scholarly journals A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Violetta Sączyńska ◽  
Katarzyna Florys-Jankowska ◽  
Anna Porębska ◽  
Violetta Cecuda-Adamczewska
Keyword(s):  
Animal Health ◽  
Fatal Outcome ◽  
High Specificity ◽  
Influenza Viruses ◽  
Screening Tests ◽  
Vector System ◽  
Epidemiologic Surveillance ◽  
Baculovirus Expression Vector ◽  
H5 Subtype ◽  
World Organization

Abstract Background H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA). Methods In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis. Results The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively. Conclusions The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

scholarly journals A Novel Epitope-blocking ELISA for Specific and Sensitive Detection of Antibodies against H5-Subtype Influenza Virus Hemagglutinin

2021 ◽  
Author(s):  
Violetta Sączyńska ◽  
Katarzyna Florys-Jankowska ◽  
Anna Porębska ◽  
Violetta Cecuda-Adamczewska
Keyword(s):  
Animal Health ◽  
Fatal Outcome ◽  
High Specificity ◽  
Influenza Viruses ◽  
Screening Tests ◽  
Vector System ◽  
Epidemiologic Surveillance ◽  
Baculovirus Expression Vector ◽  
H5 Subtype ◽  
World Organization

Abstract Background: H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).Methods: In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.Results: The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.Conclusions: The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

scholarly journals A Novel Epitope-Blocking ELISA for Specific and Sensitive Detection of Antibodies Against H5-Subtype Influenza Virus Hemagglutinin

2020 ◽  
Author(s):  
Violetta Sączyńska ◽  
Katarzyna Florys-Jankowska ◽  
Anna Porębska ◽  
Violetta Cecuda-Adamczewska
Keyword(s):  
Animal Health ◽  
Fatal Outcome ◽  
High Specificity ◽  
Influenza Viruses ◽  
Screening Tests ◽  
Vector System ◽  
Epidemiologic Surveillance ◽  
Baculovirus Expression Vector ◽  
H5 Subtype ◽  
World Organization

Abstract Background: H5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).Methods: In the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.Results: The H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.Conclusions: The H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

scholarly journals Application of the Fluorescence Polarization Assay for Detection of Caprine Antibodies to Brucella melitensis in Areas of High Prevalence and Widespread Vaccination

2007 ◽  
Vol 14 (3) ◽  
pp. 299-303 ◽  
Author(s):  
C. Ramírez-Pfeiffer ◽  
K. Nielsen ◽  
P. Smith ◽  
F. Marín-Ricalde ◽  
C. Rodríguez-Padilla ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Screening Test ◽  
Brucella Melitensis ◽  
Low Cost ◽  
Animal Health ◽  
Screening Tests ◽  
Confirmatory Test ◽  
High Prevalence ◽  
Fluorescence Polarization Assay ◽  
World Organization

ABSTRACT The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats.

scholarly journals Induction of Robust and Specific Humoral and Cellular Immune Responses by Bovine Viral Diarrhea Virus Virus-Like Particles (BVDV-VLPs) Engineered with Baculovirus Expression Vector System

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 350
Author(s):  
Zhanhui Wang ◽  
Mengyao Liu ◽  
Haoran Zhao ◽  
Pengpeng Wang ◽  
Wenge Ma ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vector System ◽  
Baculovirus Expression Vector System ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Cellular Immune Responses ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Cellular Immune ◽  
Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is an important animal pathogen that affects cattle. Infections caused by the virus have resulted in substantial economic losses and outbreaks of BVDV are reported globally. Virus-like particles (VLPs) are promising vaccine technology largely due to their safety and strong ability to elicit robust immune responses. In this study, we developed a strategy to generate BVDV-VLPs using a baculovirus expression vector system (BEVS). We were able to assemble BVDV-VLPs composed of dimerized viral proteins E2 and Erns, and the VLPs were spherical particles with the diameters of about 50 nm. Mice immunized with 15 μg of VLPs adjuvanted with ISA201 elicited higher levels of E2-specific IgG, IgG1, and IgG2a antibodies as well as higher BVDV-neutralizing activity in comparison with controls. Re-stimulation of the splenocytes collected from mice immunized with VLPs led to significantly increased levels of CD3+CD4+T cells and CD3+CD8+T cells. In addition, the splenocytes showed dramatically enhanced proliferation and the secretion of Th1-associated IFN-γ and Th2-associated IL-4 compared to that of the unstimulated control group. Taken together, our data indicate that BVDV-VLPs efficiently induced BVDV-specific humoral and cellular immune responses in mice, showing a promising potential of developing BVDV-VLP-based vaccines for the prevention of BVDV infections.

scholarly journals Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species

2021 ◽  
Vol 9 (4) ◽  
pp. 765
Author(s):  
Janika Wolff ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Real Time ◽  
Animal Health ◽  
Diagnostic Methods ◽  
Virus Species ◽  
Time Saving ◽  
Robust Detection ◽  
Highly Sensitive ◽  
Reliable Differentiation ◽  
World Organization ◽  
Real Time Qpcr

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.

