INDEL Strains Research Articles

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Phylogenetic and Molecular Analysis of the Porcine Epidemic Diarrhea Virus in Mexico during the First Reported Outbreaks (2013-2017).

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

Re-emergence of porcine epidemic diarrhea virus in a piglet-producing farm in northwestern Germany in 2019

BackgroundPorcine epidemic diarrhea (PED) is a viral enteric disease of pigs. It affects all age classes of animals but lethality is mainly seen in suckling piglets. After its first appearance in England in 1971, Porcine epidemic diarrhea virus (PEDV) has spread worldwide. While sporadic outbreaks prevailed in Europe, the disease had high impact in Asia. Following particularly severe outbreaks in 2011, high impact cases were also reported in the United States and neighboring countries in 2013. Subsequently, outbreaks were also reported in several European countries including Germany. These outbreaks were less severe. This case report describes a recent case of PED re-emergence in Germany and the sequence analyses of the causative PEDV.Case presentationIn spring 2019 5 years after re-introduction of PED into Central Europe, a piglet-producer in northwestern Germany experienced an outbreak that affected sows, their suckling piglets, and weaners. After initial confirmation of PEDV by real-time RT-PCR, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. Phylogenetic analyses showed high identities among the PEDV sequences obtained from samples of different animals and a close relation to recent strains from Hungary and France. Compared to the PEDV strains analyzed in 2014, genetic drift could be confirmed. Changes were mainly observed in the spike protein encoding S gene segment. In addition, metagenomic analyses showed multiple Picobirnavirus reads in all investigated samples.ConclusionThis case report shows that PEDV is still circulating in Europe. The causative strains are moderately virulent and are still closely related to the so-called INDEL strains reported previously in Europe, including Germany. However, a genetic drift has taken place that can be seen in a novel cluster comprising strains from Germany, Hungary and France in 2019. Relevance and impact of the detected Picobirna sequences need further investigations.

Better horizontal transmission of a US non-InDel strain compared with a French InDel strain of porcine epidemic diarrhoea virus.

From the severe porcine epidemic diarrhoea (PED) epidemics that struck in 2013 in the United States of America and other countries of North and South America, two types of porcine epidemic diarrhoea virus (PEDV) were isolated, namely the InDel and the non‐InDel strains. They are differentiated by insertions/deletions in the S1 nucleotide sequence of the S gene, and differences in virulence were observed from the clinical cases. In 2014, a PED outbreak occurred in a pig farm in France, from which an InDel strain was isolated. This study aimed at comparing, under experimental conditions, the pathogenicity and the direct and indirect transmissions between a non‐InDel strain isolated from a PED‐affected piglet in 2014 in the USA and the French InDel strain. All infected pigs showed clinical signs with the non‐InDel strain although only the inoculated and direct contact pigs showed clinical signs in the InDel strain group. Although viral RNA was detected in air samples with both strains, the indirect contact pigs remained free from infection with the InDel strain in contrast to the non‐InDel group in which airborne transmission occurred in the indirect contact pigs. All infected pigs shed virus in faeces regardless of PEDV strain with 9 of 30 pigs showing intermittent faecal shedding. The transmission rate by direct contact was found to be 2.17‐fold higher than the non‐InDel strain compared with the InDel. In conclusion, the InDel strain was less pathogenic than the non‐InDel strain in our experimental conditions. The transmission route differed between the two strains. Direct contact was the main transmission route for the InDel strain, although the non‐InDel strain was transmitted through direct contact and indirectly through the air.

