Revision as of 09:32, 27 October 2020

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow.

Biology

Description of VHSV

Viral hemorrhagic septicemia virus surface glycoprotein G is responsible for the endosomal membrane and virus envelope fusion process, which occurs when the virus enters the cell. This process helps to release the viral nucleocapsid and start the rapid replication of the virus¹.

Viral hemorrhagic septicemia virus (VHSV) infects a number of teleost fish species², yet the most susceptible is the rainbow trout³. The disease is highly impactful to the European fish industries especially, as it can cause economic losses of an estimated £40 million per year⁴. VHSV is lethal to fry and fingerlings, the mortality rates decreasing in mature fish³. The virus causes severe hemorrhages and skin lesions followed by lethargic swimming, discoloration of gills, and the destruction of the endothelial lining³. The virus enters the organism through the gill lamellae² by endocytosis, mediated by receptors. Upon entrance to the cell, the endosomal membrane and virus envelope fuse together, increasing the virus uptake into the cell and releasing the viral nucleocapsid⁵. This feature is typical of Rhabdoviruses - a family that VHSV belongs to. The low pH in the compartment of the endosome is the primary trigger of this process⁶. The virus then starts rapidly replicating in the cell. Out of six proteins coded in the virus genome, surface glycoprotein G is the one involved in the membrane fusion process¹. Glycoprotein G forms spike-like structures on the surface of the virus⁷. These spikes get inserted into the target membrane and direct further interactions of the virus and the host cells⁶. Most importantly, the antibodies against VHSV are targeted to the G protein, making it the most effective protein to induce an immune response in fish⁸. VHSV glycoprotein G can be used as an immunogenic protein for subunit vaccine production as it was already proven to work for the same purpose⁹. Yet the production of recombinant glycoproteins is problematic because it was also shown that recombinant glycoprotein G produced in E. coli is not recognized by monoclonal antibodies¹⁰. Therefore glycoproteins can be produced via DNA manipulation in S. cerevisiae to provide correct protein folding and glycosylation, which is not guaranteed in E. coli¹¹.

Results

The VHSV glycoprotein G coding sequence with a 6xHis tag on the N terminus can be successfully cloned into pfX-7 vector (Fig. 1). However, after the expression in S.cerevisiae 214, 214Δpep strains the protein does not show up on SDS-PAGE gel. The procedure yields a protein of a different size (Fig. 2-3). Glycoprotein G should be 57kDa.

Figure 1. fX-7 VHSV construct. The 6xHis tag sequence is indicated in purple. Construct sequencing results are indicated with red arrows.

Figure 2. SDS-PAGE electrophoresis results after glycoprotein G synthesis induction in S.cerevisiae 214 strain. b- sample before induction; a - sample after induction; s - supernatant sample; p - pericipate sample; ff - flow through; f1-4 - chromatography fractions,

Figure 3. SDS-PAGE electrophoresis results after glycoprotein G synthesis induction in S.cerevisiae 214Δpep strain. b- sample before induction; a - sample after induction; s - supernatant sample; p - precipitate sample; f1-5 - chromatography fractions.

References

1. Fernandez-Alonso, M. et al. Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus. Diseases of Aquatic Organisms 34, 167–176 (1998).
2. Bruno, D. E. & Ellis, A. E. Chapter 13 - Salmonid Disease Management. in Developments in Aquaculture and Fisheries Science (eds. Pennell, W. & Barton, B. A.) vol. 29 759–832 (Elsevier, 1996).
3. Skall, H. F., Olesen, N. J. & Mellergaard, S. Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming - A review. J. Fish Dis. 28, 509–529 (2005).
4. Olesen, N. J. Sanitation of viral haemorrhagic septicaemia (VHS). J. Appl. Ichthyol. 14, 173–177 (1998).
5. Lecocq-Xhonneux, F. et al. A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout. J. Gen. Virol. 75, 1579–1587 (1994).
6. Gaudin, Y., De Kinkelin, P. & Benmansour, A. Mutations in the glycoprotein of viral haemorrhagic septicaemia virus that affect virulence for fish and the pH threshold for membrane fusion. J. Gen. Virol. 80, 1221–1229 (1999).
7. G - Spike glycoprotein precursor - Viral hemorrhagic septicemia virus (strain 07-71) (VHSV) - G gene & protein. Uniprot.org (2020). at https://www.uniprot.org/uniprot/P2766
8. Lorenzen, N., Olesen, N. J. & Jørgensen, P. E. V. Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. Journal of General Virology, 71, 561–567 (1990)
9. Shin, C., Kang, Y., Kim, H.-S., Shin, Y. K. & Ko, K. Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice. Anim Cells Syst (Seoul) 23, 97–105 (2019)
10. Estepa, A. & Coll, J. Replication of rhabdovirus in trout hematopoietic cells. Investigaciones Agrarias 9, 37–44 (1994).
11. Smith, T. & Kohorn, B. Direct selection for sequences encoding proteases of known specificity. Proceedings of the National Academy of Sciences 88, 5159-5162 (1991).