Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Intestinal Enteroids
2.2. Viruses
2.3. TCID50 Assays
2.4. Virus Recovery from Foods
2.5. RNA Extraction and Purification
2.6. Detection by Real-Time RT-qPCR
2.7. Statistical Analysis
3. Results
3.1. Screening of NoV Fecal Filtrate Replication with J2 HIEs
3.2. Characterization of Culture Conditions for GII.4[P16] and GII.6[P7] Genotypes
3.3. Infection of J2 HIEs with Virus Recovered from Frozen Raspberries
3.4. Infection of J2 HIEs with Virus Recovered from Frozen Strawberries
3.5. Infection of J2 HIEs with Virus Recovered from Fresh Lettuce
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Positive (≥5-Fold Increase) | Negative (≤5-Fold Increase) | |||||
---|---|---|---|---|---|---|
Genotype: | RNA cp: | Fold-inc: | GII Genotype: | RNA cp: | GI Genotype: | RNA cp: |
GII.4 Sydney (BCM) | 3.00 × 105 | 2.9 × 103 | GII.4 (2014) * | 2.90 × 105 | GI.6[P11] * | 1.02 × 106 |
GII.4[P16] (011617) * | 1.98 × 105 | 1.4 × 104 | GII.4[P16] (1043 NVRL) | 9.50 × 104 | GI.3[P3] (#2) ^ | 3.83 × 107 |
GII.4[P16] (1036 NVRL) | 4.00 × 105 | 1.4 × 103 | GII.4 NO/Sydney (16-78)+ | 2.60 × 106 | GI.3[P3] (#3) ^ | 6.36 × 106 |
GII.4[P16] (1041 NVRL) | 4.70 × 104 | 2.1 × 103 | GII.6 (15-65)+ | 3.10 × 104 | GI.3[P3] (461-1) ^ | 5.06 × 103 |
GII.4[P16] (1046 NVRL) | 8.00 × 105 | 5.0 × 100 | GII.6[P7] (2014 vomit) * | 5.40 × 104 | GI.3[P3] (461-2) ^ | 6.38 × 105 |
GII.4 Sydney (13-39)+ | 5.60 × 105 | 2.4 × 103 | GII.6[P7] EP3 * | 1.80 × 103 | GI.3[P3] (461-3) ^ | 2.16 × 105 |
GII.4 Sydney (15-59)+ | 2.60 × 105 | 1.6 × 102 | GII.6[P7] EP4 * | 1.40 × 104 | GI * | 3.80 × 106 |
GII.6[P7] (2014 stool) * | 1.60 × 105 | 4.2 × 102 | GII.6[P7] EP5 * | 3.20 × 105 | GI (1048 NVRL) | 3.64 × 105 |
GII.6 (14-55)+ | 1.70 × 105 | 1.9 × 101 | GII.6[P7] EP6 * | 6.60 × 105 | GI (1049 NVRL) | 3.32 × 104 |
GII.6 * | 7.64 × 104 | 8.0 × 100 | GII.1 SD (2016) * | 7.20 × 105 | GI (1052 NVRL) | 5.75 × 102 |
GII.1 JW (2016) * | 4.10 × 105 | 8.4 × 101 | GII.7 (vomit) * | 1.50 × 105 | GI (1054 NVRL) | 9.62 × 106 |
GI (1257 NVRL) | 2.12 × 106 | 8.0 × 100 | GII.17 (118-2)^ | 9.92 × 104 | GI (1057 NVRL) | 1.07 × 105 |
GII (1038 NVRL) | 2.65 × 103 | GI (1249 NVRL) | 1.39 × 105 | |||
GII (1040 NVRL) | 1.42 × 105 | GI (1252 NVRL) | 8.98 × 103 |
Weigh 50 g of food and inoculate with 100 µL of 10% stool suspension (~5 × 107 gc) |
