Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins

Abstract

Grouper iridovirus (GIV) is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. The study of GIV pathogenicity has been hampered by the lack of proper immunological reagents to study the expression and function of viral proteins in the infected cells. In this study, two mouse monoclonal antibodies (mAbs) against GIV 55L and 97L proteins were produced. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen these hybridomas, resulting in the identification of two high-affinity mAbs named GIV55L-mAb-2 and GIV97L-mAb-3, respectively. Both mAbs belong to the IgG1 isotype and were effective in detecting their respective target viral protein. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analyses of GIV-infected GK cells revealed that GIV 97L is an immediate early gene, whereas GIV 55L a late one. The localization of 55L and 97L in GIV-infected cells was further characterized by immunofluorescence microscopy with the mAbs. The 55L protein mainly aggregated in the cytoplasm while 97L distributed in both the nucleus and cytoplasm of the infected cells. These studies demonstrate the validity of the two mAbs as immunodiagnostic and research reagents.

The Ambystoma tigrinum virus (ATV) RNase III gene can modulate host PKR activation and interferon production

“…However, this may be a timing and/or level of expression issue. Analysis of related iridoviruses has shown RNase III gene expression occurs between 12 and 24 hours post infection suggesting this is a late viral protein (Hu et al, 2015; Zenke and Kim, 2008) while another study has suggested this gene is a delayed early gene (Majji et al, 2009). Interestingly it has been shown previously that FV3 infected cells produce dsRNA and the level of dsRNA produced is an indicator of viral replication (Doherty et al, 2016).…”

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

“…), GIV‐55L‐mAb‐2 (500 μg mL −1 , 1:5000; Hu et al . ) or β‐actin (3.1 mg mL −1 , 1:5000; Sigma) for 2 h at 37 °C and then incubated with alkaline phosphatase‐conjugated goat anti‐mouse immunoglobulins (400 μg mL −1 , 1:5000; Santa Cruz Biotechnology) at 37 °C for 1 h. Finally, the colour band was developed in the dark using the substrate 4‐nitro‐blue tetrazolium chloride/5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT/BCIP; Sigma).…”

“…), GIV‐55L (predicted function and/or similarity to RNase III) and GIV‐97L (predicted function and/or similarity to NTPase‐helicase; Hu et al . ).…”

Canine heat shock protein 27 promotes proliferation, migration, and doxorubicin resistance in the canine cell line DTK-F

“…Mouse splenocytes were harvested and fused with NS-1 myeloma cells at a 5:1 ratio using 50% (v/v) polyethylene glycol (PEG, MW 1500) (Hu et al, 2015). The cell pellet was dispersed by gently tapping the tube and warming it to 37°C for 2 min, and 0.7 mL pre-warmed PEG was gradually added over 7 min.…”