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# Absence of Zika virus among pregnant women in Vietnam in 2008

Y.-C. Chiu<fnref n="1" />†, D. Baud<fnref n="1" />,<fnref n="2" />†, A. Fahmi<fnref n="3" />,<fnref n="4" />,<fnref n="5" />, B. Zumkehr<fnref n="3" />,<fnref n="4" />, M. Vouga<fnref n="1" />, L. Pomar<fnref n="1" />, D. Musso<fnref n="6" />,<fnref n="7" />, B. C. Thuong<fnref n="8" />, M.P. Alves<fnref n="3" />,<fnref n="4" />,<fnref n="9" /> and M. Stojanov<fnref n="1" />,<fnref n="2" />*

**Abstract**

**Background** Despite being first identified in 1947, Zika virus-related outbreaks were first described starting from 2007 culminating with the 2015 Latin American outbreak. Hypotheses indicate that the virus has been circulating in Asia for decades, but reports are scarce.

**Methods** We performed serological analysis and screened placental samples isolated in 2008 for the presence of Zika virus from pregnant women in Ho Chi Minh City (Vietnam).

**Results** None of the placental samples was positive for Zika virus. Four serum samples out of 176 (2.3%) specifically inhibited Zika virus, with variable degrees of cross-reactivity with other flaviviruses. While one of the four samples inhibited only Zika virus, cross-reactivity with other flaviviruses not included in the study could not be ruled out.

**Conclusion** Our results support the conclusion that the virus was not present among pregnant women in the Vietnamese largest city during the initial phases of the epidemic wave.

**Keywords** Zika virus, Placenta, Infection, Serology, Vietnam

**Introduction**

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused epidemics in Micronesia (2007), French Polynesia (2013) and, more recently in Latin America (2015) [1, 2]. Compared to other co-circulating arboviruses, including Dengue and Chikungunya viruses, ZIKV is associated with severe congenital anomalies in exposed foetuses such as microcephaly. ZIKV was first isolated in the Zika Forest, Uganda in 1947 [3]. Subsequently, the virus has diverged into two primary lineages, the prototypic African lineage and the Asian lineage. The latter was thought to be circulating on the Asian continent since the early 1950s [4], from which it migrated to the Yap State (Micronesia), causing the first major outbreak in 2017 [5]. ZIKV spread in the Pacific region, causing a second large outbreak in the French Polynesia [6, 7], before reaching South America between 2013 and 2015 [2], where the epidemic was declared a public health emergency of international concern by the WHO (from February to November 2016) [1]. ZIKV further spread to

<footnote>†Y.-C. Chiu and D. Baud equally contributed to the manuscript.</footnote>

<footnote>*Correspondence:
M. Stojanov
milos.stojanov@chuv.ch</footnote>

<footnote n="1">¹ Materno-Fetal and Obstetrics Research Unit, Department
Woman-Mother-Child, Lausanne University Hospital, Lausanne,
Switzerland</footnote>

<footnote n="2">² Faculty of Biology and Medicine, University of Lausanne, Lausanne,
Switzerland</footnote>

<footnote n="3">³ Institute of Virology and Immunology, Bern, Switzerland</footnote>

<footnote n="4">⁴ Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty,
University of Bern, Bern, Switzerland</footnote>

<footnote n="5">⁵ Graduate School for Cellular and Biomedical Sciences, University of Bern,
Bern, Switzerland</footnote>

<footnote n="6">⁶ Aix Marseille Université, Institut de Recherche Pour Le Développement
(IRD), Assistance Publique-Hôpitaux de Marseille, Service de Santé Des
Armées, Vecteurs-Infections Tropicales et Méditerranéennes (VITROME),
and Institut Hospitalo-Universitaire Méditerranée Infection, Marseille,
France</footnote>

<footnote n="7">⁷ Laboratoire Eurofins Labazur Guyane, Eurofins, Cayenne, French Guiana</footnote>

<footnote n="8">⁸ Tu Du Hospital, District 1, Ho Chi Minh City, Vietnam</footnote>

<footnote n="9">⁹ Multidisciplinary Center for Infectious Diseases (MCID), University
of Bern, Bern, Switzerland</footnote>
Central America, the Caribbean and reached the south-
ern states of the USA [8, 9].

