-
Pathogens which are members the family Reo-viridae can infect a wide variety of organisms, including vertebrates, invertebrates, and plants. Genus Aqureovirus, the one of the members of the family Reoviridae, mainly cause infection in aquatic animals like bony fish and shellfish (28). Specifically, some of aquareoviruse isolates can cause severe epidemic diseases in fish, such as hemorrhagic disease and pancreatitis, but the majority of isolated have been obtained during regular examination of seemingly healthy finfish and shellfish (19). Among all the aquareovirus isolates, Grass carp reovirus (GCRV), which was identified from a disastrous outbreak haemorrhage disease in fingerling and yearling grass carp from southern China, is considered to be the most pathogenic agent (24). In this regard, GCRV represents an ideal model for the study of the replication and pathogenesis of Aquareovirus.
As a member of the family Reoviridae, GCRV is a non-envelope multilayered spherical particle of icosa-hedral symmetry with a observed diameter of about 80 nm from results obtained with negative stained electron microscopy (20). The genome of GCRV enclosed in the inner core is composed of eleven segments of double stranded RNA (dsRNA). There are six or seven established sub-genogroup (Aquareovirus A-F and/or G) identified among the aquareovirus isolates, which is mainly on the basis of dsRNA genome electrophoretype and correlated RNA hybridi-zation as well as analysis of their antigenic properties (20, 24). To date, more than 50 aquareovi ruses have been isolated throughout the world, but only a few isolates have been investigated in detail, such as GCRV and SBRV (striped bass reovirus), which belong to different species in the genus (1, 20). Recent genome sequences and phylogenic analyses of Aquareovirus showed that there was a common evolutionary origin with that of the mammalian orthreovirus since they shared a high level of sequence homology. Moreover, the virions of SBRV and GCRV, which were analyzed by electron cryomicroscopy (cryoEM) and three-dimensional (3D) single particle reconstruction, also showed many similarities with that of Mammalian reovirus (MRV) (8, 22, 27). Based on above similarities on both structure resembling and genome identities, there is an argument raised on taxonomy classification or evolution origin between Orthoreoviruses and Aquareoviruses (1, 15). Orthoreoviruses and Aquareoviruses have been attributed to a distinct genus in family Reoviridae, but the similarities and differences between the two genera in both molecular divergence and structure assembly/package in their infected cells remain a great mystery. So, a detailed study on GCRV structural protein may expedite our understanding of the mechanism involved in the assembly of dsRNA viruses.
While a significant amount of structural and biochemical information is availabel about several members of the family such as Orthoreovirus (4, 13, 23, 26, 29, 30, 31), Rotavirus (5), Cypovirus (32) and Orbivirus (9), little is known about the aquareovirus at the molecular level. In an attempt to understand the infection and assembly mechanism of GCRV, it is indispensable to establish a stable system for the molecular genetic analysis of GCRV particles in vitro. In this paper, we report the construction and co-expression of Grass carp reovirus (GCRV) inner capsid non-fusion protein VP6 and enhanced green fluorescence protein (eGFP) in the insect cells for the first time, as a step moving towards an understanding of the structural basis of GCRV and its pathogenesis. Our results provide a reliable system for further in vitro expression and assembly GCRV particle by using baculovirus expression system.
Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells*
- Received Date: 04 July 2007
- Accepted Date: 08 August 2007
Abstract: Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein) genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5 days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.