Fecal and serum samples used in this study were collected from 95 pigs of 2 to 4 months of age and one 7-month-old pig from 37 different herds in six U.S. states (Arkansas, Iowa, Michigan, Missouri, North Carolina, and Oklahoma). Seventy fecal samples were obtained from pigs submitted to the Iowa State University Veterinary Diagnostic Laboratory. Sixteen serum and 10 fecal samples were obtained from pigs submitted to the University of Missouri Veterinary Medical Diagnostic Laboratory. The pigs were submitted to the Diagnostic Laboratories for a wide variety of health problems that are not related to swine HEV infection. Fecal samples from rectal swabs were resuspended in 10% calcium- and magnesium-free phosphate-buffered saline. Serum and fecal samples were stored at −70°C until analyzed.
Primer design for RT-PCR.
To develop a “universal” reverse transcription-PCR (RT-PCR) assay that is capable of detecting HEV strains with significant sequence variations, a multiple sequence alignment of the ORF2 genes of 18 different known strains of human HEV and the prototype U.S. strain of swine HEV was performed (21
). Based upon the multiple sequence alignment, two sets of degenerate HEV primers were designed for the universal nested RT-PCR assay: external primer set 3156N [forward, 5′-AATTATGCC(T)CAGTAC(T)CGG(A)GTTG-3′] and 3157N [reverse, 5′-CCCTTA(G)TCC(T)TGCTGA(C)GCATTCTC-3′] and internal primer set 3158N [forward, 5′-GTT(A)ATGCTT(C)TGCATA(T)CATGGCT-3′] and 3159N [reverse, 5′-AGCCGACGAAATCAATTCTGTC-3′]. The expected product of the universal nested RT-PCR was 348 bp.
In addition, a published nested RT-PCR assay (16
) specific for the prototype U.S. strain of swine HEV was also used in this study. The primers for the specific RT-PCR assay were based upon the published sequence of the prototype U.S. strain of swine HEV (21
): external primer set 3329 (forward, 5′-AGCTCCTGTACCTGATGTTGACTC-3′) and 3330 (reverse, 5′-CTACAGAGCGCCAGCCTTGATTGC-3′) and internal primer set 3331 (forward, 5′-GCTCACGTCATCTGTCGCTGCTGG-3′) and 3332 (reverse, 5′-GGGCTGAACCAAAATCCTGACATC-3′).
Development and standardization of the universal HEV RT-PCR assay for detection of field isolates of swine HEV.
Since HEV strains (including swine HEV) identified from different geographic regions are genetically heterogenic, a universal HEV RT-PCR assay with degenerate HEV primers was developed in this study to detect genetically divergent strains of HEV. To evaluate if the universal RT-PCR assay with degenerate HEV primers could detect known strains of HEV with significant sequence variations, total RNAs were extracted with TriZol Reagent (GIBCO-BRL) from 100 μl of the US2 strain of human HEV (4
), the Pakistani strain (Sar-55) of human HEV (38
), and the prototype U.S. strain of swine HEV (21
). Total RNAs were resuspended in DNase- and RNase-free water. RT was performed at 42°C for 60 min with Superscript II reverse transcriptase (GIBCO-BRL) using reverse primer 3157N. Five microliters of the resulting cDNA was amplified by the universal RT-PCR assay using Ampli Taq
gold DNA polymerase (Perkin-Elmer). The PCR parameters for the first-round PCR with primers 3156N and 3157N included a denaturation step at 95°C for 9 min, followed by 39 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 42°C, extension for 2 min at 72°C, and a final incubation at 72°C for 7 min. The parameters for the second-round PCR were similar, except that primers 3158N and 3159N were used.
To determine the sensitivity of the universal RT-PCR assay, the prototype U.S. strain of swine HEV (21
) with a known infectious titer of 104.5
50% pig infectious doses (PID50
) per ml was serially diluted 10-fold in phosphate-buffered saline. Total RNAs extracted from 100 μl of each dilution were tested using the universal RT-PCR assay, as well as with a published RT-PCR assay (22
) specific for the prototype U.S. strain of swine HEV. The PCR parameters for the universal PCR assay are the same as described above. The first- and second-round PCR parameters for the RT-PCR assay specific for the prototype swine HEV were similar, with an initial denaturation step at 95°C for 9 min, followed by 39 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 54°C, extension for 1 min at 72°C, and a final incubation at 72°C for 7 min.