Comparative Genomics Analysis between frog Virus 3-like Ranavirus from the First Canadian Reptile Mortality Event and Similar Viruses from Amphibians

2021 ◽  
Author(s):  
Oliver Lung ◽  
Ayooluwa J. Bolaji ◽  
Michelle Nebroski ◽  
Mat Fisher ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Animal Health ◽  
Genome Structure ◽  
Leopard Frog ◽  
The Novel ◽  
Emerging Pathogens ◽  
Frog Virus 3 ◽  
Frog Virus ◽  
Usage Patterns ◽  
The Usa ◽  
World Organization

Abstract Ranaviruses are emerging pathogens that threaten the biodiversity of wild and captive cold-blooded vertebrates. Reports of ranavirus-induced mortality events are increasing and ranavirus disease is reportable to the World Organization for Animal Health. Previous studies have suggested interclass transmission of ranaviruses and Frog virus 3 (FV3)-like viruses are of particular interest. This study presents the whole-genome assembly of a 106 kb FV3-like genome obtained from the liver tissue of a reptile (wild Chelydra serpentina, common snapping turtle) that died of ranavirus disease in Canada. The FV3-like ON turtle/2018 strain shares the highest genome-wide nucleotide identity (99.71%) with the wild-type FV3 virus detected in the USA from a Northern leopard frog and an FV3-like strain identified from a wood frog in 2017 in Alberta, Canada. The novel genome contains all 26 Iridoviridae core genes, 11 FV3-like genes, and 9 unique truncations, three of which are core Iridoviridae ORFs. Additionally, the two most closely related FV3-like strains from amphibians, were compared to a non-FV3-like amphibian infecting and a fish infecting ranavirus species that showed similar codon usage patterns. G/C-ending codons were the preferred codons for all five strains. Investigation of putative recombination events identified four potential recombination events in the FV3-like ON turtle/2018 genome consistent with this FV3-like reptile infecting strain originating from an amphibian infecting FV3-like ranavirus. Altogether, this study provides insights into the genome structure and the differences in the novel FV3-like genome compared to other ranavirus genomes.

scholarly journals Oxid Comparative Analysis of the Significance of BISAP and MEWS Score for an Early Assessment of Illness Severity and Treatment Outcome of Acute Pancreatitis

2019 ◽  
Vol 0 (0) ◽  
Author(s):  
Olivera Marinkovic ◽  
Slađana Trpkovic ◽  
Ana Sekulic ◽  
Aleksandra N. Ilic ◽  
Nataša Zdravkovc ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Intensive Care ◽  
Treatment Outcome ◽  
Prognostic Value ◽  
Fatal Outcome ◽  
High Specificity ◽  
Early Assessment ◽  
Time Points ◽  
Additional Costs ◽  
Vital Parameters

Abstract The aim of this study was to determine the significance of the use of the BISAP score, which is specific for patients with AP, in relation to the application of the MEWS score that is important for assessing the condition of critically ill patients in intensive care units, but is not specific for patients with AP. The research was conducted as a cohort prospective study and included patients of both sexes, older than 18 and diagnosed with AP. BISAP and MEWS score were monitored at least at four time points: on admission to the hospital (zero), 48 hours, 72 hours and 7 days after admission to the hospital. High levels of discrimination between patients with fatal outcome and cured patients are determined in both cases, with discrimination at MEWS being somewhat higher than BISAP score. The BISAP0 had the best discrimination for BISAP score, AUROC (0.807) and also MEWS0 for MEWS score, AUROC (0.899). In our research, the highest sensitivity was shown by BISAP7d (92.1%) and MEWS48 (88.1%), and a high specificity of 87.5% had BISAP score, 48h, 72h and MEWS score at all four points of measurement. BISAP score has a better prognostic value in relation to the form of pancreatitis, the development of complications and the outcome. However, the calculation of the MEWS score is based on monitoring the basic vital parameters so that its application is much simpler and does not require additional costs.

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