Group 3.

<p>n.t.- not tested.</p><p>Fatteners (~ 10 months old). Analysis of sera (s).</p

Summary table.

<p>*- indeterminate</p><p>**—maternal anti-HEV IgG; n.a.-not available; n.d.-not done.</p><p>HEV in minipigs.</p

Group 1.

<p>Adult minipigs “retired breeders”. Analysis of sera (s) and islets cells (i).</p

Group 2.

<p>Adult minipigs. Analysis of kidney (k) and liver (l).</p

Microorganisms tested negative in three “retired breeders” minipigs, age 24–48 months.

<p>Microorganisms tested negative in three “retired breeders” minipigs, age 24–48 months.</p

HEV genome, primers and probes, recombinant proteins and real-time RT-PCR parameters.

<p>(A) Schematic presentation of the HEV genome with three open reading frames (ORF 1–3). UTR—untranslated region. The numbers of nucleotides are given from the first nucleotide of ORF 2. Cap- cap structure, Poly (A)—poly A sequence. PCR primers (black arrows) and probes (red arrows) used in the real-time RT-PCR methods “A”, “J”, “M1”,and “M2” for the detection of HEV ORF2. Primers for methods M1 and M2 are given in brackets as 1 or 2, respectively. Numbers are given as in A. ^*Method “A” was established by Adlhoch et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref041" target="_blank">41</a>]; **Method “J” was established by Jothikumar et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.ref059" target="_blank">59</a>]. (B) Parameters of the used real-time RT-PCR methods. Standard curves and PCR efficiencies are shown. One reference plasmid was used for the real-time RT-PCR method “A” and another, for the other three methods. (C) HEV ORF 2 is coding for the 660 aa long capsid glycoprotein. Putative glycosylation sites are marked in green, a low probability glycosylation site is marked in grey. Positions of the asparagine are numbered. The immunodominant region (IDR) is given in yellow. The signal peptide is shown as a blue arrow. (D) Recombinant proteins (Prospec and GT3) used as antigens in Western blot analysis are shown. GT3 contains the entire immunodominant region (IDR). The Prospec antigen contains glutathion-S-transferase (GST) on the N-terminus. GT3 contains a 6His-tag on the N-terminus. The fragments corresponding to the ORF2 are given in green. The numbers started from the first amino acids of the capsid protein. (E) Comparative analysis of the recombinant antigens by SDS-PAGE is shown on the right. 500 ng of proteins were loaded on the gel, the gel was stained with Coomassie brilliant blue. Lane 1 –Prospec, lane 2 –GT3, M–Size markers.</p

Western blot analysis of sera from Göttingen Minipigs.

<p>(A) Western blot analysis of sera from retired breeders (Group 1). The numbers on strips corresponded to the order of animals in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139893#pone.0139893.t002" target="_blank">Table 2</a>. “+”–serum from a HEV infected pig diluted 1:300; “+ (bold)” serum from infected pig diluted 1:150. The antigen load was 300 ng/strip. Animal sera were tested twice in dilution 1:150. (B) Analysis of sera from sow-piglet pairs (Group 4). Odd numbers 1, 3, 5, 7, 9, 11 –strips incubated with sera from sows; even numbers 2, 4, 6, 8, 10, 12 –strips incubated with sera from piglets. Strip 4 was incubated with serum of piglet #319428. Strips 7, 8 (underlined) were treated with serum of the sow #314253 and serum of piglet #320282, respectively. “+” -serum from HEV infected pig; “-” serum from non-infected pig. The antigen load was 300 ng/strip. The sera specimens were diluted 1:150.</p

Analysis of the HEV sequences revealed in two sows.

<p>(A) Neighbour-joining phylogenetic tree of nucleotide sequences corresponding to a 144 nucleotides region in ORF2. Sequences from the infected sows #31453 and #314451 are given in bold. After the animal number the number of identical clones derived from the same amplicon is given in brackets as (+n). Accession numbers from the GenBank sequences are indicated. The origin of the sequences is indicated as: h-human; s-swine; wb-wild boar; rb-rabbit. The genotype and subgenotype are given as a number and a small letter, respectively. Absence of the letter means that the subgenotype is unknown. Brackets mark the HEV genotypes 3 and 4 (indicated as gt3 and gt4), respectively. The reference (Ref.) plasmid was designed using WHO standard of HEV (Acc. #AB630970). Nucleotide substitutions per 100 nucleotides are indicated. (B) Alignment of deduced amino acid sequences (48 residues) of cloned HEV sequences from sow #314451 and sow #314253 using Clustal W software. Amino acids substitutions associated with reduced infectivity of HEV (underlined in the consensus sequence) were not detected in the cloned sequences.</p

Lack of HEV in liver and kidney tissues from adult Göttingen minipigs.

<p>Three different duplex real-time RT-PCR systems “A”, “J”, and “M2” were used to screen for HEV in liver and tissue of the animals from Group 2 using 500 ng of total RNA for the analysis. Porcine cyclophilin A gene was used as a house keeping control and equal load of RNA was confirmed. Serial 10-fold dilutions (10E5-10E2) of the reference plasmid were used as a control for the system “J”. Analyses were performed in duplicates. In addition, minipigs from Group 2 were also found negative when RNA specimens were tested using a conventional semi-nested RT-PCR (data not shown).</p

Function of macroencapsulated rat islet grafts.

<p>STZ-induced diabetic minipigs were transplanted with 6,730±475 IEQ/kg BW of rat islets immobilized and integrated into the macrochamber system. ivGTT was performed prior to graft implantation, 2 weeks after implantation, and after retrieval of the graft. BW was recorded daily throughout the observation period. (<b>A</b>) Fasting blood glucose levels (black) of group one transplanted minipigs (n = 5). The graft was removed on day 30 and hyperglycemia recurred demonstrating that graft function was responsible for normoglycemia during the implantation period. Rat C-peptide levels (grey) are presented as 4^th grade polynomial curves. Error bars represent SD. <i>P</i><0.001 for comparing glucose levels during the implantation period vs the pre- and post-implantation periods (t-test). (<b>B</b>) Blood glucose levels during ivGTT of transplanted animals at two weeks (black circles) and after retrieval of the graft-containing device (black triangles). Diabetic minipigs (n = 24; double grey), naïve healthy mini-pigs (n = 11; black dashed) and naïve healthy rats (n = 36; grey dashed) served as controls. Error bars represent SD. <i>P</i><0.001 for comparing AUCs (diabetic minipigs) in the implantation period vs the pre- and post-implantation periods (t-test). (<b>C</b>) Two-day continuous glucose monitoring records of an implanted animal during week 2. (<b>D</b>) Fasting blood glucose levels (black) of group 2 transplanted minipigs (n = 3) and corresponding BW (grey). Normoglycemia was achieved rapidly after transplantation and was retained until the BW increased to a critical level of >160% of the initial BW. Data are presented as 5^th grade polynomial curves. <i>P</i><0.001 for comparing glucose levels during the first 75 days of the implantation period vs the pre-implantation period (t-test).</p