Realtime ecosystem screening for viruses

1-Despite being critical to prevent major outbreaks of emerging viruses, there is limited wildlife testing capacities in major outbreak hot spot.

2-We are proposing to develop a portable High-Throughput Sequencing facility at the heart of the Gabonese rain-forest to process the countless wildlife samples daily collected by the IRET, and use the algorithm developed by EnViro to identify viral threats before they reach human populations.

3- Zoonotic viruses represent 60% of all the human viruses. Monitoring viral dynamics in wildlife gives early signals of an outbreak, allowing to limit/prevent it. While our project focuses on the Gabonese rain-forest, there are other hot-spots all around the world (from California to Thailand, from Siberia to Chile). Current research relies extensively on a handful of labs in high-income countries. With this project, we are developing a portable strategy for viral monitoring by local teams worldwide.

Viral monitoring, especially for deadly emerging zoonotic viruses relies on complex assays, most of them being only available at BSL-4 laboratories. Due to the high cost of operation of such facilities, they are all located in high income countries. There is no such lab on the African continent, nor in South East Asia. The only lab in the Southern hemisphere is in Australia.

At the same time, viral monitoring also requires an extensive collection of wildlife sample and relies almost exclusively on local teams of conservationists and ecologists. Sending these samples in the high end laboratories faces many administrative and logistic hurdles, and sample quality decrease with time and travel.

These samples need to be studied on site. The main issue here is that any attempt to monitor viral dynamics would require an extensive network of high-end laboratories. While some project have tried this approach, they are still limited by the number of location and the high cost of operation.

Our solution is to reverse the current logic: bring the lab to the sample, not the sample to the lab. We developed a protocol that allow the mass sequencing of wildlife samples at the collection site.

High-Throughput Sequencing (HTS) is a technology that can sequence massively genomes present in a sample. Used on wildlife samples, the results give a precise image of the virus circulating in their ecosystem. This includes viruses that can infect humans and start outbreaks. 

But viruses have tiny genome, and therefore are often lost in the overwhelming bacterial and large animal genomes. The algorithm developed by EnViro International Laboratories finds the needle in the haystack. With most of the bacterial and animal sequences discarded, a regular laptop can now process the viral genome sequence. This algorithm can also detect new, previously unknown virus using a genomic signature approach.

Altogether this approach allows to have the collection, sequencing, and analysis all happening at the same place, at the same time giving a real time flow of curated information that can then be shared through internet with local (communities, hospitals, …) and global (WHO,…) stakeholders in order to prevent and respond to outbreaks.

It also provide valuable information on the health of the ecosystem and the viral diversity in the wildlife.

We are primarily working with people at the interface between rainforest and rural area in south east Gabon. Being on constant contact with wildlife and domesticated animal (that can serve as relay), these population are at high risk for zoonotic spillover.

If not adressed early, a spillover can quickly become outbreak, and in a worst case scenario, a pandemic. This is what we have seen recently with COVID-19, and a sad repeat of the emergence of  the HIV pandemic in the 70s.

Our solution provide critical information on virus circulation in the forest and its outskirt, allowing outbreak preparedness and mitigation at the local level. In Malaysia, this approach have been used to prevent Nipah Virus outbreak, and have been sucessful for the 30 years. Our project will include not one, but a whole group of emerging virus including, Ebola, Nipah, Influenza, Coronaviruses, and many other, as well as yet unknown virus with potential for spillover in humans.

Our project enhances diseases surveillance system, providing a protocol to process sample at the collection site, rather than relying on distant laboratory. It allows the analysis of a vast amount of sample through HTS, allowing for surveillance of viral dynamics at the ecosystem level. Importantly, it can be scale up world wide and is not specific to our location.  

Focusing on emergence hot-spot like the Gabonese rainforest and its interface with cultivated rural area allows preventive and mitigation measure. It is always easier and cheaper, more feasible to contain an outbreak in its early days.

This is the first-time that the High-througput sequencing will be applied on-site in the heart of the Gabonese equatorial forest. The strategy of bringing the lab to the sample will allow the screening of pathogens at ecosystem level and shorten the time for analysis and improve the quality of data. The realtime ecosystem screening for pathogens is definitely the key for preventing from pathogen spillover from wildlife into human population.

The High-througput sequencing will be applied using the device MinION supplied by Oxford Nanoppore Technologies. MinION is a portable, real-time device for DNA and RNA sequencing. It allows to:

• Direct sequencing of native DNA/RNA, or samples that have been amplified with PCR/other methods
• REAL Real-time
• No capital cost required
• Read any length of DNA/RNA - short to ultra-long
• Scalable to portable or desktop
• Simple & rapid, or automated, library prep
• High yields for large genomes

A large number of scientists,from large centralised labs to smaller
decentralised ones, are using nanopore sequencing to support rapid
sharing of SARS-CoV-2 sequence data. Rapid data sharing has been key to
the public health response, and researchers all over the world have been
fast to share the genomes they have sequenced on public databases such
as GISAID, GenBank or elsewhere.As numbers of cases increased,
laboratories have built capacity to sequence larger numbers of the
genomes, typically progressing from using the MinION device to the
GridION device for sequencing. Thousands of genomes sequenced using
nanopore technology are now available in public databases.

