From Sequencing to Awareness

Pizza Seminar • 8 October 2019

C I Mendes

Mário Ramirez Lab

The dengue virus

| Transmission & Disease

The dengue virus

doi:10.1038/nrmicro1690

| Transmission & Disease

Sequential infection increases the risk of a severe form of the infection - dengue hemorrhagic fever.

The dengue virus

Geographical distribution of dengue cases reported worldwide, July to September 2019

17 September 2019 - laboratory-confirmed autochthonous case of dengue in Barcelona, Spain

| Transmission & Disease

The dengue virus

| The virus

Dengue hemorrhagic fever:

4 different dengue serotypes, with many genotypes

After ‘primary’ infection with one serotype, ‘secondary’ infections by one or more of the other serotypes can precipitate ‘antibody dependant enhancement’ (ADE)

infection with one type gives little immune protection against the other types

The dengue virus

| The virus

DENV-1 - genotypes I-V

DENV-2 - genotypes Asian I, Asian II, Cosmopolitan, American, Asian/American & Sylvatic

DENV-3 - genotypes I-V

DENV-4 - genotypes I - III & Sylvatic

https://doi.org/10.1371/journal.pntd.0001876.g002

https://doi.org/10.1371/journal.pntd.0000757

Thailand

Viet Nam

The dengue virus

DENV: (+)ssRNA (~11Kb; 1 ORF)

The single polyprotein encodes:

Structural Proteins:
- C – capsid
- prM – pre-membrane
- M - membrane
- E - envelope
Non-Structural Proteins:
- NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5

| The virus

The dengue virus

| The virus

Empower the use of HTS to monitor the dissemination of the disease

RNA Extraction

PCR
Amplification

HTS Sequencing

➔

How do the different genotypes model transmission and infection?

| The Workflow

Ready to use but customizable
Scalable
Reproducible
Stand-alone (confidential data)
Easy to explore and share results

Requirements

A solution

A ready-to-use, reproducible, scalable workflow for the identification, serotyping, genotyping and phylogenetic analysis of DENV from HTS data

| The Workflow

DENV Identification

3830 complete DENV genomes from the NIAID Virus Pathogen Database and Analysis Resource (ViPR)
complete genome sequence
human host (exception of DENV-1 III, monkey)
collection year (1950-2018)

In Silico Typing:

161 representative sequences of all sero and genotypes.

| The Workflow

106 plasma samples (BioProject PRJNA394021)

| Test datasets

Shotgun Metagenomics dataset:

9 plasma samples
13 serum samples
1 spiked sample with the 4 serotypes
Positive and Negative controls

Targeted Metagenomics dataset:

| Test datasets

| Where to find it?

https://doi.org/10.1101/628073

Git, Nextflow (java) and a container engine (Docker, singularity, shifter...).

apt-get install git

curl -s https://get.nextflow.io | bash

apt-install docker-ce

Clone (or run remotely)

git clone https://github.com/B-UMMI/DEN-IM.git

This work was funded by: FCT - "Fundação para a Ciência e a Tecnologia" (SFRH/BD/129483/2017) and the Abel Tasman Talent Program grant from the UMCG, University of Groningen, Groningen, The Netherland

Special thanks to E Lizarazo, M P Machado, D N Silva, A Tami, M Ramirez, N Couto, J W A Rossen, J A Carriço