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1.
Chinese Veterinary Science / Zhongguo Shouyi Kexue ; 50(1):10-19, 2020.
Article in Chinese | CAB Abstracts | ID: covidwho-2056573

ABSTRACT

The aim of this study is to establish an indirect ELISA technique for detecting the SIgA antibody against porcine epidemic diarrhea virus (PEDV) to evaluate its mucosal immunity. Firstly, the S1D gene (534-789 aa) of PEDV was cloned into the pET-28a(+) vector, and induced in Escherichia coli BL21 (DE3) by IPTG, the product of which was in the form of inclusion bodies. According to Western-blot, the target protein S1D with antigenic activity was 32 ku in molecular weight and could be well detected. Then, the S1D protein was denatured by 8 mol/L urea, purified and gradient as the coating antigen to establish an indirect ELISA for detecting the PEDV specific SIgA antibody in nasal or oral mucus by optimizing conditions. And the optimal antigen coating concentration of ELISA was 2 micro g mL, the working concentrations of nasal mucus was 1:1 and the optimal blocking solution was 50 g/L skimmed milk, while the working concentrations and optimal blocking solution were 1:2 and 30 g/L BSA in oral mucus, the working concentrations of the enzyme-labeled antibody was 1:2 000 in nasal and oral mucus. Finally, 84 samples of oral and nasal mucus from immunized pigs were detected by S1D of ELISA, and the coincidence rate could reach 95.2% compared with purified PEDV of ELISA. In conclusion, the indirect ELISA established in this study provided a quick, simple, sensitive, and specific method to detect PEDV specific SigA for evaluating the level of PEDV mucosal immunity.

2.
PLoS Global Public Health ; 2(8), 2022.
Article in English | CAB Abstracts | ID: covidwho-2039236

ABSTRACT

The international tourist destination of Bali reported its first case of Coronavirus Disease 2019 or COVID-19 in March 2020. To better understand the extent of exposure of Bali's 4.3 million inhabitants to the COVID-19 virus, we performed two repeated cross-sectional serosurveys stratified by urban and rural areas. We used a highly specific multiplex assay that detects antibodies to three different viral antigens. We also assessed demographic and social risk factors and history of symptoms. Our results show that the virus was widespread in Bali by late 2020, with 16.73% (95% CI 12.22-21.12) of the population having been infected by that time. We saw no differences in seroprevalence between urban and rural areas, possibly due to extensive population mixing, and similar levels of seroprevalence by gender and among age groups, except for lower seroprevalence in the very young. We observed no difference in seroprevalence between our two closely spaced surveys. Individuals reporting symptoms in the past six months were about twice as likely to be seropositive as those not reporting symptoms. Based upon official statistics for laboratory diagnosed cases for the six months prior to the survey, we estimate that for every reported case an additional 52 cases, at least, were undetected. Our results support the hypothesis that by late 2020 the virus was widespread in Bali, but largely undetected by surveillance.

3.
Acta Veterinaria et Zootechnica Sinica ; 53(5):1536-1543, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-1994512

ABSTRACT

This study aims to investigate the protective effect of infected piglets which were immunized with different dose of inactivated porcine epidemic diarrhea virus (PEDV) vaccines. The number of infective virus particles and total virus particles of PEDV with different concentrations were determined, and the mice were immunized with different concentration vaccine prepared as antigen, respectively. The humoral and cellular immune production were determined by ELISA antibody detection method, neutralization test and ELISPOT method. Vaccine with appropriate antigen content was selected to immunize piglets, then the antibody was determined. The relationship between concentrated vaccine and protective effect was studied by challenge experiment. The results showed that, when the antigen dose was equal or greater than 8x106 pfu.mL-1, the inactive vaccine could effectively stimulate mice to produce humoral and cellular immunity. The piglets immunized with 2 mL inactivated PEDV vaccine containing 8x106 pfu.mL-1 antigen could resist diarrhea and continuous viral shedding caused by PEDV challenge. Compared with the total number of virus particles, the number of infectious virus particles was significantly correlated with antibody production (r=0.998 1), and neutralization titer was significantly correlated with piglet protection (r=0.974 7). PEDV inactivated vaccine can provide good immune protection, in which the number of infectious virus particles is the key factor to improve the antibody level. Antibody titer, as an index of humoral immunity, is an important reference for judging immune protection.