After the universal RT-PCR assay with degenerate HEV primers was standardized, 80 fecal and 16 serum samples from pigs in 37 different herds of different geographic regions (Table ) were tested by the universal RT-PCR assay. Negative and positive controls were included in each set of PCRs. The negative control was water treated the same way as the fecal suspensions and sera. The positive control was the prototype U.S. strain of swine HEV. The amplified PCR products were examined by agarose gel electrophoresis.
Detection of swine HEV RNA from fecal and serum samples of pigs of 2 to 4 months of age from different herds in the United States
The expected PCR products amplified from fecal or serum samples of pigs were purified using the glassmilk procedure with a GENECLEAN kit (Bio 101 Inc.). PCR products amplified from 27 selected pigs were directly sequenced at the Virginia Tech DNA Sequencing Facility. Sequences of the PCR products were determined for both DNA strands.
Sequence and phylogenetic analyses.
The primer sequences used to amplify the field isolates of swine HEV were excluded for the final sequence and phylogenetic analyses. The resulting 304-bp sequences in the ORF2 genes of the 27 U.S. isolates of swine HEV were analyzed and compared with the corresponding regions of other known human, swine, and avian HEV strains available in GenBank by the MacVector computer program (Oxford Molecular Inc.) (Table ). The percentages of nucleotide and amino acid sequence identities among different HEV strains were determined with the MacVector program. Phylogenetic analysis was conducted with the aid of the PAUP program (from David L. Swofford, Smithsonian Institution, Washington, D.C.; distributed by Sinauer Associates Inc., Sunderland, Mass.). A heuristic search with 1,000 replicates was used to produce a phylogenetic tree.
Pairwise comparison of the nucleotide sequences of the partial ORF2 gene of 27 swine HEV isolates identified in this study (in boldface) and other selected strains of HEV worldwidea
The geographic origins and the GenBank accession numbers of the nucleotide sequences of the HEV strains used in the phylogenetic and sequence analyses are as follows: TK78/87 (AF020608, Nepal), TK104/91 (AF020603, Nepal), TK15/92 (AF020604, Nepal), Nep4/94 (AF020607, Nepal), TK4/95 (AF020606, Nepal), Hyderabad (AF076239, India), M75 (AF093894, India), AKL-90 (AF124407, India), Y67 (AF093892, India), Madras (X99441, India), Hev037 (X98292, India), Sar-55 (M80581, Pakistan), abb-2B (U40044, Pakistan), Vietnam (AF170450), Burma (M73218), JRA1 (AP003430, Japan), JPHEV (E17109, Japan), Uigh179 (D11093, China), Lanzhou (AF141652, China), K52-87 (L25595, China), Hetian (L08816, China), Ch-T11 (AF151962, China), Ch-T21 (AF151963, China), Ch-T1 (AJ272108, China), Tw6310e (AF117279, Taiwan), Tw8e-2 (AF117275, Taiwan), Tw6196e (AF117278, Taiwan), Tw2494e (AF117276, Taiwan), Tw5483e (AF117277, Taiwan), Tw32sw (AF117280, Taiwan swine HEV), Tw74sw (AF117281, Taiwan swine HEV), Morocco (AF065061, Morocco), Mexico (M74506), VH1 (AF195061, Spain), VH2 (AF195062, Spain), E11 (AF195063, Spain), prototype swine HEV (AF082843, United States), HEV-US1 (AF060668, United States), HEV-US2 (AF060669, United States), and avian HEV (AY043166, United States).
Nucleotide sequence accession number.
The resulting sequences of the 27 swine HEV isolates described in “Nucleotide sequencing” above have been deposited with the GenBank database under accession numbers AF466659 to AF466685.