Let air dry for 1 h at RT |
Elute virus with 0.1 M Tris-HCl-0.05 M glycine-1% beef extract buffer, pH 9.2 with Polyvinylpyrrolidone (PVP), and pectinase on a shaking platform (30 min at RT) |
Clarify eluate with centrifugation at 10,000× g at 4 °C |
Concentrate the virus by ultracentrifugation at 170,000× g for 1 h at 4 °C |
Resuspend pellet in 600 µL media; sonicate and clarify particles at 4500 rpm/5 min |
Infect enteroids (100 µL/well; 1 h adsorption; 3 washes) and collect samples at times 0 and 3 dpi |
Extract RNA from cells and supernatants with TrizolQuantify virus recovery from foods and replication by RT-qPCR with in-house full-length transcripts |
Produce Matrix | Sample | Dilution | Input | 0 hpi | 72 hpi | Fold Increase (Replication +/−) |
---|---|---|---|---|---|---|
Frozen Raspberries | GII.6[P7] | None | Udt | 1.3 × 102 * | 4.2 × 104 | 3.2 × 102 (+) |
Udt | Udt | Udt | 0 (−) | |||
Udt | 3.2 × 102 | 6.7 × 104 | 2.1 × 102 (+) | |||
Udt | 3.9 × 102 | 4.2 × 106 | 1.1 × 104 (+) | |||
Udt | 5.9 × 102 | 1.9 × 107 | 3.3 × 104 (+) | |||
3.4 × 102 | 1.2 × 103 | 1.4 × 107 | 1.2 × 104 (+) | |||
GII.4[P16] | Udt | 1.3 × 102 * | 2.6 × 105 | 2.0 × 103 (+) | ||
Udt | 1.3 × 102 * | 2.0 × 105 | 1.5 × 103 (+) | |||
Udt | 6.1 × 102 | 1.0 × 108 | 1.6 × 105 (+) | |||
1.2 × 103 | 7.9 × 102 | 3.7 × 104 | 4.6 × 101 (+) | |||
Udt | 2.7 × 103 | 1.1 × 108 | 3.9 × 104 (+) | |||
Udt | 4.4 × 103 | 1.3 × 108 | 3.1 × 104 (+) | |||
1.6 × 105 | 6.4 × 103 | 6.0 × 107 | 9.4 × 103 (+) | |||
2.4 × 105 | 7.0 × 103 | 1.0 × 105 | 1.4 × 101 (+) | |||
Frozen Strawberries | GII.6[P7] | none | Udt | 4.0 × 102 | 2.6 × 107 | 6.5 × 104 (+) |
Udt | 1.5 × 103 | 1.4 × 104 | 9.5 × 100 (+) | |||
2.5 × 102 | 3.7 × 102 | 2.0 × 106 | 5.4 × 103 (+) | |||
7.1 × 102 | Udt | Udt | 0 (−) | |||
1:3 | 1.8 × 103 | 2.8 × 102 | Udt | 0 (−) | ||
1:10 | Udt | Udt | Udt | 0 (−) | ||
Udt | 1.3 × 102 * | Udt | 0 (−) | |||
Udt | 2.5 × 102 | 5.1 × 102 | 2.0 × 100 (−) | |||
5.1 × 101 | Udt | Udt | 0 (−) | |||
1:20 | Udt | 2.0 × 102 | 4.0 × 102 | 2.0 × 100 (−) | ||
4.7 × 103 | 8.8 × 102 | Udt | 0 (−) | |||
GII.4[P16] | none | 7.4 × 102 | 1.3 × 102 * | 5.9 × 107 | 4.6 × 105 (+) | |
1.1 × 103 | 1.3 × 102 * | 7.8 × 106 | 6.1 × 104 (+) | |||
5.4 × 103 | 6.8 × 102 | 1.0 × 107 | 1.5 × 104 (+) | |||
6.9 × 104 | 4.1 × 102 | 5.6 × 105 | 1.4 × 103 (+) | |||
