Development of an improved virus plaque assay based on avicel / 生物工程学报

Avicel is made of a mixture of microcrystalline cellulose (MCC) and carboxymethyl cellulose (CMC), and used for virus plaque assay. The avicel in common use is produced by FMC Biopolymer. Due to the relatively fixed proportion of MCC and CMC, avicel in common use is not suitable for plaque determination experiment of all types of viruses. In this study, we evaluated the effect of avicel made of different proportions of MCC and CMC on virus plaque assay, and developed an improved avicel virus plaque assay featured with simple and convenient operation, good practicability and high stability. To generate avicel overlays with different proportions of MCC and CMC, twelve different 2×avicel solutions were prepared. Their overall viscosity and bottom viscosity were measured to evaluate the ease of operation. The results showed that most of the 2×avicel solutions (except the 4.8% MCC+1.4% CMC and 4.8% MCC+1.0% CMC group) were easy to absorb and prepare nutrient overlap than 2×CMC solution. In order to find the best scheme to detect the titer of porcine epidemic diarrhea virus (PEDV), these avicel overlay solutions with different proportion of MCC and CMC were used as a replacement in the standard plaque assay. By comparing the size, clarity, stability and titer accuracy of virus plaque, we identified that 0.6% MCC and 0.7% CMC was the most preferable composition of avicel overlay for PEDV plaque assay. In conclusion, we developed an improved virus plaque assay based on avicel, which may facilitate the research of virus etiology, antiviral drugs and vaccines.

Development of a blocking ELISA based on a single-domain antibody target the S1 protein of porcine epidemic diarrhea virus / 生物工程学报

The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3'-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.

Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene / 生物工程学报

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus (PEDV) S1 gene can protect animals against PEDV, guinea pigs were fed with recombinant L. plantarum containing plasmid PVE5523-S1, with a dose of 2×10⁸ CFU/piece, three times a day, at 14 days intervals. Guinea pigs fed with wild type L. plantarum and the engineered L. plantarum containing empty plasmid pVE5523 were used as negative controls. For positive control, another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis (HB08+ZJ08) by intramuscular injection, with a dose of 0.2 mL/piece, three times a day, at 14 days intervals. Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d, 7 d, 14 d, 24 d, 31 d, 41 d and 48 d, respectively, and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay (ELISA). The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay. The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody, and stimulate the increase of IL-4 and IFN-γ, as well as the proliferation of spleen cells. These results indicated that the engineered L. plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs, which laid a foundation for subsequent oral vaccine development.

Immunization against porcine epidemic diarrhea virus and vaccine development / 生物工程学报

Porcine epidemic diarrhea (PED) is a major disease of pigs that inflicts heavy losses on the global pig industry. The etiologic agent is the porcine epidemic diarrhea virus (PEDV), which is assigned to the genus Alphacoronavirus in the family Coronaviridae. This review consists of five parts, the first of which provides a brief introduction to PEDV and its epidemiology. Part two outlines the passive immunity in new born piglets and the important role of colostrum, while the third part summarizes the characteristics of the immune systems of pregnant sows, discusses the concept of the "gut-mammary gland-secretory IgA(sIgA) axis" and the possible underpinning mechanisms, and proposes issues to be addressed when designing a PEDV live vaccine. The final two parts summarizes the advances in the R&D of PEDV vaccines and prospects future perspectives on prevention and control of PEDV, respectively.

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.

Generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus

The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs

PURPOSE: The first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge. MATERIALS AND METHODS: To generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test. RESULTS: The QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding. CONCLUSION: A novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.

Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014

Outbreaks of porcine epidemic diarrhea (PED) have resulted in significant economic losses in the swine industry, and another PED outbreak occurred in 2014 in Korea. Isolating and culturing PED virus (PEDV) allow investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated two PEDV isolates (QIAP1401 and QIAP1402) from naturally infected piglets at Jeju-do, Korea. Viral propagation was confirmed in Vero cells based on cytopathic effect, immunofluorescence assay, reverse transcription-polymerase chain reaction, and electron microscopic analyses. The QIAP401 isolate propagated well in Vero cells for 70 passages, with titers of 10(6.5) to 10(7.0) 50% tissue culture infectious dose/mL, which increased gradually with passaging. The nucleotide and amino acid sequences of the QIAP1401 isolate were determined and compared with those of other PEDV isolates. The QIAP1401 isolate was determined to be closely related to the USA/Minnesota271/2014 strain (> 99.9% nucleotide similarity) that was isolated in the USA in 2014. Phylogenetic analysis based on several PEDV genes suggested that a new PEDV variant is circulating in the Korean swine industry, with 93.08% similarity to the SM98 strain isolated in 1998. In addition, the QIAP1401 strain showed strong virulence in 3-day-old piglets and 11-week-old growing pigs.

