A Reverse Genetics System for the Great Lakes Strain of Viral Hemorrhagic Septicemia Virus: the NV Gene is Required for Pathogenicity

Abstract

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription

“…Recombinant VHSVs harboring the D62A and D62A E181A mutations were generated using a reverse-genetics system (53). Recombinant viruses containing the mutant M genes were viable and were compared to recombinant WT (rWT) VHSV in a series of cell-based studies.…”

“…Expression vectors for EPC MAVS and EPC IFN were obtained from Michel Brémont (French National Institute for Agricultural Research, Jouy-en-Josas, France), the VHSV L expression plasmid was reported previously (53), the Tet-mSEAP construct was obtained from Fan Dong (University of Toledo, Toledo, OH, USA), and the IFN-luciferase reporter was obtained from John Hiscott (Istituto PasteurFondazione Cenci Bolognetti, Rome, Italy). Other plasmids used included the IFN-induced transmembrane 1 (IFITM1)/luc plasmid (derived from pDW9-27CD2), SV40/␤-galactosidase (␤-Gal) (Promega), and SV40/luc (modified from SV40/␤-Gal).…”

Spring viraemia of carp virus: recent advances

“…This method involves the expression of a full-length positive-strand (antigenomic) RNA of the virus genome by vaccinia-driven T7 RNA polymerase (Fuerst et al, 1994;Schnell et al, 1986). After this recovery, many negative-strand RNA viruses have been recovered using similar technique, including human respiratory syncytial virus (Collins et al, 1995), vesicular stomatitis virus (Lawson et al, 1995) and three fish rhabdoviruses: snakehead rhabdovirus (Johnson et al, 2000), infectious hematopoietic necrosis virus (IHNV) (Biacchesi et al, 2000) and viral hemorrhagic septicemia virus (VHSV) (Ammayappan et al, 2011). However, no efforts have been continued for the development of reverse genetics system for SVCV.…”

Viral Hemorrhagic Septicemia Virus NV Gene Decreases Glycolytic Enzyme Gene Transcription

“…아울러 바이러스 의 유전자들이 바이러스 복제 과정에 필수적이기 때문에 특정 바이러스 유전자들의 결핍은 세포 수준에서가 아니라 어류 개체에서의 바이러스 유전자들의 기능을 연구하는데 활용되 고 있다 [2,17]. 또한 rVHSV 결실 실험을 이용하여 NV 유전자 가 중요한 병원성 기능을 가지고 있음이 확인되었다 [8].…”