US Porcine Epidemic Diarrhea Virus Research Articles

Molecular Characterization and Phylogenetic Analysis of a Variant Recombinant Porcine Epidemic Diarrhea Virus Strain in China

Simple SummaryWe successfully isolated and identified PEDV strain SC-YB73. The sequence analysis of the SC-YB73 genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In future research, we aim to evaluate the function of E-gene insertions using in vitro cellular culture and in vivo animal experiments.Since 2010, a variant of porcine epidemic diarrhea virus (PEDV) has re-emerged in several provinces of China, resulting in severe economic losses for the pork industry. Here, we isolated and identified a variant PEDV strain, SC-YB73, in Guangdong Province, China. The pathological observations of jejunum showed atrophy of villi and edema in the lamina propria. The sequence analysis of the viral genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. Furthermore, 50 nucleotide sites were unique in SC-YB73 compared with 27 other PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In the present study, we isolated and genetically characterized a variant PEDV strain, thus providing essential information for the control of PED outbreaks in China.

Proteases facilitate the endosomal escape of porcine epidemic diarrhea virus during entry into host cells

Exogenous and endogenous proteases play important roles in porcine epidemic diarrhea virus (PEDV) entry and replication. The roles of proteases in the viral endosomal escape and replication using trypsin (KD) or elastase (AA)-adapted US PEDV strains were studied. While PEDV KD and AA require different exogenous protease for efficient replication in cells, PEDV KD was more dependent on the protease than PEDV AA. There was no marked difference in viral trafficking between them during the entry events. Both PEDV were observed in the endosomes with or without protease at 1 h after virus inoculation. With protease, viral signals in the endosomes disappeared after 4 h, and newly synthesized viral proteins were detected in the ER after 6 h. However, without protease, viruses remained in the endosomes up to 24 h, which correlated with limited virus replication. Inhibitors of cathepsins, endogenous proteases, significantly reduced the replication of both PEDV by interfering with the viral endosomal escape.

New variants of porcine epidemic diarrhea virus with large deletions in the spike protein, identified in the United States, 2016-2017.

Four types of porcine epidemic diarrhea virus (PEDV) variants with a large deletion in the spike protein were detected, together with the original US PEDV, from pig fecal and oral fluid samples collected during 2016-2017 in the US. Two of the variants are similar to those identified in Japan: one contains a 194-aa deletion, the same as PEDV variant TTR-2/JPN/2014, while the other contains a 204-aa deletion, the same as PEDV variant JKa-292/CS1de204. Two new S1 NTD-del PEDV variants were found: one contains a 201-aa deletion located at residues 30-230 and the other contains a 202-aa deletion located at residues 24-225 of the S protein. This is the first report on coinfection of S1 NTD-del PEDV variants and the original US PEDV strain in US pigs, indicating that PEDV continues to evolve in pigs and might be responsible for disease pattern changes.

Cross protective immune responses in nursing piglets infected with a US spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original US PEDV strain

We investigated cross-protective immunity of a US spike-insertion deletion porcine epidemic diarrhea virus (PEDV) Iowa106 (S-INDEL) strain against the original US PEDV (PC21A) strain in nursing piglets. Piglets were inoculated orally with S-INDEL, PC21A or mock. At 20–29 days post-inoculation (dpi), all pigs were challenged with the PC21A strain. The S-INDEL-inoculated pigs had lower ileal IgA antibody secreting cells, serum IgA and neutralizing antibody titers compared with PC21A-inoculated pigs. No pigs in the PC21A-group developed diarrhea, whereas 81 and 100% of pigs in the S-INDEL and mock-groups had diarrhea post challenge, respectively. S-INDEL induced partial protective immunity against the original US PEDV strain.

Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage

Although porcine epidemic diarrhea (PED) has caused huge economic losses in the pork industry worldwide, an effective live, attenuated vaccine is lacking. In this study, an original US, highly virulent PED virus (PEDV) strain PC22A was serially passaged in Vero CCL81 and Vero BI cells. The virus growth kinetics in cell culture, virulence in neonatal pigs and the whole genomic sequences of selected passages were examined. Increased virus titers and sizes of syncytia were observed at the 65th passage level (P65) and P120, respectively. Based on the severity of clinical signs, histopathological lesions and the distribution of PEDV antigens in the gut, the virulence of P100 and above, but not P95C13 (CCL81), was markedly reduced in 4-day-old, caesarian-derived, colostrum-deprived piglets. Subsequently, the attenuation of P120 and P160 was confirmed in 4-day-old, conventional suckling piglets. Compared with P120, P160 replicated less efficiently in the intestine of pigs and induced a lower rate of protection after challenge. Sequence analysis revealed that the virulent viruses [P3 and P95C13 (CCL81)] had one, one, sixteen (including an early termination of nine amino acids) and two amino acid differences in non-structure protein 1 (nsp1), nsp4, spike and membrane proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives. These results suggest that PEDV attenuation can occur through multiple molecular mechanisms. The knowledge provides insights into potential molecular mechanisms of PEDV attenuation.

