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Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 10 0 TCID 50 PEDV, 1 × 10 4 TCID 50 PDCoV, and 1 × 10 2 TCID 50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Serafini Poeta Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV., Competing Interests: Declaration of Competing Interest Authors Rémi Malbec, Elisa Vandenkoornhuyse, and Christophe Audebert are employees of GD Biotech, while Vianney Souplet and Christophe Olivier are employees of Innobiochips., (Copyright © 2020. Published by Elsevier B.V.)

Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R 2 ) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEV and PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.

During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT-qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV- and PEDV-specific RT-qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT-PCR specific for the PEDV S-gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3'-terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S-gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV-infected herd seroconverted against PEDV but tested negative in a TGEV-specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT-qPCR assay or the combined use of assays targeting the PEDV S-gene and another part of the TGEV genome., (© 2016 Blackwell Verlag GmbH.)

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in the amplification step two different amplicons with sizes of 950 bp and 317 bp and (2) three pairs of internal primers in a second round of PCR (nested PCR), yielding two different amplicons with sizes of 792 bp and 208 bp for TGEV and porcine PRV-A, respectively. The genome of PEDV was not detected after the amplification step but it was detected in the second round of PCR, yielding amplicon with size of 291 bp. Multiplex nested RT-PCR can detect TGEV, PRV-A, and PEDV up to concentration 10(2) TCID(50)/mL, 10(1) TCID(50)/mL, and 27.2 microg/microl of RNA, respectively. A total of 175 field samples were collected from swine with diarrhea from January 2005 until July 2007. The samples were tested for the presence of three viruses by a multiplex nested RT-PCR. Dual infections with PEDV and PRV-A were identified in seven specimens (4%) (n = 6). Twenty-one (25%) infections were caused by PEDV and thirty-four infections (41%) were caused by PRV-A. The genome of TGEV was not detected in any of these field samples, however TGEV was detected in piglets infected experimentally. The multiplex nested RT-PCR is rapid, sensitive, and a cost-effective detection method for the detection of porcine enteric viruses., (Copyright 2010 Elsevier B.V. All rights reserved.)

A novel multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) that can detect porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (GAR) was developed. The 3 viruses (PEDV, TGEV, and porcine GAR) are major agents in viral enteric diseases of piglets. As the clinical signs of these diseases are similar, including watery diarrhea, differential detection is required for etiologic diagnosis. A mixture of 3 pairs of published primers was used for amplification of viral nucleic acids, yielding 3 different amplicons with sizes of 859 bp, 651 bp, and 309 bp for TGEV, PEDV, and porcine GAR, respectively. A total of 157 specimens (78 fecal and 79 intestinal samples) from piglets with acute gastroenteritis were collected in Korea between January 2004 and May 2005. They were tested for the presence of 3 viruses by multiplex RT-PCR. Coinfections with PEDV and porcine GAR were identified in 16 farms (43.2%). PEDV, porcine GAR, and TGEV infection were 26.3%, 13.2%, and 2.7% respectively. The relative sensitivity and specificity of multiplex RT-PCR were evaluated, with results suggesting that this assay is equal in quality to conventional single-agent RT-PCR assays (sensitivity:100%, 92.9%, 100% for TGEV, PEDV, GARs; specificity: 100% for all 3 viruses). This multiplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets.

Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly contagious enteric diseases of piglets. The clinical signs of these diseases are very similar and include watery, yellowish diarrhea. Thus, the effective differential detection of TGE virus and PED virus is required. In the present study, a duplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the differential detection of TGE and PED viruses. The primers were designed for the S gene of each virus. RNA was extracted from the intestines and stool samples that were collected from the swine with diarrhea. The RT-PCR test could detect both TGE and PED viruses with 2 TCID50/200 microl. Among 90 clinical samples, 7 TGE viruses and 2 PED viruses were detected by the duplex RT-PCR. This duplex RT-PCR may be a useful diagnostic method for the rapid, specific, and sensitive differential detection of TGE and PED viruses using clinical samples.