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Porcine epidemic diarrhea virus (PEDV) is a contagious coronavirus infecting pigs that leads to significant economic losses in the swine industry. Given that PEDV infection occurs in gut epithelial cells mainly via the fecal-oral route, induction of PEDV-specific immune responses in the mucosal compartment is required for protective immunity against viral infection. However, an effective mucosal vaccine against the currently prevalent PEDV strain is not available. In this study, we demonstrated that the N-terminal domain (NTD) of the spike (S) protein of PEDV represents a new vaccine candidate molecule to be applied via the mucosal route. We first established an Escherichia coli expression system producing the partial NTD (NTD231–501) of the PEDV S protein. Orally administered NTD231–501 protein specifically interacted with the apical area of M cells in the follicle-associated epithelium of Peyer's patch. Additionally, the NTD protein induced antigen-specific immune responses in both the systemic and mucosal immune compartments when administered orally. Collectively, we propose the NTD of the PEDV S protein to be a candidate mucosal vaccine molecule.

Molecular epidemiological analysis of ORF3 genes of PEDV endemic strains in Henan province / 中国兽医学报

To understand the genetic origin and variation of porcine epidemic diarrhea virus (PEDV) prevailing in Henan province,a total of 22 PEDV positive samples from suckling piglets with severe diarrhea were collected from 16 pig farms and amplified by RT-PCR.ORF3 genes were then cloned into pMD18-T vector and sequenced.The result of sequencing showed that the length of ORF3 genes all were 675 bp which encoded 224 amino acids.The strains in this study had eight amino acid substitutions when compared with CV777 strain.The nucleotide homologies of the 22 strains were 95.9％-100％ and amino acid homologies were 96.4％-100％.By comparing with those of European strain CV777 and vaccine strain truncated CV777,the nucleotide homologies were 97.1％-97.9％ and 94.8％-95.4％,respectively.Based on the phylogenetic relationship of ORF3 genes,the PEDV field strains and PEDV reference strains could be divided into two groups,and all the field strains identified in this study belong to group 2.It suggested that prevalent PEDV strains in this study seem to be closely related to Henan isolates in previous years,domestic strains,Janpanese strains,American strains as well as Korean strains but differ genetically from European strains or vaccine strains used in China.This experiment analyzed the ORF3 gene sequence features in prevailing of Henan region in recent years,which provides a new support of epidemiology study and protein function research of PEDV ORF3 gene.

Isolation and identification of porcine epidemic diarrhea virus strain LN-2015-1 and its S gene mutation analysis / 中国兽医学报

In order to understand the porcine epidemic diarrhea virus (PEDV) origin and variant characteristics in Liaoning province,diagnosed by PCR,separated by Vero cell,and identified by cell pathological observation,RT-PCR and S gene sequence analysis,1 PEDV strains (LN-2015-1) was successfully isolated from a pig farm of Liaoning province.Analysis of S gene sequence showed that compared withCV777 strain,there were the longest 9 bp insertion,6 bp deletion and 13 bp continuous mutation in addition to point mutation.There also were the longest 3 AA insert,2 AA deletion,and 3 AA or more continuous mutation.The epitope analysis showed that there were 16AA mutations in the 5 epitope regions.Homology analysis show that it had the highest sequence similarity of 99.2％ with HB-HA2015 strain,higher sequence similarity of 98.5％-98.8％ with the domestic and foreign representative strains isolated since 2010,and lower sequence similarity of 93.2％-95.6％ with the traditional strain isolated before 2010;the phylogenetic analysis showed that LN-2015-1 was clustered into the same group with home and abroad variation strain in recent years,and formed a small subgroup with HB-HA2015 at the same time.The evolutionary distance was far from the traditional strains.

Advances in reverse genetics to treat porcine epidemic diarrhea virus / 生物工程学报

Porcine epidemic diarrhea virus (PEDV) is one of the major etiologies responsible for the acute, highly contagious disease in the digestive tract of pigs, especially neonatal piglets. Since PEDV was first identified in Europe in the late 1970s, it has resulted in significant economic losses in many Asian swine-raising countries, including China. Recently, reverse genetics techniques including targeted RNA recombination, bacteria artificial chromosome system and in vitro ligation have been successfully used to manipulate the genome of PEDV, which providing new strategies for the clear delineation of the functions of the viral proteins, the mechanisms behind PEDV pathogenesis and the design of novel vaccines against PEDV. Here, we review the progresses of different reverse genetics platforms developed for PEDV and their applications, covering the roles of trypsin in PEDV propagation, functions of S and ORF3 protein and the development of next generation PED vaccines, and the perspectives of reverse genetics for PEDV.

