Temperature and pH requirements for viral haemorrhagic septicemia virus induced cell fusion

Abstract

The rhabdovirus causlng v~r a l haemorrhaglc septlcema (VHS), an Important disease of salmonlds, Induced forrnatlon of large syncytia in fish cell monolayers by cell fusion For maximal percentage of fus~on, not only was a pH of 5 6 needed but also a temperature of at least 14°C The fusion was dependent on the G proteln of VHSV as shown by inhibition of fuslon with speclflc anti-G polyclonal and monoclonal antibodies Part of the region of the G proteln Involved In fusion was mapped to amino acids 82 to 109 because anti-peptide antibodies blndlng to that reglon also inhlblted fuslon The fusion assay descnbed here can b e useful for studying the functionality of the G proteln of fish rhabdovlruses in the early steps of Infection and thls information can In turn, help to designing effectwe vaccination and/or therapeutic strategies against fish rhabdovlruses The descnbed fusion assay could also be a p p l~e d to other flsh rhabdoviruses such as infect~ous haematopoletic necrosls virus (IHNV) and spnng viraemia of carp vlrus (SVCV) KEY WORDS Viral haemorrhagic septicaemia Rhabdovirus Neutrahzing-enhancing a n t~b o d y Monoclonal antibodies Fusion

A Protein G Fragment from the Salmonid Viral Hemorrhagic Septicemia Rhabdovirus Induces Cell-to-Cell Fusion and Membrane Phosphatidylserine Translocation at Low pH

“…Low pH-induced Fusion Assays by Fragment 11 or pG-transfected Cells-To assay for fragment 11-induced cell spreading and fusion from without, the assay was performed in 24-well plates under similar conditions to those described before (19). Fragment 11 was added in fusion medium, RPMI 1640 (without bicarbonate) buffered with 20 mM HEPES, 20 mM MES (Sigma) at pH 6 to EPC monolayers for 30 min at 14°C.…”

“…Abs were serially diluted in fusion medium at pH 6 (optimal fusion pH) (19) and incubated overnight at 4°C. The next day, the EPC plates were washed, and the serial dilutions of Abs were pipetted into the EPC wells.…”

“…For instance, anti-p2 antibodies (Abs) inhibited phospholipid binding to VHSV (2) and VHSV-induced cell-to-cell fusion (19). In addition, p2 mediates phospholipid vesicle fusion, lipid mixing, and leakage of liposome contents and inserts itself into liposome membranes by adopting a ␤-sheet structure (1).…”

Induced fusion of VHSV persistently infected fish cells

“…Cell to cell fusion of epithelioma papulosum cyprini (EPC) cells infected with VHSV was dependent on pH (5.6) and temperature (14 °C) ( Estepa & Coll 1996, 1997). Similar results were reported in insect cells either infected with VHSV ( Lorenzen & Olesen 1995) or transfected with plasmids expressing the G protein ( Lecocq‐Xhonneux, Thiry, Dheur, Rossius, Vanderheijden, Martial & deKinkelin 1994).…”

Rainbow trout Oncorhynchus mykiss ladderlectin, but not intelectin, binds viral hemorrhagic septicemia virus IVb

“…Virus was cultured as described by Al-Hussinee et al (2010) using EPC cells (ATCC CRL-2872) and minimum essential medium with 2 mM L-glutamine, 100 units penicillin, 100 units streptomycin, 2.5 µg of amphoteracin B and 10% [v/v] certified inactivated fetal bovine serum (Invitrogen) with the initial pH adjusted to approximately 5.6 to promote fusion (Estepa & Coll 1997), based on phenyl red indicator in the media, using 7% NaHCO3. Viral propagation was then performed as per Al-Hussinee et al (2010) at a pH of 7.6.…”