ABSTRACT Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is doubtless and has increased recent years. Zoonotic spread of HEV, mainly due to genotype (gt) 3, has emerged in developed countries in the past decade. The current study has shown for the first time that domestic pigs in Bulgaria are widely infected with HEV. There is a need for a method for the cost-effective detection of the HEV infection and production of HEV immunogenic proteins that can be used as diagnostic antigens for the serological tests. Also, in this study we have compared the results from a commercial ELISA and an in-house ELISA test based on HEV open reading frame (ORF) 2 protein produced in planta, the sensitivity and the specify of the tests were evaluated. The development of a HEV diagnostic kit based on plant-derived ORF2 capsid protein is our goal and this will help to reduce the cost of such a kit and to enable diagnoses of HEV in developing countries. Serological diagnostics of hepatitis E virus infection in pigs based on capsid protein (ORF2 genotype 3) expressed in planta – sensitivity and specificity Katerina Takova 1, Tsvetoslav Koynarski 2, Valentina Toneva 1, 3, Gergana Zahmanova 1, 4 Acknowledgements In total, 433 swine blood samples were collected from 19 different pig farms and 1 slaughterhouse, located in eleven districts across Bulgaria—Pazardzhik, Plovdiv, Stara Zagora, Yambol, Ruse, Razgrad, Silistra, Shumen, Varna, Dobrich, and Burgas district. The samples were collected during 2017 (n = 75), 2018 (n = 158), and 2019 (n = 200). In 2018 samples were taken from two age-groups, 6- month-old pigs that weighed around 80–100 kg (n = 78), and approximately 3- month-old pigs that weighed around 30 kg (n = 80). The samples taken in 2017 and 2019 are all from 6-month-old pigs. In addition, 32 meat chunks from wild boar were collected for meat juice extraction. The samples were collected from animals hunted in Stara Zagora, Vratsa, Montana, and Dobrich districts during the official hunting season. CONCLUSION: This study confirmed that plant-derived HEV diagnostic antigen could provide a cost-effective and scalable alternative of the commercially available coating protein. This research was funded by the Research Fund at the University of Plovdiv, competition “Young Scientists and Doctoral Students” MU21-BF-02, the Bulgarian National Fund, Project DNTC/Russia 02-6, Project PlantaSYST. PlantaSYST has received funding from the European Union’s Horizon 2020 research and innovation programme (SGA-CSA No 664621 and No 739582 under FPA No. 664620) Figure 1. The map of Bulgaria represents the districts of origin of the farming pigs tested in grey - Pazardzhik, Plovdiv, Stara Zagora, Yambol, Ruse, Razgrad, Silistra, Shumen, Varna, Dobrich, and Burgas district. The origins of the wild boar’s testes are marked with red circles. Wild boar samples originated from Stara Zagora, Dobrich, Vratsa and Montana district. Figure 4. Determination of the optimal antigen concentration (recombinant capsid protein) for the developed ELISA. A two-fold dilution series of the recombinant capsid protein was performed to identify the optimum working concentration, and a titration experiment was performed using pig sera already with known serostatus. Results of the checkerboard titration analysis demonstrated that the most optimal and reliable results were obtained with coating protein concentration of 2 ng/ul. Figure 2. A A high HEV seroprevalence at all farms in 2019 – [98% (95% Cl 96.4 to 99.9)] compared to 2017 – [45.33% (95% CI 2.7 to 87.3)] and 2018 – [38.46% (95% CI 29.1 to 49.7) is observed. A significant year effect (2019) was found, p-value < 0.01 (Table 2). The chi-square statistic is 149.0211. The p-value is < 0.00001. The result is significant at p < 0.01. B Distribution of the observed within the farm seroprevalence of HEV among the slaughter-aged pigs (17 farms and 1 slaughterhouse with 30 % 2017- 2020, Bulgaria). The chart r