A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Viruses
- (1)
- (2)
- A leftover viremic serum sample from a patient acutely infected with HEV gt3c (National Reference Center for viral hepatitis, Sciensano, Belgium).
2.2. HEV Cell Culture Infection Experiments
2.2.1. Cell Lines and Media
2.2.2. HEV Inoculation
2.2.3. Immunofluorescence Assay
2.3. Capsid Integrity Assays
2.4. RNA Extraction, Purification and Detection
2.5. Genome Integrity Assay: Two-Step Long-Range RT-qPCR
2.6. Statistical Analysis
3. Results
3.1. Evaluation of HEV Cell Culture Models
3.1.1. A549-D3 and HuH7-S10-3 Cells Are Promising for a Cell Culture-Based HEV Infection Model
3.1.2. FBS and DMSO Positively Contribute to HEV Propagation in A549-D3 and HuH7-S10-3 Cells
3.1.3. The Peak of HEV Concentration in Supernatant of A549-D3 and HuH7-S10-3 Cells Is Reached after Five and Six Days, Respectively
3.2. PtCl4 and RNase A Combined with Conventional RT-qPCR Can Be Used as HEV Capsid Integrity Assays
3.3. Long-Range RT-qPCR Can Be Used as a Proxy for Detecting Intact HEV Genomes
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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Name | Composition |
---|---|
MMEM-0 | Maintenance * MEM with 0% FBS |
MMEM-2 | MMEM-0 + 2% FBS |
MMEM-5 | MMEM-0 + 5% FBS |
MMEM-10 | MMEM-0 + 10% FBS |
MMEM-0-D | MMEM-0 + 2% DMSO |
MMEM-2-D | MMEM-2 + 2% DMSO |
MMEM-5-D | MMEM-5 + 2% DMSO |
MMEM-10-D | MMEM-10 + 2% DMSO |
MDMEM-0 | Maintenance * DMEM with 0% FBS |
MDMEM-2 | MDMEM-0 + 2% FBS |
MDMEM-5 | MDMEM-0 + 5% FBS |
MDMEM-10 | MDMEM-0 + 10% FBS |
MDMEM-0-D | MDMEM-0 + 2% DMSO |
MDMEM-2-D | MDMEM-2 + 2% DMSO |
MDMEM-5-D | MDMEM-5 + 2% DMSO |
MDMEM-10-D | MDMEM-10 + 2% DMSO |
Pair | Target | Forward (FW) Reverse (RV) Probe (Pb) | Sequence 5′-3′ | Start (Position) | Stop (Position) | Length (Nucleotides) |
---|---|---|---|---|---|---|
1 | Pre-methyltransferase (MeT) | FW | CTACTGCCATTGAGCAGGC | 68 | 86 | 126 |
RV | GCTGCCGGGGTTGCATC | 193 | 178 | |||
Pb | CTGTGGTGGTTCGGCCGTT | 121 | 140 | |||
2 | Pre-methyltransferase (MeT) | FW | CTACTGCCATHGAGCARGC | 68 | 86 | 126 |
RV | GCTGCCGGGGTTGCATC | 193 | 178 | |||
Pb | CTGTGGTGGTTCGGCCGTT | 121 | 140 | |||
3 | Pre-methyltransferase (MeT) | FW | CTTGGCGAATGCTGTGGTG | 111 | 129 | 83 |
RV | GCTGCCGGGGTTGCATC | 193 | 178 | |||
4 | Pre-methyltransferase (MeT) | FW | ACTACTGCCATTGAGCAGGC | 67 | 86 | 80 |
RV | GATAAAAACGGCCGAACCACCAC | 146 | 124 | |||
5 | Pre-methyltransferase (MeT) | FW | GTGGTCGATGCCATGGAGG | 19 | 37 | 128 |
RV | GATAAAAACGGCCGAACCACCAC | 146 | 124 | |||
6 | Y-domain & papain-like cysteine protease (PCP) | FW | CGYCAGCTYCAGTTTTATGC | 1306 | 1325 | 173 |
RV | CAGGTRCACTCCTGCCC | 1478 | 1462 | |||
7 | Y-domain & papain-like cysteine protease (PCP) | FW | CGYCAGCTYCAGTTTTAYGC | 1306 | 1325 | 173 |
RV | CAGGTRCACTCCTGCCC | 1478 | 1462 | |||
8 | methyltransferase (MeT) [73] | FW | CTCCTGGCRTYACWACTGC | 56 | 74 | 172 |
RV | GGRTGRTTCCAIARVACYTC | 227 | 208 |
Primer Pair | Concentration Forward Primer | Concentration Reverse Primer | MasterMix | Efficiency | Slope | Y-Intercept |
---|---|---|---|---|---|---|
1 | 600 nM | 900 nM | TaqMan environmental | 1.971 | −3.394 | 33.75 |
6 | 250 nM | 250 nM | SSO advanced SYBR Green inhibitor tolerant | 1.971 | −3.393 | 32.24 |
Parameter | PtCl4 | RNase A | Long-Range RT-qPCR | Cell Culture |
---|---|---|---|---|
Infectivity assessment | Indirect (Capsid integrity) | Indirect (Capsid integrity) | Indirect (genome integrity) | Direct |
Time | +++ | +++ | ++ | + |
User friendly | +++ | +++ | + | + |
Cost price | +++ | ++ | + | + |
Sensitivity | +++ | +++ | ++ | ++ |
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Locus, T.; Lambrecht, E.; Lamoral, S.; Willems, S.; Van Gucht, S.; Vanwolleghem, T.; Peeters, M. A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment. Vet. Sci. 2023, 10, 676. https://doi.org/10.3390/vetsci10120676
Locus T, Lambrecht E, Lamoral S, Willems S, Van Gucht S, Vanwolleghem T, Peeters M. A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment. Veterinary Sciences. 2023; 10(12):676. https://doi.org/10.3390/vetsci10120676
Chicago/Turabian StyleLocus, Tatjana, Ellen Lambrecht, Sophie Lamoral, Sjarlotte Willems, Steven Van Gucht, Thomas Vanwolleghem, and Michael Peeters. 2023. "A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment" Veterinary Sciences 10, no. 12: 676. https://doi.org/10.3390/vetsci10120676