4.
SwissHerdbook Bulletin 2021. (2):72 pp. ; 2021.
Article in German | CAB Abstracts | ID: covidwho-1989219

ABSTRACT

This bulletin highlights the effects of COVID-19 in the dairy cattle sector with emphasis on milk prices and milk yield.

5.
Acta Veterinaria et Zootechnica Sinica ; 53(4):1173-1181, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-1975364

ABSTRACT

The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus (PEDV). The purified N protein was used as the coating antigen, and the ELISA reaction conditions were optimized by the chess rboard titration. A blocking ELISA method for detecting PEDV antibodies was established, and its specificity, sensitivity and repeatability tests were carried out. One hundred and forty clinical serum samples were tested, and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit. The results showed that the best antigen coating concentration was 625 ng.mL-1, and the best dilution ratio of serum was 1:1;The best dilution of the HRP-conjugated antibody working solution was 1:5 000;There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and transmissible gastroenteritis virus (TGEV). The sensitivity of PEDV positive serum was 1:16, which was equivalent to that of IDvet ELISA kit (titer 1:32). The coefficient of variation of within-run and between-run repeatability test is less than 10%, so it showed that the blocking ELISA established in this study had good repeatability and stability;the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA. The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV, epidemiological investigation and the monitoring of antibody levels after vaccine immunization.

6.
Harran Universitesi Veteriner Fakultesi Dergisi ; 11(1):120-127, 2022.
Article in Turkish | CAB Abstracts | ID: covidwho-1934962

ABSTRACT

Bovine coronavirus (BCoV) infections are widespread in newborn calf diarrhea, which is one of the critical problems in cattle breeding. This study aims to investigate BCoV infection in calves with diarrhea in Sanliurfa province. In this study, 94 calves with diarrhea (3 months) following clinical examination were sampled. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of BCoV antigen. A total of 5 stool samples were found to be BCoV positive (5.32%). This result showed that BCoV was low in calves with diarrhea in Sanliurfa province. However, considering the rapid spread of the infection in cattle populations, it is thought that it may cause significant economic losses due to treatment costs and calf deaths.

7.
Angew Chem Int Ed Engl ; 61(28): e202203662, 2022 07 11.
Article in English | MEDLINE | ID: covidwho-1819337

ABSTRACT

The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.


Subject(s)
Biosensing Techniques , COVID-19 , Antibodies, Viral , COVID-19/diagnosis , COVID-19 Testing , Humans , Immunity , Immunoglobulin G , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
8.
Journal of Heart & Lung Transplantation ; 41(4):S134-S134, 2022.
Article in English | Academic Search Complete | ID: covidwho-1783354

