Abstract
The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.
Highlights
Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae and is transmitted by Culicoides spp. of hematophagous midges
We demonstrated that BTV antigens derived from formalin-fixed and paraffin-embedded (FFPE) BTV infected cells or tissues can be reliably detected by a chromogen substrate-based immunolabeling method
Antibodies raised against BTV structural and non-structural proteins were screened by IHC detection of fixed and embedded BHK-BSR cells infected with BTV serotype 1 (BTV-1) CSIRO156 (Table 1)
Summary
Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae and is transmitted by Culicoides spp. of hematophagous midges. Various ruminant species can be infected by BTV, including cattle, sheep, deer, goats, and wild ruminants [1,2]. The clinical outcome of BTV infection is dependent on the virus strain, host species, and breed. The BTV genome consists of 10 segments of double-stranded RNA encoding 7 structural and 5 non-structural (NS) proteins [4,5]. The genomic elements are enclosed within a bi-shelled core particle made up of an inner VP3 layer and an outer layer comprised of VP7. The core structure is overlaid with a more diffuse outer coat comprising proteins VP2 and VP5 [5]. The antigenic variability within VP2, which encodes the majority of neutralizing epitopes, determines the distinct serotypes of BTV [6]
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