This work was generously supported by 1R21AI129881-01 (to T.M.C.), start-up funds from the National Emerging Infectious Diseases Laboratories, and the Boston University School of Medicine.

Serum samples used in this study were obtained from human participants of a cohort from Columbia. Sample collection was approved by the internal review board (IRB) at Universidad de Pamplona (Columbia, South America) and Los Potios Hospital8. The samples were anonymously provided and investigators had no access to patient information. The serum samples were checked for the DENV serotype. The samples were further confirmed to neutralize DENV infection in vitro. For control, serum samples f...

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Cross-reactivity of DENV antibodies leading to the ADE of other DENV serotypes has hindered the development of an effective vaccine11. ZIKV belongs to the same family, Flaviviridae, and has a considerable homology with other flaviviruses, especially DENV12. The main target of neutralizing antibodies for both ZIKV and DENV is the envelope protein, which shares a very high structural and quaternary sequence homology between the two viruses13,</sup...

Among the mosquitoes-borne viral diseases, Zika infection is one of the most clinically important1. The infection is caused by the flavivirus ZIKV which, in most cases, uses Aedes aegypti as its primary vector1,2. However, there are studies that have reported Aedes albopictus as a primary vector in some ZIKV outbreaks3. Although the infection is asymptomatic in many cases, the most common symptoms are fever, headache, and muscle pain2. There is no cure or vaccine available for ZIKV infection and the treatment available is mostly supportive. Recent outbreaks of ZIKV in South America led to severe cases of the disease and an approximately 20-fold increase in the neurodevelopmental disorder in fetuses named microcephaly2. As South America is an area endemic to several arboviruses such as DENV and West Nile virus, it is crucial to investigate whether prior immunity to other flavivirus(es) plays a role in the severity of ZIKV infections and disease.
Throughout the ages, viruses have evolved different strategies to increase their chance of infectivity in order to take over the host cell machinery and suppress the antiviral response. One of the most fascinating of all is the use of host pre-immune antibodies by viruses to enhance their replication with the phenomenon ADE4. ADE across all four serotypes of DENV has been well studied and demonstrated to increase viral titers and disease outcome5,6,7. In a previous in vitro study, we have shown significant enhancement of ZIKV replication due to pre-existing DENV immunity in primary human immune cells8. We also demonstrated a relevant in vitro method to quantify the capability of DENV pre-existing antibodies to enhance ZIKV replication in primary cells.
The protocol that we have developed uses human serum samples that are tested for DENV neutralization in TCID-50 or plaque reduction neutralization test (PRNT) assays, along with ZIKV in biologically relevant cells or cells derived from tissues that ZIKV can infect.

<table border="0" cellpadding="0" cellspacing="0" style="width:773px;" width="773">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Fetal Bovine Serum&#160;</td>
			<td style="width:119px;">GEMINI</td>
			<td style="width:119px;">100-106</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">iCycler&#160;</td>
			<td style="width:119px;">BioRad</td>
			<td style="width:119px;">785BR02188</td>
			<td>Model No. CFX96 Optics Module</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microfuge 18 Centrifuge</td>
			<td style="width:119px;">Beckman Coulter&#160;</td>
			<td style="width:119px;">367160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Nanodrop-1000</td>
			<td style="width:119px;">Thermoscientific&#160;</td>
			<td style="width:119px;">1072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Quantifast SYBR-One step RT-PCR kit&#160;</td>
			<td style="width:119px;">Qiagen&#160;</td>
			<td style="width:119px;">204154</td>
			<td>Used for 1 step RT-qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">RNeasy RNA Isolation Kit&#160;</td>
			<td style="width:119px;">Qiagen&#160;</td>
			<td style="width:119px;">74106</td>
			<td>Used for RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">RPMI-medium&#160;</td>
			<td style="width:119px;">Gibco</td>
			<td style="width:119px;">11875093</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of antibody-dependent enhancement of the zika virus in primary human cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

In Figure 1, there is a step-by-step diagrammatic illustration of all the steps involved to carry out the ADE protocol. It is a schematic diagram showing the whole procedure of ADE of ZIKV due to pre-existing immunity to DENV. Figure 2 shows how human serum samples were categorized into three different groups: DENV infection-confirmed samples are referred to as the DENV-infected group, DENV antibody-confirmed samples are referred...

