simple method of estimating fifty percent endpoints. Am J Hyg, 27:493–497.

    27. Saiz JC, Vazquez-Calvo A, Blazquez AB, Merino-Ramos T, Escribano-Romero E, Martin-Acebes MA. 2016. Zika Virus: the Latest Newcomer. Front Microbiol, 7: 496.

    28. Shi W, Zhang Z, Ling C, Carr MJ, Tong Y, Gao GF. 2016. Increasing genetic diversity of Zika virus in the Latin American outbreak. Emerg Microbes Infect, 5: e68.
        doi: 10.1038/emi.2016.68

    29. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 25: 4876–4882.
        doi: 10.1093/nar/25.24.4876

    30. Waehre T, Maagard A, Tappe D, Cadar D, Schmidt-Chanasit J. 2014. Zika virus infection after travel to Tahiti, December 2013. Emerg Infect Dis, 20: 1412–1414.
        doi: 10.3201/eid2008.140302

    31. Wang L, Valderramos SG, Wu A, Ouyang S, Li C, Brasil P, Bonaldo M, Coates T, Nielsen-Saines K, Jiang T, Aliyari R, Cheng G. 2016a. From Mosquitos to Humans: Genetic Evolution of Zika Virus. Cell Host Microbe, 19: 561–565.
        doi: 10.1016/j.chom.2016.04.006

    32. Wang Q, Yang Y, Zheng HX, Bi YH, Song JD, Li LQ, Gu DY, Wang PY, Li SH, Liu S, Zhao YZ, Liu L, Gao GF, Liu YX. 2016b. Genetic and biological characterization of Zika virus from human cases imported through Shenzhen Port. Chinese Science Bulletin, 61: 2463–2474.

    33. WHO 2016a, posting date. WHO statement on the first meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. Available: http://www.who.int/mediacentre/news/statements/2016/1st-emergency-committee-zika/en/. Accessed 18 October 2016.

    34. WHO 2016b, posting date. Zika virus and complications. Available: http://www.who.int/emergencies/zika-virus/en/. Accessed 18 October 2016.

    35. WHO 2017, posting date. Zika Situation Report. Available: http://www.who.int/emergencies/zika-virus/situation-report/05-january-2017/en/. Accessed 6 May 2017.

    36. Wolfe ND, Kilbourn AM, Karesh WB, Rahman HA, Bosi EJ, Cropp BC, Andau M, Spielman A, Gubler DJ. 2001. Sylvatic transmission of arboviruses among Bornean orangutans. Am J Trop Med Hyg, 64: 310–316.
        doi: 10.4269/ajtmh.2001.64.310

    37. Wong G, Li S, Liu L, Liu y, Bi Y. 2017. Zika virus in the testes: should we be worried? Protein Cell. 8: 162–164.
        doi: 10.1007/s13238-016-0357-3

    38. Zhang Y, Chen W, Wong G, Bi Y, Yan J, Sun Y, Chen E, Yan H, Lou X, Mao H, Xia S, Gao GF, Shi W, Chen Z. 2016. Highly diversified Zika viruses imported to China, 2016. Protein Cell, 7: 461–464.
        doi: 10.1007/s13238-016-0274-5

  • 加载中

Figures(4) / Tables(3)

Article Metrics

Article views(6831) PDF downloads(24) Cited by(0)

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages

      Corresponding author: Lei Liu, beeyh@im.ac.cn
      Corresponding author: Yingxia Liu, yingxialiu@hotmail.com
      Corresponding author: Yuhai Bi, liulei3322@aliyun.com
    • 1. Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen 518112, China
    • 2. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, Institute of Microbiology, Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing 100101, China
    • 3. China-Japan Union Hospital of Jilin University, Changchun 130033, China
    • 4. Key Laboratory for Medical Virology, NHFPC, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China
    • 5. Office of Director-General, Chinese Center for Disease Control and Prevention, Beijing 102206, China
    • 6. Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China

    Abstract: The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times.There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans.The ZIKV is genetically diverse and can be separated into Asian and African lineages.A rapid,sensitive,and specific assay is needed for the detection of ZIKV across various pandemic regions.So far,the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains.To this end,we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene.The detection limit of the assay was determined to be five RNA transcript copies and 2.94×10-3 50% tissue culture infectious doses (TCID50) of live ZIKV per reaction.The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification,when tested against other flaviviruses.The assay was also successful in testing for ZIKV in clinical samples.Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