1:3 | 1.0 × 102 | 3.2 × 102 | 2.7 × 105 | 8.4 × 102 (+) | ||
4.0 × 103 | 2.8 × 102 | 1.6 × 107 | 5.7 × 104 (+) | |||
1:10 | 6.3 × 101 | 1.3 × 102 * | 1.7 × 107 | 1.3 × 105 (+) | ||
1.8 × 102 | 1.3 × 102 * | 1.4 × 106 | 1.1 × 104 (+) | |||
2.3 × 102 | 1.3 × 102 * | 1.1 × 102 | 9.0 × 10−1 (−) | |||
3.5 × 102 | Udt | Udt | 0 (−) | |||
1:20 | 4.8 × 101 | 1.5 × 102 | 1.1 × 101 | 7.1 × 10−2 (−) | ||
4.0 × 102 | 1.4 × 102 | 1.9 × 105 | 1.4 × 103 (+) | |||
Fresh Lettuce | GII.6[P7] | None | 2.6 × 104 | 1.9 × 102 | 1.5 × 102 | 7.9 × 10−1 (−) |
3.3 × 104 | 6.1 × 102 | 5.6 × 102 | 9.3 × 10−1 (−) | |||
5.6 × 104 | 3.1 × 102 | Udt | 0 (−) | |||
9.7 × 104 | 9.6 × 102 | 3.4 × 103 | 3.5 × 100 (−) | |||
2.0 × 105 | 1.4 × 103 | 6.5 × 102 | 4.7 × 10−1 (−) | |||
3.7 × 105 | 5.7 × 102 | 1.3 × 102 | 2.3 × 10−1 (−) | |||
5.1 × 105 | 3.0 × 102 | 6.4 × 103 | 2.1 × 101 (+) | |||
1:10 | 2.0 × 104 | 2.2 × 102 | Udt | 0 (−) | ||
3.1 × 104 | 2.9 × 102 | Udt | 0 (−) | |||
4.0 × 104 | 4.1 × 102 | 1.5 × 102 | 3.6 × 10−1 (−) | |||
5.0 × 104 | 2.7 × 102 | 4.2 × 102 | 1.5 × 100 (−) | |||
GII.4[P16] | None | 6.1 × 104 | 6.3 × 102 | 4.4 × 105 | 7.0 × 102 (+) | |
7.2 × 104 | 2.0 × 102 | 2.3 × 105 | 1.2 × 103 (+) | |||
8.0 × 104 | 2.7 × 102 | 7.4 × 103 | 2.8 × 101 (+) | |||
1.3 × 105 | 1.3 × 102 * | 6.6 × 104 | 5.2 × 102 (+) | |||
1.6 × 105 | 5.1 × 102 | 7.8 × 105 | 1.5 × 103 (+) | |||
3.1 × 105 | 3.6 × 102 | Udt | 0 (−) | |||
3.4 × 105 | 4.0 × 103 | 1.1 × 106 | 2.7 × 102(+) | |||
1.2 × 106 | 9.0 × 102 | 1.8 × 107 | 1.9 × 104 (+) | |||
1:10 | 1.4 × 104 | 1.3 × 102 * | 1.3 × 102 | 1.0 × 100 (−) | ||
1.6 × 104 | 1.3 × 102 * | Udt | 0 (−) | |||
2.2 × 104 | 1.3 × 102 * | 9.4 × 104 | 7.3 × 102 (+) | |||
1.2 × 105 | 1.3 × 102 | 5.2 × 105 | 3.9 × 103 (+) |
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Wales, S.Q.; Kulka, M.; Keinard, B.; Ngo, D.; Papafragkou, E. Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce. Foods 2023, 12, 4286. https://doi.org/10.3390/foods12234286
Wales SQ, Kulka M, Keinard B, Ngo D, Papafragkou E. Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce. Foods. 2023; 12(23):4286. https://doi.org/10.3390/foods12234286
Chicago/Turabian StyleWales, Samantha Q., Michael Kulka, Brianna Keinard, Diana Ngo, and Efstathia Papafragkou. 2023. "Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce" Foods 12, no. 23: 4286. https://doi.org/10.3390/foods12234286