Research ArticleZIKA VIRUS

Rapid and specific detection of Asian- and African-lineage Zika viruses

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Science Translational Medicine  03 May 2017:
Vol. 9, Issue 388, eaag0538
DOI: 10.1126/scitranslmed.aag0538
  • Fig. 1. Design and implementation of LAMP primers.

    (A) LAMP target sequences in the Puerto Rican strain (PRVABC59) of Zika virus aligned with sequences from the Malaysia (P6-740), Uganda MR-766NIID (MR-766), and Senegal (41525) strains. Primer sequences are indicated by black bars. BIP and FIP primers are fusions of B1 and B2c and F1c and F2, respectively (B1, B2c, F1c, and F2 labels are shown). Alu I restriction sites are indicated. Sequence variations are in gray. (B) Asian-lineage–specific primers detect Zika virus RNAs exclusively from Vero cells infected with Asian strains, PRVABC59 and P6-740. (C) African-lineage–specific primers detect Zika virus RNAs exclusively from Vero cells infected with African strains, MR-766 and 41525. (B and C) PRVABC59 (circles), P6-740 (boxes), MR-766 (diamonds), and 41525 (crosses). The threshold of detection is defined by a horizontal gray line. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, relative fluorescence units (RFUs).

  • Fig. 2. Direct detection of Zika virus RNA in cells and supernatants.

    (A) Lysates of 1000 infected Vero or C6/36 cells in water. Vero cell lysate (black triangles), C6/36 cell lysate (black squares), mock-infected Vero cells (no symbols; line below threshold), mock-infected C6/36 cells (no symbols; line below threshold), infected Vero cell RNA (positive control; red circles), buffer only (no symbols; line below threshold). (B) Duplicate aliquots of infected Vero cell supernatant amplified directly (circles) or diluted 10-fold in water (squares). X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

  • Fig. 3. Direct detection of Zika virus in mosquitoes.

    A LAMP assay was performed on 2.5 μl (1/100th of total) from PRVABC59 strain–infected mosquitoes split sagittally; one-half of the mosquito carcass was homogenized in mosquito diluent (red circles) and the other half in water (blue squares). Blue and red crosses, single mosquito with low virus load (table S2, mosquito #3); x axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

  • Fig. 4. Limit of detection of Zika virus RNA.

    (A) A LAMP assay was performed on 2 μl of 10-fold dilutions of Vero cell supernatants containing 6.4 × 105 PFU/ml. Virus input: 1280 PFU (black circles), 128 PFU (black triangles), 12.8 PFU (black squares), 1.28 PFU (black diamonds), 0.128 PFU (black crosses), and 0.0128 PFU (no symbols; line below threshold). (B) A LAMP assay was performed on 10-fold dilutions of Zika virus genome copies: 105 copies (black circles), 104 copies (black triangles), 103 copies (black squares), 102 copies (black diamonds), 10 copies (black crosses), and 1 copy (line, no symbols). (A and B) Control infected Vero cell RNA (positive control; red circles) and no RNA (no symbols; line below threshold). Results are representative of a minimum of six replicates of each dilution series. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

  • Fig. 5. Detection of Zika virus spiked into human blood, plasma, saliva, urine, and semen samples.

    Healthy human biofluids were spiked with the Zika virus PRVABC59 strain from infected Vero cell supernatants at a final concentration of 106 PFU/ml. (A) Amplification of 2 μl of serial 10-fold dilutions of plasma containing 106 PFU/ml of the PRVABC59 strain followed by dilution to 1:100 or 1:1000 in water. (B) Matching samples of the 1:1000 plasma dilutions were incubated in tubes in a heat block for 70 min and examined for turbidity. (C) Tenfold dilutions of blood, saliva, urine, and semen each spiked with 106 PFU/ml of PRVABC59 Zika virus strain followed by dilution to 1:1000 in water. Virus input: (A and C) 2000 PFU (black circles), 200 PFU (black triangles), 20 PFU (black squares), 2 PFU (black diamonds), 0.2 PFU (black crosses; below threshold), 0.02 PFU (no symbols; gray line below threshold), infected Vero cell RNA (positive control; red circles), and diluted biofluids without virus (no symbols; black line below threshold). Results are representative of a minimum of six replicates of each dilution series. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

  • Table 1. Detection of Zika virus RNA in cell and supernatant RNA preparations.