      • The Zika virus (ZIKV) was first identified in 1947 from a sentinel rhesus monkey during surveillance of yellow fever in Uganda (Dick et al., 1952). The virus was subsequently isolated in humans in Uganda and Tanzania in 1952 (Macnamara, 1954). The ZIKV belongs to the Flavivirus genus within the Flaviviridae family. Similar to other flaviviruses, ZIKV is a single-stranded positive RNA virus with a genome of approximately 10.8 kb, containing a single open reading frame (ORF), flanked by two untranslated regions (UTR) located at the 5′ and 3′ ends of the genome (Kuno and Chang, 2007; Saiz et al., 2016). The single ORF encodes the viral polyprotein that is cleaved by cellular and viral proteases into three structural proteins: the capsid (C), premembrane/mem brane (prM/M), and envelope (E); and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Kuno and Chang, 2007; Saiz et al., 2016). Phylogenetic studies have shown that ZIKV can be separated into two major lineages, Asian and African, and that it shows the highest genome similarity to other mosquito-borne flaviviruses, such as dengue virus (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) (Lanciotti et al., 2008; Wang L. et al., 2016a). The reservoir for ZIKV is not clearly defined, but it is speculated that the virus is likely maintained in a primate-mosquito-primate sylvatic cycle that includes nonhuman primates and/or humans, and a broad range of mosquitoes that mainly belong to the Aedes genus (Wolfe et al., 2001; Grard et al., 2010; Musso and Gubler, 2016).

        For half a century after the discovery of ZIKV, only sporadic infections in humans were documented, mainly in Africa and Southeast Asia (Faye et al., 2014; Saiz et al., 2016). This might be partially due to the high frequency of asymptomatic infections (up to 80%), as well as the mild, self-limiting nature of ZIKV fever, with clinical manifestations that could be mistaken for other infections (Duffy et al., 2009; Saiz et al., 2016). Clinical manifestations in symptomatic cases include fever, rash, arthralgia, headache, etc. that are similar to those of other arboviral infections, such as DENV and chikungunya virus (CHIKV) (Dick et al., 1952; Hamel et al., 2016). In 2015, an outbreak of ZIKV fever originated in Brazil (Faria et al., 2016), and as of January 5, 2017, over 70 countries and territories have reported continued mosquito-borne transmission of ZIKV within their borders (World Health Organization [WHO], 2017). Accumulating evidence suggests that ZIKV infection is associated with microcephaly of the fetus in pregnant women, as well as an increased incidence of Guillain-Barré Syndrome (GBS); moreover, the infection can be sexually transmitted and poses a potential risk of testicular damage (Govero et al., 2016; Lucchese and Kanduc, 2016; Ma et al., 2016; Malkki, 2016; Mlakar et al., 2016; Parra et al., 2016; WHO, 2016b; Wong et al., 2016). These findings suggest that ZIKV is more dangerous than previously thought. As such, the WHO declared ZIKV a public health emergency of international concern (WHO, 2016a).

        Due to the general nature of disease symptoms, in addition to the co-circulation with DENV and CHIKV in many ZIKV-affected areas, accurate diagnosis of ZIKV fever is difficult (Dick et al., 1952; Hamel et al., 2016; Saiz et al., 2016). Virus isolation and serological methods are still commonly used as diagnostic tools, despite their shortcomings. For instance, ZIKV isolation is time consuming, as it requires days to grow the virus on permissible cell lines; whereas serological methods could have limited cross-reactivity with related flaviviruses (Hamel et al., 2016; Saiz et al., 2016) and are not indicative of current (active) ZIKV infection. Quantitative reverse transcription polymerase chain reaction (qRT-PCR)-based assays are known for their ability to provide rapid, sensitive, and specific pathogen detection. Currently, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains (Lanciotti et al., 2008; Faye et al., 2013; Musso and Gubler, 2016). Furthermore, ZIKV displays high genetic diversity even within the same lineage and region (Shi et al., 2016; Zhang et al., 2016). To address this problem, we describe a novel qRT-PCR assay with broad coverage of ZIKV strains, including new circulating isolates.