Sequence analyses confirmed that ZIKV isolates from
the recent outbreaks were phylogenetically related and
belonged to the Asian lineage [1]. Little is known regard-
ing ZIKV circulation prior to the recent outbreaks. Sero-
logical evidence indicates that the virus was present on
the Asian continent in the 1950s [10]. In Vietnam, ZIKV
was first documented in 1954 [11] and this remained the
only report until 2015 [12]. An outbreak was reported
at the end of 2016, mainly in the southern part of the
country, with 145 confirmed ZIKV cases [13]. Never-
theless, the effects of Zika presence in Vietnam remain
largely unrecognised due to the lack of surveillance. Due
to increased awareness from the recent outbreaks and
associated severe adverse outcomes, several studies have
proven its circulation in Asia [14–16].

In the present study, we intended to investigate the
presence of ZIKV among pregnant women in Ho Chi
Minh City (Vietnam) in 2008, the year following the first
major outbreak in the Yap State (Micronesia). We utilized
serum and placental samples obtained from pregnant
women who delivered in 2008 [17]. The materno-fetal
interface of the placenta plays a crucial role in the trans-
mission of ZIKV to the fetus [18] and infection of placen-
tal tissue was previously reported in infected pregnant
women, animal models of infection and in vitro assays
[19–22].

**Methods**

Placental and serum samples

Placental and serum samples were collected at the Tu
Du Hospital, Ho Chi Minh City (Vietnam) and stored at
-80 °C upon reception [23]. Only samples for which the
placental tissue was available were included in the pre-
sent study, for a total of 176 samples. As for the original
study, samples analysed here were collected from January
to December 2008. All patients included in the study gave
their written consent and utilization of the samples was
approved by the local ethical committees of the Tu Du
Hospital, Vietnam (clinical part) and the Lausanne Uni-
versity Hospital, Switzerland (experimental part).

Flavivirus propagation and titration

Japanese encephalitis virus (JEV, CNS769_Laos_2009; GenBank accession number KC196115.1) was kindly provided by Remi Charrel, Aix-Marseille Université, France. West Nile virus (WNV, NY99-35, GenBank accession number DQ211652.1) was kindly provided by Martin Groschup, Friedrich-Loeffler-Institute, Germany. DENV-2 was kindly provided by Dr. Katja Fink, Singapore Immunology Network, Singapore). The low passage clinical isolate of Asian lineage ZIKV (PRVABC59,

GenBank accession number KX377337) was obtained
from Public Health England (PHE). Yellow fever virus
(YFV, UVE/YFV/UNK/XX/Vaccinal strain 17D; Gen-
Bank accession number EU074025.1) was obtained at the
European Virus Archive Global (EVAg). All flaviviruses
were propagated in Vero cells (CCL-81, ATCC) cultured
in DMEM (Gibco—Thermo Fisher Scientific, Reinach,
Switzerland) supplemented with 10% FBS (Gibco) at
37 °C, 5% CO₂. Flavivirus titers were determined in Vero
cells using an immunoperoxidase assay using the anti-
flavivirus group antigen antibody 4G2 (clone D1-4G2-
4–15, ATCC, HB-112). Virus titers were calculated and
expressed as 50% tissue culture infective dose per ml
(TCID₅₀/ml) using the Reed and Muench method [24].

Serum neutralization assay

Serial twofold dilutions of heat-inactivated sera (30 min, 56 °C) were prepared in serum-free DMEM medium, with a starting dilution of 1:4 for ZIKV and 1:8 for JEV, WNV and YFV. Serial serum dilutions were incubated at 37 °C for 60 min with an equal volume of the corresponding virus to provide 100 PFU per 100 µl (1000 PFU per 100 µl in the case of DENV2). Serum-virus mixture was added to monolayers of Vero cells in 96-well flat-bottom tissue culture-treated microtiter plates (TPP) and incubated at 37 °C with 5% CO₂ for two hours, after which DMEM containing 2% FBS 1% PenStrep (Gibco) was added to the wells. After 72 h, the culture plates were washed with 300 µl/well of cold PBS (Gibco) and fixed for 20 min at RT with 4% PFA. Then, the plates were washed once with 300 µl/well of 0.1% saponin (AppliChem GmbH, Darmstadt, Germany). A 100 µl/well of primary antibody mix was added and incubated for 30 min at 37 °C. The plates were washed twice with 200 µl/well of 0.1% saponin. Secondary antibody mix (rabbit anti-mouse HRP, Dako) was prepared in 0.3% saponin with a dilution factor of 1:250. A 100 µl/well of secondary antibody was added for 30 min at 37 °C in the dark. The plates were washed twice with 200 µl/well of 0.1% saponin and 80 µl/well of AEC substrate solution was added and incubated for 15 min at RT in the dark. When the plaques appeared with the desired coloration, the reaction was stopped with 100 µl/well of tap water. The microtiter plates were scanned with an ImmunoSpot analyzer (Cellular Technology Limited).