    ND, not determined.

    Virus RNAAsian Zika
    LAMP
    African Zika
    LAMP
    RT-PCR
    (primer
    specificity)
    LAMP
    (primer
    specificity)
    PRVABC59*++ (Zika)− (WNV, DENV,
    YFV)
    P6-740++ (Zika)− (WNV, DENV,
    YFV)
    41525++ (Zika)− (WNV, DENV,
    YFV)
    MR-766++ (Zika)− (WNV, DENV,
    YFV)
    WNVND+ (Pan-flavi)+ (WNV)
    DENV-2ND+ (Pan-flavi)+ (DENV)
    YFVND+ (Pan-flavi)+ (YFV)
    BussuquaraND+ (Pan-flavi)ND
    St. Louis
    encephalitis
    ND+ (Pan-flavi)ND
    LangatND+ (Pan-flavi)ND
    Deer tickND+ (Pan-flavi)ND
    IlheusND+ (Pan-flavi)ND
    CHIKVND+ (CHIKV)ND

    *PRVABC59-infected Vero cell total RNA and C6/36 cell total RNA.

    • Table 2. Direct detection of the PRVABC59 Zika virus strain in cell lysates and mosquito tissue homogenates.

      dpi, days post-infection; MD, mosquito diluent.

      SampleZika LAMP (number of positive/total)PFU
      Mock-infected cultured cells*0
      Infected cultured cells*+0
      Infected Vero cell supernatant+6.4 × 106 PFU/ml
      Infected C6/36 cell supernatant+7.4 × 106 PFU/ml
      Mock-infected mosquitoes− (0/3)− (3/3)
      Infected mosquitoes (7 dpi)+ (15/15)+ (14/15)
      Infected mosquitoes (14 dpi)+ (MD, 9/10; H2O, 10/10)+ (10/10)
      Infected mosquito midgut+ (4/5)+ (2/5)
      Infected mosquito salivary gland+ (4/5)+ (2/5)
      Infected mosquito carcass+ (1/5)+ (1/5)

      *Vero cells or C6/36 cells, lysed in water.

      †Positive for plaque formation.

      • Table 3. LOD (30) of Zika virus RNA with the LAMP assay (n ≥ 6 replicates).

        NA, not applicable.

        SampleAssayLOD95% CIPFU/ml*
        Vero cell supernatantLAMP0.43 PFU0.24–4.89214
        Vero cell supernatantqRT-PCR0.17 PFU0.08–6.5587
        Genome equivalentsLAMP111 copies58–1148NA
        Genome equivalentsqRT-PCR53 copies38–98NA
        Plasma (1:100)LAMP0.28 PFU0.19–0.8731
        Plasma (1:1000)LAMP0.05 PFU0.03–0.4221
        Plasma (1:1000)LAMP (turbidity)0.17 PFU0.08–2.5286
        Spiked bloodLAMP0.05 PFU0.03–0.2524
        Spiked urineLAMP0.32 PFU0.22–3.26159
        Spiked salivaLAMP0.13 PFU0.09–0.8663
        Spiked semenLAMP5.57 PFU2.18–608.142785

        *Based on a 2-μl sample volume and rounded to nearest whole number.

        †Final dilution in water.

        • Table 4. Serum samples from Zika virus–infected patients in Nicaragua (20 positive and 12 negative by qRT-PCR).
          Sera (n = 32)Serum RNA preparation (n = 32)
          Direct LAMPqRT-PCRLAMP
          PositiveNegativePositiveNegativePositiveNegative
          qRT-PCRPositive18 (90%)2 (10%)19 (95%)1 (5%)19 (95%)1 (5%)
          Negative4 (33%)8 (67%)012 (100%)1 (8%)11 (92%)
        • Table 5. Plasma samples from Zika virus–infected patients in Brazil (31 positive and 18 negative by qRT-PCR and serology).
          Plasma (n = 49)RNA (n = 26)RNA and plasma combined
          qRT-PCRLAMPLAMPLAMP
          PositiveNegativePositiveNegativePositiveNegativePositiveNegative
          qRT-PCR and serologyPositive25 (81%)6 (19%)20 (65%)10 (32%)16 (70%)7 (30%)26 (84%)5 (16%)
          Negative018 (100%)12 (67%)6 (33%)2 (9%)1 (33%)12 (67%)6 (33%)
        • Table 6. Zika virus RNA–positive semen samples from male U.S. travelers confirmed to be infected with Zika virus.
          Semen (five samples)
          Sample numberRNA copies/ml (log10)LAMPDNase + LAMP
          15.4++
          24.3+
          33.2+
          44.1++
          53.4++

        Supplementary Materials

        • www.sciencetranslationalmedicine.org/cgi/content/full/9/388/eaag0538/DC1

          Fig. S1. Confirmation of Zika-specific, RNA-dependent LAMP amplification.

