Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains

BACKGROUND: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. RESULTS: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. CONCLUSIONS: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains

Effects of Medium Chain Fatty Acid Application in Swine Feed on Porcine Epidemic Diarrhea Virus

Medium chain fatty acid (MCFA) application has been identified as a promising strategy to decrease viral pathogen transmission in swine feed. Four experiments were conducted to: 1) determine if MCFAs are effective when applied to feed both prior to and after porcine epidemic diarrhea virus (PEDV) inoculation measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), 2) evaluate the effects of varying amounts and combinations of MCFA measured by qRT-PCR, and 3) evaluate selected MCFA treatments in a bioassay. In Exp. 1, treatments were arranged in a 2 × 2 + 1 factorial with the main effects of chemical treatment (0.3% Sal CURB [Kemin Industries, Des Moines, IA] or 1% MCFA blend of 1:1:1 C6:C8:C10 [PMI, Arden Hills, MN]) and timing of chemical treatment (pre or post-inoculation with PEDV), plus a positive control (feed inoculated with PEDV and no chemical treatment). Feed was treated with the respective treatment either before or after inoculation at which point it remained at ambient temperature for 24 h and then was analyzed via qRT-PCR. The analyzed values represent cycle threshold (Ct), for which a lower number indicates greater detection of viral nucleic acid. Results demonstrated that all combinations of chemical treatment and timing increased Ct compared to the positive control (P \u3c 0.05). Additionally, treatment of feed pre-PEDV inoculation resulted in increased Ct value compared to post- inoculation treatment (P = 0.009) and Sal CURB increased Ct in comparison with 1% MCFA (P \u3c 0.0001). In Exp. 2, the chemical treatments were applied pre-inoculation and consisted of:1) positive control, 2) 0.3% Sal CURB, 3) 0.125% C6, 4) 0.25% C6, 5) 0.33% C6,6) 0.125% C8, 7) 0.25% C8, 8) 0.33% C8, 9) 0.125% C10, 10) 0.25% C10, 11) 0.33% C10, 12) 0.125% C5, 13) 0.25% C5, 14) 0.33% C5, and 15) 0.66% C5, which were analyzed via qRT-PCR. Treatment of feed with 0.33% C8 resulted in increased (P \u3c 0.05) Ct values compared to all other levels of MCFA and the positive control feed. Further, Sal CURB, 0.25% C6, 0.33% C6, all levels of C8, 0.25% C10, 0.33% C10, or 0.66% C5 all had increased Ct values compared to positive control feed (P \u3c 0.05). Increasing amounts of each individual MCFA resulted in increased Ct (P \u3c 0.045). In Exp. 3, the chemical treatments were applied pre-inoculation and consisted of: 1) positive control; 2) 0.3% Sal CURB; 3) 0.25% MCFA blend; 4) 0.375% MCFA blend; 5) 0.500% MCFA blend; 6) 0.750% MCFA blend; 7) 1.0% MCFA blend; 8) 0.125% C6 + 0.125% C8; 9) 0.25% C6 + 0.25% C8; 10) 0.33% C6 + 0.33% C8; 11) 0.125% C6 + 0.125% C10; 12) 0.25% C6 + 0.25% C10; 13) 0.33% C6 + 0.33% C10; 14) 0.125% C8 + 0.125% C10; 15) 0.25% C8 + 0.25% C10; and 16) 0.33% C8 + 0.33% C10, which were analyzed via qRT-PCR. Treating feed with Sal CURB, 0.500% blend, 0.750% blend, 1.0% blend, all levels of the C6 + C8, 0.25% C6 + 0.25% C10, 0.33% C6 + 0.33% C10, 0.25% C8 + 0.25% C10, or 0.33% C8 + 0.33% C10 resulted in increased Ct compared to the positive control (P \u3c 0.05). Lastly, in Exp. 4, feed was treated pre-inoculation with either 1) no treatment (positive control); 2) 0.3% Sal CURB; 3) 0.5% MCFA blend; or 4) 0.3% C8 and samples were analyzed via qRT-PCR and bioassay. Adding either 0.5% MCFA blend or 0.3% C8 resulted in increased Ct compared to the positive control. Further, only the positive control resulted in a positive in vivo bioassay. This set of experiments demonstrates that MCFA and Sal CURB are effective at decreasing detection of PEDV in feed both prior to and post-inoculation. Additionally, inclusion of lower levels of MCFA than previously evaluated may provide protection against PEDV transmission through feed

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains

Background At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. Results A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. Conclusions These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.This article is published as Chen, Qi, Joseph T. Thomas, Luis G. Giménez-Lirola, John M. Hardham, Qinshan Gao, Priscilla F. Gerber, Tanja Opriessnig et al. "Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains." BMC Veterinary Research 12, no. 1 (2016): 1-10. DOI: 10.1186/s12917-016-0697-5. Copyright 2016 Chen et al. Attribution 4.0 International (CC BY 4.0). Posted with permission

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains

Background: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. Results: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. Conclusions: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains

Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naïve Conventional Neonatal and Weaned Pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID50/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID50/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID50/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID50/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID50/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.This article is published as Thomas, Joseph T., Qi Chen, Phillip C. Gauger, Luis G. Giménez-Lirola, Avanti Sinha, Karen M. Harmon, Darin M. Madson et al. "Effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs." PloS one 10, no. 10 (2015): e0139266. doi: https://doi.org/10.1371/journal.pone.0139266. Copyright: © 2015 Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License

Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naïve Conventional Neonatal and Weaned Pigs.

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID50/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560-0.056 TCID50/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2-37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID50/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560-5.6 TCID50/ml (Ct 24.2-31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56-0.0056 TCID50/ml (Ct values 35.3 ->45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms

Infectious titers, PCR Ct values and genomic copies/ml of serial dilutions of PEDV stock.

<p>* Average values of triplicate PCR reactions for each dilution.</p><p>Infectious titers, PCR Ct values and genomic copies/ml of serial dilutions of PEDV stock.</p