While the attention of public health authorities has been rivetted on the current COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, many other viruses have been stealthily circling the globe, causing serious disease and death in humans and other animals.

One such pathogen is a virus responsible for African Swine Fever (ASF). The highly contagious, often lethal disease has devastated swine populations around the world and is currently spreading rapidly across Africa, Europe, Asia, and the Americas.

No treatment or vaccine exists for ASF. Swine herds must be continuously monitored for any signs of the disease. If ASF is suspected, laboratory confirmation is carried out. This is often a costly and time-consuming undertaking, (typically a few days from sample collection to test result output), which makes containing ASF outbreaks challenging. Positive test results are usually followed by the slaughtering of all animals affected or exposed.

In a new study, Arizona State University researcher Chao Wang and his colleagues advance an innovative method for rapidly diagnosing the disease, which could revolutionize the process and help safeguard millions of animals worldwide. Wang is a researcher with the Biodesign Center for Molecular Design and Biomimetics and the School of Electrical, Computer & Energy Engineering.

The project is a collaboration between ASU and Centro de Investigación en Sanidad Animal in Spain, which is also the European Union reference laboratory and the Food and Agriculture Organization reference Centre for ASF.

Wang and his colleagues have received about $750,000 in funding from the Agriculture and Food Research Initiative program of the National Institute of Food and Agriculture of U.S. Department of Agriculture to pursue their diagnostic test, dubbed NaSRED, (for nanoparticle-supported rapid, electronic detection), a highly accurate means of detecting ASFV, the virus responsible for African Swine Fever.

NaSRED offers several advantages over existing methods. The test can be performed at drastically reduced cost, yielding results in minutes rather than hours or days. It can easily be carried out in the field, without recourse to the sophisticated laboratory facilities required for processing similar diagnostic tests.

Remarkably, NaSRED's diagnostic sensitivity is estimated to exceed that of enzyme-linked immunosorbent assays (ELISA), the current gold standard for such tests, by more than 10 times and surpass ELISA's dynamic range by over 100 times. (The dynamic range is the range from the lowest to the highest quantities a diagnostic test can measure.)

The basic technique has already shown impressive results, where an earlier prototype was used to pinpoint the presence of two global human pathogens, SARS-CoV-2 and Ebola.

I came across the ASF virus and was immediately amazed by its complexity. The virus particle itself is protected with multiple layers and decorated with more than 50 structural proteins, in contrast to the SARS-CoV-2 virus particle with only one single lipid bilayer and a few surface proteins. This makes it extremely difficult to understand how the virus works to cause the damage. This is also one big reason there is no effective vaccine available. Therefore, virus diagnostics for ASF are of paramount importance."

Chao Wang, Researcher, Arizona State University

Invisible threat

ASF is an extremely contagious viral disease, often spreading like a wildfire throughout swine populations and inflicting mortality rates of 95-100%. The virus causes severe disease in both domestic and wild pigs.

ASF was first detected in East Africa in the early 1900s, later spreading to Europe in the late 1950s. More recently, ASF has carved a swath of destruction through many Asian countries. China has been especially hard hit, losing roughly 50% of its pigs to the disease since its first appearance there in 2018, causing direct economic losses of $141 billion in just one year.

The disease is causing considerable socioeconomic hardship in countries that export live pigs and pork products, as well as in countries where pigs are important food sources in the diet.

While the U.S., including Puerto Rico and the U.S. Virgin Islands, is currently ASF-free, it is believed to be at increased risk of importing the disease through other countries in the Americas where ASF is already present, including in the Dominican Republic and Haiti.

The effect of ASF reaching the states could be devastating as the U.S. is the world's third largest pig producer, with more than 11.5 million tons of pork produced each year, and it's also the world's second largest pork exporter. The U.S. may face far-reaching economic consequences if ASF is introduced.

Meeting the challenge

Existing diagnostic tests for ASF involve the detection of nucleic acid, antibodies or antigens specific to the ASF virus. The most sensitive tests, including real time polymerase chain reaction (PCR) and ELISA require laboratory facilities and trained personnel to perform. They also often involve long turnaround times for results, a significant drawback when tracking a fast-moving, explosively contagious disease like ASF.

Ideally, swine populations should be under continual surveillance by means of a portable diagnostic, stockpiled near animal pens, enabling earlier and faster disease detection at reduced cost. Such a method would also permit widespread, high-frequency testing, a crucial factor in disrupting the transmission chain of the virus and containing an ASF outbreak.

The project involves the design and validation of NaSRED, a portable diagnostic sensing device, using metal nanoparticles. The NaSRED test uses collections of metal nanoparticles with different optical characteristics to ferret out ASF biomarkers-;telltale signals of the presence of the ASF virus.

The project proposal describes detection of a specific ASF protein known as p72 and two ASF antibodies, anti-p54, anti-p30, making for a much more accurate and sensitive test, compared with diagnostics that only test for one of these disease signatures.

The technology uses metal nanoparticles affixed with specific molecular binders known as ligands, which seek out proteins and antibodies associated with the ASF virus. When the nanoparticles encounter ASF proteins and antibodies in a sample of blood, the disease targets stick like glue to the nanoparticle sensors.

As the ASF proteins and antibodies adhere to the nanoparticles, they aggregate to form clusters. The increased weight of these clusters causes them to sink to the bottom of the test tube, causing a detectable color change in the solution. The unique optical properties of these clusters, which signal positive identification of the ASF virus, are observed when light is passed through the solution.

Using simple and inexpensive electronic circuits, the system can easily convert the optical signals produced by the metal nanoparticles into a quantitative electronic readout of test results. The collected signals will be automatically recorded by circuitry and transmitted to computers or phone, where negative or positive results will be displayed, making the testing an even easier task that could eventually be performed by farmers.

The final sensing system, ideal for pen-side testing, will be hand-held and the cost of materials is expected to be around $20, while individual tests can be performed for under $1. Rapid testing, requiring less than a microliter of blood, will deliver results within minutes.

The technology is a particularly attractive option for testing swine in resource-limited settings and promises to dramatically enhance efforts to combat this global scourge.