scholarly journals Isolation, characterization and neutralizing activity of porcine epidemic diarrhea viruses from Vietnam

2021 ◽  
Vol 61 (3) ◽  
pp. e23
Author(s):  
Van Tan Do ◽  
Quang Lam Truong ◽  
Hoai Thu Dao ◽  
Thi Lan Nguyen ◽  
Jini Kim ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Vero Cells ◽  
Watery Diarrhea ◽  
Sequence Comparisons ◽  
Neonatal Piglets ◽  
Homologous Strain ◽  
Porcine Epidemic Diarrhea ◽  
High Virus ◽  
Virus Isolates

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were successfully propagated in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene revealed that all 3 isolates belong to genogroup 2a, which is closely related to other prevalent Asian strains. Amino acid sequence comparisons revealed 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013–2015, suggesting that field PED viruses (PEDVs) evolve continuously. Experiments in animals demonstrated that antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log2) of neutralizing antibody against the homologous strain, and showed a relatively lower level of neutralizing antibody against the field isolates. This finding would be helpful in choosing a PEDV strain for vaccine development.

THE OUTBREAK OF PORCINE EPIDEMIC DIARRHEA IN TAIWAN

2014 ◽  
Vol 40 (03) ◽  
pp. 115-121 ◽  
Author(s):  
Ming-Chung Deng ◽  
Chia-Yi Chang ◽  
Tien-Shine Huang ◽  
Shu-Ting Kuo ◽  
Hsiang-Jung Tsai ◽  
...  
Keyword(s):  
Animal Health ◽  
Vero Cells ◽  
Transmissible Gastroenteritis Virus ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
Diarrhea Virus ◽  
Severe Diarrhea ◽  
Porcine Epidemic Diarrhea ◽  
Transmissible Gastroenteritis ◽  
Group 2

Between January 20 and April 30 of 2014, a total of 103 diarrhea cases from 47 herds in 13 counties were submitted to the Animal Health Research Institute. In 20 of the 25 herds with detail history, severe diarrhea and vomiting occurred in pigs of all ages, with mortality approaching 100% in suckling pigs. The differential etiologies, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine group A rotavirus (GARV), were tested by reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR of PEDV was positive in 79 cases of 34 herds. Attempts to isolate PEDV in Vero cells revealed that only 7 specimens from 7 herds showed the cytopathic effects (CPEs) of fusion and syncytia. These CPEs were indeed caused by PEDV, as confirmed by RT-PCR, sequencing, and electron microscopy. Sequence comparisons of diarrhea samples and isolated PEDV were assayed by MEGA 5.2 software. The newly isolated PEDV/Taiwan/2014 strains were clustered in group 2 as novel PEDV, together with strains PEDV/USA/2013, PEDV/China/2011–2013, PEDV/Thailand/2007–2008, and PEDV/Korea/2008–2009, whereas the classical CV777 strain was placed in a separate group 1. These results indicated that a novel PEDV was the cause of the recent new outbreak of diarrhea in Taiwan.

scholarly journals Optimization of Vero Cells Grown on a Polymer Fiber Carrier in a Disposable Bioreactor for Inactivated Coxsackievirus A16 Vaccine Development

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 613
Author(s):  
Keda Chen ◽  
Chaonan Li ◽  
Ying Wang ◽  
Zhenwei Shen ◽  
Yikai Guo ◽  
...  
Keyword(s):  
Large Scale ◽  
Vaccine Development ◽  
Virus Titer ◽  
Foot And Mouth Disease ◽  
Neutralizing Antibody ◽  
Vero Cells ◽  
Coxsackie Virus ◽  
Scale Production ◽  
Polymer Fiber ◽  
Culture Process

