Johns Hopkins APL Researchers Take Steps to Rapidly Characterize Emerging Biothreats

Bird flu has recently infected scores of wild and domestic birds, sea lions, mink and bears, raising concerns about the virus’s ability to mutate and spread into new populations. Increased spread of viruses transmitted between animals and people, referred to as zoonotic diseases, is seen in parts of the world where people live in proximity to animal species. Scientists estimate that three out of every four new infectious diseases in people originate from an animal, and approximately three out of four new diseases every year are zoonotic.

The Johns Hopkins Applied Physics Laboratory (APL) in Laurel, Maryland, is taking steps to fend off these emerging biothreats. APL has been sequencing influenza viruses since 2014 and in the past few years has transitioned its capabilities to laboratories around the world, including the Institut Pasteur du Cambodge (IPC).

“Novel viruses can present significant threats to national security,” said Sarah Herman, who manages the Biological and Chemical Sciences program in APL’s Research and Exploratory Development Department (REDD). “Leveraging our team’s expertise in biology and collaborations with researchers worldwide, we are enabling time-critical responses to get ahead of the next biothreat.”

“To combat global pathogen threats and enhance global security, we need sustainable, agile surveillance,” added Peter Thielen, an APL molecular biologist who leads viral genomic surveillance projects. “Intentional, ongoing pathogen genome surveillance — in regions of the world where animals and humans frequently interact — increases our ability to detect emerging zoonotic diseases, informing outbreak responses and reducing the risk of future pandemics.”

Enabling Rapid Public Health Response

This past February, a young Cambodian girl and her father were infected with a highly pathogenic H5N1 avian influenza virus — the first time the virus had infected humans in Cambodia since 2014.

The IPC used molecular biology and computational capabilities, developed and optimized by APL researchers in REDD, to swiftly identify the virus and mount a response. Genome sequencing revealed that the virus belonged to a genetic subgroup of highly pathogenic H5N1 avian influenza endemic to Cambodia — meaning it was a variant that had circulated in birds within the country for some time. This virus group is genetically distinct from highly pathogenic H5N1 avian influenza viruses circulating in much of the world, which have been widely responsible for recent animal outbreaks and sporadic human infections.

Once the IPC sequenced the virus, data immediately became available for analysis in Cambodia and at APL through coordinated data sharing. While the girl, who was 11 years old, unfortunately succumbed to the infection, this genetic information informed Cambodia’s local public health response and enabled officials to minimize further spread within the community.

Since 2019, the APL team has worked with groups such as the IPC in more than 25 countries to establish sustainable pathogen genomic sequencing capabilities. Using customized sequencing protocols and APL’s Basestack bioinformatics platform, the APL team has trained researchers around the globe to use handheld sequencers and bespoke analysis software. The ability of public health workers to complete the process themselves makes disease surveillance more accessible and user-friendly. Researchers and public health workers on location upload data they gather into shared, public and semipublic data repositories such as GISAID and the National Center for Biotechnology Information for further analysis by the infectious disease surveillance community.

There are many steps between the collection of a sample and the release of a genome sequence, and it can be a time-consuming and labor-intensive process. A few years ago, it could take many weeks or months to go from sample collection to sequence data. With the massive increase in pathogen genomic surveillance since the beginning of the COVID-19 pandemic, some specialized laboratories can generate pathogen sequences in a few days from sample to data. APL researchers optimized each step in the influenza sequencing process, condensing the timeline into a single workday.

Thielen credits the rapid sequencing to APL’s direct collaborations with clinics and nonprofit research organizations that don’t typically have the resources to develop new molecular or informatics resources from scratch.

“Our international collaborations enable around-the-clock research,” Thielen said. “Laboratory professionals on the other side of the world can share data when their day is ending, and our group can then take it to the next level of the analysis process. With teams of people working around the world to better understand global data, we can take a broad look at biothreats and help decision makers take action more confidently. The rapid response in Cambodia is a demonstration of those capabilities.”

Safely Characterizing Emerging Zoonotic Threats in the Laboratory

Once a virus is sequenced, APL researchers conduct virus threat characterization. This process can help determine whether a virus can transmit to humans, can spread from person to person, and is different from other known variants.

“There is an unmet need to assess new viruses as they emerge from nature,” said Claire Marie Filone, an APL virologist helping to lead virus characterization. “Our goal is to identify if previously uncharacterized viruses are a cause for concern. Those are the viruses we need to consider for greater intervention or rapid detection tests.”

In the Cambodian case, virus characterization took place after the individuals were infected, but APL is tapping into its data pipeline and partnerships to characterize new viruses before they cause infections.

To characterize a virus, researchers take parts of its genome — letters that make up the virus’s genetic material — to create safe, noninfectious surrogates. A surrogate lacks the ability to replicate or spread in cells but can still enter cells and produce a signature of infection. Surrogates also eliminate the need to ship viruses across the globe, which can help expedite the public health response. Once created, surrogates can be exposed to different types of cells, such as human cells and animal cells, to evaluate what species they might be able to infect.

“Think of a lock and a key,” Filone explained. “The virus is the key, and it needs to find its lock, a compatible host cell. When we characterize a virus, we look at human cells to understand if there are any locks present on those cells. If there is not a matching lock, it’s less likely the virus could spread through a community. If there is a matching lock, it is a red flag that there needs to be more analysis with further samples.”

When a surrogate successfully locks with a cell, it expresses a protein that glows fluorescent green. If the protein glows when the surrogate is exposed to human cells, there is a risk that the virus could spread through human-to-human transmission.

As APL looks to the future of genomic surveillance, the team is using next-generation analytical tools and computational models to understand potential evolutionary trajectories of viruses. This work was recently published in the journal Virus Evolution. It adds to a toolkit of approaches that can be used to safely characterize pathogen genetic changes and their potential impact on humans and other animals.

“Biothreats don’t care about international borders,” Thielen said. “For health and national security, it is critical to identify when zoonotic transmission is likely and to characterize it in place prior to its spread. Rapid surveillance and characterization allow us to better anticipate and counter these emerging threats.”