Ecosystem-wide Outbreak Prevention

This project is submitted by EnViro International Laboratories, in collaboration with the Institute for Tropical Ecology Research (ITER) in Gabon.

EnViro International Laboratories is a non-profit organization that studies viral dynamics in emergence hot areas, and provides resources for outbreak preparedness at the local level. The project will be supervised by Dr. Olivier Pernet (EnViro International Laboratories) with Dr. Chimene Nze Nkogue (ITER)

Dr. Pernet is a molecular virologist with 15 years of research experience in emerging zoonotic viruses, with a special attention on bat related diseases (Nipah, Ebola, SARS-CoV-1, SARS-CoV-2,...). Dr. Pernet work focuses on host-virus interactions and their use in biomedical applications (outbreak preparedness, high-throughput serology, ...).

Dr. Nze Nkogue is a field virologist with over 5 years of experience in Moukalaba-Doudou National Park, working in viruses infecting wildlife, from the reservoir of Marburg virus in bats, to Adenoviruses and Bocaparvoviruses in gorillas.

Zoonoses are diseases transmitted between species. They represent half of all the viral human diseases, and include Ebola, Nipah, Rabies, Influenzas, Coronaviruses, ... They are responsible for devastating human outbreaks (Ebola, Coronaviruses), but also affect domestic/farm animals (rabies, influenza), and threaten a wildlife already weakened (Ebola is a major killer of Gorillas)

Unfortunately, with the notable exception of influenza, too little is done to monitor these highly dynamics viruses. This project ambitions to use inexpensive, portable, high-throughput assays in viral emergence hot spots to identify and anticipate viral threats. For this project, we will specifically work with the Institute for Tropical Ecology Research (ITER) in the Moukalaba-Doudou National Park, Gabon, an Ebola emergence hot-spot and also a sanctuary for engendered primates.

The goal is not only to prevent outbreaks in the area, but also to create a model for locally-adapted zoonoses monitoring strategy that can be replicated elsewhere.

The best way to thwart an outbreak is to act early, or even better: to anticipate it. If not addressed early, a spillover can quickly become an outbreak, and in a worst case scenario, a pandemic. This is what we have seen recently with COVID-19. Unfortunately, optimal viral dynamics surveillance requires an ecosystem wide approach that cannot be achieve with current strategies.

Most viral emergence hot spots are located in areas that lacks high level biosafety laboratories (BSL-3/4). Samples often have to cross borders to be safely processed in an appropriate laboratory. There is no BSL-4 laboratory in Africa, South America, or South East Asia, where highly pathogenic viruses abound in wild life. Not only these laboratory are rare and located far from the hot spots, but they are extremely costly to operate and require a specifically trained personnel.

To solve this issue, we have developed surveillance protocols that can be run on-site by local scientists during their routine wildlife monitoring. This simple, safe, inexpensive, high-throughput approach allows for the processing of vast amount of samples, yielding a clear image of viral dynamics in the ecosystem and provide early warning signals for viral outbreak.

Step-1: Using our high-throughput, local-condition adapted, inexpensive antibody based assays to monitor viruses in the samples collected by the ITER in the Moukalaba-Doudou National Park, Gabon. The idea is to find the needle in the haystack, the early variation in viral dynamics that indicate a virus is spreading. While we expect most samples to be negative or baseline, a sudden increase of positive samples in a specific location is a clear signal we should focus on.

Step-2: Once a positive sample has been identified, we are planning to use molecular analysis (viral genetic sequencing) to identify exactly what virus is spreading. This relies on portable high-throughput sequencing systems that are commercially available (Nanopore MinION). The sequencing results will be analysed by a new algorithm developed by EnViro International Laboratories. This algorithms detect viral genetic signatures, and is therefore not specific to one pathogen but a whole family. This will gives us critical information on the various circulating strains and mutants.

Step-3: Real-time feedback to the communities, government, and other stack-holders. The goal is to make our results available to front-line public health professionals for immediate and local response against an emerging virus.

The project will provide data for the communities living in the area of the Moukalaba-Doudou National Park. Moukalaba-Doudou NP is located at the junction of the Ogooué-Maritime, Ngounié, and Nyanga Provinces in Gabon, cumulating 250,000 inhabitants. Specifically, we are primarily serving people living at the interface between rainforest and rural area in south west Gabon. Being on constant contact with wildlife and domesticated animal (that can serve as relay), these populations are at high risk for zoonotic spillover.

But outbreaks and pandemics are a global problem. The key to fight outbreaks is to deal with it early, at the local level. We are working with the hope that it will serves as a model to the multitude of area that are on the front-line of viral emergence.

