Pathology of tumors in fish associated with retroviruses: a review.

Thirteen proliferative diseases in fish have been associated in the literature with 1 or more retroviruses. Typically, these occur as seasonal epizootics affecting farmed and wild fish, and most lesions resolve spontaneously. Spontaneous resolution and lifelong resistance to reinfection are 2 features of some piscine retrovirus-induced tumors that have stimulated research interest in this field. The purpose of this review is to present the reader with the epidemiological and morphological features of proliferative diseases in fish that have been associated with retroviruses by 1 or more of the following methods: detection of C-type retrovirus-like particles or reverse transcriptase activity in tumor tissues; successful tumor transmission trials using well-characterized, tumor-derived, cell-free inocula; or molecular characterization of the virus from spontaneous and experimentally induced tumors. Two of the diseases included in this review, European smelt spawning papillomatosis and bicolor damselfish neurofibromatosis, at one time were attributed to a retroviral etiology, but both are now believed to involve additional viral agents based on more recent investigations. We include the latter 2 entities to update the reader about these developments.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Detection of viral hemorrhagic septicemia in round gobies in New York State (USA) waters of Lake Ontario and the St. Lawrence River.

In May 2006 a large mortality of several thousand round gobies Neogobius melanostomus (Pallas, 1814) occurred in New York waters of the St. Lawrence River and Lake Ontario. Necropsies of sampled fish from these areas showed pallor of the liver and gills, and hemorrhagic areas in many organs. Histopathologic examination of affected tissues revealed areas of necrosis and hemorrhage. Inoculations of fathead minnow Pimephales promelas (Rafinesque, 1820) cell cultures with dilutions of tissue samples from the necropsied gobies produced a cytopathic effect within 5 d post-inoculation. Samples of cell culture supernatant were tested using RT-PCR and confirmed the presence of viral hemorrhagic septicemia virus (VHSV). Sequence analysis of the VHSV isolate resulted in its assignment to the type-IVb subgroup. The detection of VHSV in a relatively recent invasive fish species in the Great Lakes and the potential impact of VHSV on the ecology and economy of the area will require further investigation and careful management considerations.

Greenhouse gas emissions from conventional, agri-environmental scheme, and organic Irish suckler-beef units.

The problems of overproduction within the European Union countries and the environmental impact of agriculture have lead to the introduction of schemes that aim to reduce both. Beef (Bos taurus) production forms a large component of the Irish agricultural industry and accounts for more than one quarter of agricultural economic output. Recently, the European CAP (Common Agricultural Policy) has been re-evaluated to include supplementary measures that encompass the environmental role of agriculture rather than just the production role. A life cycle assessment (LCA) method was adopted to estimate emissions per kilogram of CO2 equivalent per kilogram of live weight (LW) leaving the farm gate per annum (kg CO2 kg(-1) LW yr(-1)) and per hectare (kg CO2 ha(-1) yr(-1)). Fifteen units engaged in suckler-beef production (five conventional, five in an Irish agri-environmental scheme, and five organic units) were evaluated for emissions per unit product and area. The average emissions from the conventional units were 13.0 kg CO2 kg(-1) LW yr(-1), from the agri-environmental scheme units 12.2 kg CO2 kg(-1) LW yr(-1), and from the organic units 11.1 kg CO2 kg LW yr(-1). The average emissions per unit area from the conventional units was 5346 kg CO2 ha(-1) yr(-1), from the agri-environmental scheme units 4372 kg CO2 ha(-1) yr(-1), and from the organic units 2302 kg CO2 ha(-1) yr(-1). Results indicated that moving toward extensive production could reduce emissions per unit product and area but live weight production per hectare would be reduced.

Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 10(10) viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 10(10) inoculum, or approximately 10(7) viral RNA copies.

The relationship between greenhouse gas emissions and the intensity of milk production in Ireland.