**Serological analysis**

Serology for ZIKV was performed with the “Anti-Zika
Virus ELISA (IgG)” kit (EI 2668–9601 G, EUROIM-
MUN Schweiz AG, Luzern, Switzerland), according to
the manufacturer’s specifications. Confirmation of posi-
tive samples was performed using a custom designed fla-
vivirus mosaic Indirect Immunofluorescence Test (IIFT)
(EUROIMMUN Schweiz AG, Luzern, Switzerland), in which cells infected with Zika, Dengue (I-IV), West Nile, Yellow fever and Japanese encephalitis viruses were used to detect their antigens.

## Detection of ZIKV in placental samples

For the analysis of the presence of ZIKV in placental samples, approximately 25 mgs of tissue were homogenized using the Molecular Grinding Resin (G-Biosciences, St. Louis, MO, USA) and subsequently processed with Nucelospin RNA II extraction kit for total RNA extraction according to the manufacturer's protocol. Complementary DNA (cDNA) was obtained with the SuperScript II Reverse Transcriptase kit (Invitrogen Life Technologies, Carlsbad, CA, USA) by using Random Primer Hexamers (Invitrogen Life Technologies) and following the manufacturer's instructions. ZIKV RNA levels were assessed by quantitative RT-PCR using the iTaq Universal Probes Supermix (Bio-Rad, Reinach, Switzerland) and the Rotor Gene 6000 thermocycler (Corbett Research, Sydney, Australia) as previously described [25]. As positive controls, placental homogenates were spiked with different inocula of ZIKV strain PRVABC59.

## Results

The 176 placental samples included in the study were tested for the presence of ZIKV RNA by RT-qPCR. None of the samples resulted positive, while viral RNA was successfully detected in all positive controls which were spiked with viral preparations.

Among the 176 samples included in the study, four (2.3%) were positive for ZIKV serology (ELISA analysis repeated twice for each sample). These samples were further tested for cross-reactivity against other flaviviruses, including Dengue (DENV, serotypes I-IV), West Nile (WNV), Yellow fever (YFV) and Japanese encephalitis (JEV) viruses. As depicted in Table 1, the four samples showed a high degree of cross-reactivity with all other flaviviruses. This can be further observed in Supplementary Fig. 1, with sample 2180 as an example.

To further characterise seropositive samples, we performed the serum neutralization assay (SNA), to assess serum ability to neutralize ZIKV virions. In parallel, SNA was done for DENV, JEV, WNV and YFV to monitor the extent of cross-reactivity sera samples (Table 2).

Serum sample 2054 was shown to specifically inhibit the infection by ZIKV, with no effect on the other flaviviruses included in the analysis (only one DENV serotype was tested). Cross-reactivity was observed for sample 2028, with the highest value for JEV. The remaining two serum samples (2087 and 2180) were not able to neutralize ZIKV.

## Discussion

Little is known about ZIKV circulation in Asia prior the recent major outbreak in Latin America. Samples used in this study were collected throughout the whole year, thus avoiding issues related with the seasonality of the presence of *Aedes* vectors in southern Vietnam. As expected, high levels of cross-reactivity between flaviviruses, which are endemic in Vietnam, were observed in our samples. In our sera collection from 176 pregnant women, samples from four patients (2.3%) were able to neutralize ZIKV in the serum neutralization assay. All the four pregnancies were uneventful. Interestingly, one sample did not show cross-reactivity with the flaviviruses tested here, although this could not exclude cross-reactivity with other flaviviruses, especially other

<table><caption>**Table 2** – Serum neutralization assay performed with sera of seropositive samples</caption><thead><tr><th>Sample</th><th>ZIKV</th><th>DENV</th><th>JEV</th><th>WNV</th><th>YFV</th></tr></thead><tbody><tr><td>2028</td><td>1/64</td><td>1/1024</td><td>1/2048</td><td>neg</td><td>neg</td></tr><tr><td>2054</td><td>1/128</td><td>neg</td><td>neg</td><td>neg</td><td>neg</td></tr><tr><td>2087</td><td>neg</td><td>1/64</td><td>1/32</td><td>neg</td><td>neg</td></tr><tr><td>2180</td><td>neg</td><td>1/128</td><td>neg</td><td>neg</td><td>neg</td></tr><tr><td>Control 1<sup>a</sup></td><td>neg</td><td>neg</td><td>neg</td><td>neg</td><td>1/256</td></tr><tr><td>Control 2<sup>b</sup></td><td>neg</td><td>neg</td><td>1/64</td><td>neg</td><td>1/64</td></tr></tbody></table>

