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Table of Contents
Year : 2019  |  Volume : 56  |  Issue : 2  |  Page : 166-169

Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit

1 Regional Blood Center of Ribeirão Preto; Department of Internal Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil
2 Regional Blood Center of Ribeiräo Preto, Ribeiräo Preto, Sao Paulo, Brazil

Date of Submission02-Feb-2018
Date of Acceptance01-Mar-2019
Date of Web Publication31-Jul-2019

Correspondence Address:
Svetoslav Nanev Slavov
Laboratory of Molecular Biology, Blood Center of Ribeiräo Preto, University of Säo Paulo, CEP 14051-140, Ribeiräo Preto, Säo Paulo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.263724

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During zika and dengue viruse (ZIKV and DENV) outbreaks, the majority of the infected individuals remain clinically asymptomatic. Such asymptomatic individuals may occasionally acquire both arboviruses, donate blood, and contaminate haemoderivatives. The aim of this study was to characterize a ZIKV/DENV-4 coinfection in asymptomatic blood donor who donated blood during a large mixed ZIKV/DENV outbreak in the Säo Paulo State, Brazil. On the basis of post-donation information, one blood donor sample was found positive for ZIKV and DENV RNA. The DENV molecular serotyping was performed by molecular testing. The sample was also titrated on VERO E6 cells in order to define the presence of infectious arboviruses. The real-time PCR testing of the blood donor sample demonstrated very high viral load for both ZIKV and DENV. Further, molecular serotyping of DENV showed that the presence of DENV-4. The viral titration in cell culture indicated a titre of 2.75x10[6] PFU/ml which was concordant with the presence of infectious viruses in the blood donation. This is an interesting report for the simultaneous presence of infectious ZIKV and DENV-4 in asymptomatic blood sample. Special attention must be paid during mixed arboviral outbreaks for the possibility of transfusion-transmission of multiple arboviral agents.

Keywords: Coinfection; dengue virus; serotyping; zika virus

How to cite this article:
Slavov SN, Ferreira FU, Rodrigues ES, Gomes R, Covas DT, Kashima S. Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit. J Vector Borne Dis 2019;56:166-9

How to cite this URL:
Slavov SN, Ferreira FU, Rodrigues ES, Gomes R, Covas DT, Kashima S. Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit. J Vector Borne Dis [serial online] 2019 [cited 2020 Jul 6];56:166-9. Available from: http://www.jvbd.org/text.asp?2019/56/2/166/263724

During zika virus (ZIKV) outbreaks, up to 80% of the infected individuals may remain asymptomatic[1]. This situation is similar to the closely related dengue viruses 1–4 (DENV 1–4), which can also cause high percentage of asymptomatic infections[2]. In endemic areas, different fla-viviruses including DENV 1–4, West Nile virus (WNV)[3], and ZIKV can circulate simultaneously. Such cocircula- tion may result in arboviral coinfections in asymptomatic individuals, who may donate blood and be responsible for the transfusion-transmission of these viruses as reported previously[2],[4].

In tropical regions, mosquito vectors like Aedes ae- gypti and Ae. albopictus can simultaneously host and transmit ZIKV, DENV and/or Chikungunya virus (CHIKV)[5],[6]. Therefore, a concomitant human infection by different arboviruses is possible. Such cases have been described for ZIKV/CHIKV[7], ZIKV/DENV[8] and DENV/ CHIKV[6] during outbreaks in Colombia, Haiti and Mexico. In the above mentioned cases, the arboviruses were isolated from body fluids (urine, saliva and blood), and the viral RNA detection was performed in patients with clinical symptoms of arboviral disease (travelers returning from endemic areas; patients with haemorrhagic fever). Differently, in asymptomatic blood donors the concomi- tant presence of different arboviruses represents threat for the transfusion practice as both viral infections can be transmitted by application of the obtained haemoderivatives.

In this communication, we report a simultaneous DENV/ZIKV RNA detection by TaqMan® real-time PCR in a donated blood (plasma) unit obtained from a pre-symptomatic Brazilian blood donor, with subsequent viral isolation and tittering by plaque assay[9]. At the time of blood donation, the volunteer donor was completely asymptomatic, however, three days later she reported a clinical picture compatible with arboviral disease (fever, conjunctivitis and rash).