ABSTRACT

Exosomes are nanosized vesicles released by cells into body fluids. We have demonstrated the presence of circulating exosomes containing viral antigens in lung transplant recipients (LTxR) undergoing rejection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an important risk factor for LTxR undergoing immunosuppression. Our goal is to determine whether exosomes with SARS-CoV-2 spike protein are induced in LTxR with SARS-CoV-2 infection and exosomes are immunogenic in mice, inducing immune responses to the spike protein We analyzed 67 patients with SARS-CoV-2 infection for the induction of circulating exosomes with SARS-CoV-2 spike protein. Exosomes were isolated from plasma by an exosome precipitation protocol followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were first analyzed by western blot with specific antibodies to SARS-CoV-2 spike and its nucleoprotein. Eluted proteins from the gel were analyzed by mass spectrometry. Exosomes were subjected to transmission electron microscopy (TEM) to detect spike and nucleocapsid antigens. To determine the immunogenicity of isolated exosomes, C57BL/6 mice were immunized with exosomes carrying SARS-CoV-2 spike protein. Exosomes from SARS-CoV-2 infected LTxR expressed SARS-CoV-2 spike protein S2 and increased levels of RNA related to SARS-CoV-2. Peptides specific for SARS-CoV-2 spike protein in exosomes were confirmed by mass spectroscopy. TEM also revealed the expression of spike protein and nucleocapsid antigens on the exosome surface. Mice immunized with exosomes carrying the spike protein, developed antibodies to SARS-CoV-2 spike antigens. Severe inflammation and lesions were also demonstrated in the lungs of mice immunized with exosomes carrying SARS-CoV-2 spike protein. Splenic lymphocytes from mice immunized with exosomes carrying SARS-CoV-2 spike antigen also demonstrated increased frequency of T-cells which are spike protein antigen specific and secreting IFN-γ and TNF-α. SARS-CoV-2 infected LTxR induce circulating exosomes with spike protein and nucleic acids related to SARS-CoV-2. Since the induced exosomes are highly immunogenic, we propose that the exosomes induced by SARS-CoV-2 will have immunological consequences relevant to the COVID19 disease process. [ FROM AUTHOR] Copyright of Journal of Heart & Lung Transplantation is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

9.
Vaccine ; 39(49): 7175-7181, 2021 12 03.
Article in English | MEDLINE | ID: covidwho-1508202

ABSTRACT

The development of new, low-cost vaccines and effective gene therapies requires accurate delivery and high-level expression of candidate genes. We developed a plasmid vector, pIDV-II, that allows for both easy manipulation and high expression of exogenous genes in mammalian cells. This plasmid is based upon the pVax1 plasmid and shares a common structure with typical mammalian transcription units. It is composed of a chicken ß-actin promoter (CAG), followed by an intron and flanked by two restriction sites, and also includes a post-transcriptional regulatory element, followed by a transcriptional termination signal. While the modification of pVax1 elements either decreased eGFP expression levels or had no effect at all, replacement of the promoter, the poly-A signal, deletion of the T7 and AmpR promoters, and inversion of the ORI-Neo/Kan cassette, significantly increased in vitro eGFP expression with the modified plasmid called pIDV-II. To further evaluate our vector, expression levels of three viral antigens were compared in cell lines transfected either with pVax1 or pCAGGS backbones as controls. Higher transgene expression was consistently observed with pIDV-II. The humoral and cellular responses generated in mice immunized with pIDV-II vs pVax1 expressing each viral antigen individually were superior by 2-fold or more as measured by ELISA and ELISPOT assays. Overall these results indicate that pIDV-II induces robust transgene expression, with concomitant improved cellular and humoral immune responses against the transgene of interest over pVax1. The new vector, pIDV-II, offers an additional alternative for DNA based vaccination and gene therapy for animal and human use.


Subject(s)
Vaccines, DNA , Animals , DNA , Immunity, Humoral , Mice , Mice, Inbred BALB C , Transgenes , Vaccines, DNA/genetics
10.
J Biol Chem ; 296: 100745, 2021.
Article in English | MEDLINE | ID: covidwho-1213326

ABSTRACT

Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics.


Subject(s)
Adaptive Immunity , Antibodies, Viral/chemistry , Antigens, Viral/chemistry , Immunity, Innate , Receptors, Antigen, T-Cell/chemistry , Receptors, Cytokine/chemistry , SARS-CoV-2/immunology , Allergy and Immunology/history , Animals , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibody Specificity , Antigen Presentation , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/virology , Crystallography/history , Crystallography/methods , History, 20th Century , History, 21st Century , Humans , Protein Folding , Protein Interaction Domains and Motifs , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Cytokine/genetics , Receptors, Cytokine/immunology , SARS-CoV-2/pathogenicity , V(D)J Recombination
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