Background
Methods
Construction of a full-length cDNA clone of HEV
Primer ID | Sequence (5′~ 3′) | Product | Length (bp) | Reference |
---|---|---|---|---|
S11 | AGGCTCCTGGCRTYACTACTG | F1 | 1163 bp | 2 |
S12 | GCCYTGGCGAATGCTGTG | |||
A11 | GGCCRGGAATGTAATCACG | |||
A12 | GCGGCACTGGGCRTAAAACT | |||
HEV-A1 | AAAGGAATGAAGAGGCTGGAG | F2 | 1520 bp | This study |
HEV-A3 | GAAAAGTCTGGCCGTGATTAC | |||
HEV-B2 | TCCTCAGTAATAGTAAGGGC | |||
HEV-B4 | AGGTCGATGGTTACGTTCCC | |||
HEV-C1 | TGCCTGTTGGGCTGAGTTTTGATG | F3 | 1394 bp | This study |
HEV-C3 | GCCAGCCATAGCTTGGTTTGAAG | |||
D1R | AAGGTCTTGCTCCACGCAGATATC | |||
D3R | CTGGAAGAATGTTATACGAGACAC | |||
D2F | CTTGTGGAGGCCATGGTGGAGAA | F4 | 1243 bp | This study |
D4F | ATGGTGGAGAAAGGCCAGGAT | |||
ER1 | TCACGCCAAGCGGAGCCGAGT | |||
ER2 | GAAGGGGTTGGTTGGATGAAT | |||
EF1 | TTTCTGGGGTGACCGGGTTGATT | F5 | 1791 bp | This study |
EF2 | CTATATTCATCCAACCAACCCCT | |||
HEV31 | CAGGGAGCGCGGAACGCAGAAAAGA | |||
HEV32 | TCAATACTCCCGAGTTTTACCCACC | |||
5' Race-A1 | GCAGTGARTARAGYGCAAYCCCHGTCT | 5' RACE | 501 bp | 2 |
5' Race-A2 | CGRGCCATYGCCTCNGCRACATC | |||
3' Race-S1 | ACYACNACTGCTGCYACACGBTTYATGA | 3' RACR | 936 bp | 2 |
3' Race-S2 | CTYTGTTYAAYCTTGCTGAYACGCTKCTC | |||
T7P1 | GCCTAGCTAGCTAGTCTAGATAATACGACTCACTATA | 5′ terminal | – | This study |
PAP2 | CGGTCGACCGATCGATTTTTTTTTTTTTTTTTTTTTTTTTCAGGGAGCGC | 3′ terminal | – | This study |
HEV1 | AATTATGCC(T)CAGTAC(T)CGG(A)GTTG | Positive strand of HEV RNA | 348 bp | [23] |
HEV2 | CCCTTA(G)TCC(T)TGCTGA(C)GCATTCTC | |||
HEV3 | GTT(A)ATGCTT(C)TGCATA(T)CATGGCT | |||
HEV4 | AGCCGACGAAATCAATTCTGTC | |||
HEV6 | AGCTCCTGTACCTGATGATGTTGACTC | Negative strand of HEV RNA | 266 bp | [24] |
HEV7 | CTACAGAGCGCCAGCCTTGATTGC | |||
HEV8 | GCTCACGTCATCTGTCGCTGCTGG | |||
HEV9 | GGGCTGAACCAAAATCCTGACATC | |||
WHO-F | GGTGGTTTCTGGGGTGAC | qRT-PCR | 70 bp | [25] |
WHO-R | AGGGGTTGGTTGGATGAA |
In vitro transcription of capped full-length HEV genomic RNA
Cell transfection and immunofluorescence assay
Inoculation of BALB/c mice with capped RNA transcripts
Detection of HEV RNA in feces, serum and tissues by nested RT-PCR and quantitative RT-PCR
Determination of HEV antibodies by ELISA
Profile liver biochemistry in serum
Detection of HEV ORF2 protein in tissues by Western blotting
Infection of A549 cells with viruses recovered from mouse inoculated with capped RNA transcripts of swine HEV clones
Histopathologic examination
Statistical analysis
Results
A full-length cDNA clone of swine HEV is infectious
Capped RNA transcripts of pGEM-HEV was infectious in BALB/c mice
Sample | Strand | HEV RNA transcripts | Control | |
---|---|---|---|---|
Feces | 0 dpi | Positive | – | – |
3 dpi | Positive | +(1/3) | +(0/3) | |
4 dpi | Positive | +(2/3) | +(0/3) | |
7 dpi | Positive | +(3/3) | +(0/3) | |
14 dpi | Positive | +(3/3) | +(0/3) | |
21 dpi | Positive | +(3/3) | +(0/3) | |
28 dpi | Positive | +(3/3) | +(0/3) | |
Serum | 0 dpi | Positive | +(0/3) | +(0/3) |
Negative | +(0/3) | +(0/3) | ||
7 dpi | Positive | +(3/3) | +(0/3) | |
Negative | +(1/3) | +(0/3) | ||
14 dpi | Positive | +(3/3) | +(0/3) | |
Negative | +(2/3) | +(0/3) | ||
21 dpi | Positive | +(3/3) | +(0/3) | |
Negative | +(3/3) | +(0/3) | ||
28 dpi | Positive | +(3/3) | +(0/3) | |
Negative | +(2/3) | +(0/3) | ||
Tissues | Liver | Positive | +(3/3) | +(0/3) |
Negative | +(3/3) | +(0/3) | ||
Spleen | Positive | +(3/3) | +(0/3) | |
Negative | +(3/3) | +(0/3) | ||
Kidney | Positive | +(2/3) | +(0/3) | |
Negative | +(2/3) | +(0/3) | ||
Colon | Positive | +(0/3) | +(0/3) | |
Negative | +(1/3) | +(0/3) |