Sequence analysis of the spike gene of Porcine epidemic diarrhea virus isolated from South China during 2011–2015

The spike gene of porcine epidemic diarrhea virus (PEDV) was sequenced from 55 South China field strains isolated from pigs with symptoms of diarrhea. The sequences were compared within the set of field strains as well as with reference strains available in GenBank. Within the 55 South China PEDV field strains, the deduced amino acid sequence identities ranged from 93.8% to 99.9 % and ranged from 90.7% to 99.5% when compared with the foreign reference strains in GenBank. Our phylogenetic analysis showed that 10 of the 55 South China PEDV strains belonged to G1b and 45 belonged to G2b.

Antiviral Activity of Corylus heterophylla Fisch Against Porcine Epidemic Diarrhea Virus Infection

The porcine epidemic diarrhea virus (PEDV) has recently been shown to cause huge economic losses in the global pork industry. Our results demonstrated that the extract dose-dependently inhibited the replication of PEDV and reduced the visible cytopathic effect (CPE). Treatment with C. heterophylla Fisch extract resulted in marked reduction of PEDVinduced cytokine and chemokine expression. The antiviral activity of C. heterophylla Fisch extract on PEDV replication was found to be primarily exerted at the early stages after infection. Taken together, our data indicate that C. heterophylla Fisch extract may be a good therapeutic agent for use against PEDV and also a potential candidate to be evaluated against other human and animal coronaviruses.

Recent vaccine technology in industrial animals

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.

Genetic diversity of nucleocapsid genes of recent porcine epidemic diarrhea viruses isolated in Korea

Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets, resulting in large economic losses because of high mortality. In November 2013, PEDV reemerged in Korea, and these outbreaks have since continuously occurred. In the present study, we determined the full-length nucleocapsid (N) gene sequences of three Korean PEDV field isolates collected in 2014-2015. Sequence and phylogenetic analysis of N genes revealed that recent prevalent Korean PEDV isolates were very closely related to the US PEDV isolates in 2013. Interestingly, the phylogenetic tree based on the nucleotide sequencing of the PEDV N gene was similar to the tree topology of the PEDV complete genomes. Therefore, our data provide a better understanding of the genetic diversity and contribute to the accurate diagnosis and development of vaccines against PEDV.

Reemergence of porcine epidemic diarrhea virus on Jeju Island / 대한수의학회지

Porcine epidemic diarrhea virus (PEDV) strains responsible for recent outbreaks in the United States have been occurring in Mainland Korea since late 2013. Over the past 10 years, PEDV outbreaks have not been reported on Jeju Island. However, in late March of 2014, PEDV re-emerged on Jeju Island and was found to be genetically identical to PEDV strains currently circulating in Mainland Korea. The present study was conducted to provide a better understanding of the epidemiology of PEDV and more effective preventive measures against PED.

Reemergence of porcine epidemic diarrhea virus on Jeju Island / 대한수의학회지

Porcine epidemic diarrhea virus (PEDV) strains responsible for recent outbreaks in the United States have been occurring in Mainland Korea since late 2013. Over the past 10 years, PEDV outbreaks have not been reported on Jeju Island. However, in late March of 2014, PEDV re-emerged on Jeju Island and was found to be genetically identical to PEDV strains currently circulating in Mainland Korea. The present study was conducted to provide a better understanding of the epidemiology of PEDV and more effective preventive measures against PED.

Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection

An indirect porcine epidemic diarrhea (PED) virus (PEDV) enzyme-linked immunosorbent assay (ELISA) was compared with the serum neutralization (SN) test by testing 46 samples from experimentally infected sows, 73 samples from naive sows, and 1, 024 field sow samples from 48 commercial swine farms of undefined PED status. The SN test and the ELISA were performed using PEDV, KPEDV-9 strain. Viral proteins as a coating antigen of PEDV ELISA were extracted from the cytoplasm of PEDV-infected Vero cells using a non-ionic detergent, Triton X-100, and a simple protocol of PEDV ELISA was followed. The presence of antibodies in these experimental samples was confirmed by SN and ELISA in which the sensitivity of the ELISA was 89.1%, and the corresponding specificity was 94.5%. On testing 1, 024 field samples, an overall agreement of 84.2% was generated between the SN and ELISA. This study demonstrates that the PEDV ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against PEDV.