      • The DENV 1–4 strains were provided by Prof. Chengfeng Qin. Strains of the ZIKV isolates MR_766 and PRVABC59 were provided by Prof. Mifang Liang, and the PLCal_ZV strain was provided by Prof. Gary Kobinger (University of Laval and Public Health Agency of Canada). The yellow fever virus (YFV) BJ01 strain and ZIKV SZ_SMGC-1 and CAS01 strains were isolated by our group from previously imported cases to China. Viral stocks were prepared using Vero or C6/36 cell lines. The RNA was extracted from viral stocks using the QIAamp RNA Viral Kit (Qiagen, Heiden, Germany) and MagaBio plus Virus RNA Purification Kit (Automatic Nucleic Acid Purification System NPA-32+, BIOER, China) according to the manufacturer instructions. The RNA was washed with buffers AW1 and AW2, and eluted in 50 µL of AVE buffer and stored at –80 °C for subsequent use.

      • Vero cells in 96-well plates were grown to 90% confluence and infected with 10-fold serial dilutions of the cell supernatant for 1 h at 37 °C. The inoculum was then removed, and cells were overlaid with fresh DMEM plus 2% FBS. At 6 days post infection (dpi), plates were assessed for the lowest dilution at which 50% of the wells exhibited cytopathology. The TCID50 values were calculated according to the Reed-Muench method (Reed and Muench, 1938).

      • The E gene of ZIKV was chosen as the target for the primer design because of its unique characteristic that facilitates its differentiation from those of other fla- viviruses. All sequences of the 81 ZIKV strains used in the present study were downloaded from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/Zika/), and aligned using the Clustal X program (Thompson et al., 1997). Conserved ZIKV-specific sequences that were divergent from other flaviviruses were identified, and the primers (ZIKV-F/ZIKV-R) were designed using the Primer Premier 5 software. The probe (ZIKV-P) was labeled with the fluorescent reporter dye 6-carboxyfluorescein (FAM) at the 5′-end and the fluorescent quencher dye 6-carboxytetramethylrhodamin (TAMRA) at the 3′-end. The sequences and genome positions of the primer and probe set are shown in Figure 1.

        Figure 1.  Conserved sequence selection for primer and probe design. The E genes of 81 ZIKVs were downloaded for alignment; the most conserved regions that were divergent from other flaviviruses were selected for the primer and probe design. Alignment of the regions of designed primers and probe with reference strains of ZIKVs and other flaviviruses are shown. Dots indicate identity with the consensus sequence on top of the alignment.

      • A section of the E gene, 98 base pairs in size, was amplified with the primers ZIKV-F/ZIKV-R, and the product was purified using the Gene JET PCR Purification Kit (Thermo, MA, USA) and ligated to the pGEM-T vector (Promega, Madison, USA). In vitro transcription was performed using the MEGAscript T7 Transcription Kit (Thermo, MA, USA) and quantified using the Nanodrop 2000 Spectrophotometer (Thermo, MA, USA). The RNA copy number (molecules/µL) was calculated using the following equation: [C × A / 330 × L], where C rep- resents the concentration of RNA (g/mL) assessed by the optical density measurement; A is the Avogadro number (6.023 × 1023); L is the length of the synthetic RNA (number of nucleotides); and 330 is an approximation of the molecular weight of a nucleotide (g/mol).

      • Urine and serum samples were obtained from two confirmed cases of ZIKV infection in China (Deng C. et al., 2016a; Deng YQ. et al., 2016b; Liu et al., 2016; Wang Q. et al., 2016b). The RNA was extracted using the QIAamp RNA Viral Kit (Qiagen, Heiden, Germany) according to manufacturer recommendations.

