Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Téllez, Yolanda; Sahoo, Malaya K.; Balmaseda, Ángel; Harris, Eva; Pinsky, Benjamin A.

doi:10.3201/eid2207.160326

Cited by 162 publications

(166 citation statements)

References 13 publications

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“…DNA was removed on column by using 30 U per sample PureLink DNAse (Invitrogen) for 15 min at room temperature. ZIKV RNA in serum was quantified using the ZCD assay with RNA standards 43 . ZIKV RNA in PBMCs was quantified using a modified ZIKV real-time RT-PCR singleplex assay originally developed by the CDC 44 .…”

Section: Methodsmentioning

confidence: 99%

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua

et al. 2017

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The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as ‘Trojan horses’ and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14+CD16+ monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14+ monocytes was significantly decreased, while the CD14+CD16+ monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14+CD16+ monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14+CD16+ monocytes in particular.

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Section: Methodsmentioning

confidence: 99%

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua

et al. 2017

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“…They were classified as DENV immune if they either entered the cohort with detectable anti-DENV EI antibodies or entered the cohort study with no detectable anti-DENV antibodies and had one or more documented DENV infections during their time in the cohort. All suspected ZIKV cases were confirmed by RT-PCR in serum and/or urine using triplex assays that simultaneously screen for DENV and CHIKV infections (ZCD assay [46], CDC Trioplexor and, in some cases, the CDC ZIKV monoplex assay [20], in parallel with a DENV-CHIKV multiplex assay [47]). The PDCS was approved by the IRB of the Nicaraguan Ministry of Health and the University of California, Berkeley.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were tested for ZIKV and/or DENV using the ZCD assay, as previously described (46). DENV-positive samples were serotyped using a serotype-specific DENV multiplex assay (46,52). In some laboratories, samples were tested by RT-PCR for ZIKV as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

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Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

Grifoni

Pham

Sidney

et al. 2017

J Virol

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While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here, we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether preexisting dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with tetravalent dengue attenuated vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors but declines in DENV-preexposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells from DENV-preexposed donors selectively upregulated granzyme B and PD1, unlike DENV-naive donors. Finally, we discovered that ZIKV

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“…The PDCS is a community-based prospective study of children 2-14 y of age that has been ongoing since August 2004 in Managua, Nicaragua (13). All Zika suspected cases were confirmed by RT-PCR in serum and/or urine, using triplex assays that simultaneously screen for DENV and CHIKV infections [ZCD assay (20), CDC Trioplex (21), or in some cases the CDC ZIKV monoplex assay (8), in parallel with a DENV-CHIKV multiplex assay (22)]. As a control group, a total of 146 patients with dengue confirmed by RT-PCR (23,24) between 2005 and 2013 were included, of whom 115 (41 primary cases and 74 secondary cases) belonged to the PDCS and 31 (18 primary cases and 13 secondary cases) were participants in the Nicaraguan Hospital-based Dengue Hospital at the National Pediatric Reference Hospital in Nicaragua (25), which has been ongoing since 1998.…”

Section: Methodsmentioning

confidence: 99%

Antibody-based assay discriminates Zika virus infection from other flaviviruses

Balmaseda

Stettler

Medialdea-Carrera

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/ plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENVnaive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.is a mosquito-borne flavivirus that is spread via the bite of infected Aedes mosquitoes or by sexual transmission and is responsible for the explosive 2015-2017 epidemic in the Americas. ZIKV infection during pregnancy is linked to devastating birth defects and associated anomalies, designated congenital Zika syndrome (1, 2), whereas in adults, ZIKV infection has been associated with Guillain Barré syndrome (3). Flaviviruses are enveloped RNA viruses containing an ∼11-kb positive-stranded RNA genome that encodes three structural and seven nonstructural proteins. Cells infected by flaviviruses secrete nonstructural protein 1 (NS1), which has multiple roles in immune evasion and pathogenesis (4, 5).Antibody responses generated in response to flavivirus infections are notoriously cross-reactive, representing a significant obstacle for the specific diagnosis of infection using serological assays. Multiple RT-PCR-based assays for the detection of ZIKV RNA are available, but their use is limited to the narrow window when viral RNA is detectable in body fluids. This is highly variable among patients and subject to reporting error, as symptoms are mild, and thus patients may take less notice of the day of onset, but in most cases, it is up to 7 d in serum, ...

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Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

Abstract: Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

Cited by 162 publications

References 13 publications

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

Antibody-based assay discriminates Zika virus infection from other flaviviruses

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