Measurement of bluetongue virus binding to a mammalian cell surface receptor by an in situ immune fluorescent staining technique.

A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been shown to function as a receptor for a number of viruses, its role as a receptor for BTV was evaluated with the in situ IFA. Binding of BTV to both CHO and CPAE cells was inhibited in a dose dependent manner by HS. In addition, HS deficient CHO cells showed greatly diminished binding of BTV when compared to the parental cell line. The IFA protocol will find application, as a non-isotopic, quantifiable technique, to study virus-cell receptor interactions. Information gained from such studies will expand our understanding of the early steps in virus replication.

In situ immune infrared fluorescent staining for detection and quantitation of bluetongue virus in Culicoides insect cell culture.

Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from Culicoides sonorensis; however, techniques to detect and quantitate viable virus directly in these insect cells are lacking. In situ immune infrared fluorescent staining techniques were developed to visualize and quantitate BTV infection in Culicoides cell culture by both an endpoint titration and an agarose overlay fluorescent focus assay. Insect cell cultures infected with BTV were fixed, permeabilized and reacted with virus-specific monoclonal antibody and fluorescent-labeled secondary antibody. Virus replication in the infected cells was visualized and quantitated by measuring fluorescence with an infrared imager. The sensitivity of virus detection in insect cell culture using these techniques was comparable to or better than detection by standard techniques in vertebrate cell culture.

Identification, expression and characterisation of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses.

Salivary proteins of Culicoides biting midges are thought to play a key role in summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification, expression and clinical relevance of a candidate allergen of the North American midge Culicoides sonorensis. Immunoblot analysis of midge saliva revealed a 66 kDa protein (Cul s 1) that was bound by IgE from several SE-affected (SE+) horses. Further characterisation by fragmentation, mass spectrometry and bioinformatics identified Cul s 1 as maltase, an enzyme involved in sugar meal digestion. A cDNA encoding Cul s 1 was isolated and expressed as a polyhistidine-tagged fusion protein in a baculovirus/insect cell expression system. The clinical relevance of the affinity-purified recombinant Cul s 1 (rCul s 1) was investigated by immunoblotting, histamine release testing (HRT) and intradermal testing (IDT) in eight SE+ and eight control horses. Seven SE+ horses had rCul s 1-specific IgE, whereas only one control animal had IgE directed against this allergen. Furthermore, the HRT showed rCul s 1 induced basophil degranulation in samples from seven of eight SE+ horses but in none of the control animals. rCul s 1 also induced immediate (7/8), late-phase (8/8) and delayed (1/8) skin reactivity in IDT on all SE+ horses that had a positive test with the whole body extract (WBE) of C. sonorensis. None of the control horses showed immediate or delayed skin reactivity with rCul s 1, and only one control horse had a positive late-phase response, while several non-specific late-phase reactions were observed with the insect WBE. Thus, we believe rCul s 1 is the first specific salivary allergen of C. sonorensis to be described that promises to advance both in vitro and in vivo diagnosis and may contribute to the development of immunotherapy for SE in horses.

Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease.

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Characterization of cell lines developed from field populations of Culicoides sonorensis (Diptera: Ceratopogonidae).

Two cell lines, ABADRL-Cs-W3 (W3) and ABADRL-Cs-W8A (W8), were developed from a field population of Culicoides sonorensis Wirth & Jones. The cell lines were characterized by isozyme phenotyping and the ability to support the replication of bluetongue virus (BLU) and epizootic hemorrhagic disease virus (EHDV) (Orbivirus, Reoviridae). Comparison of isozymes found in the cell lines with those found in adult C. sonorensis colony insects confirmed that the cell lines were of C. sonorensis origin. There was, however, sufficient isozyme variation present in the cell lines to construct a unique isozyme profile for each cell line. Although both cell lines supported BLU and EHDV replication to the same level, one-step growth curves for BLU indicated that virus replication was faster and attained a peak titer earlier in the W3 cell line than in the W8 cell line. Viral proteins and RNA were detected earlier in the W3 cell line as well. The accelerated virus growth kinetics observed in the W3 cell line and the adherent nature of the cells makes it more suitable for certain Orbivirus studies.