A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/µL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/µL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus increases disease severity, cell trophism and earlier upregulation of IFN-α and IL12.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.

Effective inactivation of porcine epidemic diarrhea virus on contaminated surgery masks by low-concentrated sodium hypochlorite dispersion.

Coronaviruses present a considerable concern for humans and animals. The current world- wide pandemic of SARS-CoV-2 virus showed many gaps in understanding of coronaviruses spread and transmission. Because of lack of effective vaccine against SARS-CoV-2 the only preventive measures are represented by wearing protective masks and gloves thus limiting potential risk of contact with the airborne virus. Inversely, the limited time of protective function of the masks presents another drawback of their use. Therefore, the application of disinfection agent dispersed on the surface of protective masks may enhance their effectivity and safety of their application. The aim of the study was to examine the virucidal efficacy of low-concentra- ted sodium hypochlorite dispersed using ultrasonic humidifier on the surface of surgery masks. The study was conducted using SARS-CoV-2 surrogate virus, namely porcine epidemic diarrhea virus (PEDV) representing a model with similar biophysical properties and genomic structure to human coronaviruses. Five different concentrations of the disinfectant with different content of sodium hypochlorite were selected for the study. A final concentration of 0.228 g/L sodium hypochlorite effectively inactivated the PED virus and may support the biosafety of masks usage.

Isolation and characterization of a variant subgroup GII-a porcine epidemic diarrhea virus strain in China.

BACKGROUND: Highly virulent variants of porcine epidemic diarrhea virus (PEDV) have been closely associated with recent outbreaks of porcine epidemic diarrhea (PED) in China, which have resulted in severe economic losses to the pork industry. METHODS: In the current study, the variant PEDV strain HM2017 was isolated and purified and a viral growth curve was constructed according to the median tissue culture infective dose (TCID50). HM2017 were amplify with RT-PCR and analyzed by phylogeny analysis. Animal pathogenicity experiment was carried to evaluate the HM2017 clinical assessment. RESULTS: Genome-based phylogenetic analysis revealed that PEDV strain HM2017 was clustered into the variant subgroup GII-a that is currently circulating in pig populations in China. The highest median tissue culture infectious dose of strain HM2017 after 15 passages in Vero cells was 1.33 × 107 viral particles/mL. Strain HM2017 was highly virulent to suckling piglets, which exhibited clinical symptoms at 12 h post-infection (hpi) (i.e., weight loss at 12-84 hpi, increased body temperatures at 24-48 hpi, high viral loads in the jejunum and ileum, and 100% mortality by 84 hpi). CONCLUSION: The present study reports a variant subgroup GII-a PEDV HM2017 strain in China and characterize its pathogenicity. PEDV strain HM2017 of subgroup GII-a presents a promising vaccine candidate for the control of PED outbreaks in China.

Establishment of method for dual simultaneous detection of PEDV and TGEV by combination of magnetic micro-particles and nanoparticles.

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.

Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/µL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.

Immune responses to porcine epidemic diarrhea virus (PEDV) in swine and protection against subsequent infection.

Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan.

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full-length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016-2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild-type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.

Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 101 copies/µL, 102 copies/µL and 102 copies/µL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s<5%) and specificities (100%), and possessed sensitivities of 92.24%, 92.19% and 91.23% for PEDV, PDCoV and SADS-CoV respectively. In summary, the established microfluidic-RT-LAMP chip detection system could satisfy the demanding in field diagnoses, which was suitable for promotion in remote areas due to its fast, portable and cost-effective characters.

Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR.

Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 µl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 µl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 µl and the Upper LQ (ULQ) 108 copies/5 µl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.

EuNPs-mAb fluorescent probe based immunochromatographic strip for rapid and sensitive detection of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED), induced by porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets, resulting in significant economic losses in the pig industries. In this study, an immunochromatographic assay (ICA) based on a EuNPs-mAb fluorescent probe was developed and optimized for rapid detection of PEDV. The limit of detection (LOD) of the ICA was 0.218 µg/mL (2.725 × 103 TCID50/mL) and its linear detection range was 0.03125-8 µg/mL (3.91 × 102-105 TCID50/mL). The ICA was also validated for the detection of PEDV in swine stool samples. 60 swine stool samples from southern China were analyzed by the ICA and RT-PCR, and the results showed that the coincidence rate of the ICA to RT-PCR was 86.67%, which was significantly higher than that of AuNPs based ICA. The ICA is sensitive and specific and can achieve on-site rapid detection of swine stool samples. Therefore, the ICA has a great potential for PED diagnosis and prevention.