Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows

Abstract

Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein.Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100 % mortality at dilutions up to 10 -4 in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10 % when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease.

Real-Time Dissecting the Entry and Intracellular Dynamics of Single Reovirus Particle

“…For preparing the anti-VP7 antibody, the recombinant plasmid pET-32a-VP7 was transformed into an E. Coli strain BL21 (DE3) for protein expression. Then the fusion protein was purified and used to immunize mice as previously reported (Liu J. et al, 2016 ). The experiment were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.…”

“…The protein expression was tested with western blotting as described previously (Liu J. et al, 2016 ). The anti-NS25 antibody acted as the primer antibody, at a dilution of 1:500, and alkaline phosphatase (AP)-coupled goat anti-mouse IgG was used as the secondary antibody (Promega).…”

“…The genome encode 7 structural proteins (VP1–VP7) and 5 nonstructural proteins. The outer-capsid proteins of reovirus are responsible for initiating infection, stimulate the host immune system and the acid-activated penetration (Liemann et al, 2002 ; Danthi et al, 2010 ; Liu J. et al, 2016 ). The inner capsid proteins possess the enzymatic activities necessary for viral transcription (Odegard et al, 2004 ).…”

Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production

“…Comparing the protein sequences of the three genotypes of GCRV, it is found that the similarity is less than 20%, so the functions of the encoded proteins are also distinct. For example, the S11 segment of GCRV-I and GCRV-III is predicted to encode non-structured proteins (NS26 and VP8/VP15, respectively), while the S11 fragment in GCRV-II is predicted to encode a 35-kDa protein (VP35) with a conserved putative zinc-binding motif and acts as a putative outer-clamp protein (7)(8)(9)(10). In recent years, more and more research studies are focused on the understanding of GCRV's involvement in pathogenesis and immune response (11).…”

A Novel Subunit Vaccine Based on Outer Capsid Proteins of Grass Carp Reovirus (GCRV) Provides Protective Immunity against GCRV Infection in Rare Minnow (Gobiocypris rarus)

“…The VP35 protein encoded by the S11 gene fragment of GCRV type II and VP4 protein (outer capsid protein) encoded by the S6 gene fragment [9,10] play an important role during virus entry into cells and can induce an immune response in the host [11,12], so they can be used as candidate protein vaccines [13]. expressed recombinant VP35 protein in prokaryotic cells and evaluated the immune protection of the recombinant VP35 protein by performing a series of experiments on grass carps.…”