SHIC Funded Study Develops DNA Sensors for Infectious Swine Virus Detection

By Abbey Canon

Swine viral pathogens such as PRRSV and PEDV pose significant economic challenges to the swine industry. The Swine Health Information Center funded a study to investigate novel diagnostic tools for differentiating infectious versus non-infectious swine viruses to use on-site for real-time detection. The study, led by Dr. Yi Lu at University of Texas Austin, aimed to develop a novel method for differentiating intact viruses versus those virus particles rendered noninfectious by disinfection. Novel diagnostic tools such as the one investigated in this study could provide valuable information to pork producers and veterinarians by evaluating cleaning and disinfection efficacy after a viral disease outbreak.

Find the industry summary of project #22-003 here.

While diagnostic methods such as PCR and cell culture are available to detect viral genetic material and assess infectivity, respectively, inherent limitations to these tests include required sample pretreatment and a skilled operator for processes performed on costly equipment. Further, the tests require hours to days to be conducted in a professional laboratory, making these tests unsuitable for on-site detection. The goal of the study described herein was to develop a novel method for direct detection of intact viruses without any sample pretreatment using a handheld portable meter.

The novel method under investigation is based on DNA aptamers that can be selected to bind and differentiate infectious swine viruses from noninfectious and other viruses. By immobilizing the aptamers into a nanopore, only infectious PRRSV or PEDV produce electrochemical signal changes and thus can be detected and quantified using a handheld meter. This innovative direct detection method utilizing DNA aptamers integrated into solid-state nanopores has previously demonstrated success in detecting a wide range of human viruses, including and human adenovirus and SARS-CoV-2.

There were two primary objectives of the study to apply the technology to swine viruses:

1. To obtain DNA aptamers that can bind infectious PRRSV and PEDV through in vitro selection and counter selection processes, with the aim of enhancing selectivity.
2. To design and validate DNA aptamer-nanopore sensors for the direct detection of infectious PRRSV and PEDV, both within controlled cell cultures and real-world field samples.

To conduct this study, PRRSV and PEDV samples were prepared in the laboratory of Dr. Ying Fang at the University of Illinois at Urbana-Champaign. A portion of the PRRSV and PEDV samples were rendered noninfectious by adding binary ethylenimine or UV light and then purified. Researchers conducted 10 SELEX (systematic evolution of ligands by exponential enrichment) experiments under varying conditions to optimize aptamer selection for PRRSV and PEDV. The SELEX process is designed to identify aptamers which are short, single-stranded DNA or RNA molecules that bind selectively to target viruses.

In previous work by this research group, SELEX projects targeted other viruses, such as human adenovirus and SARS-CoV-2. Using lessons learned from previous work, researchers optimized aptamer selection conditions by varying factors such as virus concentration, purity, and reaction times. Initial findings indicated that virus purity was a critical factor affecting the success of selections. Researchers focused on conducting SELEX experiments using ultra-centrifugally purified PRRSV and PEDV. This approach yielded promising results where shifts in melting temperatures that mirrored previous successes with other viral targets were observed.

After performing NGS on these more refined samples, data was analyzed, and several promising DNA sequences were obtained. Biochemical studies identified aptamers with the highest selectivity for either infectious PRRSV or PEDV. Currently, researchers have identified three different aptamer candidates for PRRSV. First, candidate 5 demonstrated binding to active PRRSV II and inactive PRRSV II, with no binding to active PEDV or active PRRSV I. Second, candidate 7 exhibited binding to active PRRSV II but no binding to inactive PRRSV II, active PEDV, or active PRRSV I. Third, candidate 9 showed binding to both active PRRSV II and active PRRSV I, while no binding was observed with inactive PRRSV II or active PEDV.

Despite these promising results, challenges with reproducibility and consistency in binding assays occurred in this initial pilot study. Additional optimization or complementary techniques may be required for field applications. The insights gained from this study underscore the importance of sample purity and the need to further enhance aptamer selectivity to improve detection reliability in applied settings. Further optimization of the DNA aptamer sequences, and assay conditions are required to develop this system into a practical sensor for on-site and real-time detection.