Molecular evolution of porcine epidemic diarrhea virus and porcine deltacoronavirus strains in Central China

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are epizootic swine viruses. To detect and study the evolution of PEDV and PDCoV in central China (Shanxi, Henan, Hubei province), 70 clinical intestinal and fecal samples from piglets with severe watery diarrhea during August 2015 and June 2016 were collected, tested and analyzed. PEDV was more frequently detected by PCR than PDCoV. Phylogenetic analysis of S genes showed that the 10 PEDV strains from this study clustered into G2a (n = 7) and G2b (n = 3) groups. Additionally, the three G2b strains (PEDV S2△) contained the same specific 3 nt deletion in S2 as other reference strains in G2b. Interestingly, complete genome analysis indicated that CH/hubei/2016 was closer to the US INDEL strain and G2a group. CH/hubei/2016 had one recombination event in S2 gene which may have resulted from AH2012-12 (from G2b group) and CH-ZMDZY-11 (from G2a group). Furthermore, 10 purifying selection sites in S gene indicated an adaptive evolution of PEDV in central China swine herds. These results suggested that Pandemic G2a and G2b are predominant PEDV genotype circulating in central China. In addition, the deletion and recombination identified in S gene suggested PEDV strains of central exhibited an evolutionary variety. However, whether these changes affect the pathogenicity and antigenicity of wild PEDV is unknown and is worth for further investigation.

Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013\u20132016 and PEDVs collected from recurrent outbreaks

BackgroundSince late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013–2016, were sequenced and analyzed.ResultsPhylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics.ConclusionsThese results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.

Identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the United States.

Two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the USA: US prototype (also called non-S INDEL) and S INDEL PEDVs. In February 2017, a PEDV variant (USA/OK10240-8/2017) was identified in a rectal swab from a sow farm in Oklahoma, USA. Complete genome sequence analyses indicated this PEDV variant was genetically similar to US non-S INDEL strain but had a continuous 600-nt (200-aa) deletion in the N-terminal domain of the spike gene compared to non-S INDEL PEDVs. This is the first report of detecting PEDV bearing large spike gene deletion in clinical swine samples in the USA.

Investigation of three outbreaks of Porcine Epidemic Diarrhea in Germany in 2016 demonstrates age dependent differences in the development of humoral immune response

Porcine epidemic diarrhea (PED) has reemerged in Europe since 2014. Characterized by a rapid onset of diarrhea in pigs of all ages, morbidity can reach up to 100% whereas mortality is variable. The virus strains involved in the recent European outbreaks all cluster together with US strains (S INDEL) that lead to less severe clinical signs. In this study, fattening pigs and suckling piglets (n = 105) on farms with no prior PED history were monitored after an acute outbreak of the disease, caused by an S INDEL strain of PED virus (PEDV). For diagnostic investigations in the affected farms, real time RT-PCR was performed to detect PEDV RNA in individually taken fecal samples, and two commercial ELISA kits, both based on the N protein of PEDV, were used to detect IgG in serum samples of pigs experiencing acute signs of the disease. PEDV RNA could be detected in fecal samples up to 14 days after initial sampling. Comparing both ELISAs by Cohens Kappa showed substantial agreement (κ = 0,771). Antibodies were detectable in all fattening pigs (100%) within 10 days after the occurrence of first clinical signs and remained detectable for about two months at least in 20.6% (farm 1) and 45.7% (farm 2) of the animals, respectively. In contrast, only 18 of 34 (52.9%) suckling piglets seroconverted. Although, PEDV RNA was found in fecal samples of all piglets, 13 piglets did not demonstrate antibodies at any sampling day. PCR to detect PEDV RNA in fecal samples seems to be a reliable diagnostic tool during and after the acute outbreak. In the present study, IgG ELISA kits proved to be a feasible diagnostic tool, but age dependent differences in detection rate and persistence of antibodies need to be considered.

Molecular characterization of the spike gene of the porcine epidemic diarrhea virus in Mexico, 2013-2016.

