Recombinant Sleeping disease virus Structural polyprotein

What is Sleeping disease virus and how is it classified taxonomically?

Sleeping disease virus is a member of the Salmonid alphavirus genus within the Togaviridae family. The virus has a single-stranded RNA genome that is 11,894 nucleotides long, excluding the 3′ poly(A) tail. Although its genome structure is similar to classical alphaviruses such as Sindbis virus and Semliki Forest virus, the nucleotide and predicted amino acid sequences of SDV are only distantly related to other alphaviruses. Furthermore, SDV has the shortest 5′UTR and 3′UTR described among alphaviruses, with the 5′UTR being only 27 nucleotides long—approximately half or a third of the size found in other alphavirus genomes .

What are the primary host species affected by Sleeping disease virus?

Sleeping disease primarily affects rainbow trout (Oncorhynchus mykiss), where the disease is characterized by an abnormal behavior of the fish, which remain on their sides at the bottom of tanks, appearing to be in a "sleeping state"—hence the name of the disease. A related disease also affects farmed Atlantic salmon (Salmo salar L.) . The disease was initially suspected to have a viral etiology, which was later confirmed through isolation and characterization of the causative agent .

How does the structural polyprotein of SDV compare with other alphaviruses?

While the search results don't provide specific details about the SDV structural polyprotein comparison, we can infer from alphavirus biology that the structural polyprotein contains genes encoding the capsid protein and envelope glycoproteins. In SDV, these structural components would include the fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) protein, which are integral membrane proteins that play crucial roles in viral entry and assembly. Unlike some other paramyxoviruses, SDV has some unique characteristics in its genome organization and protein expression that distinguish it from classical alphaviruses .

What methods have been developed for generating recombinant Sleeping disease virus?

Researchers have successfully developed a system for generating recombinant SDV (rSDV) using a full-length cDNA approach. The process involves:

• Generation of a complete cDNA copy of the SDV genome

• Fusion of this cDNA to a hammerhead ribozyme sequence at the 5′ end

• Insertion into a transcription plasmid (pcDNA3) backbone, yielding the construct pSDV

• Transfection of pSDV into fish cells, specifically vTF7-3-infected BF-2 cells

The methodology differs from those used for mammalian alphaviruses, as attempts to generate infectious RNA through in vitro transcription of SDV cDNA directly fused to a T7 or SP6 promoter failed. Similarly, transfection of a plasmid construct with a T7 promoter fused to SDV cDNA into vTF7-3-infected cells did not result in recombinant virus recovery. The successful approach required the hammerhead ribozyme sequence to ensure proper 5′ end formation .

How can successful recovery of recombinant SDV be verified experimentally?

Verification of successful rSDV recovery involves multiple experimental approaches:

• Indirect immunofluorescence assay on rSDV-infected cells using a panel of monoclonal antibodies (MAbs) directed against nonstructural and structural SDV proteins

• Detection of cell-to-cell spreading of the recombinant virus through observation of increasing focus size over time (7-10 days post-transfection)

• Confirmation that supernatant from transfected cells can infect fresh cells

• Molecular verification through detection of genetic tags (such as a BlpI restriction enzyme site) introduced into the rSDV RNA genome by RT-PCR and restriction enzyme digestion

• Verification of proper maturation of viral proteins, demonstrated by staining live rSDV-infected cells with neutralizing MAbs (e.g., anti-E2 neutralizing 17H23 and D20 MAbs)

What challenges exist in adapting recombinant SDV to different growth conditions?

One significant challenge in SDV research is temperature adaptation. Wild-type SDV and standard recombinant SDV replicate at low temperatures (10°C) in fish-derived cells such as RTG-2, CHSE-214, and BF-2 cells. BF-2 cells have been identified as the most appropriate cell line for producing SDV at high titers (>10^8 PFU/ml).

Researchers have successfully adapted rSDV to grow at a higher temperature (14°C instead of 10°C), resulting in a variant called rSDV 14. This temperature adaptation process involves multiple passages at incrementally higher temperatures and leads to genetic changes. Interestingly, this temperature adaptation can significantly alter the virus's pathogenicity—while standard rSDV is attenuated in trout, rSDV 14 becomes pathogenic. Comparative genomic analysis of wild-type SDV, rSDV, and rSDV 14 has revealed several amino acid changes that may be linked to this shift in pathogenicity .

How can recombinant SDV be used as a gene vector system?

Recombinant SDV has been successfully developed as a gene vector system capable of accommodating and expressing foreign genes. The experimental approach involves:

• Modification of the pSDV construct to incorporate reporter genes

• Addition of an SDV RNA subgenomic promoter fused to the foreign gene

• Strategic placement of the foreign gene either upstream of the structural genes (pGFP-SDV) or downstream from the stop codon of the structural genes (pSDV-GFP)

Researchers have determined that a 100-nucleotide-long DNA fragment in the nsP4 sequence upstream of the junction region likely contains the promoter sequence necessary for expression. Expression efficiency differs based on the placement of the foreign gene, with higher expression observed when the gene is located downstream of the structural genes in the SDV genome.

What mutation strategies can be employed to study SDV protein function?

The development of an infectious cDNA clone of SDV has enabled targeted mutation studies, particularly focusing on the nsP2 protein. Researchers can:

• Introduce specific mutations into the cDNA construct using standard molecular biology techniques

• Transfect cells with the mutated constructs to recover mutant viruses

• Assess the impact of these mutations on viral replication through viral growth curves, protein expression analysis, and cytopathic effect observations

This approach has been used to evaluate the impact of various targeted mutations in nsP2 on viral replication, providing insights into the functional domains of this important viral protein. Similar strategies can be applied to study the structural polyprotein and its components, though specific examples are not detailed in the provided search results .