      • The RNA samples were tested by qRT-PCR in an ABI QuantStudio 7 Real-Time cycler (Applied Biosystems, Foster City, USA). The One Step PrimeScriptTM RT-PCR Kit (Takara, Dalian, China) was used as follows: 0.8 µL enzyme mixture (including reverse transcriptase [RT] and Taq polymerase), 10 µL 2 × One Step RT-PCR buffer III, 0.4 µL of each primer and probe (20 µmol/L), 0.4 µL ROX Reference Dye II, 2.6 µL RNase free water, and 5 µL RNA (total 20 µL/reaction mixture). Each qRT-PCR run contained one negative and one positive control. The negative control consisted of water in place of the RNA sample. The positive control was ZIKV nucleic acid extracted from viral stocks, as described above. The qRT-PCR assay conditions were as follows: reverse transcription for 5 min at 42 °C; 10 s at 95 °C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 cycles of 5 s at 95 °C; and 30 s at 55 °C (annealing-extension step). The data were analyzed using the QuantStudioTM Real-Time PCR Software (Applied Biosystems, Foster City, USA). Commercial qRT-PCR kits were also used for the detection of DENV, YFV, and ZIKV (Da An Gene Co., Ltd., Guangdong, China) following manufacturer instructions. All samples were analyzed in triplicate with three independent runs.

      • The genomes of all ZIKVs were downloaded from the NCBI database for alignment. After systematic analysis, we identified a highly conserved region of 98 nucleotides (nt) in length on the E gene that was specific to ZIKV, but divergent from other flaviviruses. Based on the analysis, we designed the following primer-probe set for this conserved region: ZIKV-F (5′-TGAYAAGCAR- TCAGACAC-3′), ZIKV-R (5′-TCACCARRCTCCCT- TTGC-3′) and ZIKV-P (5′-FAM-GTGGAYAGAGG- YTGGGGAAA-TAMRA-3′), which hybridized to po sitions 1222–1239, 1302–1319, and 1265–1284, respectively, in the ZIKV genome (Figure 1, as calculated from GenBank accession number AY632535). The primer-probe set was then used for ZIKV detection by the one-step qRT-PCR method, as described in the Materials and Methods section.

      • To test the specificity of our qRT-PCR method, different flaviviruses including DENV 1–4, YFV, and several ZIKV strains were used. All five ZIKVs, including both African and Asian lineages (Figure 2) could have been detected by our primer-probe set (Table 1). In addition, with our primer-probe set, amplification was not ob served in any of the RNA preparations from the DENV and YFV strains, or gene fragments of WNV. The novel qRT-PCR with the new primer-probe set displayed high specificity for ZIKVs without any amplification of other flaviviruses.

        Figure 2.  Phylogenetic analysis of ZIKVs. Genomes of ZIKVs were analyzed using the Mega 7 software. Strains of ZIKV imported into China and reference strains used in the present study are colored in green and red, respectively.

        Flavivirus species Reference Lineage GenBank no. Virus titer (TCID50/mL) RT-qPCR assays
        This publication
        ZIKV MR_766 African KX377335 2.1 × 106 14.75 ± 0.22
        ZIKV PRVABC59 Asian KX377337 3.2 × 104 20.22 ± 0.16
        ZIKV PLCal_ZV Asian KF993678 3.2 × 105 17.59 ± 0.22
        ZIKV SZ_SMGC-1 Asian KX266255 3.2 × 105 17.96 ± 0.44
        ZIKV CAS01 Asian NA 2.7 × 105 18.59 ± 0.23
        DENV CN/GZ27/2014 Serotype 1 KP723473 NAc Ud
        DENV DENV2-43 Serotype 2 AF204178 NA U
        DENV YN02 Serotype 3 KF824903 NA U
        DENV GZB5 Serotype 4 AF289029 NA U
        YFV BJ01/2016 b KY495641 NA U
        WNVa NY99 b NC_009942 1 × 106(copies/μL) NA
        Note: ZIKV, Zika virus; DENV, dengue virus; YFV, yellow fever virus; WNV, West Nile virus. a The synthetic envelope gene was used instead of the live virus; b Not applicable; c Not available; d Undetected. The Ct values of the commercial kits for DENVs (CN/GZ27/2014, DENV2-43, YN02, and GZB5) and YFV were 17.22, 12.18, 15.23, 16.42, and 15.59, respectively.

