A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Phylogenomic characterization of two novel members of the genus Megalocytivirus from archived ornamental fish samples.

The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

Characterization of an outbreak of astroviral diarrhea in a group of cheetahs (Acinonyx jubatus).

A Mamastrovirus was identified in an outbreak of diarrhea in cheetahs (Acinonyx jubatus). Five young adult and two adult cheetahs presented with lethargy, anorexia, watery diarrhea and regurgitation over an 11-day period. Fecal samples were submitted for electron microscopy and culture. Electron microscopy results revealed particles morphologically consistent with an astrovirus, and no other viral pathogens or significant bacterial pathogens were identified. The astrovirus was confirmed and sequenced using consensus astroviral PCR, resulting in a 367 base pair partial RNA-dependent-RNA polymerase (RdRp) product and a 628 base pair partial capsid product. Bayesian and maximum likelihood phylogenetic analyses were performed on both the RdRp and the capsid protein segments. All animals were monitored and treated with bismuth subsalicylate tablets (524mg PO BID for 5 days), and recovered without additional intervention. This is the first report we are aware of documenting an astrovirus outbreak in cheetah.

Viruses of pet fish.

Investigation of a fish kill should begin with water quality analysis and use of standard necropsy techniques to rule out the role of parasites or bacteria. The presence of a virus does not mean necessarily that it is a pathogen. Another point to consider is the potential influence of water temperature on pathogenicity; this may be an important factor in developing disease. A list of laboratories that is approved by the USDA to conduct diagnostic testing of aquaculture species can be found at: http://www.aphis.usda.gov/vs/nvsl/labcertification/aquaapplab.htm.

First isolation of largemouth bass virus.

The first reported fish kill caused by largemouth bass virus (LMBV) occurred in 1995 in Santee-Cooper Reservoir, South Carolina, USA. Subsequently, this iridovirus has been implicated in additional fish kills and has also been found in clinically healthy fish in numerous locations in the southeastern USA. We compared the virus from Santee-Cooper Reservoir with a virus isolated in 1991 from large-mouth bass, Micropterus salmoides, from Lake Weir, Florida. Restriction fragment length polymorphisms and the DNA sequence of a portion of the major capsid protein gene were identical for the South Carolina and Florida isolates. These results establish that LMBV was first found in Florida, rather than South Carolina. We propose that the name largemouth bass virus continue to be used for this virus, rather than alternative names based on geographical origin.