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Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major concern for swine health. Isolating PRRSV is essential for identifying infectious viruses and for vaccine formulation. This study evaluated the potential of using tongue fluid (TF) from perinatal piglet mortalities for PRRSV isolation. Four collection protocols were tested: extracting TF from fresh tissues using phosphate-buffered saline (PBS group), extracting TF from fresh tissues using virus transportation medium (VTM group), extracting TF from freeze-thawed tissue (freeze-thaw group), and using tissue homogenates (homogenate group). Two cell lines (ZMAC and MARC-145) and primary alveolar macrophages (PAM) were evaluated for their effect on successful PRRSV isolation. An eligible PRRSV-positive unstable breeding herd in Midwestern USA was chosen for the study. Tongues were collected in 20 batches (~30 mortalities per batch). Within each batch, each tongue tissue was cut into four quarters, with each quarter randomly assigned to one of the four collection protocols and RT-qPCR tested. Virus isolation (VI) was attempted on 10 batches. The mean RT-qPCR cycle threshold (Ct) values for the PBS, VTM, freeze-thaw, and homogenate groups were 21.9, 21.8, 22.6, and 24.8, respectively. The VI success rate was 22.6%, 12.1%, 2.8%, and 2.8% in the PBS, VTM, freeze-thaw, and homogenate groups, respectively. The probability of successful VI was 3.1% and 21.0% in the MARC-145 and ZMAC cell lines, respectively, and 4.8% in the PAM cells. TF from perinatal mortalities is an option for PRRS VI, aiding in PRRSV monitoring and control programs.

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen affecting the global swine industry. The first open reading frame 5 (ORF5)-based genetic lineage classification system describing global PRRSV-2 genetic diversity was introduced over a decade ago. Although refinements have been proposed for the predominant lineage in the USA (lineage 1), PRRSV-2 phylogenetic classification system at international levels has not been thoroughly evaluated and updated since 2010. In this study, based on analysis of 82,237 global ORF5 sequences reported during 1989–2021, we classified PRRSV-2 into 11 genetic lineages (L1‒L11) and 21 sublineages (L1A‒L1F, L1H‒L1J, L5A‒L5B, L8A‒L8E, and L9A‒L9E). The proposed classification system is flexible for growth if additional lineages, sublineages, or more granular classifications are needed. For example, for more fine-scale epidemiological investigation, L1C was further divided into five groups (L1C.1‒L1C.5), with L1C.5 corresponding to the recently emerged L1C variant. Comparison between restriction fragment length polymorphism (RFLP) typing and phylogenetic classification revealed the inaccuracy of using RFLP to determine PRRSV-2 genetic relatedness in most scenarios. Genetic homology of six commercial PRRSV-2 vaccines to each lineage/sublineage and detection frequency of vaccine-like viruses were determined. Global geographic distribution of each lineage/sublineage was presented. Temporal dynamic changes of PRRSV-2 in the USA during 1989–2021 were investigated by analyzing 73,092 ORF5 sequences. In summary, this study refined PRRSV-2 ORF5-based phylogenetic classification and investigated the geographic distribution and temporal changes of PRRSV-2 at the lineage/sublineage levels. The refined classification system and molecular epidemiology data in this study will be invaluable for future characterization of PRRSV-2. IMPORTANCE In this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

A safe and efficacious live-attenuated vaccine for porcine epidemic diarrhea virus (PEDV) is not commercially available in the United States yet. Two major PEDV strains are currently circulating in US swine: highly virulent non-S-INDEL strain and milder virulent S-INDEL strain. In this study, the safety and protective efficacy of a plaque-purified S-INDEL PEDV isolate formulated as a vaccine candidate was evaluated. Ten pregnant gilts were divided into three groups and orally inoculated at 79 days of gestation and then boosted at 100 days gestation (T01: n = 4, vaccination/challenge; T02: n = 4, non-vaccination/challenge; T03: n = 2, non-vaccination/non-challenge). None of the gilts had adverse clinical signs after vaccination. Only one T01 gilt (#5026) had viral replication and detectible viral RNA in feces. The same gilt had consistent levels of PEDV-specific IgG and IgA antibodies in serum and colostrum/milk. Farrowed piglets at 3 to 5 days of age from T01 and T02 gilts were orally challenged with 103 TCID50/pig of the virulent non-S-INDEL PEDV while T03 piglets were orally inoculated with virus-negative medium. T01 litters had overall lower mortality than T02 (T01 36.4% vs. T02 74.4%). Specifically, there was 0% litter mortality from T01 gilt 5026. Overall, it appears that vaccination of pregnant gilts with S-INDEL PEDV can passively protect piglets if there is virus replication and immune response induction in the pregnant gilts.

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.