        Table 1.  ZIKV and other flavivirus strains used in the present study

      • The detection limit of the novel qRT-PCR assay was evaluated using the quantitative RNA standards from a pGEM-T vector expressing the target sequence of the E gene, and viral RNAs prepared from serial ten-fold dilutions of the five ZIKV stocks. Assays were per- formed in triplicate for both methods. The qRT-PCR method using the RNA standards as a template showed that cycle threshold (Ct) values were linear between 1 (mean Ct value = 39.14) and 1 × 108 molecules (mean Ct value = 14.92). The regression coefficient (R2 = 0.999) indicated that over this range, the assay was both accurate and precise (Figure 3A). The detection limit was determined to be five RNA transcript copies per reaction, based on the standard curve (Figure 3A) and specific amplification curves (Figure 3B). Moreover, RNA samples were extracted from ten-fold serial dilutions of stock ZIKV ranging from 3.2 × 105 to 2.1 × 10–2 TCID50/mL, and were used to test the detection limit. Results showed that the detection limit of the qRT-PCR assay was similar among the five ZIKV strains under evaluation, that is, between 2.94 × 10–3 and 4.48 × 10–3 TCID50 per reaction (2.94 × 10–3 TCID50 for MR_766; 3.78 × 10–3 TCID50 for CAS01; and 4.48 × 10–3 TCID50 for the other three strains). Viral titers were shown to correlate well with the obtained Ct values, ranging from 17.71 to 38.74 (Figure 4A-4E, Table 2). The Ct values across the quantitative range showed a standard deviation ranging from 0.01 to 0.58 (Table 2). According to our results, the specimen was considered positive if the Ct value was less than 38, and negative if it was undetermined. Any Ct values between 38 and 40 with typical amplification curves (Figure 3B) were considered indeterminate and positive if the repeat results were similar to the previous results, and this was confirmed by sequencing the amplicon.

        Figure 3.  Sensitivity of the qRT- PCR assay using synthetic ZIKV RNA. (A) Standard curve for ten-fold serial dilution of synthetic ZIKV RNA. The log number of ZIKV RNA transcripts (Copies/µL) is expressed linearly on the x-axis, whereas Ct values obtained from qRT-PCR are expressed linearly on the y-axis. (B) Representative amplification curves of the different concentrations of synthetic ZIKV RNA.

        Figure 4.  Sensitivity of the qRT- PCR assay using ZIKV viral RNAs. (A–F) Standard curves for ten-fold serial dilutions of stock ZIKV strains PRVABC59, MR_766, CAS01, PLCal_ZV, and SZ_SMGC-1, respectively. The log number of live ZIKV (TCID50/mL) is expressed linearly on the x-axis, whereas Ct values are expressed linearly on the y-axis.

        Serial dilutiona Ct Values
        MR_766 CAS01 PLCal_ZV SZ_SMGC-1 PRVABC59
        Viral stock 17.92 ± 0.04 18.69 ± 0.23 17.71 ± 0.40 18.16 ± 0.39 20.32 ± 0.06
        10–1 21.13 ± 0.18 21.80 ± 0.25 20.95 ± 0.01 21.36 ± 0.42 23.62 ± 0.18
        10–2 24.49 ± 0.06 25.15 ± 0.05 24.19 ± 0.13 24.62 ± 0.48 26.96 ± 0.02
        10–3 27.71 ± 0.11 28.51 ± 0.09 27.80 ± 0.10 27.86 ± 0.45 30.26 ± 0.04
        10–4 31.01 ± 0.18 32.25 ± 0.10 31.21 ± 0.05 31.51 ± 0.61 34.06 ± 0.10
        10–5 34.52 ± 0.01 35.89 ± 0.58 34.64 ± 0.10 34.86 ± 0.36 37.03 ± 0.31
        10–6 37.75 ± 0.22 38.66 ± 0.54 38.74 ± 0.25 37.48 ± 0.13 Neg
        10–7 Negb Neg Neg Neg Neg
        Note: aThe titers of stock ZIKV were 2.1 × 105 TCID50/mL (MR_766), 2.7 × 105 TCID50/mL (CAS01), 3.2 × 105 TCID50/mL (SZ_SMGC-1), 3.2 × 105 TCID50/mL (PLCal_ZV) and 3.2 × 104 TCID50/mL (PRVABC59), respectively; b Negative.

        Table 2.  Sensitivity of the qRT-PCR assay for ZIKV RNA detection

      • To assess the performance of the qRT-PCR assay in a clinical setting, tests were conducted on urine and serum samples from two confirmed ZIKV-infected cases imported to China, as mentioned in the Materials and Methods section. As expected, all samples tested positive for ZIKV and displayed comparatively lower Ct values with the qRT-PCR assay than those obtained with the commercial detection kit for ZIKV. However, no statistically significant differences were observed between the two results (Table 3).