          Fig. S2. Control samples containing each virus RNA.

          Fig. S3. Alignment of Zika LAMP primer sequences with target sequences in all known Zika virus sequences.

          Fig. S4. Direct detection of Zika virus in mosquitoes.

          Fig. S5. Confirmation of Zika-specific LAMP amplification from mosquito homogenate.

          Fig. S6. Zika LAMP assay is dependent upon RNA.

          Fig. S7. Visual LAMP assay of mosquito homogenates.

          Fig. S8. Limits of PRVABC59-LAMP detection match RT-PCR.

          Fig. S9. LAMP assay detects PRVABC59 virus in human biofluids.

          Table S1. Zika LAMP primers.

          Table S2. Plaque titration of split, infected mosquitoes.

          Table S3. Plaque titration of midguts, salivary glands, and carcass.

          Table S4A. LAMP assay LOD for genome copies.

          Table S4B. TaqMan LOD for genome copies.

          Table S5A. Plasma 1:1000 LOD.

          Table S5B. Plasma 1:1000 turbidity LOD.

          Table S6. Blood 1:1000 LOD.

          Table S7. Urine 1:1000 LOD.

          Table S8. Saliva 1:1000 LOD.

          Table S9. Semen 1:1000 LOD.

          Table S10. Nicaraguan serum and RNA samples.

          Table S11. Brazilian plasma and RNA samples.

        • Supplementary Material for:

          Rapid and specific detection of Asian- and African-lineage Zika viruses

          Nunya Chotiwan, Connie D. Brewster, Tereza Magalhaes, James Weger-Lucarelli, Nisha K. Duggal, Claudia Rückert, Chilinh Nguyen, Selene M. Garcia Luna, Joseph R. Fauver, Barb Andre, Meg Gray, William C. Black IV, Rebekah C. Kading, Gregory D. Ebel, Guillermina Kuan, Angel Balmaseda, Thomas Jaenisch, Ernesto T. A. Marques, Aaron C. Brault, Eva Harris, Brian D. Foy, Sandra L. Quackenbush, Rushika Perera, Joel Rovnak*

          *Corresponding author. Email: joel.rovnak{at}colostate.edu

          Published 3 May 2017, Sci. Transl. Med. 9, eaag0538 (2017)
          DOI: 10.1126/scitranslmed.aag0538

          This PDF file includes:

          • Fig. S1. Confirmation of Zika-specific, RNA-dependent LAMP amplification.
          • Fig. S2. Control samples containing each virus RNA.
          • Fig. S3. Alignment of Zika LAMP primer sequences with target sequences in all known Zika virus sequences.
          • Fig. S4. Direct detection of Zika virus in mosquitoes.
          • Fig. S5. Confirmation of Zika-specific LAMP amplification from mosquito homogenate.
          • Fig. S6. Zika LAMP assay is dependent upon RNA.
          • Fig. S7. Visual LAMP assay of mosquito homogenates.
          • Fig. S8. Limits of PRVABC59-LAMP detection match RT-PCR.
          • Fig. S9. LAMP assay detects PRVABC59 virus in human biofluids.
          • Table S1. Zika LAMP primers.
          • Table S2. Plaque titration of split, infected mosquitoes.
          • Table S3. Plaque titration of midguts, salivary glands, and carcass.
          • Table S4A. LAMP assay LOD for genome copies.
          • Table S4B. TaqMan LOD for genome copies.
          • Table S5A. Plasma 1:1000 LOD.
          • Table S5B. Plasma 1:1000 turbidity LOD.
          • Table S6. Blood 1:1000 LOD.
          • Table S7. Urine 1:1000 LOD.
          • Table S8. Saliva 1:1000 LOD.
          • Table S9. Semen 1:1000 LOD.
          • Table S10. Nicaraguan serum and RNA samples.
          • Table S11. Brazilian plasma and RNA samples.

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