James W. Casey

Genomic Variation of the Fibropapilloma-Associated Marine Turtle Herpesvirus across Seven Geographic Areas and Three Host Species

ABSTRACT Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.

Identification and Characterization of an Exogenous Retrovirus from Atlantic Salmon Swim Bladder Sarcomas

ABSTRACT A novel piscine retrovirus has been identified in association with an outbreak of leiomyosarcoma in the swim bladders of Atlantic salmon. The complete nucleotide sequence of the Atlantic salmon swim bladder sarcoma virus (SSSV) provirus is 10.9 kb in length and shares a structure and transcriptional profile similar to those of murine leukemia virus-like simple retroviruses. SSSV appears unique to simple retroviruses by not harboring sequences in the Atlantic salmon genome. Additionally, SSSV differs from other retroviruses in potentially utilizing a methionine tRNA primer binding site. SSSV-associated tumors contain high proviral copy numbers (greater than 30 per cell) and a polyclonal integration pattern. Phylogenetic analysis based on reverse transcriptase places SSSV with zebrafish endogenous retrovirus (ZFERV) between the Gammaretrovirus and Epsilonretrovirus genera. Large regions of continuous homology between SSSV and ZFERV Gag, Pol, and Env suggest that these viruses represent a new group of related piscine retroviruses.

In situ hybridization and immunohistochemical study of walleye dermal sarcoma virus (WDSV) nucleic acids and proteins in spontaneous sarcomas of adult walleyes (Stizostedion vitreum).

Twenty-two anatomically independent dermal sarcomas from six adult walleye fish (Stizostedion vitreum) collected during the spring from Oneida Lake, New York, were examined by in situ hybridization and immunohistochemistry for the presence of walleye dermal sarcoma virus (WDSV). The viral RNA, DNA, and 90-kd protein were localized at the cellular level. Riboprobes complementary to the 5′ terminal region of WDSV genome were used to detect viral nucleic acids. Rabbit polyclonal antiserum was generated against the 90-kd virus-associated antigen, presumably a product of the env gene, for immunohistochemical studies. Viral transcripts were detected in the neoplastic cells of all dermal sarcomas, in which they were generally abundant. Rare mononuclear inflammatory cells and cells within the epidermis also expressed viral RNA. In all sarcomas, low to moderate levels of viral DNA were present in all neoplastic and most mononuclear inflammatory and epidermal cells. Many neoplastic cells were immunopositive for the virus-associated protein. The distribution of immunopositive neoplastic cells mimicked approximately that of cells containing viral transcripts. The number of neoplastic cells with transcripts exceeded that of cells with protein, suggesting that productively infected neoplastic cells constituted a subset of the neoplastic cells that expressed WDSV transcripts. The viral antigen was also present within many mononuclear inflammatory cells. These data suggested that 1) dermal sarcomas were associated with elevated transcriptional activity of WDSV in the neoplastic cells and 2) the cell tropism of WDSV extended beyond the mesenchymal fibroblast-like neoplastic cells and included at least mononuclear inflammatory and epidermal cells.

PCR and RT-PCR Analysis of Infection and Transcriptional Activity of Walleye Dermal Sarcoma Virus (WDSV) in Organs of Adult Walleyes (Stizostedion vitreum)

The pathogenesis of walleye dermal sarcoma virus (WDSV) infection was investigated in adult walleyes (Stizostedion vitreum). Three tumor-bearing and three tumor-free walleyes were collected in the spring from Oneida Lake, New York, and analyzed for viral infection and transcriptional activity. Specifically, the target organs for viral infection and supporting viral transcriptional activity were determined by assessing for the presence of WDSV DNA and RNA in the brain, liver, kidney, skin, and spleen. For each organ, WDSV DNA and RNA were detected using the polymerase chain reaction (PCR) and reverse transcription PCR (RTPCR) respectively. Quantitative estimates of the number of viral DNA and RNA copies were obtained in each case by comparing the signal intensity of the sample to that of external controls. WDSV RNA/DNA ratios, based on those quantitative estimates, were computed for each organ. An RNA/DNA ratio of 3 was arbitrarily chosen as the threshold above which there was viral transcriptional activity. Viral DNA was found in all the organs examined from the three tumor-free walleyes. In those three tumor-free walleyes, low levels of WDSV RNA were detected in only one kidney and two spleen samples. In the three tumor-bearing walleyes, viral DNA was found in one brain, one kidney, two liver, and two skin samples. In contrast to the few organs from tumor-free walleyes in which WDSV RNA was detected, in tumor-bearing walleyes WDSV RNA was present in the one brain examined and in 2/3 kidney, 2/3 liver, 3/3 skin, and 3/3 spleen samples. A WDSV RNA/DNA ratio above 3 was obtained in all three tumor-bearing walleyes but in only one tumor-free fish. These data indicated that 1) both tumor-bearing and tumor-free walleyes were infected by WDSV, 2) many cell types were targeted by WDSV and supported viral transcription, and 3) tumor-bearing walleyes harbored a transcriptionally active WDSV, whereas tumor-free walleyes contained mostly silent WDSV DNA.

