In VitroNeutralization of Viral Hemorrhagic Septicemia Virus by Plasma from Immunized Zebrafish

Abstract

We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish.

Characterization and Vaccine Potential of Membrane Vesicles Produced by Francisella noatunensis subsp. orientalis in an Adult Zebrafish Model

“…The expression and antiviral activity of IFNs and Mxa have been extensively described in zebrafish. Viral hemorrhagic septicemia virus (VHSV)-infected zebrafish showed an increase in ifna , ifnb , and mxa gene expression, reaching a maximum after 72 h ( 47 ). In our case, we did not detect significant changes in the expression of ifnphi1-3 genes.…”

“…However, the small amount of serum obtainable from small fish such as zebrafish precludes such assays. Studies in zebrafish infected with rhabdovirus, such as viral hemorrhagic septicemia virus (VHSV), have shown that plasma from surviving zebrafish has a neutralizing activity in fish cells in vitro and that the neutralization correlated with the level of in vivo protection ( 47 ). Synergy between antibodies, cytokines, and phagocytes against intracellular pathogen appears to be critical for clearance of an infection.…”

Innate Multigene Family Memories Are Implicated in the Viral-Survivor Zebrafish Phenotype

“…To obtain concentrated VHSV for neutralizing assays, supernatants were centrifuged at 60.000 g for 180 min at 4°C, and pellets were frozen at -70°C until use. VHSV was titrated by the focus forming units (ffu) assay [ 29 ].…”

Perspectives on antigen presenting cells in zebrafish

“…Although it has been well demonstrated that teleost B cells proliferate and secrete pathogen-specific antibody (Zhang et al, 2010; Xu et al, 2013; Cain et al, 2002; Castro et al, 2013; Chinchilla et al, 2013), the importance of humoral immunity in controlling infections in zebrafish is unclear. For example, zebrafish B cells appear to have little to no role in the response against Edwardsiella tarda (Yang et al, 2013).…”