Standard Virus Neutralization Test Research Articles

Detection of anti-enterovirus IgG in human sera by ELISA method using the KTL-510 peptide

Enterovirus (EV) infections occur frequently in humans. In some geographical areas they are more common. These viruses cause diseases with varying degrees of severity, from a simple respiratory tract infection to severe diseases. Since EVs include more than 70 serotypes currently circulating in the population, a methodology that detects most of them is needed. ELISA is a rapid, sensitive, and economical diagnostic method for the identification of EV serotypes and can also be used as a retrospective diagnostic tool or in the investigation of outbreaks of infection. Commercial EV-ELISAs often appear and gradually disappear from the market supply. We have used the KTL-510 peptide, a synthetic viral protein of poliovirus VP1, as an antigen in a peptide-based ELISA for the detection of a broader spectrum of anti-EV antibodies. We aimed to design, optimize, and standardize this in-house ELISA with the peptide, and implement the method for routine detection of anti-EV IgG in human sera. For determining the cut-off value, we used 100 patients’ sera which were previously tested negative for IgG antibodies against EVs using a commercial ELISA kit available. We monitored patients’ sera samples sent for serological testing of anti-coxsackievirus antibodies to the National Reference Center for the Identification of Enteric Viruses between 2018–2022. These serum samples were examined using a standard virus neutralization test as well as the newly developed ELISA method.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Epidemiological Study of Multiple Zoonotic Mosquito-Borne Alphaviruses in Horses in Queensland, Australia (2018-2020).

The increased frequency of extreme weather events due to climate change has complicated the epidemiological pattern of mosquito-borne diseases, as the host and vector dynamics shift to adapt. However, little is known about the seroprevalence of common mosquito-borne virus infections in horses in Australia. In this study, serological surveys for multiple alphaviruses were performed on samples taken from 622 horses across two horse populations (racehorses and horses residing on The University of Queensland (UQ) campus) in Queensland using the gold standard virus neutralization test. As is the case in humans across Australia, Ross River virus (RRV) is the most common arbovirus infection in horses, followed by Barmah Forest virus, with an overall apparent seroprevalence of 48.6% (302/622) and 4.3% (26/607), respectively. Horses aged over 6 years old (OR 1.86, p = 0.01) and residing at UQ (OR 5.8, p < 0.001) were significantly associated with seroconversion to RRV. A significant medium correlation (r = 0.626, p < 0.001) between RRV and Getah virus (GETV) neutralizing antibody titers was identified. Collectively, these results advance the current epidemiological knowledge of arbovirus exposure in a susceptible host in Australia. The potential use of horses as sentinels for arbovirus monitoring should be considered. Furthermore, since GETV is currently exotic to Australia, antibodies cross-reactivity between RRV and GETV should be further investigated for cross-protection, which may also help to inform vaccine developments.

An Investigation of Bovine Viral Diarrhoea Virus (BVDV) and Bovine Parainfluenza Type 3 Virus (BPI3V) Infections in Small-Scale Cattle Herds in Afyonkarahisar Province

Respiratory and reproductive system disorders are the most important problems of ruminant breeding and cause great economic loss. Bovine viral diarrhea and bovine parainfluenza 3 infections are among the leading respiratory pathogens and are very common in our country. The aim of this study is to serologically investigate the status in family-type small-scale enterprises of these two virus infections throughout Afyonkarahisar province. For this purpose, 1.279 adult cattle blood samples were collected from all the districts, which were not vaccinated recently for these infections. Sera samples were controlled using standard virus neutralization test and seropositive samples were re-tested to determine antibody titers. Pestivirus-specific antibody presence was detected in 978 (74.9%) cattle, and the rates varied between 50.7% (Ihsaniye) and 92.8% (Cay) according to the districts. For BPI3V, the least positivity was found in Sinansa (58.8%) while the highest rate was detected in Emirdag with 97.5%. In total, 82.7% (1.058 / 1.279) seropositivity was determined. As a result, the determined values for these economically important infections are not very different from those obtained in the country, but are higher than expected due to the target population. Considering that the domestic animal population has decreased, supporting of small producers with national or regional preventive practices is gaining importance.

Prevalência de anticorpos contra o vírus da arterite equina nas mesorregiões Noroeste, Centro Ocidental e Norte Central do Paraná

RESUMO: O objetivo deste estudo foi investigar a prevalência de anticorpos contra o vírus da arterite viral equina (EVAV) em equinos sadios criados nas mesorregiões Noroeste, Centro Ocidental e Norte Central do estado do Paraná. Após o cálculo do tamanho amostral, foram analisadas, utilizando a técnica de soroneutralização, amostras de soro sanguíneo de 653 equinos. Nenhum animal sororreagente foi encontrado nas mesorregiões Noroeste (0/236) e Centro Ocidental (0/99). Na mesorregião Norte Central a prevalência foi de 0,62% (2/318), totalizando 0,30%. Pode-se concluir que a arterite viral equina (AVE) ainda não representa um problema de importância epidemiológica nos equinos criados nas mesorregiões paranaenses estudadas.

Baculovirus expression and diagnostic utility of the glycoprotein E of bovine herpesvirus-1.1 Egyptian strain “Abu-Hammad”

A recombinant baculovirus construct expressing glycoprotein E (gE) of the Egyptian BoHV-1.1 Abu-Hammad strain (rBac/gE-AbuH) was generated and characterized. The recombinant gE (rgE) secreting protein in culture medium of infected insect cells was used as a coating antigen in an indirect enzyme-linked immunosorbent assay (ELISA) to test its utility for detection of antibody against gE of BoHV-1. Indirect gE-ELISA was compared to standard virus neutralization test and commercial blocking gE-ELISA for detection of anti-gE antibody in a panel of bovine sera. Antibody titers estimated by both ELISAs were closely correlated with those determined by virus neutralization test. In conclusion, the developed indirect gE-ELISA was a reliable candidate for inexpensive detection of anti-gE antibody in control and experimental bovine sera with high specificity and sensitivity. Moreover, it emphasized the diagnostic utility of gE based ELISAs to distinguish cattle infected with BoHV-1 from those vaccinated with the gE negative mutants.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

Virus Neutralizing Anitbodies to Bovine Herpes Virus Type 1 (BHV-1), Bovine Viral Diarrhoea (BVD) and Rinderpest (RPV) Viruses in Smallholder East African Zebu Cattle in Coastal Kenya

A total of 511 sera samples were collected from Small East African Zebu (EAZ) cattle on 32 smallholder farms in Malindi Division, Kilifi District, Coast Province of Kenya. They were assayed using the standard virus neutralization test (VNT) for antibodies to rinderpest (RPV) and some rinderpest-like diseases namely bovine viral diarrhoea (BVD) and bovine herpes virus type 1 9BHV-1). The overall estimated prevalences were 10.2%, 45.8% and 28.6%, respectively. A significant increase (p The Kenya Veterinarian Vol. 18 (1) 1994: pp. 21-24