At present, there are no vaccines available for hand, foot, and mouth disease, which is caused by Coxsackie virus A16 (CVA16) infection. In the present study, we isolated epidemic strains of CVA16 and optimized the production of the virus in Vero cells. The system comprised growing the infected cells on polymer fiber paper carriers in a serum-free medium containing 0.5% (w/v) lactalbumin hydrolysate a mini bioreactor. Disposable Bioflo310 and AmProtein Current perfusion bioreactors were used to monitor virus infection and Vero cell culture. The total number of cells increased from 1.5 × 109 to 3.0 × 1010. In our optimized culture process, the virus titer reached 7.8 × 107 TCID50/mL at three days after infection. The inactivated CVA16 prepared from our optimized culture procedure elicited a slightly higher neutralizing antibody titer compared with that derived from routine culture procedures. These results will promote the large-scale production of inactivated CVA16 vaccines using nonwoven polymer fiber paper cell cultures.

scholarly journals Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Akitsu Masuda ◽  
Jae Man Lee ◽  
Takeshi Miyata ◽  
Takeru Ebihara ◽  
Kohei Kakino ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vector System ◽  
Watery Diarrhea ◽  
Small Scale ◽  
Efficient Production ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

AbstractPorcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

scholarly journals Construction of Porcine Epidemic Diarrhea Virus-Like Particles and Its Immunogenicity in Mice

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 370
Author(s):  
Jihee Kim ◽  
Jaewon Yoon ◽  
Jung-Eun Park
Keyword(s):  
Immune Responses ◽  
Genetic Material ◽  
Vero Cells ◽  
Swine Industry ◽  
E Proteins ◽  
Virus Like Particles ◽  
Diarrhea Virus ◽  
Humoral Immune ◽  
Neonatal Piglets ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED), a highly contagious and lethal enteric disease in piglets, is characterized by diarrhea, vomiting, and dehydration, with high mortality in neonatal piglets. Despite the nationwide use of attenuated and inactivated vaccines, the outbreak of PED is still a major problem in the swine industry. Virus-like particles (VLPs) are artificial nanoparticles similar to viruses that are devoid of genetic material and are unable to replicate. VLPs have good safety profiles and elicit robust cellular and humoral immune responses. Here, we generated PED VLPs in eukaryotic cells and examined their immune responses in mice. We found that the M protein is essential for the formation of PED VLPs. Interestingly, PED VLP formation was decreased in the presence of E proteins and increased in the presence of N proteins. Both IgG and IgA antibodies were induced in mice immunized with PED VLPs. Moreover, these antibodies protected against PED virus infection in Vero cells. PED VLPs immunization induced Th2-dominant immune responses in mice. Our results indicate that PED VLPs induce strong immune responses in mice, suggesting that the VLP-based vaccine is a promising vaccine candidate.

scholarly journals Dengue 2 PDK-53 Virus as a Chimeric Carrier for Tetravalent Dengue Vaccine Development

2003 ◽  
Vol 77 (21) ◽  
pp. 11436-11447 ◽  
Author(s):  
Claire Y.-H. Huang ◽  
Siritorn Butrapet ◽  
Kiyotaka R. Tsuchiya ◽  
Natth Bhamarapravati ◽  
Duane J. Gubler ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Vero Cells ◽  
Vaccine Virus ◽  
Wild Type ◽  
Dengue Vaccine ◽  
Virus Challenge ◽  
Viral Interference ◽  
Antibody Titers ◽  
Virus Expression

ABSTRACT Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK2 cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.

scholarly journals The Accessory Protein ORF3 Contributes to Porcine Epidemic Diarrhea Virus Replication by Direct Binding to the Spike Protein

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 399 ◽  
Author(s):  
Challika Kaewborisuth ◽  
Qigai He ◽  
Anan Jongkaewwattana
Keyword(s):  
Virus Replication ◽  
Porcine Epidemic Diarrhea Virus ◽  
Watery Diarrhea ◽  
Accessory Protein ◽  
Structural Protein ◽  
S Protein ◽  
Diarrhea Virus ◽  
Neonatal Piglets ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.

