Isolated and characterized U.S. (IFA) assays using the prototype and S-INDEL-variant strains as signal viruses; 2) trojan neutralization (VN) lab tests against the prototype and JNK-IN-7 S-INDEL-variant infections; 3) PEDV prototype stress whole virus structured ELISA; 4) PEDV prototype stress S1-structured ELISA; and 5) PEDV S-INDEL-variant stress S1-structured ELISA. The positive antisera against the prototype stress reacted to and neutralized both prototype and S-INDEL-variant infections, as well as the positive antisera against the S-INDEL-variant stress reacted to and neutralized both prototype and S-INDEL-variant infections also, as analyzed by IFA antibody JNK-IN-7 assays and VN lab tests. Antibodies against both PEDV strains could possibly be discovered by all three ELISAs although recognition rates varied to some extent. Conclusions These data suggest which the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-neutralized and cross-reacted both JNK-IN-7 strains in vitro. The existing serological assays predicated on U.S. PEDV prototype stress can identify antibodies against both U.S. PEDV strains. [5]. The PEDV genome is 28 approximately?kb long and includes ORF1a and ORF1b encoding the replicase polyproteins and various other opening reading structures (ORFs) encoding four structural protein [spike (S), envelope (E), membrane (M), and nucleocapsid (N)] and a single nonstructural proteins NS3B (encoded by ORF3) [1]. In the U.S., an extremely virulent PEDV stress (U.S. PEDV prototype strain) was recognized during the initial PED outbreaks [2, 6, 7]. Lately, a PEDV variant strain having insertions and deletions (INDEL) in the spike gene compared to the U.S. prototype strain was recognized in U.S. swine with slight clinical signs based on field observations [8]. This U.S. PEDV variant strain, also known as S INDEL strain [4], formed a distinct phylogenetic cluster compared to U.S. PEDV prototype strains [4, 8, 9]. One PEDV isolate (Personal computer177) possessing a 197-aa deletion in the N-terminal S protein was found out during JNK-IN-7 PEDV isolation in cell tradition; however, this PEDV isolate still phylogenetically clustered with the U.S. PEDV prototype strains and was not considered as one of the S-INDEL-variant strains [10]. Marthaler et al [11] reported a third strain of PEDV (Minnesota188) in U.S. swine that experienced 6 nucleotide deletions (2 amino acid deletions) in the spike gene (different from the U.S. S-INDEL-variant strains). However, the PEDV Minnesota188 was genetically very closely related to the U.S. PEDV prototype strains and it is arguable whether it should be called a third strain of PEDV in the U.S. The PEDV Personal computer177 and Minnesota188 are probably the mutants of the U.S. PEDV prototype strains. Consequently, there are at least two genetically different PEDV strains currently circulating in U.S. swine: U.S. PEDV prototype strain and S-INDEL-variant strain. The U.S. PEDV prototype strains have been successfully isolated and propagated in cell tradition by several organizations [7, 10, 12, 13]. A number of serological assays, including an indirect fluorescent antibody (IFA) assay, a computer virus neutralization (VN) test, a whole virus-based enzyme-linked immunosorbent CYSLTR2 assay (ELISA), a recombinant S1 protein-based ELISA, and recombinant nucleocapsid protein-based ELISAs, have been developed for the detection of PEDV-specific antibodies [14C18]. All of these serological assays are based on the U.S. PEDV prototype strains. In this study, we isolated a U.S. PEDV S-INDEL-variant strain in cell tradition. Pigs were experimentally inoculated having a U.S. PEDV prototype strain and the newly isolated U.S. PEDV S-INDEL-variant strain, respectively, to generate strain-specific antisera. Subsequently, the generated swine antisera were subjected to an in vitro evaluation for serological cross-reactivity and cross-neutralization between the two strains. Specifically, 1) PEDV IFA.