In Mexico, the first outbreaks suggestive of the circulation of the porcine epidemic diarrhea virus (PEDV) were identified at the beginning of July 2013. To identify the molecular characteristics of the PEDV Spike (S) gene in Mexico, 116 samples of the intestine and diarrhea of piglets with clinical signs of porcine epidemic diarrhea (PED) were obtained. Samples were collected from 14 farms located in six states of Mexico (Jalisco, Puebla, Sonora, Veracruz, Guanajuato, and Michoacán) from 2013 to 2016. To identify PEDV, we used real-time RT-PCR to discriminate between non-INDEL and INDEL strains. We chose samples according to state and year to characterize the S gene. After amplification of the S gene, the obtained products were sequenced and assembled. The complete amino acid sequences of the spike protein were used to perform an epitope analysis, which was used to determine null mutations in regions SS2, SS6, and 2C10 compared to the sequences of G2. A phylogenetic analysis determined the circulation of G2b and INDEL strains in Mexico. However, several mutations were recorded in the collagenase equivalent (COE) region that were related to the change in polarity and charge of the amino acid residues. The PEDV strain circulating in Jalisco in 2016 has an insertion of three amino acids (232LGL234) and one change in the antigenic site of the COE region, and strains from the years 2015 and 2016 changed the index of the surface probability, which could be related to the re-emergence of disease outbreaks.

Isolation and experimental inoculation of an S INDEL strain of porcine epidemic diarrhea virus in Japan

In 2013, porcine epidemic diarrhea (PED) was reported in Japan for the first time in 7years and caused significant economic losses. In the present study, we isolated PED virus (PEDV) circulating in Japan using Vero cell cultures and analyzed sequences of S1 genes of these PEDV isolates. Sequence analysis revealed that one of these strains contained distinct insertion and deletions in the S gene (i.e., S INDEL). Furthermore, inoculation of PEDV into 1-week-old pigs demonstrated that the S INDEL strain had a lower pathogenicity than the North American (NA) prototype strain. This is the first report comparing pathogenicity of an S INDEL strain with the NA prototype strain following experimental inoculation. Excretion of PEDV in the feces of S INDEL strain-inoculated pigs occurred later than in NA prototype strain-inoculated pigs. Thus, our findings suggested that the S INDEL strain had different viral dynamics than the NA prototype strain.

Emergence of porcine epidemic diarrhea virus in southern Germany.

BackgroundOver the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany.Case presentationEpidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia.ConclusionPhylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.

Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014.

We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan.

New variants of porcine epidemic diarrhoea virus (PEDV), which emerged in Taiwan in late 2013, have caused a high morbidity and mortality in neonatal piglets. To investigate the molecular characteristics of the spike (S) gene of the emerging Taiwan PEDV strains for a better understanding of the genetic diversity and relationship among the Taiwan new variants and the global PEDVs, full-length S genes of PEDVs from nine 1-7day-old piglets from three pig farms in the central and southern Taiwan were sequenced and analysed. The result of phylogenetic analysis of the S gene showed that all the Taiwan PEDV strains were closely related to the non-S INDEL strains from US, Canada and China, suggesting a common ancestor for these strains. As compared with the historic PEDVs and CV777-based vaccine strains, the nine Taiwan PEDV variants shared almost the same genetic signatures as the global non-S INDEL strains, including a series of insertions, deletions and mutations in the amino terminal as well as identical mutations in the neutralizing epitopes of the S gene. The high similarity of the S protein among the Taiwan and the globally emerged non-S INDEL PEDV strains suggests that the Taiwan new variants may share similar pathogenesis and immunogenicity as the global outbreak variants. The development of a novel vaccine based on the Taiwan or the global non-S INDEL strains may be contributive to the control of the current global porcine epidemic diarrhoea outbreaks.

Distinct characteristics and complex evolution of PEDV strains, North America, May 2013-February 2014.

Porcine epidemic diarrhea virus (PEDV), which emerged in the United States in 2013, has spread throughout North America. Limited availability of PEDV complete genomes worldwide has impeded our understanding of PEDV introduction into the United States. To determine the relationship between the North American strains and global emerging and historic PEDV strains, we sequenced and analyzed complete genomes of 74 strains from North America; the strains clustered into 2 distinct clades. Compared with the initially reported virulent US PEDV strains, 7 (9.7%) strains from 4 states contained insertions and deletions in the spike gene (S INDELs). These S INDEL strains share 99.8%-100% nt identity with each other and 96.2%-96.7% nt identity with the initial US strains. Furthermore, the S INDEL strains form a distinct cluster within North American clade II, sharing 98.6%-100% nt identity overall. In the United States, the S INDEL and original PEDV strains are co-circulating and could have been introduced simultaneously.