What systems have been developed to study SDV pathogenesis in vivo?

Experimental systems for studying SDV pathogenesis in vivo primarily use juvenile trout as a model organism. The methodological approach includes:

• Infecting juvenile trout by immersion in a water bath containing the virus

• Monitoring fish for mortality and disease signs over time

• Challenging previously infected fish with virulent virus to assess protection

• Comparing pathogenicity between different viral strains (e.g., wild-type SDV, rSDV, and rSDV 14)

Using this system, researchers have observed significant differences in pathogenicity: wild-type SDV causes high mortality (up to 80%), while recombinant wild-type virus-like rSDV causes no deaths or disease signs, though fish are readily infected. This system has also demonstrated that rSDV can provide long-lasting protection when fish are challenged with wild-type SDV 3 and 5 months post-infection, suggesting potential vaccine applications .

How does recombinant SDV differ from wild-type SDV in terms of pathogenicity?

Despite having a similar genetic structure, recombinant SDV derived from cDNA (rSDV) exhibits significantly attenuated pathogenicity compared to wild-type SDV (wtSDV). Experimental infection of juvenile trout with rSDV results in asymptomatic infection with no mortality, whereas wtSDV infection leads to approximately 80% cumulative mortality. This striking difference in pathogenicity occurs despite rSDV successfully infecting the fish, as demonstrated by virus detection methods.

The molecular basis for this attenuation is not fully characterized but likely involves subtle changes in the viral genome introduced during the cloning process or differences in the dynamics of viral replication. Nucleotide sequence comparisons between wtSDV, rSDV, and the pathogenic temperature-adapted variant rSDV 14 have identified several amino acid changes that may be linked to pathogenicity in trout .

What is the mechanism of protective immunity induced by recombinant SDV?

While the search results don't provide detailed information about the specific immunological mechanisms, they do demonstrate that infection with the attenuated rSDV induces long-lasting protection against challenge with virulent wtSDV. Fish infected with rSDV and subsequently challenged with wtSDV 3 and 5 months post-infection showed significant protection against disease.

This protection likely involves both humoral immunity (antibody-mediated) targeting the viral structural proteins, particularly the surface glycoproteins, and cellular immune responses. The ability of rSDV to replicate in fish without causing disease while inducing protective immunity makes it a promising candidate for vaccine development against Sleeping disease .

How do nucleocytoplasmic trafficking mechanisms affect viral protein function in SDV infection?

While the provided search results don't specifically address nucleocytoplasmic trafficking in SDV, search result discusses the importance of matrix protein trafficking in Respiratory Syncytial Virus (RSV), another negative-sense RNA virus. By analogy, we can infer that similar mechanisms might be relevant for SDV.

In RSV, the matrix protein traffics into and out of the nucleus at specific times during the infectious cycle and can inhibit transcription, which may be key to viral pathogenesis. Nuclear export of the matrix protein is Crm1-dependent, and inhibiting this export with compounds like LMB reduces virus production.

For SDV research, investigating whether structural or non-structural proteins undergo similar nucleocytoplasmic trafficking would be valuable. If similar mechanisms exist in SDV, they could potentially:

• Regulate viral gene expression

• Influence host cell transcription

• Affect viral assembly and budding

• Provide targets for antiviral intervention

What factors determine the genetic stability of recombinant SDV carrying additional genetic material?

The development of rSDV as a gene vector system has revealed challenges related to genetic stability. While SDV can accommodate substantial additional genetic material (at least 2.7 kilobases, representing a 20% increase in genome size), the stability of these constructs decreases with serial passage.

For example, rSDV carrying three copies of the "GFP unit" lost GFP expression after one additional passage in cell culture, despite the cells remaining infected with the virus. This suggests selective pressure against maintaining the larger genome during replication.

Factors that likely influence genetic stability include:

• The size of the inserted sequence

• The location of the insertion within the viral genome

• The nature of the inserted sequence and its potential impact on viral RNA structures

• The expression level of the inserted gene and its impact on viral protein balance

• Selection pressures during viral replication and assembly

Further research examining these factors would help optimize SDV-based vector systems for applications requiring stable expression of foreign genes .

What approaches can be used to elucidate the role of specific domains within the SDV structural polyprotein?

Advanced research into SDV structural polyprotein domains would benefit from several complementary approaches:

• Site-directed mutagenesis: Using the infectious cDNA clone to introduce specific mutations in the structural polyprotein genes, followed by analysis of the resulting phenotypes in terms of viral assembly, infectivity, and pathogenicity.

• Domain swapping: Replacing specific domains in the SDV structural polyprotein with corresponding domains from related viruses to identify regions responsible for specific functions or host interactions.

• Protein-protein interaction studies: Identifying interactions between structural proteins and between structural and non-structural proteins using techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity ligation assays.

• Structural biology approaches: Determining the three-dimensional structure of individual structural proteins or the assembled virion using X-ray crystallography, cryo-electron microscopy, or other structural techniques.

• In vivo pathogenesis studies: Testing mutant viruses with specific alterations in the structural polyprotein for changes in tissue tropism, virulence, or immune responses in the fish host.

These approaches would provide insights into how the structural polyprotein is processed, assembled, and functions in the viral life cycle, potentially identifying targets for intervention strategies .