KEY TAKEAWAYS:
- Research projects seek more sensitive and specific hepatitis A virus tests.
- One research team is using pretreatments to chew up or bind to non-infectious RNA fragments, making them unavailable to detection.
- The other is looking at an enzyme pretreatment as well as CRISPR-based technology to create a more sensitive, less expensive and faster test. Similar CRISPR technology was used to create at-home COVID tests.
Referenced CPS Research:
Two research teams are taking slightly different approaches to modifying hepatitis A virus (HAV) screening tests so they’re more sensitive and results better correlate to potential infectivity.
“If we can identify infectious virus that can cause disease, not just virus RNA, we expect we can avoid unnecessary recalls,” said Jose Santos García, Ph.D., at the Universidad Autónoma de Nuevo León in Mexico. “It also will support better prevention and, more importantly, help the industry.”
The goal of improving current HAV tests is to overcome the challenges of cultivating the virus in cell culture. Unlike bacterial pathogens, the viral pathogen’s inability to be grown on laboratory media is a major obstacle.
As a result, testing for potential viral contamination typically relies on a standardized ISO test that first entails concentrating and purifying the virus from the sample, and then uses RT-qPCR, a molecular technique, to detect virus RNA.
It is well recognized that incomplete or inactivated viruses, and small fragments of virus RNA, can persist in the environment even after the virus is no longer infectious, so a positive test result doesn’t necessarily mean a produce item poses a public health risk.
Lee-Ann Jaykus, Ph.D., Emerita, at North Carolina State University, is leading research that uses enzyme and/or chemical pretreatments before RT-qPCR to bind to or chew up RNA fragments, making them unavailable to detection.
Garcia is spearheading efforts to develop an enzyme pretreatment that destroys non-infectious fragments while retaining the infectious capsid, or protein-armored genetic material. His group also is developing a second method that uses the pretreatment coupled with CRISPR gene-edited technology rather than the traditional RT-qPCR step to create a more sensitive, faster and less expensive test.
The Center for Produce Safety has funded both projects for one year.
Chemical Pretreatments
Jaykus and her team are looking at two different ways to pretreat the sample concentrate prior to RT-qPCR. The first is to use enzymes that “chew up” naked virus RNA so it cannot be detected by RT-qPCR.
The other involves using chemical compounds that can enter damaged capsids, bind to virus RNA and prevent PCR amplification from occurring.
“So we’re trying to target all potential ways that non-infectious virus can present itself in samples,” she said.
Joining Jaykus as co-investigators are Benjamin Chapman, Ph.D., and Lynette Johnson, Ph.D., both with NCSU.
Once the researchers have optimized the pretreatments, they will test them using the only HAV strain that is laboratory cultivatable.
They plan to expose this strain to heat or chlorine to produce virus suspensions having different proportions of infectious to non-infectious virus. They chose two different inactivation methods because HAV may react differently to a physical treatment than to a chemical one.
The researchers will then directly compare their optimized pretreatment methods and RT-qPCR to cell culture infectivity assays applied to the suspensions of fully infectious, partially inactivated or fully inactivated HAV.
From there, they plan to evaluate the efficacy of the top one or two pretreatments to artificially contaminated soft fruits, having different proportions of infectious to non-infectious HAV. They will process them using the standardized ISO protocol.
Testing their candidates in the real matrix to which the method will be applied will allow the researchers to assess its efficacy on real-world samples, Jaykus said.
Enzymes + CRISPR
García is working to validate two methods to detect infectious HAV and discriminate against non-infectious virus including free RNA. Joining him as a co-investigator is Norma L. Heredia, Ph.D., also at the Universidad Autónoma de Nuevo León.
They chose to focus on both frozen berries and irrigation water because of the increased risks posed by using contaminated irrigation water, García said.
The first method uses the standardized ISO method to which the researchers have added an enzyme pretreatment that discriminates against non-infectious free RNA or RNA fragments.
The second method uses the same enzyme pretreatment but will replace the RT-qPCR amplification step with one that uses CRISPR-Cas. A similar CRISPR technology was used to develop quick at-home COVID tests during the pandemic, García said.
The beauty of a CRISPR-based HAV assay is its simplicity, he said. It doesn’t require expensive, complicated lab equipment and a well-trained technician to produce results like traditional RT-qPCR does. The CRISPR test results also can be produced in as few as 36 minutes compared to about two hours for RT-qPCR.
The researchers currently are working with laboratories at the University of Delaware and Emory University to validate the techniques. They also plan to develop an instructional manual to guide users through the new techniques.
In addition, García said he hoped to partner with at least one company to commercialize the HAV quick test. This project is being conducted in collaboration with Kalmia Kniel, Ph.D., at the University of Delaware, and Juan León, Ph.D., at Emory University.