Evaluation of Viral Replication by Tonate Virus (TONV) and Zika Virus (ZIKV), Within an ex Vivo Trophoblast and Placental Model

NCT05589012 | Unknown

• 1 other identifier: 2020-A01318-31
• Study Type: observational
• Target: 8
• Locations: 0 countries
• Sites: N/A

Brief Summary

Prospective, non-interventional study carried out after culturing placental trophoblastic tissue ex vivo and infection with Zika and Tonate

Conditions

Zika Virus Infection

Keywords

trophoblast; placenta

Outcome Measures

Primary Outcomes (1)

• To assess the replication potential of TONV and ZIKV after placenta infection ex vivo.

  Determine the viral load (RT-qtPCR) of TONV and ZIKV after placenta infection, ex vivo in RNA copies/mL

  inclusion

Secondary Outcomes (10)

• Validate the ex vivo placental model for TONV.

  Inclusion

• Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).

  first trimester

• Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).

  first trimester

• Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).

  first trimester

• Compare viral infection of the placenta at different terms of pregnancy (first trimester and term).

  term

Study Arms (1)

Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies.

The placentas will be collected following a birth by caesarean section after oral and written information by delivery of the information note for women who have had a normal singleton pregnancy at term. Similarly, trophoblasts are collected following endo-uterine aspiration in the context of voluntary termination of pregnancy. * Cell line and virus * Infection of human placental culture explants * Determination of viral load in tissues by qRT-PCR

Biological: Cell line and virus; Biological: Infection of human placental culture explants; Biological: Determination of viral load in tissues by qRT-PCR

Interventions

Cell line and virus BIOLOGICAL

C6/36 cells will be cultured in L15 culture medium (Leibovitz's L-15 medium), supplemented with 5% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin at 28°C in a humidified atmosphere containing 5% C02. ZIKV and TONV will be expanded and titrated using C6/36 cells. The viral stock will be aliquoted in 100 µL aliquots and stored at -80°C.

Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies.

Infection of human placental culture explants BIOLOGICAL

Placental tissues will be handled within one hour of delivery. The chorionic villi will be dissected into 5mm sections and the tissues washed abundantly, minimum 3 times with a standard culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% L-Glutamine and 1% Penicillin/Streptomycin) to remove maternal blood, membranes and blood clots. Collagen gel sponges will be placed in 6-well plate wells containing 3mL of culture medium (RPMI-1640 supplemented with 15% heat-inactivated fetal bovine serum (FCS), 1% Penicillin/streptomycin, 0 1% Gentamycin, 1% Amphotericin B, 1% L-Glutamine, 1% non-essential amino acid, 1% sodium pyruvate) per well. Chorionic villi will be dissected into 5mm sections and placed on top of collagen gel sponges at the interface between culture medium and air.

Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies.

Determination of viral load in tissues by qRT-PCR BIOLOGICAL

The tissues will be lysed by mechanical disruption in a lysis buffer + 4%TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) (Machery-Nagel ref: 740395.107) with the Precellys system. The lysed tissues will be diluted in 100 mg/mL of Macherey-Nagel lysis buffer, aliquoted and stored at -80°C until extraction. Total RNA will be extracted in duplicate using the Nucleospin 96 RNA Core kit (Macherey Nagel ref: 740466.4), following the supplier's instructions. Standard samples, controls and ZIKV and TONV viral RNA will be extracted and tested in parallel under the same conditions. The extracted RNA will be subjected to reverse transcriptase, using the Superscript One-Step RT-qPCR kit (Invitrogen ref: 11732088) according to the manufacturer's recommendations, with a probe and primer specific for ZIKV and TONV.

Human placentas and trophoblasts (biological waste) from uncomplicated pregnancies.

Eligibility Criteria

• Age: 18 Years+
• Sex: female
• Healthy Volunteers: No
• Age Groups: Adult (18-64), Older Adult (65+)
• Sampling Method: Non-Probability Sample

Study Population

Pregnant women

You may qualify if:

• Patient over 18 years old
• Singleton pregnancy of normal course, at term, birth by caesarean section OR
• Trophoblast from Voluntary Termination of Pregnancy (IVG), after endo-uterine aspiration

You may not qualify if:

• Need for pathological, genetic or bacteriological examination of the placenta
• Multiple pregnancy
• HBV+ (Hepatitis B positive), HCV+ (Hepatitis C positive) , known CMV seroconversion during pregnancy (CytoMégaloVirus)
• Immunosuppression (drugs, corticosteroids, etc.)
• Diabetes
• Pre eclampsia
• Intrauterine growth retardation (IUGR),
• Vascular or placental pathology.
• Refusal to participate
• Patient under guardianship / curatorship / security measure
• Patient under AME (state medical aid)

Sponsors & Collaborators

• Assistance Publique - Hôpitaux de Paris: lead

MeSH Terms

Conditions

Zika Virus Infection

Condition Hierarchy (Ancestors)

Mosquito-Borne Diseases; Vector Borne Diseases; Infections; Arbovirus Infections; Virus Diseases; Flavivirus Infections; Flaviviridae Infections; RNA Virus Infections

Central Study Contacts

PICONE Olivier

CONTACT

01 47 60 66 36; olivier.picone@aphp.fr

DECHANET Aline

CONTACT

aline.dechanet@aphp.fr

Study Design

• Study Type: observational
• Observational Model: COHORT
• Time Perspective: PROSPECTIVE
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: August 22, 2022
• First Posted: October 21, 2022
• Study Start: November 1, 2023
• Primary Completion: April 1, 2024
• Study Completion: April 1, 2024
• Last Updated: October 21, 2022

Record last verified: 2022-08

Data Sharing

• IPD Sharing: Will not share