Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolation and Growing of Primary Hepatocytes
2.2. Immune Peroxidase Monolayer Assay (IPMA)
2.3. Inoculation of Primary Hepatocytes
2.4. RNA Extraction/Isolation and Real-Time RT-PCR
2.5. Sensitivity of the System Compared to a Pig Model
3. Results
3.1. Isolation and Culturing of the Primary Hepatocytes
3.2. Sensitivity of the System Compared to the Animal Pig Model
Concentration of the Inoculum (Genomic Copies/mL) | Concentration of the Inoculum (Log Genomic Copies/mL) | Log Genome Copies/mL at D0 | Log Genome Copies/mL at D6 | Primary Hepatocytes Model (Increase Log Copies/mL at D6 after Inoculation) | Animal Model (Total Animals/Tested Positive) |
---|---|---|---|---|---|
107 | 7.00 | 1.29 (SD = 0.00) | 5.14 (SD = 0.12) | 3.47 (SD = 0.66) | 6/6 |
4 × 105 | 5.60 | 1.50 (SD = 0.30) | 4.32 (SD = 0.01) | 3.52 (SD = 0.38) | Nt |
8 × 104 | 4.90 | 1.30 (SD = 0.02) | 4.32 (SD = 0.01) | 3.02 (SD = 0.03) | Nt |
104 | 4.00 | 1.68 (SD = 0.46) | 3.33 (SD = 0.06) | 1.65 (SD = 0.52 | 6/6 |
3.33 × 103 | 3.52 | 1.29 (SD = 0.00) | 2.73 (SD = 0.73) | 1.45 (SD = 0.34) | Nt |
2 × 103 | 3.30 | 1.29 (SD = 0.00) | 2.16 (SD = 0.15) | 0.87 (SD = 0.15) | Nt |
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Origin (Sample Type and HEV Genotype) | Log Genome Copies/mL of the Tested Sample | Country of Origin | Reference | Log Genome Copies/mL at D0 | Log Genome Copies/mL at D6 | Log Increase in Genome Copies/mL |
---|---|---|---|---|---|---|
Positive control (HEV PCR-positive hepatocytes HEV-3f) | 6.42 | The Netherlands | WBVR | 1.99 (SD = 0.68) | 5.44 (SD = 0.14) | 3.45 (SD = 0.54) |
Liver HEV-3f | 5.10 | The Netherlands | Bouwknegt et al. [22] | 2.96 (SD = 0.68) | 5.1 (SD = 0.23) | 2.06 (SD = 0.91) |
Liver HEV-4 | 3.66 | Belgium | Hakze et al. [23] | 1.29 (SD = 0.00) | 2.51 (SD = 0.07) | 1.22 (SD = 0.07) |
Liver HEV-3f in different dilutions | 7.37 | Denmark | WBVR | See Table 2 | See Table 2 | See Table 2 |
Sausage | 3.49 | France | Berto et al. [10] | 1.29 (SD = 0.00) | 2.5 (SD = 0.00) | 1.21 (SD = 0.00) |
Faeces HEV-3c | 6.49 | The Netherlands | WBVR, 2019 | 3.58 (SD = 0.09) | 4.67 (0.27) | 1.1 (SD = 0.17) |
Liver 2019A HEV-3c | 7.16 | The Netherlands | Boxman et al. [24] | 3.14 (SD = 0.25) | 4.04 (SD = 0.06) | 0.63 (SD = 0.19) |
Liver 2019B HEV-3c | 6.39 | The Netherlands | Boxman et al. [24] | 3.04 (SD = 0.14) | 3.52 (SD = 0.11) | 0.48 (SD = 0.03) |
Liver 2022A HEV-3c | 4.39 | The Netherlands | WFSR 2022 | 1.36 (SD = 0.10) | 2.45 (SD = 0.07) | 1.09 (SD = 0.03) |
Liver 2022B HEV-3c | 6.58 | The Netherlands | WFSR 2022 | 2.64 (SD = 0.47) | 3.16 (SD = 0.02) | 0.52 (SD = 0.49) |
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Hakze-van der Honing, R.W.; van Oort, S.; Dirks, R.A.M.; van der Poel, W.H.M. Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples. Pathogens 2023, 12, 1231. https://doi.org/10.3390/pathogens12101231
Hakze-van der Honing RW, van Oort S, Dirks RAM, van der Poel WHM. Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples. Pathogens. 2023; 12(10):1231. https://doi.org/10.3390/pathogens12101231
Chicago/Turabian StyleHakze-van der Honing, Renate W., Sophie van Oort, René A. M. Dirks, and Wim H. M. van der Poel. 2023. "Development of an Ex Vivo Assay for Identification of Infectious Hepatitis E Virus in Different Kinds of Food Samples" Pathogens 12, no. 10: 1231. https://doi.org/10.3390/pathogens12101231