Swimbladder Leiomyosarcoma in Atlantic Salmon (Salmo salar) in North America

Leiomyosarcoma with associated retrovirus were found in North America for the first time in adult Atlantic salmon (Salmo salar) held in a quarantine facility at the North Attleboro National Fish Hatchery (NANFH), Massachusetts, USA. The fish had been collected as age 1–2 yr animals from the Pleasant River, Maine, and were to be used as brood stock in a population augmentation program for that river. Neoplastic disease was observed at NANFH initially in older (age 4 yr) fish, followed by age 3 yr fish. Disease was not observed in age 2 yr fish. The mortality pattern was chronic.

Low prevalence of Cyprinid Herpesvirus 3 Found in common carp (Cyprinus carpio carpio) collected from nine locations in the Great Lakes.

Cyprinid herpesvirus 3 (CyHV3) is a viral disease of fish first detected in the United States in 1998. Since that time, mortality events in common carp (Cyprinus carpio carpio) have occurred in several locations within the Great Lakes basin, but not within the Great Lakes themselves. We sampled 675 carp from 20 sites across the Great Lakes and Lake St. Clair, Michigan, USA, between 19 July and 26 September 2010. We tested the gill and a pooled internal organ sample from each fish for CyHV3 with the use of a quantitative polymerase chain reaction (qPCR) assay. Virus was detected in 18 fish from nine sites in four lakes (Lakes Michigan, Huron, St. Clair, and Ontario). Tissues from these 18 fish were also tested for CyHV3 with the use of the PCR assay recommended by the World Organization for Animal Health; amplification was achieved from two fish and confirmation by sequencing of CyHV3 from one fish collected in Lake St. Clair. The results of this study suggest that CyHV3 is present in the Great Lakes.

Cancers Induced by Piscine Retroviruses

Retroviruses have been detected in the majority of vertebrate species analyzed to date. In fish, thirteen proliferative diseases have been associated with the presence of retroviruses. The etiologic relationship of retroviruses with these diseases is primarily based on tumor-associated retrovirus-like particles, the presence of reverse transcriptase activity in neoplastic tissues, and the ability to transmit disease with tumor extracts. Increased epizootics of cancers in fish, particularly in farm-reared or hatchery facilities, have prompted the awareness and further study of the suspected retroviruses. Many proliferative diseases in fish develop and regress seasonally and provide unique models for the study of cancer development and regression. The complete proviral sequences of six fish retroviruses have revealed unique genome organizations and expression patterns. Two simple retroviruses have been identified: an exogenous virus from Atlantic salmon swim-bladder tumors and an endogenous retrovirus in the zebrafish genome. A complex retrovirus was isolated from a cultured snakehead-fish cell-line, although an association with disease has not been shown. Three complex retroviruses isolated from walleye skin-proliferative diseases display a similar genomic structure and encode three novel accessory proteins that contribute to oncogenesis and tumor regression. The accessory proteins from walleye dermal-sarcoma virus (WDSV) function in the regulation of host and viral-gene expression by altering cell-signaling pathways and induction of apoptosis. A new retroviral genus, Epsilonretroviruses, has been established based on the distinctive sequence and structure of the walleye viruses. Phylogenetic analyses show a high degree of heterogeneity within the piscine retroviruses.