Activities

1-Ecosystem sampling for virus detection (arthopod vector collection, non invasive sampling of wildlife)

2-High-throughput sequencing and analysis of viral sequences diversity

3- data sharing with locals (community, stakeholders)

Outputs
1- Arthropod vectors and wildlife species are identified in the study site

2- DNA and RNA viruses infecting wildlife are sequenced and identified.

3- The local stakeholders are informed about the viruses circulating in the area

Short term outcomes

The virus dynamics is known in the study site

Long term outcomes

The technology is expanded to broader sites for a the surveillance of emerging infectious diseases.

The knowledge of known and unknown viruses infecting wildlife in Moukalaba-Doudou National Park will promote the conservation of these animals in the sense it allows preventive actions and on the other hand could help preventing from pathogen spill over into human population. At first the local communities inhabiting the interface human/animals (boundaries forest/village) and involved in activities in the forest. But also researchers, ecologists, conservationists involved in wildlife related activites.Finally, the general population. The people served by the project will increase will increase gradually.

The main goal in the next five years is to be able to monitor the ecosystem for viruses on site using HTS. Once the technology is set, we would like to expand it at the country level and why not at the regional level in Congo Basin.

The main limitation is funding.

We will continue applying for grants in order to implement this project.

My organization is a research institution (The Research Institute in Tropical Ecology(Gabon)) and the solution is part of a larger research project of wildlife health monitoring in Moukalaba-Doudou National Park, Gabon, in collaboration with EnViro International Laboratories, an international NGO.

The project Reat-time monitoring of pathgens involves 3 full time researchers and 5 field workers.
Eventually, we could include some master students on the course of the project depending on the budget.

The project is led by Chimene NZE NKOGUE, PhD in Veterinary Science, IRET Researcher. She will coordinate project activities, data collection and analysis and report writing. She will design and perform virus screening experiments. Dr NZE NKOGUE is a field virologist with over 5 years of experience working in viruses infecting wildlife. From the study of the reservoir of Marburg virus in bats, to the diversity of Adenoviruses and bocaparvoviruses in gorillas, Dr NZE NKOGUE had gained a good expertise in performing scientific research in remote areas together with local people and foreign collaborators. As a researcher, she is interested in virus diversity and evolution in primates. She has described a new genotype of Primate bocaparvovirus in infant gorillas of Moukalaba-Doudou National Park (Gabon). The named Gorilla bocaparvovirus 2 is the first non-human primate bocaparvovirus member of the Primate bocaparvovirus 2. Her lab skills include basic molecular biology, virology, serology, and data analysis (sequences, phylogeny, etc). Her language skills (fluent in english, french, japanese, and spanish) associated to her sociality increase her hability to manage people and adapt in new environment and conditions. The NGS setting will be operated by Olivier PERNET, PhD, UCLA Virology Instructor. He will train IRET researchers on the use of High Throughput Sequencing  and President of EnViro International Laboratories. He will participate to the sequences and data analysis as well. He will also provide expertise in molecular virology, emerging infectious diseases, and project management. Dr PERNET is teaching Molecular Biology and Virology  at UCLA.

Currently we partner with EnViro International Laboratories, an international  non-profit organization led by Dr Olivier Pernet.

Dr PERNET is teaching Molecular Biology and Virology  at UCLA. As a researcher, his work is focused on 2 topics: 1-emerging infectious diseases and 2-HIV cure.  In 2014, he has coordinated the project that documented the first case of human Henipavirus in Cameroon. His lab skills include basic molecular biology (sequencing, PCR, cloning...), virology (BSL-4 work, infection, viral production and titration, development of recombinant system and pseudotyping), biochemistry, serology, and data analysis (sequences, phylogeny, etc). He is also supervising students (mostly undergraduate students, but also graduate students, and post-docs).  Dr. Pernet is also involved in the COVID-19 response at the University of Southern California (USC), and recently developed a saliva-based assay for SARS-CoV-2 detection. Finally, he is developing new projects related to virus screening in endangered wildlife.

Dr Pernet will train researcher of IRET in using the High-througput sequencing.

I believe this project will provide reliable data on viruses circulating in wildlife in equatorial Gabonese forest. These informations shared with all the stakeholders will contribute to mitigate the impact of emerging infectious diseases on endangered wildlife and on humans at the interface with animals.

We are a team of researchers and we want to set up a long term study which will need monitoring and evaluation skills.

Currently we are partners with EnViro International Laboratories, an international NGO which is expert in developping assays for emerging virus screening.

We will be happy to partner with Nanopore or any other institution interested in our project.