Riikka Holopainen

Ranavirus in wild edible frogs Pelophylax kl. esculentus in Denmark

A survey for the amphibian pathogens ranavirus and Batrachochytrium dendrobatidis (Bd) was conducted in Denmark during August and September 2008. The public was encouraged via the media to register unusual mortalities in a web-based survey. All members of the public that registered cases were interviewed by phone and 10 cases were examined on suspicion of disease-induced mortality. All samples were negative for Bd. Ranavirus was isolated from 2 samples of recently dead frogs collected during a mass mortality event in an artificial pond near Slagelse, Denmark. The identity of the virus was confirmed by immunofluorescent antibody test. Sequencing of the major capsid protein gene showed the isolate had more than 97.3% nucleotide homology to 6 other ranaviruses.

Comparative study of ranavirus isolates from cod (Gadus morhua) and turbot (Psetta maxima) with reference to other ranaviruses

Two iridovirus isolates recovered from cod (Gadus morhua) and turbot (Psetta maxima) in Denmark were examined in parallel with a panel of other ranaviruses including frog virus 3 (FV3), the reference strain for the genus Ranavirus. The isolates were assessed according to their reactivity in immunofluoresent antibody tests (IFAT) using both homologous and heterologous antisera and their amplification in PCR using primers targeting five genomic regions. The corresponding PCR fragments were sequenced, and the sequences obtained were used in phylogenetic analysis. In addition, the pathogenicity to rainbow trout under experimental challenge conditions was investigated. The viruses were serologically and genetically closely related to highly pathogenic ranaviruses such as European catfish iridovirus (ECV), European sheatfish iridovirus (ESV) and epizootic haematopoietic necrosis virus (EHNV). The challenge trials indicate that rainbow trout fry cultured at 15°C are not target species for the virus isolates in the present panel. We suggest that the two isolates belong in the genus Ranavirus and propose the name Ranavirus maxima (Rmax) for the turbot isolate.

Quantitation of ranaviruses in cell culture and tissue samples

A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.

Characterization of perch rhabdovirus (PRV) in farmed grayling Thymallus thymallus

Two Finnish fish farms experienced elevated mortality rates in farmed grayling Thymallus thymallus fry during the summer months, most typically in July. The mortalities occurred during several years and were connected with a few neurological disorders and peritonitis. Virological investigation detected an infection with an unknown rhabdovirus. Based on the entire glycoprotein (G) and partial RNA polymerase (L) gene sequences, the virus was classified as a perch rhabdovirus (PRV). Pairwise comparisons of the G and L gene regions of grayling isolates revealed that all isolates were very closely related, with 99 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis demonstrated that they were closely related to the strain isolated from perch Perca fluviatilis and sea trout Salmo trutta trutta caught from the Baltic Sea. The entire G gene sequences revealed that all Finnish grayling isolates, and both the perch and sea trout isolates, were most closely related to a PRV isolated in France in 2004. According to the partial L gene sequences, all of the Finnish grayling isolates were most closely related to the Danish isolate DK5533 from pike. The genetic analysis of entire G gene and partial L gene sequences showed that the Finnish brown trout isolate ka907_87 shared only approximately 67 and 78% identity, respectively, with our grayling isolates. The grayling isolates were also analysed by an immunofluorescence antibody test. This is the first report of a PRV causing disease in grayling in Finland.

Genome Sequence of a Ranavirus Isolated from Pike-Perch Sander lucioperca

ABSTRACT The pike-perch iridovirus (PPIV) was isolated in Finland from apparently healthy pike-perch fingerlings during routine disease surveillance. Our phylogenomic analysis revealed that PPIV is the first fish member of a clade of ranaviruses previously described from European and Chinese amphibians.

Infectious pancreatic necrosis virus (IPNV) strain with genetic properties associated with low pathogenicity at Finnish fish farms

Infectious pancreatic necrosis (IPN) is a contagious viral disease of fish that causes economic losses in aquaculture worldwide. In Finland, IPN virus (IPNV) has been isolated since 1987 from adult fish showing no signs of clinical disease at fish farms located in the coastal areas of the Baltic Sea. The inland area of Finland, however, remained free of IPN until 2012, when fish on several rainbow trout farms were diagnosed IPNV-positive. The fish mortalities detected at the farms were low, but clinical signs and histopathological changes typical for IPNV infection were seen in juvenile salmonids. IPNV was isolated at high water temperatures up to 22°C. In 2013 and 2014, IPNV detections continued at inland farms, indicating that infections have spread. The aim of this study was to describe the epidemiology of the outbreak and to characterise the Finnish inland IPNV isolates using histopathological, immunohistochemical and genetic approaches. In order to determine the epidemiological origin of the inland IPNV infections, the partial viral capsid protein (VP2) gene sequences of the inland IPNV isolates were compared with the sequences of the isolates from the coastal farms. Based on the genetic analysis, the inland isolates belong to IPNV Genogroup 2 (Serotype A3/Ab), and the origin of the isolates appears to be one or several coastal fish farms.

Molecular characterisation of infectious pancreatic necrosis viruses isolated from farmed fish in Finland

Infectious pancreatic necrosis virus (IPNV) has been isolated annually since 1987 from salmonids without clinical signs at coastal fish farms in Finland. In the inland area, viral isolations were rare until 2012, when IPNV was detected at several freshwater fish farms. Between 2013 and 2015, the infection spread and IPNV was continuously isolated from several farms, both inland and on the coast. The aim of this study was to genetically characterise the IPNV isolates collected from Finnish coastal and inland fish farms over the last 15 years, and to detect genetic changes that may have occurred in the virus populations during the study period. The partial VP2 gene sequence from 88 isolates was analysed. In addition, a complete genomic coding sequence was obtained from 11 isolates. Based on the genetic analyses, Finnish IPNV isolates belong to three genogroups: 2, 5 and 6. The genetic properties of the isolates appear to vary between inland farms producing juveniles and food fish farms in the coastal region: the inland farms harboured genogroup 2 isolates, whereas at coastal farms, all three genogroups were detected. Little genetic variation was observed within the Finnish genogroup 2 and 5 isolates, whereas among the genogroup 6 isolates, two subgroups were detected. All isolates studied demonstrated amino acid patterns in the viral VP2 gene previously associated with avirulence. However, increased mortality was detected at some of the farms, indicating that more research is needed to clarify the relationship between the pathogenicity and genetic properties of IPNV isolates from different genogroups.

An Outbreak of Rabbit Hemorrhagic Disease in Finland

Abstract Rabbit hemorrhagic disease (RHD) was detected in European rabbits (Oryctolagus cuniculus) for the first time ever in Finland in 2016. Reports of dead feral rabbits in Helsinki started to accumulate from April 2016. The Finnish Food Safety Authority Evira received the first animals in late April, and the main necropsy finding was severe, acute necrotizing hepatitis. Genetic material from RHD virus (RHDV) was detected in the liver and was further characterized as RHDV2. The Finnish virus did not group with RHDV strains from a concurrent outbreak in neighboring Sweden, suggesting another origin. The outbreak peaked in May and lasted until August, after which sightings of both live and dead rabbits became rare. No major outbreaks in domestic rabbits were observed, although infection in one pet rabbit was confirmed.