Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro.

Ying Zhong,Xiaoqian Tang,Jing Xing,Wenbin Zhan,Xiuzhen Sheng

doi:10.3390/ijms19092536

Abstract

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV–host interaction and might be promising inhibitors of LCDV infection in fish.

Highlights

Lymphocystis disease (LCD) is a viral disease occurring in marine, brackish and freshwater teleost fish with worldwide distribution [1], which is characterized by the appearance of pearl-like nodules on the skin, fins, tail and the internal organ of the affected fish [2]
The E. coli strain BL21 that contained recombinant plasmid pET-32a-ORF038 was cultured in the LB medium and the expression of recombinant VAP (rVAP) was induced by isopropyl-β-D thiogalactopyranoside (IPTG)
For lymphocystis disease virus (LCDV) studies, the monoclonal antibodies (MAbs) against LCDV are produced [21] and a 27.8 kDa cellular receptor are identified through immunoprecipication using anti-LCDV MAbs [13], and thereafter, a 32 kDa envelop protein of LCDV is identified as a viral attachment protein (VAP) to interact with the 27.8 kDa receptor protein by using anti-27.8 kDa receptor MAbs [16]

Summary

Introduction

Lymphocystis disease (LCD) is a viral disease occurring in marine, brackish and freshwater teleost fish with worldwide distribution [1], which is characterized by the appearance of pearl-like nodules on the skin, fins, tail and the internal organ of the affected fish [2]. The causative agent of LCD is lymphocystis disease virus (LCDV), a large icosahedral DNA virus in the genus Lymphocystivirus of the family Iridoviridae [4], with double-layered capsid, an outer envelope and a fringe consisting of fibril-like external protrusions [2,5,6]. A 27.8 kDa membrane protein from flounder (P. olivaceus) gill (FG) cells has been recently identified as a cellular receptor to mediate LCDV binding and infection [13,14,15], and a 32 kDa envelop protein of LCDV, encoded by the open reading frame (ORF) 038 gene in LCDV-C, is found as a viral attachment protein (VAP) to interact with the 27.8 kDa receptor protein [16]. No effective methods are developed for the prevention and cure of fish lymphocystis disease [17,18], and the molecular mechanism underlying LCDV infection and pathogenesis, especially the LCDV–host interaction, needs to be further clarified

Methods

Results

Conclusion