Schematic diagrams of genomic structure, the strategy for genomic cDNA cloning, and molecular characterization of unique features of three emergent U.S.

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Schematic diagrams of genomic structure, the strategy for genomic cDNA cloning, and molecular characterization of unique features of three emergent U.S. PEDV strains (MN, IA1, and IA2) isolated in Minnesota and Iowa in 2013. Schematic diagrams of genomic structure, the strategy for genomic cDNA cloning, and molecular characterization of unique features of three emergent U.S. PEDV strains (MN, IA1, and IA2) isolated in Minnesota and Iowa in 2013. (A) Eight overlapping cDNA fragments covering the entire PEDV genome for each of the three U.S. strains, represented by thick lines, were amplified by RT-PCR from total RNAs extracted from fecal or small intestine samples. The names of each fragment are indicated. The location and primer sequences for the RT-PCR and the RACE-PCR used to generate the overlapping RT-PCR products are available in Table S1 in the supplemental material. The numbers on the scale bar indicate distances from the 5′ end of the genome. (B) Organization of the PEDV genome and locations of unique amino acid (aa) changes identified in the three U.S. strains. The approximate positions and sizes of genes in the PEDV genome that correspond to the scale bar are shown in panel A. The putative S1/S2 boundary (amino acid positions) of the S protein is also shown. Nucleotide (nt) and amino acid differences between the 3 emergent U.S. PEDV sequences and the consensus sequences of 23 other known PEDV strains and their positions are depicted. Con, PEDV consensus sequences; US, unique sequences in three U.S. PEDV strains; *, unique amino acids and nucleotides shared by the three U.S. strains and one Chinese AH2012 strain; UTR, untranslated region; nsp, nonstructural protein; Ac, acidic domain; PLP, papain-like proteinase; X, X domain (ADP-ribose-1′-phosphatase [ADRP]); Y, unknown Y domain; NTD, N-terminal domain of the spike gene. (C) Comparison of antigenic index profiles of the NTD (aa 1 to 380) of S protein between the prototype strain CV777 (genogroup 1 [G1]) and U.S. strain MN (genogroup 2 [G2]). The corresponding alignment of amino acid sequences and positions of the two regions containing amino acid deletions/insertions (indicated by dashes and shaded) are shown. DR, region containing deletions. Identical amino acids are marked in blue, whereas mismatches are marked in red. Favorable amino acid mismatches are displayed as colons, whereas neutral mismatches are depicted as periods. The predicted N-linked glycosylation sites in DR1 and DR2 are indicated by arrowheads. (D) Comparisons of the primary sequences and the predicted secondary structures of a 5′-proximal region in the 5′-UTR between Belgian PEDV/CV777 (nt 42 to 133), U.S. PEDV/MN (nt 42 to 129), and a bat coronavirus BtCoV/512/2005 (nt 44 to 131). Three deletions of nucleotides (no. 1 to 3) are indicated by dashes and shaded in the primary sequences and are indicated by arrows in the secondary structures. The core sequences (CUAAAC) of leader transcription-regulating sequences (CS-L) are boxed by dashed lines. Two stem-loops (SL2 and SL4) conserved in all coronaviruses are indicated. (There is no SL3 in alphacoronaviruses.) Nucleotides in the stems are marked in blue, whereas nucleotides in the loops are marked in red. Yao-Wei Huang et al. mBio 2013; doi:10.1128/mBio.00737-13