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.

In Vivo and In Vitro Characterization of the Recently Emergent PRRSV 1-4-4 L1C Variant (L1C.5) in Comparison with Other PRRSV-2 Lineage 1 Isolates.

The recently emerged PRRSV 1-4-4 L1C variant (L1C.5) was in vivo and in vitro characterized in this study in comparison with three other contemporary 1-4-4 isolates (L1C.1, L1A, and L1H) and one 1-7-4 L1A isolate. Seventy-two 3-week-old PRRSV-naive pigs were divided into six groups with twelve pigs/group. Forty-eight pigs (eight/group) were for inoculation, and 24 pigs (four/group) served as contact pigs. Pigs in pen A of each room were inoculated with the corresponding virus or negative media. At two days post inoculation (DPI), contact pigs were added to pen B adjacent to pen A in each room. Pigs were necropsied at 10 and 28 DPI. Compared to other virus-inoculated groups, the L1C.5-inoculated pigs exhibited more severe anorexia and lethargy, higher mortality, a higher fraction of pigs with fever (>40 °C), higher average temperature at several DPIs, and higher viremia levels at 2 DPI. A higher percentage of the contact pigs in the L1C.5 group became viremic at two days post contact, implying the higher transmissibility of this virus strain. It was also found that some PRRSV isolates caused brain infection in inoculation pigs and/or contact pigs. The complete genome sequences and growth characteristics in ZMAC cells of five PRRSV-2 isolates were further compared. Collectively, this study confirms that the PRRSV 1-4-4 L1C variant (L1C.5) is highly virulent with potential higher transmissibility, but the genetic determinants of virulence remain to be elucidated.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Porcine Respiratory Coronavirus (PRCV): Isolation and Characterization of a Variant PRCV from USA Pigs.

Porcine respiratory coronavirus (PRCV), a mutant of the transmissible gastroenteritis virus (TGEV), was first reported in Belgium in 1984. PRCV typically replicates and induces mild lesions in the respiratory tract, distinct from the enteric tropism of TGEV. In the past 30 years, PRCV has rarely been studied, and most cited information is on traditional isolates obtained during the 1980s and 1990s. Little is known about the genetic makeup and pathogenicity of recent PRCV isolates. The objective of this study was to obtain a contemporary PRCV isolate from US pigs for genetic characterization. In total, 1245 lung homogenate samples from pigs in various US states were tested via real-time PCR targeting PRCV and TGEV RNA. Overall, PRCV RNA was detected in five samples, and a single isolate (ISU20-92330) was successfully cultured and sequenced for its full-length genome. The isolate clustered with a new group of variant TGEVs and differed in various genomic regions compared to traditional PRCV isolates. Pathogens, such as PRCV, commonly circulate in pig herds without causing major disease. There may be value in tracking genomic changes and regularly updating the diagnostic methods for such viruses to be better prepared for the emergence of variants in ecology and pathogenicity.

Development and Clinical Applications of a 5-Plex Real-Time RT-PCR for Swine Enteric Coronaviruses.

A PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and validated for the simultaneous detection and differentiation of four swine enteric coronaviruses (PEDV, PDCoV, TGEV and SADS-CoV) in one PCR reaction (XIPC serves as an exogenous internal positive control). The 5-plex PCR had excellent analytical specificity, analytical sensitivity, and repeatability based on the testing of various viral and bacterial pathogens, serial dilutions of virus isolates, and in vitro transcribed RNAs. The 5-plex PCR had comparable diagnostic performance to a commercial PEDV/TGEV/PDCoV reference PCR, based on the testing of 219 clinical samples. Subsequently, 1807 clinical samples collected from various U.S. states during 2019-2021 were tested by the 5-plex PCR to investigate the presence of SADS-CoV in U.S. swine and the frequency of detecting swine enteric CoVs. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. Although there is no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance to detect and differentiate these viruses in swine samples and other host species samples as some of these coronaviruses can cause cross-species infection.