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.
 |
Further ReadingYou can view the full report by clicking here. |
