Molecular Detection and Characterization of Porcine Epidemic Diarrhea Virus and Porcine Aichivirus C Coinfection in México

Swine enteric viral infections are responsible for substantial economic losses in the pork industry worldwide. Porcine epidemic diarrhea (PEDV) is one of the main causative agents of diarrhea in lactating pigs, and reports of PEDV coinfection with other enteric viruses highlight the importance of viral interactions for disease presentation and outcomes. Using next-generation sequencing (NGS) and sequence analyses from samples taken from piglets with acute diarrhea, we explored the possible interactions between PEDV and other less reported pathogens. PEDV coinfection with porcine kobuvirus (PKV) was detected in 36.4% (27/74) of samples. Full genomes from porcine coronavirus and kobuvirus were obtained, as was a partial porcine sapovirus genome (PSaV). The phylogenetic results show the clustering of these strains corresponding to the geographical relationship. To our knowledge, this is the first full genome and isolation report for porcine kobuvirus in México, as well as the first phylogenetic analysis for porcine sapovirus in the country. The NGS approach provides a better perspective of circulating viruses and other pathogens in affected production units.

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Gene Variations in Cis-Acting Elements between the Taiwan and Prototype Strains of Porcine Epidemic Diarrhea Virus Alter Viral Gene Expression

Genes ◽

10.3390/genes9120591 ◽

2018 ◽

Vol 9 (12) ◽

pp. 591

Author(s):

Tsung-Lin Tsai ◽

Chen-Chang Su ◽

Ching-Chi Hsieh ◽

Chao-Nan Lin ◽

Hui-Wen Chang ◽

...

Keyword(s):

Gene Expression ◽

Porcine Epidemic Diarrhea Virus ◽

The Body ◽

Economic Losses ◽

Viral Gene ◽

Sequence Motif ◽

Cis Acting ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea ◽

In Cis

In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the cis-acting elements between the porcine epidemic diarrhea virus (PEDV) Taiwan (TW) strain and the prototype strain CV777 alter gene expression. For this aim, we analyzed the variations of the cis-acting elements in the 5’ and 3’ untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.

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EGR1 suppresses porcine epidemic diarrhea virus replication by regulating IRAV to degrade viral nucleocapsid protein

Journal of Virology ◽

10.1128/jvi.00645-21 ◽

2021 ◽

Author(s):

Hua Wang ◽

Ning Kong ◽

Yajuan Jiao ◽

Sujie Dong ◽

Dage Sun ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Nucleocapsid Protein ◽

E3 Ubiquitin Ligase ◽

Economic Losses ◽

Restriction Factor ◽

N Protein ◽

Host Restriction ◽

Diarrhea Virus ◽

Protein Ubiquitination ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus that has re-emerged lately, resulting in large economic losses. During viral infection, interferon (IFN-I) plays a vital role in the antiviral innate immunity. However, PEDV has evolved strategies to limit IFN-I production. To suppress virus replication, the host must activate the IFN-stimulated genes and some host restriction factors to circumvent viral replication. This study observed that PEDV infection-induced early growth response gene 1 (EGR1) expression in PEDV-permissive cells. EGR1 overexpression remarkably suppressed PEDV replication. In contrast, depletion of EGR1 led to a significant increase in viral replication. EGR1 suppressed PEDV replication by directly binding to the IFN-regulated antiviral (IRAV) promoter and upregulating IRAV expression. A detailed analysis revealed that IRAV interacts and colocalizes with the PEDV nucleocapsid (N) protein, inducing N protein degradation via E3 ubiquitin ligase MARCH8 to catalyze N protein ubiquitination. Knockdown of endogenous MARCH8 significantly reversed IRAV-mediated N protein degradation. The collective findings demonstrate a new mechanism of EGR1-mediated viral restriction, in which EGR1 upregulates the expression of IRAV to degrade PEDV N protein through MARCH8. IMPORTANCE PEDV is a highly contagious enteric coronavirus that has rapidly emerged worldwide and caused severe economic losses. No currently available drugs or vaccines could effectively control PEDV. PEDV has evolved many strategies to limit IFN-1 production. We identified EGR1 as a novel host restriction factor and demonstrated that EGR1 suppresses PEDV replication by directly binding to the IRAV promoter and upregulating the expression of IRAV, which interacts and degrades the PEDV N protein via E3 ubiquitin ligase MARCH8 to catalyze nucleocapsid protein ubiquitination, which adds another layer of complexity to innate antiviral immunity of this newly identified restriction factor. A better understanding of the innate immune response to PEDV infection will aid the development of novel therapeutic targets and more effective vaccines against virus infection.

