Assessment of temperature and time on the survivability of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces.

Fomites might be responsible for virus introduction in swine farms, highlighting the importance of implementing practices to minimize the probability of virus introduction. The study's objective was to assess the efficacy of different combinations of temperatures and holding-times on detecting live PRRSV and PEDV on surfaces commonly found in supply entry rooms in swine farms. Two PRRSV isolates (MN 184 and 1-4-4 L1C variant) and one PEDV isolate (NC 49469/2013) were inoculated on cardboard and aluminum. An experimental study tested combinations of four temperatures (20°C, 30°C, 40°C, and 50°C) and six holding-times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours) for the presence of the viruses on each surface type. After virus titration, virus presence was assessed by assessing the cytopathic effects and immunofluorescence staining. The titers were expressed as log10 TCID50/ml, and regression models; half-lives equations were calculated to assess differences between treatments and time to not detect the live virus. The results suggest that the minimum time that surfaces should be held to not detect the virus at 30°C was 24 hours, 40°C required 12 hours, and 50°C required 6 hours; aluminum surfaces took longer to reach the desired temperature compared to cardboard. The results suggest that PRRSV 1-4-4 L1C variant had higher half-lives at higher temperatures than PRRSV MN 184. In conclusion, time and temperature combinations effectively decrease the concentration of PRRSV and PEDV on different surfaces found in supply entry rooms in swine farms.

Phylogenomic assessment of 23 equid alphaherpesvirus 1 isolates obtained from USA-based equids.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates. METHODS: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses. RESULTS: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates. CONCLUSIONS: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.