Reverse transcription-PCR assays for the differentiation of various US porcine epidemic diarrhea virus strains

Concurrently, several porcine epidemic diarrhea virus (PEDV) variants are circulating in US swine farms, including the original US and the spike insertion-deletion (S-INDEL) strains. In this study, reverse transcription (RT)-PCR assays for the detection and differentiation of different US PEDV variants were developed based on the differences in the S1 domain of the spike (S) gene. This assay successfully differentiated three PEDV strains: PC22A (the original US virulent), Iowa106 (S-INDEL), and PC177 (S-197DEL) that was derived from cell culture adaptation and has a 197 amino acid-deletion in the S1 domain. The assays did not amplify the porcine deltacoronavirus OH-FD22 strain or transmissible gastroenteritis virus Miller strain. It is the first report on the development of RT-PCR assays allowing the detection and differentiation of all major types of US PEDV variants.

Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection.

Although the original US porcine epidemic diarrhea virus (PEDV) was confirmed as highly virulent by multiple studies, the virulence of spike-insertion deletion (S-INDEL) PEDV strains is undefined. In this study, 3–4 day-old conventional suckling piglets were inoculated with S-INDEL PEDV Iowa106 (4 pig litters) to study its virulence. Two litters of age-matched piglets were inoculated with either the original US PEDV PC21A or mock as positive and negative controls, respectively. Subsequently, all pigs were challenged with the original US PEDV PC21A on 21–29 days post-inoculation (dpi) to assess cross-protection. All S-INDEL Iowa106- and the original US PC21A-inoculated piglets developed diarrhea. However, the severity of clinical signs, mortality (0–75%) and fecal PEDV RNA shedding titers varied among the four S-INDEL Iowa106-inoculated litters. Compared with the original PC21A, piglets euthanized/died acutely from S-INDEL Iowa106 infection had relatively milder villous atrophy, lower antigen scores and more limited intestinal infection. Two of four S-INDEL Iowa106-infected sows and the original PC21A-infected sow showed anorexia and watery diarrhea for 1–4 days. After the original PC21A challenge, a subset (13/16) of S-INDEL Iowa106-inoculated piglets developed diarrhea, whereas all (5/5) and no (0/4) pigs in the mock and original PC21A-inoculated pigs had diarrhea, respectively. Our results suggest that the virulence of S-INDEL PEDV Iowa106 was less than the original US PEDV PC21A in suckling pigs, with 100% morbidity and 18% (6/33) overall (0–75%) mortality in suckling pigs depending on factors such as the sow’s health and lactation and the piglets’ birth weight. Prior infection by S-INDEL Iowa106 provided partial cross-protection to piglets against the original PC21A challenge at 21–29 dpi.

Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.

Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs

Our study demonstrated potential mechanisms by which porcine epidemic diarrhea virus (PEDV) infection induces greater disease severity of nursing vs. weaned conventional pigs. Twenty-six-day-old weaned [PEDV-inoculated (n=11); mock (n=9)] and 9-day-old nursing pigs [PEDV-inoculated (n=9); mock (n=11)] were inoculated orally [8.9 log10 genomic equivalents (GE)/pig] with PC21A strain or mock (MEM). Pigs were monitored for clinical signs and PEDV RNA titers in feces and serum. For pathology and immunofluorescence staining for Ki67 (marker for crypt proliferation) and LGR5 (marker for crypt stem cell), 3–4 pigs were euthanized at postinoculation days (PIDs) 1, 3 and 5. Severe watery diarrhea and atrophic enteritis with moderate to high PEDV RNA titers in feces (7.5–12.2 log10 GE/ml) and low viral RNA titers in serum (5.6–8.6 log10 GE/ml) were observed in all inoculated nursing piglets at PIDs 1–5. In contrast, weaned pigs did not show evidence of PEDV infection at PID 1. Pigs exhibited high fecal shedding titers at PIDs 2–5 and mild to severe atrophic enteritis at PIDs 3–5, indicating a longer incubation for PEDV infection. While uninoculated or inoculated 27–31-day-old pigs showed large numbers of Ki67- or LGR5-positive cells in the intestinal crypts, there was a lack of LGR5-positive cells and low proliferation of crypts in jejunum of uninoculated 10–14-day-old piglets, possibly causing a slower turnover of enterocytes; however, the number of LGR5-positive cells and proliferation of intestinal crypts increased remarkably at 3–5 days after inoculation. Biologic mediators that promote crypt stem cell regeneration would be targets to improve the intestinal epithelium renewal during PEDV infection.

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.

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1mL of 1×103 PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.

Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene

The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n=7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5–6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains.

Comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in Korea

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is an enveloped, positive-sense, single-stranded RNA virus, which causes severe diarrhea and dehydration in suckling pigs. We detected three PEDV strains from ten small intestine samples from piglets with acute diarrhea and we determined the complete genome sequences of the reemerging Korean PEDV field isolates, except for the noncoding regions from both ends. The complete genome sequences of the strains were identical or almost identical (one synonymous single-nucleotide polymorphism (SNP) in the ORF1a/1b genomic sequence). Interestingly, comparative genome analysis of recent Korean PEDV isolates and other strains revealed that the complete genome sequences of recent Korean strains were almost identical (99.9%) to those of the US PEDV strains isolated in 2013. These results suggest that the three reemerging Korean strains are distinct from previous endemic Korean PEDV strains and has been recently introduced into Korea from oversea with high likelihood.

Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs.

To understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged US strain, PC21A, of the virus. At 24–48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. These findings confirm that strain PC21A is highly enteropathogenic.