Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay To Detect Antibodies to Viral Hemorrhagic Septicemia Virus

Abstract

f Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Temporal Variation in Viral Hemorrhagic Septicemia Virus Antibodies in Freshwater Drum (Aplodinotus grunniens) Indicates Cyclic Transmission in Lake Winnebago, Wisconsin

“…After blood collection, the fish were euthanized by immersion in 200 mg/liter MS-222 for 10 min and immediately placed on ice in separate labeled plastic bags. The serum was heated at 45°C for 30 min to inactivate complement (22).…”

“…We used a recently developed virus neutralization (VN) assay to detect neutralizing antibodies in serum (22). Twofold serial dilutions of serum were mixed with 100 times the 50% tissue culture infective dose (TCID 50 ) of the virus (25,26) and incubated at 15°C for 24 h. The serum-virus mixtures were then inoculated onto 7% polyethylene glycol (PEG)-treated epithelioma papulosum cyprinid (EPC) cells and incubated at 15°C for 5 days (27).…”

Viral haemorrhagic septicaemia virus (VHSV Id) infections are detected more consistently using syndromic vs. active surveillance

“…qRT-PCR could also be used as the primary diagnostic screening test for fish farms, but a positive result should be confirmed by other methods such as antibody testing. ELISA or plaque neutralisation tests have been reported to be good tools to screen for anti bodies against VHSV in Europe and the USA (FregenedaGrandes & Olesen 2007, Fregeneda-Grandes et al 2009, Schyth et al 2012, Millard et al 2014, Wilson et al 2014. Our study also proved ELISA to be a useful method for screening, since VHSV in fection could be detected several months after a clinical disease outbreak occurred.…”

The Effect of Crude Protein Halimeda Sp. On Cyprinus Carpio Infected Koi Herpes Virus on Expression of Major Histocompatibility Complex Class-1

“…The elevated specific antibody titers will increase the protection function in the fish. Antibody titer of 1/16 -1/80 able to neutralizing effect of Viral Septicemia Hemorrhagic Virus in the fish [12]. Conditions of fish in a healthy state due to the induction of PCP Halimeda sp capable of triggering the production of adhesion molecules and cytokines by immune cells.…”