Our solution focus on outbreak preparedness and response. It provide critical information on virus circulation in the rainforest and its outskirt, allowing outbreak preparedness and mitigation at the local level. In Malaysia, similar approach have been used to prevent Nipah Virus outbreak, and have been successful for the last 30 years. Our project will include not only Nipah Virus, but a whole group of emerging virus including Ebola, Influenza, Coronaviruses, and many other, as well as yet unknown viruses with potential for outbreak.

Importantly, our project could serve as a model for other local initiative on outbreak preparedness worldwide.

When I was a post-doc at UCLA, I worked with human and wildlife samples from Cameroon. Similarly to Africa, there is no BSL-4 in California, so I had to develop a low biosafety-level protocol. Not only the project was a success (one of the assay we are planning to use in Gabon derived from this work), but we identified a new Henipavirus as well as risk factors for wildlife to human transmission.

While Henipaviruses are well documented in Australia and Asia, where they cause deadly encephalitis outbreaks, they had never been documented in African populations. But in some Cameroonian communities we worked with, 3% of the tests were seropositive. This raised important questions: how many other infections do we miss or misdiagnose? What is the pandemic potential for these viruses?

Therefore, I then worked on two front: on the molecular side, I modified my protocols to be able to target and identify a broad range of emerging viruses with pandemic potential. I also worked with field scientists to understand what was there needs, and how my project could benefit to them. This is when I realised the samples they collect daily could be the basis of outbreak prevention.

In 2009-2010, I organized field sample collections. I was collecting bat samples for viral analysis. I collaborated with field scientists and was impressed by their dedication toward bats, an animal that is usually associated with diseases. Importantly, they showed me the roles and place of the bats in their ecosystems. It resonated with my work on host-virus molecular interactions. What I used to see at the cellular levels, they would observed it at the ecosystem level. Therefore, I spent the last 10 years advocating for studies of these delicate host-virus interaction at the ecosystem level, and how human activities endangered this balance.

While the message was welcome in conservationist societies, molecular biologists were rarely interested at the beginning. However, opinions changed overtime. With the massive Ebola outbreaks in 2014-16 and 2018-20, the rise of Zika Virus, and the COVID-19 pandemic, the topic now receive the attention it deserve.

I am passionate about my project because this is not just about a virus in a laboratory. This is about a whole ecosystem and the viruses that circulate in it. The results may prevent a local outbreak, the extinction of an endangered species, or stop the next pandemic.

This project rely on a tight collaboration with field scientists in Gabon, combining expertise in molecular biology and emerging diseases with high quality samples.

The Moukalaba-Doudou National Park (MDNP) in Gabon is remarkable by its biodiversity and the interactions between wildlife and human activities (farms, tourism). It is also located in the Ebola hot-spots belt. It is a sanctuary for a multitude of species of interest for emerging diseases: from the endangered gorillas to highly diverse bat groups ; from rodent species to tsé-tsé flies ; and of course humans. This make Moukalaba-Doudou National Park one of the most interesting place in the world to study emerging diseases. The vast amount of sample collected there by field scientists with years of experience in this ecosystem is invaluable.

Unfortunately, the quality of the samples would degrade overtime if they were to be shipped for analysis in an American or European BSL-4 laboratory. Moreover, there is not enough of these laboratories, and not enough trained personnel. It would be impossible to process the wealth of samples collected in MDNP in a timely fashion.

By processing the sample on-site, we can bypass these issue and significantly increase the amount of sample analysed

In 2012, I developed an assay to safely study in a regular laboratory (BSL-1/2) the deadly Henipavirus isolated from Cameroonian bat populations. There was no negative control available, no healthy bat blood for sale. That alone should have prevent me to validate my assay for bats, a major stroke considering how critical bats are in the field of emerging diseases.

To solve this issue, I contacted all the zoos in the US that hosted bats of that specific species for several generations. I ultimately found my healthy bat at the Brevard Zoo in Melbourne, Florida. I discussed with the veterinarians at the zoo, and they provided my with an aliquot of blood they collected during a recent check up.

Thanks to these samples, the assay was validated and we showed that our results were comparable to the results obtained by the CDC with their gold standard protocol in BSL-4.

When COVID-19 emerged earlier this year, I immediately worked with the University of Southern California (USC / Keck Medicine) Department of Pediatrics, to adapt the protocol used for HIV diagnostic to SARS-CoV-2. By mid-March, I had developed saliva based assay and my lab set up a small “drive-through” testing centre for hospital personnel. In the early day of the outbreak, when testing was scarce, that allowed nurses and doctors to be tested until the hospital Pathology Department could take over the effort and scale up testing.