scholarly journals A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity

2021 ◽  
Author(s):  
Vincent Legros ◽  
Solène Denolly ◽  
Manon Vogrig ◽  
Bertrand Boson ◽  
Eglantine Siret ◽  
...  
Keyword(s):  
Intensive Care ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Surface Protein ◽  
Humoral Response ◽  
Neutralizing Antibody ◽  
Rapid Decline ◽  
Serum Samples ◽  
Immune Correlates ◽  
Human Coronaviruses

AbstractUnderstanding the immune responses elicited by SARS-CoV-2 infection is critical in terms of protection against reinfection and, thus, for public health policy and vaccine development for COVID-19. In this study, using either live SARS-CoV-2 particles or retroviruses pseudotyped with the SARS-CoV-2 S viral surface protein (Spike), we studied the neutralizing antibody (nAb) response in serum samples from a cohort of 140 SARS-CoV-2 qPCR-confirmed infections, including patients with mild symptoms and also more severe forms, including those that required intensive care. We show that nAb titers correlated strongly with disease severity and with anti-spike IgG levels. Indeed, patients from intensive care units exhibited high nAb titers; conversely, patients with milder disease symptoms had heterogeneous nAb titers, and asymptomatic or exclusive outpatient-care patients had no or low nAbs. We found that nAb activity in SARS-CoV-2-infected patients displayed a relatively rapid decline after recovery compared to individuals infected with other coronaviruses. Moreover, we found an absence of cross-neutralization between endemic coronaviruses and SARS-CoV-2, indicating that previous infection by human coronaviruses may not generate protective nAbs against SARS-CoV-2. Finally, we found that the D614G mutation in the spike protein, which has recently been identified as the current major variant in Europe, does not allow neutralization escape. Altogether, our results contribute to our understanding of the immune correlates of SARS-CoV-2-induced disease, and rapid evaluation of the role of the humoral response in the pathogenesis of SARS-CoV-2 is warranted.

scholarly journals Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Receptor Binding Domain ◽  
Proof Of Concept ◽  
Reference Standard ◽  
Improve Measurement ◽  
Antibody Titers

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates

2021 ◽  
Vol 13 (583) ◽  
pp. eabe5449
Author(s):  
Nicole Darricarrère ◽  
Yu Qiu ◽  
Masaru Kanekiyo ◽  
Adrian Creanga ◽  
Rebecca A. Gillespie ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Nonhuman Primates ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Neutralizing Antibody ◽  
Gene Families ◽  
Influenza Viruses ◽  
Public Health Importance ◽  
Water Emulsion

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus–associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

scholarly journals Antibody titers measured by commercial assays are correlated with neutralizing antibody titers calibrated by international standards

2021 ◽  
Author(s):  
Yu-An Kung ◽  
Chung-Guei Huang ◽  
Sheng-Yu Huang ◽  
Kuan-Ting Liu ◽  
Peng-Nien Huang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Diagnostic Techniques ◽  
International Standards ◽  
World Health ◽  
Serum Samples ◽  
Antibody Titers ◽  
Health Organization

The World Health Organization (WHO) has highlighted the importance of an international standard (IS) for SARS-CoV-2 neutralizing antibody titer detection, with the aim of calibrating different diagnostic techniques. In this study, IS was applied to calibrate neutralizing antibody titers (IU/mL) and binding antibody titers (BAU/mL) in response to SARS-CoV-2 vaccines. Serum samples were collected from participants receiving the Moderna (n = 20) and Pfizer (n = 20) vaccines at three time points: pre-vaccination, after one dose, and after two doses. We obtained geometric mean titers of 1404.16 and 928.75 IU/mL for neutralizing antibodies after two doses of the Moderna and Pfizer vaccines, respectively. These values provide an important baseline for vaccine development and the implementation of non-inferiority trials. We also compared three commercially available kits from Roche, Abbott, and MeDiPro for the detection of COVID-19 antibodies based on binding affinity to S1 and/or RBD. Our results demonstrated that antibody titers measured by commercial assays are highly correlated with neutralizing antibody titers calibrated by IS.

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