In this research,
genetic relationships among members of genus Ranavirus, including several known and less studied
isolates, were investigated. Sequences of several viral genes, including major capsid protein (MCP),
DNA polymerase (DNApol), neurofilament triplet H1-like protein (NF-H1), ribonuclease reductase
alpha and beta subunits (RNR-alpha and -beta) and RNase III, were obtained and the phylogenetic analyses based
on the sequence data were performed.
The results confirm that the genus Ranavirus encompasses genetically divergent isolates. Most of the ranaviruses characterized to date are closely related to epizootic haematopoietic necrosis virus (EHNV) and frog virus 3 (FV3). Santee-Cooper ranaviruses and grouper iridoviruses cluster separately from the main group of ranaviruses.
Methods based on PCR and subsequent restriction enzyme analysis (REA) of viral DNApol and NF-H1 genes were developed for the detection and differentiation of ranaviruses. DNApol PCR was able to detect 13 different ranavirus isolates.
The most divergent isolates were distinguished with REA of the DNApol gene, whereas differentiation of the more closely related ranaviruses was accomplished with REA of the NF-H1 gene. Quantitative PCR (qPCR) based on the viral DNApol gene was developed in order to detect a wide range of ranaviruses and to estimate the number of virions by using a standard curve. Another qPCR method targeting the fish glucokinase (GK) gene was developed for the quantitation of piscine host cells. Using both DNApol and GK qPCR, it was possible to obtain a viral load value, virions per host cell, from ranavirus-infected cell cultures and fish tissues.
Pathogenicity of nine different ranavirus isolates to rainbow trout (Oncorhynchus mykiss) was studied. None of the isolates caused signs of disease, but virus was isolated from few of the fish collected during the challenge trial. The results indicate that persistent ranavirus infections may occur in certain conditions and that rainbow trout could act as a vector species for ranaviruses.
In addition, fish epithelial cells were infected with four different ranaviruses in order to study the host immune response to ranavirus infection. The mRNA expression of five host immune response genes was measured with qPCR at seven time points after viral inoculation. Based on the results, even though all ranavirus isolates induced apoptotic changes in the epithelial cells, the immune response elicited varied between different ranaviruses. A strong pro-inflammatory response was elicited by FV3 and EHNV, whereas European catfish virus and doctor fish virus induced a mild increase in the expression of regulatory cytokine TGF-beta Gene expression of beta-2 microglobulin was enhanced by all viral isolates, indicating that a pathogen presentation pathway based on MHC class I molecules was activated due to ranavirus infection.
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