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Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

Journal of Virology ◽

10.1128/jvi.00406-19 ◽

2019 ◽

Vol 93 (15) ◽

Cited By ~ 10

Author(s):

Yixuan Hou ◽

Hanzhong Ke ◽

Jineui Kim ◽

Dongwan Yoo ◽

Yunfang Su ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Vaccine Candidate ◽

Spike Protein ◽

Economic Losses ◽

High Dose ◽

Type I ◽

Viral Pathogen ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

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Nanoparticle-based vaccine development and evaluation against viral infections in pigs

Veterinary Research ◽

10.1186/s13567-019-0712-5 ◽

2019 ◽

Vol 50 (1) ◽

Cited By ~ 8

Author(s):

Santosh Dhakal ◽

Gourapura J. Renukaradhya

Keyword(s):

Immune Responses ◽

Viral Infections ◽

Protective Efficacy ◽

Vaccine Delivery ◽

Field Conditions ◽

Economic Losses ◽

Virus Infections ◽

Trade Restrictions ◽

Diarrhea Virus ◽

Mouth Disease Virus

Abstract Virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. The economic burden caused by virus infections such as Porcine Reproductive and Respiratory Syndrome Virus, Swine influenza virus, Porcine Epidemic Diarrhea Virus, Porcine Circovirus 2, Foot and Mouth Disease Virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. Pigs can also have a role in zoonotic transmission of some viral infections to humans. Inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. Thus, improvements over existing vaccines are necessary to: (1) Increase the breadth of protection against evolving viral strains and subtypes; (2) Control of emerging and re-emerging viruses; (3) Eradicate viruses localized in different geographic areas; and (4) Differentiate infected from vaccinated animals to improve disease control programs. Nanoparticles (NPs) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. NPs help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. Some of the NPs-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (M) cells and dendritic cells (DCs). In conclusion, NPs-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. This review highlights the recent developments in NPs-based vaccines against porcine viral pathogens and how the NPs-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy.

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Investigation of the Role of the Spike Protein in Reversing the Virulence of the Highly Virulent Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strains and Its Attenuated Counterpart

Viruses ◽

10.3390/v12010041 ◽

2019 ◽

Vol 12 (1) ◽

pp. 41 ◽

Cited By ~ 2

Author(s):

Chi-Fei Kao ◽

Hui-Wen Chang

Keyword(s):

Mortality Rate ◽

Porcine Epidemic Diarrhea Virus ◽

Rational Design ◽

Economic Losses ◽

Diarrhea Virus ◽

Viral Shedding ◽

S Gene ◽

Porcine Epidemic Diarrhea ◽

Highly Virulent

Porcine epidemic diarrhea virus (PEDV) has continuously caused severe economic losses to the global swine industries; however, no successful vaccine against PEDV has been developed. In this study, we generated four autologous recombinant viruses, including the highly virulent iPEDVPT-P5, attenuated iPEDVPT-P96, and two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) with the reciprocally exchanged spike (S) gene, to study the role of the S gene in PEDV pathogenesis. A deeper understanding of PEDV attenuation will aid in the rational design of a live attenuated vaccine (LAV) using reverse genetics system. Our results showed that replacing the S gene from the highly virulent iPEDVPT-P5 led to complete restoration of virulence of the attenuated iPEDVPT-P96, with nearly identical viral shedding, diarrhea pattern, and mortality rate as the parental iPEDVPT-P5. In contrast, substitution of the S gene with that from the attenuated iPEDVPT-P96 resulted in partial attenuation of iPEDVPT-P5, exhibiting similar viral shedding and diarrhea patterns as the parental iPEDVPT-P96 with slightly severe histological lesions and higher mortality rate. Collectively, our data confirmed that the attenuation of the PEDVPT-P96 virus is primarily attributed to mutations in the S gene. However, mutation in S gene alone could not fully attenuate the virulence of iPEDVPT-P5. Gene (s) other than S gene might also play a role in determining virulence.