During the rainy season of 2016 (January-May), the City of Ribeiräo Preto (northeastern part of the São Paulo State, Brazil) experienced an explosive ZIKV/DENV outbreak with 5309 ZIKV-suspected and 34,912 DENV- confirmed cases, according to the Bulletin of the Municipal Epidemiological Surveillance Agency, Ribeiräo Preto (http://www. saude.ribeiraopreto.sp.gov. br/ssaude/pdf/ dengue_balanco.pdf). Differently from DENV, the diagnosis and confirmation of ZIKV fever was performed only on clinical basis (low grade fever, lymphadenopathy, extensive itchy exanthema, weak to moderate arthralgia and myalgia, conjunctivitis) as by the time of the outbreak and due to its sudden onset, the health authorities were not prepared to respond with urgent ZIKV detection. Therefore, many ZIKV cases might have been misdiagnosed, not only because of the overlapping clinical symptoms of the arboviral fevers, but also due to the mainly asymptomatic course of ZIKV fever.

All the volunteer blood donors, who donate blood in Blood Center of Ribeirão Preto, Brazil are orientated to communicate adverse effects which occur after donation, i.e. post-donation information. During January-May 2016, on the basis of post-donation information obtained from the blood donors (presence of fever, exanthema, arthralgia, retro-orbital pain, myalgia, and flu-like symptoms), 80 collected plasma donations (units) were deferred from transfusion. All of them were screened for ZIKV and DENV RNA.

Viral RNA was extracted from 140 μl plasma using the QIAamp viral RNA mini kit (QIAGEN) following the manufacturer’s instructions. ZIKV/DENV RNA was detected by TaqMan® real-time PCR using primers and probes available in the literature[10],[11]. The identification of DENV 1-4 serotypes was performed by molecular serotyping using TaqMan® real-time PCR available from the literature[12]. Viral RNA was quantified using in-house standard curves for ZIKV/DENV obtained after serial dilutions of the respective fragments which were cloned into TOPO® TA Cloning Vector (Thermo Fisher Scientific). The infectivity of the detected viruses was confirmed by isolation in Vero E6 cell culture. In brief, the donor plasma was concentrated by ultra centrifugation (48,000 x g, 90 min), and diluted to 1 : 50 in DMEM medium without fetal bovine serum (FBS). The diluted samples were inoculated on the cell line with confluence 70-80% and gently agitated at 37 °C for 1 h. Consequently, DMEM with 2% FBS was added and the infected cells were monitored for the appearance of cytopathic effect. From the Day 4 (appearance of visible cytopathic effect), the supernatant was tested for the presence ofZIKV/DENV RNA and changes in the viral load.

The plasma samples were also tit rated by viral plaque assay as described previously[9]. In brief, Vero E6 cells were plated, in 24-well plates, at concentration of 2x105 cells/500 μl/well, and infected when reached 100% confluence. Subsequently, the medium was removed, cells were washed with PBS, and incubated with serial dilutions of the positive for DENV/ZIKV sample conditioned in FBS-free DMEM. After 60 min of incubation under gentle agitation, the medium was removed, cells were washed with PBS, and cultured in 1.5% carboxy-methyl- cellulose supplemented with 1% FBS DMEM. After 5-6 days of culture, the cells were fixed overnight with 4% formaldehyde and stained with 1% crystal violet for 1 h. Viral plaques were counted and the viral titre was calculated and expressed as plaque forming unit per milliliter (PFU/ml).

During the testing of the deferred blood units for DENV and ZIKV RNA, the presence ofZIKV and DENV RNA was detected concomitantly in one of the donations. The obtained blood unit belonged to a 32 yr-old female, repeat blood donor of Afro-Brazilian descent who was O+ and RH positive. She was approved on all blood donation interviews on January 11, 2016 and was considered eligible for donation. However, three days later she complaint the Blood Center of Ribeirão Preto (during the quarantine of the obtained blood) about clinical symptoms relating to a febrile condition accompanied by conjunctivitis and itching confluent exanthema covering the whole body including the scalp. Hence, the donation was discarded and further transfusion-transmission of both arboviruses was prevented.

The quantification of the viral RNA in the blood unit demonstrated 13,064,384 copies/ml for ZIKV and 5,173,561.02 copies/ml for DENV RNA. Further, molecular serotyping of DENV demonstrated that the detected strain belongs to DENV-4 serotype. Cytopathic effect, presented as extensive cell death accompanied by detachment and opaque view of the nucleus of the infected cells (for cytopathic changes see [Figure 1]a and [Figure 1]b was observed from the Day 5 after plasma inoculation. Cell supernatant was collected and tested for ZIKV/ DENV RNA. The obtained molecular results were also consistent with the presence of RNA both arboviruses in cell culture with high viral loads. To further confirm the viral infectivity, plaque assay was performed with viral titration demonstrating a titre of 1.1x106 PFU/ml at the Day 12; and 2.75x106 PFU/ml at Day 16, without virus differentiation [Figure 2]a and [Figure 2]b.
Figure 1: Phase-contrast microscopic images of VERO E6 cell culture infected by zika and dengue viruses obtained from a plasma of a pre-symptomatic blood donor—(a) Negative control represented by mock-infected VERO E6 cells with plasma of a dengue and zika negative blood donor; and (b) VERO E6 cells infected with dengue and zika viruses plasma (positive) with pronounced cytopathic effect represented by cell detachment and opaque view of the infected cells (100 x magnification).