        Samples qRT-PCR assays Pa
        Our method Commercial kit
        Patient 1 Serum 29.67 ± 0.82 30.63 ± 0.13 0.1146
        Urine 29.54 ± 0.61 30.47 ± 0.12 0.0606
        Patient 2 Serum 26.71 ± 0.58 27.66 ± 0.84 0.1823
        Note: aData were analyzed using the Student’s t-test, and P-values represent a comparison of our method with that of the commercial kit.

        Table 3.  Detection of ZIKV in clinical samples

      • In the present study, the detection limit was determined to be as low as five RNA transcript copies from a pGEM-T vector expressing the target sequence, and 2.94 × 10–3 TCID50 of live ZIKV per reaction, with high accuracy and precision (R2 = 0.999). These findings reflect greater sensitivity than the previously developed qRT-PCR targeting NS5 gene and other traditional RT-PCR assays that target the E gene (Lanciotti et al., 2008; Faye et al., 2013; Basarab et al., 2016). The difference in abundance of the E and NS5 genes in the ZIKV virion might in- fluence the sensitivity of qRT-PCR detection. Currently, diagnosis of ZIKV infection is mainly based on the detection of ZIKV RNA during the first few days after the onset of symptoms (Musso and Gubler, 2016). According to previous studies, ZIKV RNA can be detected in several types of bodily fluids, including blood, urine, saliva, breast milk, and semen (Musso and Gubler, 2016). In symptomatic patients, viremia ranges from 7.28 × 106 to 9.3 × 108 copies/mL, and in asymptomatic patients, from 2.5 × 103 to 8 × 106 copies/mL (Besnard et al., 2014; Waehre et al., 2014; Aubry et al., 2016; Musso and Gubler, 2016). In urine, viral loads range from 3.8 × 103 to 2.2 × 108 copies/mL with a greater persistence than in serum (10 to > 20 days, and > 7 days, once it becomes undetectable in serum) (Besnard et al., 2014; Gourinat et al., 2015; Musso et al., 2015). Accordingly, our assay qualifies for the surveillance of ZIKV from various types of clinical samples without nonspecific amplification of other flaviviruses. In comparison to qRT-PCR, ELISAs are limited by cross-reactivity with other flaviviruses, due to the close relatedness to, and co-circulation of other flaviviruses in ZIKV endemic regions. Furthermore, detection of ZIKV is best achieved during the acute-phase; however, it is difficult to determine the period of the onset of symptoms, as the majority of cases are asymptomatic (Hamel et al., 2016; Saiz et al., 2016).

        In conclusion, we have developed a rapid, specific qRT-PCR assay with high sensitivity and broad coverage of circulating ZIKV strains (both African and Asian lineages). This assay would be of value in surveillance efforts across various regions of the outbreak.

      • This work was supported by the National Science and Technology Major Project (2016ZX10004222), the Sanming Project of Medicine in Shenzhen (ZDSYS2015 04301534057), the Key specialized fund for infectious diseases in Shenzhen City (No. 201161), the intramural special grant for influenza virus research from the Chinese Academy of Sciences (KJZD-EW-L09 and KJZD-EW-L15), and the Shenzhen Science and Technology Research and Development Project (JCYJ20160427151920801 and JCYJ20160427153238750). G.F.G. is a leading principal investigator of the National Natural Science Foundation of China (NSFC) Innovative Research Group (81621091). Y.B. is supported by the Youth Innovation Promotion Association of Chinese Academy of Sciences (CAS) (2017122). G.W. is the recipient of a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (CIHR) and the President’s Interna tional Fellowship Initiative from the CAS. We thank Dr. Chengfeng Qin and Gary Kobinger for supplying strains of DENVs and ZIKVs.

      • The authors declared that they have no conflict of interest. The studies have been approved by our institutional research ethics committee, and written informed consent was obtained from all patients.

      • YHB and YY designed the experiments. YY, GW, BGY, YHB, SHL, SQL, HXZ and QW carried out the experiments. YY, YHB and GW analyzed the data. YXL, LL, MFL and GFG acquired the clinical samples and provided scientific input. YY, GW and YHB wrote the paper. All authors read and approved the final manuscript.

    Figure (4)  Table (3) Reference (38) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return