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1.
Fig 9

Fig 9. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Effect of S17 insert on Sar55 successful transfection of S10-3 and BHK-21 cells. The efficiency of transfection of S10-3 (A) and BHK-21 (B) cells was monitored by flow cytometry. (C) Immunofluorescence microscopy of transfected BHK-21 cells stained for ORF2 protein at day 5 posttransfection.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
2.
Fig 1

Fig 1. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Diagram of the HEV genome summarizing the location and size, in amino acids, of nonviral sequences inserted into the HVR region. S17, human S17 ribosomal protein fragment in the Kernow strain; GTPase, GTPase-activating protein fragment in the Kernow strain; S19, S19 ribosomal protein fragment in the LBPR strain (21).

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
3.
Fig 8

Fig 8. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Genomes or viruses encoded by p6 can replicate in, and infect swine LLC-PK1 cells. (A) Swine cells transfected with transcripts from p6delS17 or p6 containing the S17 insert were assayed by flow cytometry at day 5 posttransfection. (B) Triplicate samples of p6 virus harvested from the medium of transfected HepG2/C3A cells were titered in parallel on HepG2/C3A cells (open bars) and LLP-CK1 cells (stippled bars) under code.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
4.
Fig 4

Fig 4. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Removal of S17 sequence from p6 eliminates the adaptive effect of most point mutations. S10-3 cells were transfected with p1 and p6 plasmids with or without S17 sequence. Triplicate samples were analyzed by flow cytometry at day 4 posttransfection. P values were all <0.0001 except for p1/S17 versus p6delS17. Error bars denote the standard deviations.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
5.
Fig 6

Fig 6. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Synonymous mutations in the S17 insert have little effect on the efficiency of successful transfections. Mutations that preserved the amino sequence were introduced into the third base of 54/58 codons (mutant 1) or 41/58 codons (mutant 2) in theS17 insert, and RNA transcripts were transfected into S10-3 cells. The efficiency of successful transfection was determined by flow cytometry of triplicate samples at 6 days posttransfection.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
6.
Fig 3

Fig 3. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Reversion of aa 882, 904, and 965 in the X region reduces the level of successful transfection. S10-3 cells were transfected with the 5′ORF1 plasmid lacking the CCA and X region mutations (fragment 671-2182), the ORF1/CCA plasmid containing both the CCA and X region mutations (fragment 2182-3063), p6, and a revertant plasmid containing the CCA but not the X region mutations (p6/882; 904; 965 revert). Cells in triplicate samples were immunostained and analyzed by flow cytometry at 6 days posttransfection. P values are given, and error bars denote the standard deviations.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
7.
Fig 10

Fig 10. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Lack of the ORF3 protein does not inhibit cell-to-cell spread in HepG2/C3A cultures. HepG2/C3A cells were electroporated with transcripts from p6 or p6/ORF3-null plasmids, mixed with naive HepG2/C3A cells and cultured at 37°C. Triplicate samples were harvested on each of indicated days, fixed with methanol, and stored at −80°C until assayed by flow cytometry. The results of representative flow cytometry scans are shown. Error bars indicate the standard deviation. P = 0.74 for day 5 values of the two viruses, indicating that a similar number of cells had been successfully transfected with each construct.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
8.
Fig 5

Fig 5. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Expression of luciferase from ORF2 is substantial and prolonged in the presence of the S17 insert. The ORF2 viral capsid protein was replaced with the gaussia luciferase gene in p6 genomes lacking the S17 insert or the X gene region mutations. After transfection of S10-3 cells, culture medium was completely replaced every 24 h. (A) The ratio of luciferase units produced by p6/luc genomes with (solid bars) or without (hatched bars) the S17 insert is shown in parentheses above each time point. Error bars are standard deviation. (B) The average luciferase production from genomes encoded by two independent cDNA clones (stippled and crosshatched bars) lacking the three X gene mutations was decreased 2.3- to 5.1-fold compared to that from p6/luc genomes (ratios are shown in parentheses above each time point).

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
9.
Fig 2

Fig 2. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Transfection of S10-3 cells with sequential plasmid constructs. Representative flow cytometry profiles of nontransfected and transfected cells are shown on the left. Numbers beneath each clone indicate the fragment position on the genome that was replaced with the corresponding fragment amplified from the passage 6 virus quasispecies; the parentheses indicate gene regions included in the fragment. The new construct served as the background plasmid for the next replacement, and the procedure was repeated until all of the p1 had been replaced with p6 sequences. Finally, all plasmids were transcribed, transfected and immunostained for ORF2 protein in the same experiment: triplicate samples were harvested and tested by flow cytometry at 3 days posttransfection. Student t test P values are given for adjacent samples. P ≤ 0.05 was considered significant. Error bars indicate the standard deviations.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.
10.
Fig 7

Fig 7. From: Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination.

Comparison of efficiency of successful transfection by p6 genomic transcripts encoding different HVRs. Triplicate samples were subjected to flow cytometry at day 5 posttransfection. Error bars represent the standard deviation, and brackets denote Student t test P values. (A) The 174 nt encoding the 58-aa S17 insert were deleted or replaced with the 114-nt GTPase insert from passage 1 or with the 3′-terminal 174 nt of green fluorescent protein (GFP). #1 and #2 are two independent clones. P values for p6 versus any other genome were ≤0.0003. (B) 5′ 174 nt encoding the first 58 aa of GFP. # 1 to #3 are independent clones. P values for p6 versus any other genome were ≤0.001. (C) The 5′ half, middle, or 3′ half (87 nt) of the S17 insert replaced full-length S17. P values for p6 versus any other genome were ≤0.0002. (D) 117 nt S19 ribosomal protein gene insert. #1 and 2 are independent clones. The P values for p6 versus any other genome were ≤0.001, and the P values among the three GFP clones were ≤0.27.

P. Shukla, et al. J Virol. 2012 May;86(10):5697-5707.

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