Transmissible gastroenteritis virus (TGEV), a coronavirus, replicates in intestinal enterocytes and causes diarrhea in young pigs. Porcine respiratory coronavirus (PRCV), a spike (S) gene natural deletion mutant of TGEV, has a respiratory tissue tropism and causes mild or subclinical respiratory infections. Conventional antigen-based diagnostic tests fail to differentiate TGEV and PRCV, and a blocking ELISA test to serologically differentiate TGEV/PRCV-infected pigs is conducted on convalescent serum retrospectively after disease outbreaks. A reverse transcription (RT)-nested polymerase chain reaction (PCR) with primers targeted to the S gene deletion region to differentiate TGEV/PRCV was developed. The specificity of the RT-nested PCR was confirmed with reference and recent field strains of TGEV/PRCV, and its sensitivity was analyzed by testing nasal and fecal samples collected from pigs at various days postinoculation (DPI) with TGEV or PRCV. Specific PCR products for TGEV/PRCV were detected only with the homologous reference or field coronaviruses and for 10-14 DPI of pigs with TGEV (feces) or PRCV (nasal samples). The RT-nested PCR assay was more sensitive than antigen-based assays on the basis of duration of virus detection in experimentally infected pigs and was directly applicable to nasal as well as fecal specimens from the field.

Three hundred and forty-seven serum samples from 22 Iowa swine herds were screened for TGEV/PRCV neutralizing antibody. Ninety-one percent of the sera and all 22 herds were positive. These sera were then tested by the blocking ELISA test to distinguish TGEV and PRCV antibody. The ELISA test confirmed the high percentage of TGEV/PRCV positive sera. By the blocking ELISA test, 12 herds were PRCV positive, 6 herds were TGEV positive and 4 herds were mixed with sera either positive for TGEV or PRCV antibody. The results suggest a recent increase in TGEV/PRCV seroprevalence in Iowa swine most likely due to subclinical PRCV infections.

An RT-PCR method was developed that amplified genetic material from the 5' end of the S protein gene of both transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), but discriminated between the two by the size of the product generated. A number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. The assay was shown to detect viral RNA from all of the 26 different TGEV and PRCV isolates examined, covering a period from 1946 to 1996. Detection of TGEV in clinical specimens was possible using a spin column method to extract RNA and sensitivity was compared to virus isolation and antigen detection ELISA. The method could provide a means of confirming positive results from immunological screening tests such as FAT and ELISA, reducing the need for virus isolation and convalescent serology.

Transmissible gastroenteritis (TGE) is an enteric disease of swine caused by a coronavirus, designated as transmissible gastroenteritis virus (TGEV). Commonly used methods for TGEV detection include viral isolation and detection of the viral antigen by indirect immunofluorescence (IFA), immunoperoxidase, and immunogold silver staining. Each of these techniques has some advantages and disadvantages. In general IFA and immunohistochemistry are preferred over viral isolation as TGEV isolation is not very reliable because not all field isolates replicate in cell cultures. The diagnosis of TGEV has become more complicated since the emergence of porcine respiratory coronavirus (PRCV). PRCV is believed to be a TGEV mutant, and can not be easily differentiated from TGEV by immunological tests. Nucleic acid probes and polymerase chain reaction (PCR) have successfully been used to detect and differentiate these viruses. These techniques can detect viral nucleic acids in the specimen but do not provide information on the cell types infected by these viruses. Recently we have developed isotopic and nonisotopic in situ hybridization techniques (ISH) for the detection of these viral nucleic acids in formalin-fixed paraffin-embedded tissues. Furthermore, this procedure can differentiate between TGEV- and PRCV-infected cells. By ISH, TGEV is detected in the mature absorptive enterocytes of tissues infected by TGEV and the crypt epithelial cells are also infected but to a lesser extent. For PRCV, the main infected cells are epithelial cells of the bronchioles, type II pneumocytes, and alveolar and septal macrophages. ISH is an excellent tool for studying molecular pathogenesis of these two viruses especially when used in combination with immunohistochemistry.