. 2022 Dec 13;11(1):e03872-22. doi: 10.1128/spectrum.03872-22

TABLE 3.

Point mutation primers for constructing transfer vectors used to rescue recombinant PEDVs

Recombinant virus	Transfer vector	Point mutation template	Primer name	Primer location (positions)^a	Primer sequence (5′–3′)^b	Mutant amino acid site(s)
r-S^parA^att	p-PEDV-S^parA^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant1	1807–1840	TACCCTGAGTTCGGTAGTGGTGTTAAGTTTACGT	605, 612
r-S^parA^att	p-PEDV-S^parA^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant2	1881–1907	CACGCCTAAACCACTTCAAGGTGTCAC	633, 635

r-S^parB^att	p-PEDV-S^parB^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant3	1984–2008	CTTACAAATTCTAGCTTTTTGGCAG	667
r-S^parB^att	p-PEDV-S^parB^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant4	2101–2126	GAGCAGGCTGCATATGTTGATGATG	707

r-S^parC^att	p-PEDV-S^parC^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant5	2278–2302	GGCTATGTTCCACTTCAGGATGGCC	764, 766

r-S^parD^att	p-PEDV-S^parD^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant6	2306–2334	GTCAAGATTGCACCCATGGTTACTGGG	774
			PEDmutant7	2407–2429	GTTGATTGTGTTACATATGTTTG	806
			PEDmutant8	2616–2637	CTAATGTGCTGGGTGTTTCCGTG	877

r-S^parE^att	p-PEDV-S^parE^att-ΔORF3/RLuc	p-PEDV-S^par-ΔORF3/RLuc	PEDmutant9	2662–2688	TGGTACAAAAAGGGTCTTTTATTGAAG	891, 893
r-S^attA^par	p-PEDV-S^attA^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant11	1807–1840	TACCCTGCGTTCGGTAGTGGTGTTAAGTTGACGT	605, 612
r-S^attA^par	p-PEDV-S^attA^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant12	1881–1907	CACGCCTAAACCACTTGAAGGTATCAC	633, 635

r-S^attB^par	p-PEDV-S^attB^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant13	1984–2008	CTTACAAATTCTAGCATTTTGGCAG	667
r-S^attB^par	p-PEDV-S^attB^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant14	2101–2126	GAGCAGGCTGCATATGTTAATGATG	707

r-S^attC^par	p-PEDV-S^attC^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant15	2278–2302	GGCTATGTTCCACCTCAGTATGGCC	764, 766

r-S^attD^par	p-PEDV-S^attD^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant16	2306–2334	GTCAAGATTGCACCCACGGTTACTGGG	774
			PEDmutant17	2407–2429	GTTGATTGTGCTACATATGTTTG	806
			PEDmutant18	2616–2637	CTAATGTGCTGGGTGCTTCCGTG	877

r-S^attE^par	p-PEDV-S^attE^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant19	2660–2688	TGGTACAAAAAAGGTCTGTTATTGAAG	891, 893

r-S^parAB^att	p-PEDV-S^parAB^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant3	1984–2008	CTTACAAATTCTAGCTTTTTGGCAG	667
r-S^parAB^att	p-PEDV-S^parAB^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant4	2101–2126	GAGCAGGCTGCATATGTTGATGATG	707

r-S^parAC^att	p-PEDV-S^parAC^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant5	2278–2302	GGCTATGTTCCACTTCAGGATGGCC	764, 766

r-S^parAD^att	p-PEDV-S^parAD^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant6	2306–2334	GTCAAGATTGCACCCATGGTTACTGGG	774
			PEDmutant7	2407–2429	GTTGATTGTGTTACATATGTTTG	806
			PEDmutant8	2616–2637	CTAATGTGCTGGGTGTTTCCGTG	877

r-S^parAE^att	p-PEDV-S^parAE^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant9	2660–2688	TGGTACAAAAAGGGTCTTTTATTGAAG	891, 893

r-S^attAE^par	p-PEDV-S^attAE^par-ΔORF3/RLuc	p-PEDV-S^attA^par-ΔORF3/RLuc	PEDmutant19	2660–2688	TGGTACAAAAAAGGTCTGTTATTGAAG	891, 893

r-S^parA^att891^att	p-PEDV-S^parA^att891^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant10	2662–2686	GTACAAAAAGGGTCTGTTATTGAAG	891

r-S^parA^att893^att	p-PEDV-S^parA^att893^att-ΔORF3/RLuc	p-PEDV-S^parA^att-ΔORF3/RLuc	PEDmutant24	2662–2686	GTACAAAAAGGGTCTTTTATTGAAG	893

r-S^par(612, 633, 635, 891)^att	p-PEDV-S^par(612, 633, 635, 891)^att-ΔORF3/RLuc	p-PEDV-S^parA^att891^att-ΔORF3/RLuc	PEDmutant20	1807–1840	TACCCTGCGTTCGGTAGTGGTGTTAAGTTTACGT	605

r-S^par(605, 633, 635, 891)^att	p-PEDV-S^par(605, 633, 635, 891)^att-ΔORF3/RLuc	p-PEDV-S^parA^att891^att-ΔORF3/RLuc	PEDmutant21	1807–1840	TACCCTGAGTTCGGTAGTGGTGTTAAGTTGACGT	612

r-S^par(605, 612, 635, 891)^att	p-PEDV-S^par(605, 612, 635, 891)^att-ΔORF3/RLuc	p-PEDV-S^parA^att891^att-ΔORF3/RLuc	PEDmutant22	1881–1907	CACGCCTAAACCACTTGAAGGTGTCAC	633

r-S^par(605, 612, 633, 891)^att	p-PEDV-S^par(605, 612, 633, 891)^att-ΔORF3/RLuc	p-PEDV-S^parA^att891^att-ΔORF3/RLuc	PEDmutant23	1881–1907	CACGCCTAAACCACTTCAAGGTATCAC	635

r-S^par(605, 633, 891)^att	p-PEDV-S^par(605, 633, 891)^att-ΔORF3/RLuc	p-PEDV-S^par(605, 633, 635, 891)^att-ΔORF3/RLuc	PEDmutant23	1881–1907	CACGCCTAAACCACTTCAAGGTATCAC	635

r-S^att(605, 633, 891)^par	p-PEDV-S^att(605, 633, 891)^par-ΔORF3/RLuc	p-PEDV-S^att-ΔORF3/RLuc	PEDmutant20	1807–1840	TACCCTGCGTTCGGTAGTGGTGTTAAGTTTACGT	605
			PEDmutant22	1881–1907	CACGCCTAAACCACTTGAAGGTGTCAC	633
			PEDmutant24	2662–2686	GTACAAAAAGGGTCTTTTATTGAAG	891

The location of the primers is relative to the S gene sequence of the PEDV DR13^par strain (GenBank accession number DQ862099).

The mutated nucleotides of the primers are marked in boldface type.