<table><caption>**Table 1** – Serological analysis of the four ZIKV-positive samples with other flaviviruses. Analysis cut-offs are shown (Pos)</caption><thead><tr><th></th><th>ZIKV<br>Pos: ≥ 1:100</th><th>DENV-1<br>IgG (Titer 1:)</th><th>DENV-2</th><th>DENV-3</th><th>DENV-4</th><th>YFV</th><th>JEV</th><th>WNV</th></tr></thead><tbody><tr><td>2028</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td><td>≥ 3200</td></tr><tr><td>2054</td><td>3200</td><td>10,000</td><td>10,000</td><td>10,000</td><td>10,000</td><td>1000</td><td>1000</td><td>1000</td></tr><tr><td>2087</td><td>10,000</td><td>32,000</td><td>32,000</td><td>32,000</td><td>32,000</td><td>10,000</td><td>10,000</td><td>10,000</td></tr><tr><td>2180</td><td>1000</td><td>10,000</td><td>10,000</td><td>10,000</td><td>3200</td><td>1000</td><td>1000</td><td>1000</td></tr></tbody></table>

<footnote><sup>a</sup> Serum from vaccinated patient with YFV</footnote>

<footnote><sup>b</sup> Serum from vaccinated patient with YFV, JEV</footnote>
DENV serotypes. While multiple reports indicate the
circulation of ZIKV in Asia since the 1960s, very little
is known about the extent and the characteristics of
Asian clade strains prior to the recent outbreaks. Phy-
logenetic analyses showed that the ZIKV strain present
in Micronesia in 2007 was related to a strain isolated
in 2010 in Southeast Asia (Cambodia) [9]. It would be
therefore of interest to perform analyses on samples
isolated prior 2007 in Southeast Asian countries to gain
more insight on the origin of the epidemic ZIKV clade.
A 2017 retrospective study indicated that ZIKV was
present in Vietnam prior to its identification in 2016
[26], however, the prevalence was very low (0.035%).
Our results should be confirmed using a larger sample
collection. Nevertheless, serological studies are compli-
cated by the co-circulation of other flaviviruses in the
endemic regions such as Vietnam and Southeast Asia
more generally, as indicated by our results.

Importantly, ZIKV RNA could not be detected from
placental samples included in this study, although
transient infection of the placenta has been previously
described in approximately 20% of infected women
[17]. Nevertheless, we could not completely exclude
the presence of the virus in the placental tissue, as only
a fraction of the organ was used for RNA extraction.
Moreover, we could not exclude degradation of viral
RNA due to the relatively long period of storage.

Taken together, our results suggest that ZIKV was
not present among pregnant women in Ho Chi Minh
City (Vietnam) during the first epidemic episodes in the
Pacific.

**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s40794-023-00189-7.

**Additional file 1: Supplementary figure 1.** Cross-reactivity of Zika positive samples with other flaviviruses based on antigen detection in virus-infected cell lines. The results of sample 2180 are shown as an example, by using serum dilution at 1:1000. The mock control consists of mock infected cells.

**Authors' contributions**

Conceptualisation, D.B. and M.S.; methodology, D.B. and M.S.; formal analysis, Y.-C. C., D.B., A.F., B.Z., M.A. and M.S.; Original draft preparation, Y.-C. C., D.B. and M.S.; Review and editing, M.V., L.P., D.M., T.B.C. All authors have read and agreed to the published version of the manuscript.

**Funding**

Open access funding provided by University of Lausanne. This work was supported by the Department of Obstetrics and Gynecology, Lausanne University Hospital, Switzerland and by the SNSF grant numbers 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributed to David Baud. David Baud is also supported by the "Fondation Leenaards" through the "Bourse pour la relève académique", by the "Fondation Divesa" and by the "Loterie Romande".

**Availability of data and materials**

Data will be made available upon request to the authors.

**Declarations**

**Ethics approval and consent to participate**

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of Tu Du Hospital, Vietnam (clinical part) and the Lausanne University Hospital, Switzerland (experimental part).

**Competing interests**

The authors declare no conflict of interest.

Received: 12 October 2022 Accepted: 6 February 2023

Published online: 01 March 2023

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