Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets

AbstractPorcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2009–2010 and reported in United States swine for the first time in February 2014. However, diagnostic tools other than polymerase chain reaction for PDCoV detection were lacking and Koch׳s postulates had not been fulfilled to confirm the pathogenic potential of PDCoV. In the present study, PDCoV peptide-specific rabbit antisera were developed and used in immunofluorescence and immunohistochemistry assays to assist PDCoV diagnostics. The pathogenicity and pathogenesis of PDCoV was investigated following orogastric inoculation of 5-day-old piglets with a plaque-purified PDCoV cell culture isolate (3×104 TCID50 per pig). The PDCoV-inoculated piglets developed mild to moderate diarrhea, shed increasing amount of virus in rectal swabs from 2 to 7 days post inoculation, and developed macroscopic and microscopic lesions in small intestines with viral antigen confirmed by immunohistochemistry staining. This study experimentally confirmed PDCoV pathogenicity and characterized PDCoV pathogenesis in neonatal piglets

First retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in Argentina

Background: (TGEV) was made by the NationalServices of Animal Health of Argentina (SENASA) to the World Organization of Animal Health (OIE). The notificationwas based on a serological diagnosis in a small farm with a morbidity rate of 2.3% without enteric clinical signs. Inorder to determine if TGEV was circulating before the official report, a retrospective study on cases of neonataldiarrhea was performed. The selection criteria was a sudden increase in mortality in 1- to 21-day-old piglets withwatery diarrhea that did not respond to antibiotics. Based on these criteria, three clinical cases were identifiedduring 2010?2015.Results: All animals that were evaluated presented histological lesions consistent with enteric viral infection. Thefeces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence ofround particles of approximately 80 nm in size and characterized by finely granular electrodense nucleoidsconsistent with complete particles of coronavirus. The presence of the TGEV antigen was confirmed by monoclonalspecific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situhybridization to detect a TGE mRNA encoding spike protein. All sections evaluated in this case were negative forPEDV and rotavirus A.Conclusions: This is the first case series describing neonatal mortality with etiological confirmation of TGEV inArgentina. The clinical diagnosis of TGEV infections in endemic regions is challenging due to the epidemiologicaldistribution and coinfection with other enteric pathogens that mask the clinical presentation.Fil: Piñeyro Piñeiro, Pablo Enrique. University Of Iowa. Ames Laboratory; Estados Unidos. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Patología. Cátedra de Patología Especial Veterinaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lozada, María Inés. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Patología. Laboratorio de Patología Especial Veterinaria "Dr. Bernardo Epstein"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alarcon, Laura Valeria. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Sanguinetti, Ramon. Ministerio de Agricultura, Ganadería, Pesca y Alimento; ArgentinaFil: Cappuccio, Javier Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Pérez, Estefanía Marisol. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología Especial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vannucci, Fabio. University Of Minnesota. School Of Public Health.; Estados UnidosFil: Armocida, Alberto Domingo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Madson, Darin Michael. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología Especial; ArgentinaFil: Perfumo, Carlos Juan. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología Especial; ArgentinaFil: Quiroga, María Alejandra. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología Especial; Argentin

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

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

Serum antibody responses in 3-week-old pigs inoculated with serial dilutions of PEDV.

<p>IFA antibody titers (A), serum virus neutralizing antibody titers (B), and PEDV whole virus-based ELISA antibody titers (C) were shown for 3 pigs in each group that went through 28 days post inoculation.</p

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

PEDV shedding in rectal swabs of the weaned pigs.^*

<p>* 3 pigs were necropsied at 7 DPI with the remaining 3 being necropsied at 28 DPI.</p><p>^‡ For groups 4, 5, 6, and 7, rectal swabs from all pigs were negative by PEDV rRT-PCR. To save space, 3 pigs are shown in one row.</p><p>PEDV shedding in rectal swabs of the weaned pigs.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139266#t005fn001" target="_blank">*</a>}</p

Virus shedding in rectal swabs of the 5-day-old pig study (A) and of the 3-week-old pig study (B) as determined by a quantitative PEDV N gene-based rRT-PCR.

<p>The virus titers (Log₁₀Genomic copies/ml) at each time points were mean virus titers of all available pigs (both PCR positive and negative pigs) except the 10⁻⁸ group in the 5-day-old pig study where only one out of 4 pigs was positive in the study period and included in figure (A).</p

PEDV shedding in rectal swabs of the neonatal piglets.^*

<p>*All pigs PCR positive on rectal swabs were necropsied on day 4 due to the severity of clinical signs</p><p>PEDV shedding in rectal swabs of the neonatal piglets.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139266#t003fn001" target="_blank">*</a>}</p