Infection with hepatitis E virus (HEV) is responsible for acute hepatitis leading to approximately 3.3 million new symptomatic cases and over 44,000 deaths annually (World Health Organization, 2022). HEV strains that infect humans belong to the Hepeviridae family and the genus Paslahepevirus (Purdy, et al. 2022). Paslahepeviruses are categorized into eight genotypes, from which HEV-1, -2, -3, -4, and -7 infect humans (Ahmed and Nasheri. 2023). HEV-1 and HEV-2 are mainly waterborne and endemic in several low- and middle-income countries (LMICs) (Raji, et al. 2021). HEV-3, -4, and -7 are zoonotic, and exposure to infected animals or consumption of their undercooked meat can cause infection to humans (Ahmed and Nasheri. 2023). HEV-3 is more common in industrialized countries and is mostly attributed to the consumption of pork products, or wild boars (Sooryanarain and Meng. 2020). In recent years the number of HEV-3 infections has been steadily increasing in Europe, and HEV-3 is becoming an emerging concern for the agro-food safety (European Centre for Disease Prevention and Control. 2017).

HEV particles are icosahedral, non-enveloped in feces and bile but wrapped in a lipid envelope in blood and cell culture supernatant, and thus HEV is considered a quasi-enveloped virus (Yin, et al. 2016). The HEV genome is a single-stranded RNA with positive polarity that encodes three open reading frames (ORFs) (Kenney and Meng. 2019). ORF 1 codes for the viral non-structural proteins including the RNA-dependent RNA polymerase (RdRP) (Oechslin, et al. 2022). ORF 2 codes for the capsid protein, which is the main target for neutralizing antibodies (Kenney and Meng. 2019). The protein coded by ORF3, is believed to be involved in acquisition of lipid bilayers to form the quasi-envelope virion, allowing it to evade the immune system (Ju and Ding. 2019).

While adaptations from the ISO 15216 method have been employed for the detection of HEV, no standard method for the isolation and detection of HEV from contaminated food, water or wastewater exists (Cook et al., 2022, Miura et al., 2016). Additionally, the existing methods are tedious, do not report on the viral infectivity, and are sensitive to the presence of RT-PCR inhibitors (Cook, et al. 2022). While there are assays based on magnetic beads for the isolation of other foodborne viruses such as norovirus and hepatitis A virus (Tian et al., 2008, Nasheri et al., 2020), no such assays have been developed for HEV yet. Viral isolation assays based on magnetic beads provide several advantages compared to the traditional methods based on viral precipitation: 1) faster turnaround times; 2) adaptability to automation settings; 3) ability to capture undamaged viral particles and 4) higher efficiency in removing potential RT-PCR inhibitors (Nasheri et al., 2020, Suresh et al., 2019). The goal of this study is to compare the efficiency of different types of magnetic beads in isolation of HEV-3 in solution.

One of the methods that we examined was immunoprecipitation, using several monoclonal antibodies conjugated to Dynabeads M-270 Epoxy beads, which are polystyrene beads with an epoxy coating and a diameter of 2.8 µm. The epoxy coating on the Dynabeads allows for formation of covalent bond with the amino group in monoclonal antibodies (mAbs). The other type of magnetic bead employed in this study was the Nanotrap Microbiome A beads, which are hydrogel particles made of N-isopropylacrylamide copolymers with an average diameter of 0.3 µm. Their capture mechanism is through the chemical affinity baits in the form of reactive dyes such as Reactive Red 120, Reactive Yellow 86, and Cibracon Blue (Lin et al., 2020, Shafagati et al., 2014). Reactive Red 120, for example, contains an Azo-dye chromophore linked to a trichlorotriazine ring, which is negatively charged and as such allows for electrostatic interaction with the positively charged amine groups found in proteins (National Center for Biotechnology Information). Thus, these nanoparticles bind to viral proteins like capsid proteins and surface glycoproteins through electrostatic and hydrophobic interactions at high affinity (Lin et al., 2020, Andersen et al., 2023) as seen in Fig. 1.

In this study, we explored different bead-based options for the detection of HEV-3 by comparing the efficiency of several types of mAb coated Dynabeads and Nanotrap Microbiome A beads in capturing the virus.