Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 10²to 2 × 10⁵50% tissue culture infective doses (TCID₅₀)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

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.

Porcine deltacoronavirus (PDCoV) was detected in feces from diarrheic sows during an epidemic of acute and transmissible diarrhea. No transmissible gastroenteritis virus or porcine epidemic diarrhea virus was detected. The PDCoV USA/IA/2014/8734 from the herd was sequenced for full-length genomic RNA to further characterize PDCoV in U.S. swine.

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.