The innovation is to process the sample on-site, not in a distant laboratory. This is made possible by 2 critical and innovative aspects of the project: portability and high-throughput processing.

The first step of this project relies on a novel high-throughput seroneutralization assay (HT-SNA) developed specifically to safely study deadly pathogens in a minimal, portable setting. Importantly, this approach allow the detection a whole family of virus, not just one. The safety has already been reviewed and approved by the CDC.

The sequencing/genomic part relies on the new Nanopore MinION technology that allows high-throughput sequencing with a device the size of a cell phone and can be transported directly to a remote site for immediate analysis and response. A new sequences analysis algorithm developed by EnViro International Laboratories and optimized for emerging viruses will be used to find not only one specific virus, but a whole family of virus, allowing the discovery of new virus with pandemic potential before they can cause an outbreak.

Together, the portability allows the project to work independently of the major BSL-4 laboratories located in limited location, and bring the sample processing and analysis on the site of the viral emergence. The high-throughput component allows to process the multitude of samples collect on a daily basis by the field scientists.

The main issue with outbreak prevention is that we react to slowly when a new virus emerge. By the time a threat is recognized by the major players in global health, it is often too late. This is due to the disconnection between the emergence sites and the locations of laboratories capable of such analyses (BSL3/4). There is no BSL-4 laboratory in Africa, South America, or South East Asia, where highly pathogenic viruses abound in wildlife and human activities accelerate their emergence.

While field scientists collect an incredible amount of samples of high quality, the lack of laboratory prevent the complete analysis that would identify outbreak early signals. Reciprocally, for virologists working in BSL-3/4 laboratories, it is challenging to get access to samples and process them in optimal conditions: international shipping is strictly regulated, transport degrades the samples, and working in a BSL-3/4 is extremely onerous. Therefore only a limited subset of samples can be studied.

Ideally, outbreak response should occurs early, at the local level. It took 4 months for the WHO to confirm the Ebola outbreak in West Africa because the samples needed to be analysed in Europe. But it took only a few days to confirm Ebola in DRC/Équateur where diagnostic assays were readily available. Unfortunately, this is an exceptional case. Moreover, virus monitoring uses assays that focus only on one virus. A efficient solution should be local and broad spectrum.

Therefore, we developed new protocols that allow to safely study viral dynamics at the local scale for a local response. Our project allows a hot spot like the Ogooué-Maritime in Gabon to monitor viral movement between species, provide early signals for viral emergence, and allow a rapid, optimal outbreak response. It is important that this model can be adapted to any location, any ecosystems. It is also important that the results will be easily accessible in real time through a web application by the concerned communities, public health professionals, and international organizations.

This project may prevent a local outbreak, the extinction of an endangered species, or stop early the next pandemic.

We are currently working with a small group of Gabonese scientists (~10-20 people) in the fields of virology, conservation, and ecology.

After the first year, the viral monitoring should be ongoing and will provide data for the communities living in the area of the Moukalaba-Doudou National Park. Moukalaba-Doudou is located at the junction of the Ogooué-Maritime, Ngounié, and Nyanga Provinces in Gabon, cumulating 250,000 inhabitants.

In five years the process should be working on a routine basis providing data not only to the local communities (250,000 inhabitants) but also to public health related international organizations.

After the first year, the viral monitoring should be ongoing and will
provide data on viral dynamics for the communities living in the area of the
Moukalaba-Doudou National Park.

In five years the process should be working on a routine basis providing
data not only to the local communities but also international organization.

One of our main goal is to create a model that can be replicated elsewhere. We do not ambition to become another focal point that will centralize all the sample processing/analysis. Our success will not to become the biggest, but to become the most copied, adapted, and connected.

Because we are focusing on virus with outbreak and pandemic potential,
it is difficult to estimate the number of people that will ultimately
benefit from the project. This is the whole point of this project: by serving the local communities around the Moukalaba-Doudou National Park, we also serving the rest of the world.

Financial barrier: although the sero-neutralization assay is designed with cost in mind ( less than 1 USD per reaction), the second step relies on a relatively expensive technology. While Nanopore MinIONs are cheaper than alternative technologies and perfectly adapted to this project, their cost remain high and will require most of the budget.

Regulation: regulation governing shipping of technology and biological material is complex. Regulations compliance is a major and time consuming component of this project.

