A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells

ABSTRACT Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43. IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.

Standardization and Validation of an Immunoperoxidase Assay for the Detection of African Horse Sickness Virus in Formalin-Fixed, Paraffin-Embedded Tissues

An immunoperoxidase assay for the detection of African horse sickness virus (AHSV) in formalin-fixed tissues is a valuable tool in the study of the pathogenesis of the disease, as well as a useful addition to existing diagnostic tests when only preserved tissues are available. An assay that uses Hamblin antiserum in a basic avidin-biotin complex detection system was standardized and validated in accordance with the guidelines of the American Association of Veterinary Laboratory Diagnosticians Subcommittee on Standardization of Immunohistochemistry. Using 128 positive cases of African horse sickness confirmed by viral isolation and serotyping and 119 negative cases from countries where the disease has never occurred, diagnostic sensitivity and diagnostic specificity were 100% in the prime target tissues of heart and lung. There was no variation in the ability of the assay to detect all 9 serotypes of AHSV, and there was no cross-reactivity with other orbiviruses in formalin-fixed tissues. The only cross-reactivity observed was in the lungs of 2 negative cases infected with Rhodococcus equi. The assay gave good results on tissues that had been fixed in formalin for up to 365 days. Nonspecific staining was minimal provided that the standard procedures for processing and staining tissues were followed. Good immunohistochemical results were also obtained on samples fixed as long as 24 hr after death. The assay, therefore, provides a robust diagnostic tool for detection of AHSV in formalin-fixed tissues, provided the analysis is done by an experienced pathologist.

Setaria digitata infections in cattle: parasite load, microfilaraemia status and relationship to immune response

A total of 110 cattle were examined in an area endemic for Bancroftian filariasis for the prevalence of infection of the bovine filarial parasite Setaria digitata. About 12.5% of cattle were found to harbour both adult worms in the peritoneum and microfilariae (mf) in circulation; 70% of the cattle were amicrofilaraemic but with an adult worm infection. A third group of cattle (16.5%) was free of detectable mf and adult worms. The presence of adult worms and/or mf did not influence the antibody levels to any of the four antigen preparations of S. digitata. However, there was a significant inverse relationship between the presence of antibodies to microfilarial sheaths and the absence of circulating mf as shown by the immunoperoxidase assay. Cattle immunoglobulin containing high titres of anti-sheath antibodies cleared circulating microfilariae very effectively in Mastomys coucha thus demonstrating the protective nature of anti-sheath antibodies in eliminating circulating microfilariae in vivo.