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The Roles of Apoptosis in Swine Response to Viral Infection and Pathogenesis of Swine Enteropathogenic Coronaviruses

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.572425 ◽

2020 ◽

Vol 7 ◽

Author(s):

Zhichao Xu ◽

Yun Zhang ◽

Yongchang Cao

Keyword(s):

Viral Infections ◽

System Development ◽

Biological Evolution ◽

Host Responses ◽

Economic Losses ◽

Transmissible Gastroenteritis Virus ◽

Swine Industry ◽

Animal Virus ◽

Diarrhea Virus ◽

Severe Diarrhea

Apoptosis is a tightly regulated mechanism of cell death that plays important roles in various biological processes including biological evolution, multiple system development, anticancer, and viral infections. Swine enteropathogenic coronaviruses invade and damage villous epithelial cells of the small intestine causing severe diarrhea with high mortality rate in suckling piglets. Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), Porcine deltacoronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV) are on the top list of commonly-seen swine coronaviruses with a feature of diarrhea, resulting in significant economic losses to the swine industry worldwide. Apoptosis has been shown to be involved in the pathogenesis process of animal virus infectious diseases. Understanding the roles of apoptosis in host responses against swine enteropathogenic coronaviruses infection contribute to disease prevention and control. Here we summarize the recent findings that focus on the apoptosis during swine coronaviruses infection, in particular, TGEV, PEDV, PDCoV, and SADS-CoV.

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A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus

Journal of Nanobiotechnology ◽

10.1186/s12951-019-0531-x ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 4

Author(s):

Zhiqian Ma ◽

Tianyu Wang ◽

Zhiwei Li ◽

Xuyang Guo ◽

Yangsheng Tian ◽

...

Keyword(s):

Phage Display ◽

Porcine Epidemic Diarrhea Virus ◽

Diagnostic Methods ◽

Watery Diarrhea ◽

Economic Losses ◽

Serum Samples ◽

Phage Display Technology ◽

Diarrhea Virus ◽

Blocking Elisa ◽

Porcine Epidemic Diarrhea

Abstract Background Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum. Results Using phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%. Conclusions In brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.

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Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs

BMC Veterinary Research ◽

10.1186/s12917-019-2123-2 ◽

2019 ◽

Vol 15 (1) ◽

Cited By ~ 1

Author(s):

Ohnmar Myint ◽

Ayako Yoshida ◽

Satoshi Sekiguchi ◽

Nguyen Van Diep ◽

Naoyuki Fuke ◽

...

Keyword(s):

Sensitivity And Specificity ◽

High Mortality ◽

Porcine Epidemic Diarrhea Virus ◽

Indirect Elisa ◽

Neutralization Test ◽

Elisa Test ◽

Watery Diarrhea ◽

Economic Losses ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Abstract Background Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. Results One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80–100%, respectively. Conclusions The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

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Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain

Virology Journal ◽

10.1186/s12985-019-1232-7 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 2

Author(s):

Yu Wu ◽

Wei Li ◽

Qingfeng Zhou ◽

Qunhui Li ◽

Zhichao Xu ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Clinical Symptoms ◽

Vero Cells ◽

Economic Losses ◽

Protective Effects ◽

Diarrhea Virus ◽

Cytopathic Effects ◽

Porcine Epidemic Diarrhea ◽

Piglet Survival

Abstract Background Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. Methods In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. Results Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. Conclusions Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.

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A Comprehensive View on the Host Factors and Viral Proteins Associated With Porcine Epidemic Diarrhea Virus Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.762358 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yi Hu ◽

Xiaohong Xie ◽

Lingchen Yang ◽

Aibing Wang

Keyword(s):

Virus Infection ◽

Porcine Epidemic Diarrhea Virus ◽

Molecular Mechanisms ◽

Viral Proteins ◽

Host Factors ◽

Watery Diarrhea ◽

Economic Losses ◽

Target Cells ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a coronavirus pathogen of the pig intestinal tract, can cause fatal watery diarrhea in piglets, thereby causing huge economic losses to swine industries around the world. The pathogenesis of PEDV has intensively been studied; however, the viral proteins of PEDV and the host factors in target cells, as well as their interactions, which are the foundation of the molecular mechanisms of viral infection, remain to be summarized and updated. PEDV has multiple important structural and functional proteins, which play various roles in the process of virus infection. Among them, the S and N proteins play vital roles in biological processes related to PEDV survival via interacting with the host cell proteins. Meanwhile, a number of host factors including receptors are required for the infection of PEDV via interacting with the viral proteins, thereby affecting the reproduction of PEDV and contributing to its life cycle. In this review, we provide an updated understanding of viral proteins and host factors, as well as their interactions in terms of PEDV infection. Additionally, the effects of cellular factors, events, and signaling pathways on PEDV infection are also discussed. Thus, these comprehensive and profound insights should facilitate for the further investigations, control, and prevention of PEDV infection.

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