Financial barriers: the easiest answer would be to continuously apply for research grants to overcome this issue. The data yield by this project will not only allow a quick response against outbreak, it will also yield important information regarding ecology and virology and open new possibilities of funding.

However, as research funds are getting scarce in the current context, it is important to have alternative strategies. In the future, we might operate following the university "core" model: This project ambitions to bring high-throughput sequencing capacity in south west Gabon. While this technology is critical for our project, such capacity can be useful to other projects developed by third part organization outside our field of expertise (soil metagenomic in agriculture for example). It is therefore possible to share the equipment and reagents costs to lower the overall operational costs and expand the genomic sequencing capacities in the area.

Regulation issues: this is partly covered by Dr. Pernet experience as biosafety officer in various academic institutions, as well as his experience working in BSL-4 with select agents in different countries. During his career, Dr. Pernet built a strong network of expert in this field that could provided their help if needed.

Institute for Tropical Ecology Research (ITER), Gabon :

The whole project rely on the collaboration between molecular virologists at EnViro International Laboratories and the field scientists at ITER. EnViro International Laboratories develops tools adapted to their specific field condition. These cover protocols for high-throughput sero-neutralization assays, high-throughput sequencing (Nanopore MinION) and sequencing analysis.

We provide protocol and assay adapted to the local conditions.

Importantly, we provide a model that can be replicated anywhere in the world that face potential viral emergences. Our approach bypasses the centralization issues associated with the current system relying on a handful of high biosafety-level laboratories present exclusively in developed countries.  

We provide a network connecting local field scientists with molecular virologists, epidemiologists, computer scientists, and mathematicians. We promote a translational approach of these issues involving local and global actors (One Health approach).

We will provide our results to impacted communities, local governments, and public health professional, in order to maximize their impact. The ultimate goal is to be able to respond immediately, at the local level to an outbreak threat to prevent it to become a pandemic.

We are a non profit organization founded by academics. We currently rely exclusively on grants and donations.

In the future, we might also operate following the university "core" model. For example, this project ambitions to bring high-throughput sequencing capacity in south west Gabon. While this technology is critical for our project, such capacity can be useful to other project developed by third part organizations (soil metagenomic in agriculture for example).

It is therefore possible to share the equipment and reagents costs to lower the overall operational costs and expand the genomic sequencing capacities in the area.

N/A

We are focusing on grants and have submitted several proposals either focusing on specific aspect of the project (virus threatening great apes for example, sample collection) or more general topics (developing genomic capacities in the Ogooué Maritime area, emerging virus discoveries).

.................................Year 1        Year 2
Reagents/Assays

Nanopore MinIONs...........45000...........90000
Seroneutralization assay.....5000...........10000

Salary

Lab technician..............30000............30000

Equipment

Biosafety cabinet............20000

Cell culture incubator........5000

Fluorescence reader.........25000

Indirect costs.................20000............20000

Total..........................150,000.......150,000

Financial issues: The Elevate Prize will allow us to afford the sequencing technology required by the Step-2 of the project. As described in the budget, this is a major cost. The Elevate Prize will also allows us to hire a dedicated scientist working on this project.

Regulatory issues: As mentioned before, regulations on technology and biological materials exports can be extremely complex. Despite Dr. Pernet experience in the area, we hope The Elevate Prize could provide help in navigating international regulations on the topic. 

As we explained in the other sections, on of our main goal is to create a model that can be replicated elsewhere. We do not ambition to become another focal point that will centralize all the sample processing/analysis. Our success will not to become the biggest, but to become the most copied, adapted, and connected.

For that to happen, we need to be able to help the scientists who will want to emulate our project. We need to be able to ship reagent, protocols, and technologies to other part of the world. This is where we need The Elevate Prize: helping us navigating the administrative regulation covering these exchange between countries in order to build a strong network of laboratories working worldwide on emergence hot spots.

Although right now we are focusing on our current project in Gabon, there are organizations that we think would be able to help us in the future.

For example, the ALIMA (https://www.alima-ngo.org/en/) is an association answering a similar question regarding the lack of high biosaftey-level laboratories (BSL-3/4). However, instead of adapting the current protocols to low-safety laboratories (as we do), they develop solutions to bring temporary BSL-3/4 on the sites of outbreaks. Their work during the recent Ebola outbreaks  was remarkable, and we think our project might benefit in the long term by working with this organization and their complementary approach of the issue.

Once our project yield data usable data for public health, we hope The Elevate Prize network could help us to publicize our work and reach the international organizations that have the manpower and the experience in outbreak response.