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Figure 2: Plaque formation assay for determination of the viral titre: (a) Twelve days of cultivation with viral titre of 1.1 x 106 PFUs/ ml; and (b) Viral plaques observed at Day 16 of cultivation with a titre of 2.75 x 106 PFUs/ml.

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An important issue was presence of ZIKV and DENV in urinary excretion of the infected blood donor. However, as this study was retrospective and it tested only blood donations prepared for transfusion, it was impossible to test the urine of the donor for ZIKV and DENV RNA.

This is a pioneer report, which demonstrates the simultaneous presence of infectious ZIKV and DENV-4 in plasma from a pre-symptomatic blood donor. Such issue raises concerns about the simultaneous transfusiontransmission of both the arboviruses, especially during outbreaks. The importance of this study is not uniquely restricted to tropical Latin American countries; many Asian, African and European countries beyond DENV and/or ZIKV circulation, demonstrate urban outbreaks of Yel-low fever virus[13], WNV[14],[15] and CHIKV1[16],[17], therefore the transfusion-transmission of arboviral co-infections gains a worldwide significance.

It is not completely clear if Aedes mosquito vectors can transmit simultaneously ZIKV and DENV infections in humans. However, this study demonstrates that asymptomatic blood donors can become double viraemic with infectious virus and consequently contaminate hae- moderivatives. Moreover, the blood donation was performed in January 2016 which coincided with the highest number of reported cases of DENV and ZIKV, and density of the transmitting vectors due to high precipitation volume which might have favoured the presence of mixed arbovirus infection in the respective blood donor.

In the present case, the contaminated blood unit was deferred from donation due to donor’s post-donation information and transfusion-transmission was thus prevented. However, this coinfection was detected only occasionally, due to the availability of post-donation information. There is no routine testing of DENV and ZIKV RNA in the Brazilian blood banks despite of the endemicity and frequent outbreaks of both the viruses. Due to the large proportions of the outbreak in Ribeirão Preto city region and the absence of routine DENV/ZIKV nucleic acid test (NAT) diagnosis in Brazil, it is possible that transfusiontransmission of these arboviruses may have occurred.

This short report provides a valuable information on the concomitant presence of DENV/ZIKV viremia in asymptomatic blood donor and a possible transmission of both the viruses by blood transfusion. However, given the fact, that concomitant circulation of other arboviruses may also exist in addition to DENV and ZIKV, like CHIKV and WNV, there is a possibility of an expanding range of the coinfections and their potential impact on the blood transfusion safety.

The obtained results open the question for introduction of possible measures to prevent transfusion-transmission of arboviruses. A very efficient measure is to halt the blood donation in an area which presents an on-going arboviral outbreak, which in this case appears impossible since the examined region cannot depend on an external blood collection and supplies very large area with hae- moderivatives (almost the whole western part of the Säo Paulo state). Aparticularly interesting measure is an application of a routine nucleic acids testing for DENV, ZIKV and CHIKV RNA in the blood banks, however, such a measure must be carefully balanced against other health priorities of Brazil due to its high cost, unpredictable emergence of the outbreaks and difficulty to differentiate vectors from transfusion-transmission of the arboviruses[18]. Moreover, further studies are necessary to evaluate the impact of the simultaneous presence of DENV/ZIKV in blood units on the transfusion safety.

Conflict of interest

The authors declare no conflict of interest.

Ethical statement: Not applicable.

  Acknowledgements Top

The authors are grateful to Sandra Navarro Bresciani for the graphical designs. This study was supported by Fundação de Amparo e Pesquisa do Estado de São Paulo-FAPESP, Brazil (Grant No. 2009/16623-1, CTC- 2013/081352), Conselho Nacional do Desenvolvim- ento Científico e Tecnológico, Brazil (INCTC-573.754/ 2008-0 and INCTC-2008/57.877-3), and FINEP, Brazil (245/2016).

  References Top

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