Search for genetic markers for PCR-RV indication of the causative agent of viral hemorrhagic septicemia in fish

Viral hemorrhagic septicemia is one of the most dangerous fish diseases, causing significant damage to the economic development of aquaculture. In the Russian Federation, diagnostic measures to identify this pathogen are usually based on serological approaches. However, the fastest and the most accurate methods are molecular genetic diagnostic methods based on the polymerase chain reaction. The aim of this work was to develop a combination of primers and probe suitable for specific PCR indication of hemorrhagic septicemia virus in real time. At the first stage of the work, a bioinformatic analysis of the currently available nucleotide sequences of the genomes of the viral hemorrhagic septicemia causative agent was carried out. After analyzing the sequences of this virus, a marker locus of 180 bp was determined inside of the N gene encoding the nucleoprotein. Inside of this locus, primers and a probe suitable for detecting isolates of viral hemorrhagic septicemia in the real time polymerase chain reaction were developed. As a result of a series of experiments conducted on cDNAs isolated from VHSV CF 1.2 and VHSV 22-ARM virus cultures, the 1.2 and VHSV 22-ARM virus cultures, the amplification program and the composition of the reaction mixture were determined. It has been established that the developed primers have 100% specificity, and sensitivity can reach 12 copies of virus particles per microliter. Thus, the nucleotide markers selected as part of that work and the primers and probe constructed on their basis have all the characteristics necessary for their use as the basis of a test system that allows for rapid PCR indication of the virus to detect and identify the causative agent of viral hemorrhagic septicemia.

1. Yusuff S., Kurath G., Kim M.S., Tesfaye T.M., Li J., McKenney D.G., Vakharia V.N. The glycoprotein, non-virion protein, and polymerase of viral hemorrhagic septicemia virus are not determinants of host-specific virulence in rainbow trout. Virol J. 2019; 16 (1):31. https://doi.org/10.1186/s12985-019-1139-3

2. Baillon L., Mérour E., Cabon J., Louboutin L., Vigouroux E, Alencar ALF, Cuenca A, Blanchard Y, Olesen NJ, Panzarin V, Morin T, Brémont M, Biacchesi S. The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss). Front Microbiol. 2020; 11:574231. https://doi.org/10.3389/fmicb.2020.574231.

3. Kim H.J., Cuenca A., Olesen N. J Validation of a novel one-step reverse transcription polymerase chain reaction method for detecting viral haemorrhagic septicaemia virus. Aquaculture. 2018; 492:170-183. https://doi.org/10.1016/j.aquaculture.2018.03.047.

4. Ito T., Kurita J., Mori K., Olesen N.J. Virulence of viral haemorrhagic septicaemia virus (VHSV) genotype III in rainbow trout. Vet Res.; 47:4. https://doi.org/10.1186/s13567-015-0303-z

5. Pylnov V.A., Burlachenko I.V., Bychkova L.I. Application of the nest PCR variant (RT -PCR) for the diagnosis of hemorrhagic septicemia virus in salmon fish. Current issues of freshwater aquaculture : Collection of scientific papers of Branch for the freshwater fisheries of the Federal State Budget Scientific Institution “Russian Federal Research Institute of Fisheries and oceanography”. 2023; 94:122-129 (In Russ.).

6. Anisimova E.A., Dodonova E.A., Mirgazov D.A., Fakhrutdinov N.A., Elizarova I.A., Pankova E.V., Osyanin K.A. Selection of optimal conditions of PCR setup to identify canine brucellosis causative agent. Bulletin of Altai State Agricultural University. 2023; 12(230):55-59. https://doi.org/10.53083/1996-4277-2023-230-12- 55-59 (In Russ.).

7. Gromova E.A., Mirgazov D.A., Dodonova E.A., Elizarova I.A., Gorbunova M.E., Osyanin K.A. Development of a multiplex RTPCR test system for detecting the causative agent of brucellosis. The Veterinarian. 2024; 3:34-40. https://doi.org/10.33632/1998-698X_2024_3_34 (In Russ.).

8. Gromova E.A., Osyanin K.A., Dodonova E.A., Gorbunova M.E., Khammadov N.I., Makaeva A.R. Genetic markers for PCR indication of salmonid infectious hematopoietic necrosis virus. Bulletin of Kursk State Agricultural Academy. 2024; 5:88-94 (In Russ.).

9. Chico V., Gomez N., Estepa A., Perez L. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J Virol Methods. 2006; 132(1- 2):154-9. https://doi.org/10.1016/j.jviromet.2005.10.005

10. Warg J.V, Clement T., Cornwell E.R, Cruz A., Getchell R.G, Giray C., Goodwin A.E, Groocock G.H., Faisal M., Kim R., Merry G.E., Phelps N.B., Reising M.M., Standish I., Zhang Y., Toohey-Kurth K. Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols. Dis Aquat Organ. 2014; 111(1):15-22. https://doi.org/10.3354/dao02758

11. Tarasova A. S., Perchun A. V., Melnikov V. P. Polymerase chain reaction for detection of some highly dangerous viral fish disease agents. Veterinary Science Today. 2020; 1(32):11-16. https://doi.org/10.29326/2304-196X-2020-1-32-11-16

12. Perchun A.V., Vorobyov V.V., Melnikov V.P. Detection of particularly dangerous viral diseases of salmon fish (Salmo salar) using polymerase chain reaction. Fisheries. 2022; 5:88-93. https://doi.org/10.37663/0131-6184-2022-5-88-93