Watch this Scientific Journal Video about Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells at JoVE.com

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

This method can be used to evaluate the effects of pre-existing immunity against dengue virus on Zika virus infection, using actual human patient serum and primary human immune cells. The use of actual human patient serum samples and human primary cells provide deeper insight into the antibody dependent enhancement of Zika virus by pre-immune sera and antibodies. This method provides insight into the ability of antibodies within a patient's blood to enhance a virus infection in other human cells.It is important to ensure that the viral infection levels are high enough for detection but low enough not to overwhelm the cells or to interfere with the data interpretation. Begin by seeding three times ten to the fourth cells in 100 microliters of cell culture medium per well in a sterile, flat bottom 96-well plate. When all the cells have been plated, place the plate in a 37 degree Celsius and 5%carbon dioxide incubator and make 10 fold serial dilutions of thawed human serum samples in serum free medium.Be sure to keep the serum dilutions and dilution factor constant for all the pre-immune serums before adding the dilutions to the virus solutions and the cells. Next, thaw Zika virus strains stock solution in a 37 degree Celsius water bath for one to two minutes before quickly transferring the stock to ice. Add a 0.1 multiplicity of infection equivalent volume of Zika virus to the serum aliquots and place the plate in the cell culture incubator for one hour to allow the dengue virus antibodies to form complexes with the Zika virions.At the end of the incubation, wash the cells with 100 microliters of PBS per well and add 50 microliters of immune complex to each well. After the two hour incubation, remove the medium and wash the wells twice with 100 microliters of PBS per well to completely remove the immune complexes and any unattached virions. Then feed the cells with 100 microliters per well of cell culture medium supplemented with 10%fetal bovine serum.Then return the cells to the cell culture incubator for 48 hours. Two days after the infection, wash the cells two times with 100 microliters of sterile PBS per well before adding 250 microliters of cell lysis buffer with 10%beta mercaptoethanol to each well. Pipette up and down at least five times with scratching to speed up the lysis process and transfer the cell lysates to sterile labeled 1.5 milliliter tubes.Add an equal volume of 70%ethanol to each tube and pipette the lysate solution up and down four to five times. When the lysate suspensions are clear, transfer each solution into labeled silica-based columns in two milliliter collection tubes for centrifugation. Discard the flow-through keeping the columns in the same collection tubes and add 700 microliters of Wash Buffer 1 to each column for a second centrifugation.After discarding the flow-through, rinse the columns two more times with 500 microliters of Wash Buffer two per wash. After the second wash, transfer the columns to new two milliliter collection tubes for another centrifugation. Add 30 microliters of 42 degree Celsius RNase-Free water to the center of each column for centrifugation and recover the eluted RNA.For quantitative real-time polymerase chain reaction or QRTPCR analysis add one microliter of gene specific forward and reverse primers from 10 micromolar stocks along with 0.25 microliters of reverse transcriptase mix per reaction. Note that primers which recognize Zika virus genetic material will detect viral genes and allow for infection quantification. Next, add 12.5 microliters of SYBR Green Mix followed by 25 microliters of water to each reaction adding 15 microliters of Master Mix in each well of 96-well QPCR plate.When using a Master Mix, add 100 nanograms of the RNA sample to the mix and pipette the Master Mix into individual wells of the QRTPCR plate. Note that samples should be run in triplicate on the same QRTPCR plate. Then run the samples on a quantitative PCR machine according to the parameters outlined in the table.Clicking the melt curve tab in the system software to monitor the melt curve. The melt curve will show single peak in all of the samples for a particular gene to confirm the presence of only one amplicon. For optimum results, use 0.5 degree Celsius temperature increments between steps and a minimum holding time of 10 seconds in the melt curve protocol.Then click the quantification data tab to obtain a quantitative cycle for each sample and export the data to a spreadsheet for further quantification analysis. The human serum samples can be categorized into three different groups:dengue virus infection confirmed samples, dengue virus antibody confirmed samples, and healthy sera with no dengue virus neutralizing antibodies or RNA. After 48 hours of infection, QRTPCR analysis demonstrates that most sera containing dengue virus serotype one to four antibodies are able to enhance Zika virus replication at different levels.The highest increase in Zika virus titres is found in macrophages treated with sera-containing dengue virus serotype two and four antibodies compared to serotype one and three which show a relatively lower induction of Zika virus. It is important to maintain a sterile environment, to wear the proper personal protection equipment, and to always keep the thawed virus serum samples and QPCR reagents on ice. This protocol can easily be modified to study antibody-dependent enhancement of other flavivirus such as yellow fever virus, dengue virus, or West Nile virus using a panel of patient serum.This technique will be highly useful for carrying out future antibody-dependent enhancement studies in the arbovirus or virology fields. Proper biosafety level two personal protection equipment should be used at all times and all viral waste should be decontaminated in 10%bleach for 30 minutes before disposal.

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Immunology and Infection

8.6K vistas.  Boston University School of Medicine. Este método se puede utilizar para evaluar los efectos de la inmunidad preexistente contra el virus del dengue en la infección por el virus del Zika, utilizando el suero real del paciente humano y las células inmunes humanas primarias. El uso de muestras de suero reales para pacientes humanos y células primarias humanas proporciona una visión más profunda de la mejora dependiente de los anticuerpos del virus del Zika mediante sueros y anticuerpos preinmunes. Este método proporciona inf...