Three kinds of treatment with Homoharringtonine, Hydroxychloroquine or shRNA and their combination against coronavirus PEDV in intro

Abstract Background: Porcine epidemic diarrhea virus (PEDV) of the family Coronaviridae has caused substantial economic losses in the swine husbandry industry. There’s currently no specific drug available for treatment of coronaviruses or PEDV. Method: In the current study, we use coronavirus PEDV as a model to study antiviral agents. Briefly, a fusion inhibitor tHR2, recombinant lentivirus-delivered shRNAs targeted to conserved M and N sequences, homoharringtonine (HHT), and hydroxychloroquine (HCQ) were surveyed for their antiviral effects. Results: Treatment with HCQ at 50 mM and HHT at 150 nM reduced virus titer in TCID50 by 30 and 3.5 fold respectively, and the combination reduced virus titer in TCID50 by 200 fold. Conclusion: Our report demonstrates that the combination of HHT and HCQ exhibited higher antiviral activity than either HHT or HCQ exhibited. The information may contribute to the development of antiviral strategies effective in controlling PEDV infection.

Three kinds of treatment and their combination against coronavirus PEDV

10.21203/rs.2.23990/v1 ◽

2020 ◽

Author(s):

Cuicui Li ◽

Xiao-Jia Wang

Keyword(s):

Low Dose ◽

Antiviral Agents ◽

Virus Titer ◽

Economic Losses ◽

Diarrhea Virus ◽

Antiviral Effects ◽

Low Concentrations ◽

The Family ◽

Coronavirus Infection ◽

Abstract Background Porcine epidemic diarrhea virus (PEDV) of the family Coronaviridae has caused substantial economic losses in the swine husbandry industry. The currently available vaccines are only partially effective and there is no specific drug available for treatment of viral infection.Method In the current study, we use animal coronavirus PEDV as a model to study antiviral agents. Briefly, a fusion inhibitor tHR2, recombinant lentivirus-delivered shRNAs targeted to conserved M and N sequences, homoharringtonine (HHT), and hydroxychloroquine (HCQ) were surveyed for their antiviral effects. Results Treatment with HCQ at 50 mM reduced virus titer in TCID50 by 30 fold, and the combination with HHT at 150 nM reduced virus titer in TCID50 by 200 fold. Conclusion The combination of two drugs with low dose can more effectively inhibit PEDV infection compared to one alone. The present study offers information to contribute to the development of effective antiviral strategies against coronavirus infection, and demonstrates that the combination treatment, even at low concentrations, presents more potent antiviral activity than the separate components acting alone.

In vitro Assessment of Antiviral Effect of Natural Compounds on Porcine Epidemic Diarrhea Coronavirus

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.652000 ◽

2021 ◽

Vol 8 ◽

Author(s):

Manuel Gómez-García ◽

Héctor Puente ◽

Héctor Argüello ◽

Óscar Mencía-Ares ◽

Pedro Rubio ◽

...

Keyword(s):

Antiviral Activity ◽

Formic Acid ◽

Antiviral Agents ◽

Antiviral Effect ◽

Vero Cells ◽

Optical Microscope ◽

Dependent Manner ◽

Diarrhea Virus ◽

Organic acid and essential oils (EOs), well-known antimicrobials, could also possess antiviral activity, a characteristic which has not been completely addressed up to now. In this study, the effect of two organic acids (formic acid and sodium salt of coconut fatty acid distillates) and two single EO compounds (thymol and cinnamaldehye) was evaluated against porcine epidemic diarrhea virus (PEDV). The concentration used for each compound was established by cytotoxicity assays in Vero cells. The antiviral activity was then evaluated at three multiplicities of infection (MOIs) through visual cytopathic effect (CPE) evaluation and an alamarBlue assay as well as real-time reverse-transcription PCR (RT-qPCR) and viral titration of cell supernatants. Formic acid at at a dose of 1,200 ppm was the only compound which showed antiviral activity, with a weak reduction of CPE caused by PEDV. Through the alamarBlue fluorescence assay, we showed a significant anti-CPE effect of formic acid which could not be observed by using an inverted optical microscope. RT-qPCR and infectivity analysis also showed that formic acid significantly reduced viral RNA and viral titers in a PEDV MOI-dependent manner. Our results suggest that the antiviral activity of formic acid could be associated to its inhibitory effect on viral replication. Further studies are required to explore the anti-PEDV activity of formic acid under field conditions alone or together with other antiviral agents.

Caerin1.1 Suppresses the Growth of Porcine Epidemic Diarrhea Virus In Vitro via Direct Binding to the Virus

Viruses ◽

10.3390/v10090507 ◽

2018 ◽

Vol 10 (9) ◽

pp. 507 ◽

Cited By ~ 3

Author(s):

Nan Guo ◽

Bingzhou Zhang ◽

Han Hu ◽

Shiyi Ye ◽

Fangzhou Chen ◽

...

Keyword(s):

Antiviral Activity ◽

Antiviral Drug ◽

Vero Cells ◽

Economic Losses ◽

Diarrhea Virus ◽

Direct Binding ◽

Porcine Epidemic Diarrhea ◽

And Control

Porcine epidemic diarrhea (PED) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. Therefore, it is urgent for us to find out efficient ways to prevent and control this disease. In this study, the antiviral activity of a cationic amphibian antimicrobial peptide Caerin1.1 against porcine epidemic diarrhea virus (PEDV) was evaluated by an in vitro system using Vero cells. We found that even at a very low concentration, Caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in PEDV infections. In addition, Caerin1.1 showed powerful antiviral activity without interfering with the binding progress between PEDV and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of PEDV.

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 ◽

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.

Short communication: antiviral activity of porcine IFN-λ3 against porcine epidemic diarrhea virus in vitro

Virus Genes ◽

10.1007/s11262-016-1374-2 ◽

2016 ◽

Vol 52 (6) ◽

pp. 877-882 ◽

Cited By ~ 9

Author(s):

Haiyan Shen ◽

Chunhong Zhang ◽

Pengju Guo ◽

Zhicheng Liu ◽

Minhua Sun ◽

...

Keyword(s):

Antiviral Activity ◽

Short Communication ◽

Diarrhea Virus ◽

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 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.

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):

Vaccine Development ◽

Vaccine Candidate ◽

Spike Protein ◽

Economic Losses ◽

High Dose ◽

Type I ◽

Viral Pathogen ◽

Diarrhea Virus ◽

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.

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 ◽

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.

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 ◽

Diagnostic Methods ◽

Watery Diarrhea ◽

Economic Losses ◽

Serum Samples ◽

Phage Display Technology ◽

Diarrhea Virus ◽

Blocking Elisa ◽

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.

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 ◽

Indirect Elisa ◽

Neutralization Test ◽

Elisa Test ◽

Watery Diarrhea ◽

Economic Losses ◽

Diarrhea Virus ◽

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.