European Union agri-environmental schemes aim to reduce the environmental impact of agricultural production, but were developed before consideration of greenhouse gas emissions from agriculture. Life cycle assessment methodology provided a framework for comparing emissions as kg CO2 equivalent per kg of energy corrected milk (ECM) (kg CO2 kg(-1) ECM yr(-1)) and per hectare (kg CO2 ha(-1) yr(-1)) for farms both within and outside the Irish agri-environmental scheme. The agri-environmental scheme farms operate extensive systems from 40 to 120 cows producing between 3032 and 5946 kg ECM cow(-1) lactation(-1). The cows are fed on grass, conserved silage, and concentrates. Supplementation ranged between 250 and 620 kg cow(-1) yr(-1). The conventional farms had between 30 and 77 milking cows producing 4736 to 6944 kg ECM cow(-1) lactation(-1). Supplementation ranged from 400 to 1000 kg cow(-1) yr(-1). The emissions from each unit were estimated using published emissions factors and possible error was evaluated by using ranges for each factor. Calculated emissions ranged from 0.92 to 1.51 kg CO2 kg(-1) ECM yr(-1) and 5924 to 8323 kg CO2 ha(-1). On average, total emissions from conventional farms were around 18% (p = 0.01) greater than the agri-environmental scheme farms and emissions per hectare (total area required) were 17% greater (p = 0.02) but there was no significant difference (p = 0.335) in terms of emission per unit milk produced. To evaluate greenhouse gas emissions for each farm in terms of the system intensity it was necessary to define a measure of intensification and area per liter of milk produced that was best.

Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma.

Real-time quantitative polymerase chain reaction (qPCR) was used to quantify viral loads of human herpesviruses (HHVs) at diagnosis in 61 samples of malignant B cells: 21 chronic lymphocytic leukemia (B-CLL), 29 acute lymphoblastic leukemia (B-ALL) and 11 multiple myeloma (MM); control samples were blasts from 16 acute myeloid leukemia (AML) and 24 blood or bone marrow samples from healthy donors. The majority of samples from healthy donors and patients (B-ALL, B-CLL or AML, but not MM) was positive for EBV and contained <100 ebv copies/100 ng dna. ebv loads were occasionally high (>500 copies/100 ng DNA) in B-ALL (2/16) and in B-CLL (2/21) samples. The fractions of samples positive for HHV-8 and HHV-6A, less than 10% for MM patients, were high for adults with B-ALL (18.8% HHV-8+, 43.8% HHV-6A+) or B-CLL (28.6% HHV-8+, 52.4% HHV-6A+). B-ALL, B-CLL and MM samples were rarely positive for HHV-6B and HHV-7. Lastly, 75% of B-ALL samples were positive for CMV, and CMV loads were significantly higher in B-ALL samples than in MM, B-CLL or AML samples. We also used PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) to look for novel HHVs in B cell samples: no sequence indicative of a new HHV was detected. Altogether, the data indicate that the presence of multiple HHVs, including EBV and CMV at high loads, is not rare in B-ALL and B-CLL cell samples. Future prospective studies should determine whether patients with high EBV/CMV loads at diagnosis in tumor samples face a higher risk of delayed hematological recovery, virus-related complications or relapse.

Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 1010 viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 1010 inoculum, or approximately 107 viral RNA copies.

Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR.

Quantitative real-time PCR has been used to measure fibropapilloma-associated turtle herpesvirus (FPTHV) pol DNA loads in fibropapillomas, fibromas, and uninvolved tissues of green, loggerhead, and olive ridley turtles from Hawaii, Florida, Costa Rica, Australia, Mexico, and the West Indies. The viral DNA loads from tumors obtained from terminal animals were relatively homogeneous (range 2-20 copies/cell), whereas DNA copy numbers from biopsied tumors and skin of otherwise healthy turtles displayed a wide variation (range 0.001-170 copies/cell) and may reflect the stage of tumor development. FPTHV DNA loads in tumors were 2.5-4.5 logs higher than in uninvolved skin from the same animal regardless of geographic location, further implying a role for FPTHV in the etiology of fibropapillomatosis. Although FPTHV pol sequences amplified from tumors are highly related to each other, single signature amino acid substitutions distinguish the Australia/Hawaii, Mexico/Costa Rica, and Florida/Caribbean groups.

Intracellular targeting of walleye dermal sarcoma virus Orf A (rv-cyclin).

Walleye dermal sarcoma virus (WDSV) induces tumors and allows or possibly directs tumor regression. WDSV encodes a putative cyclin homologue, Orf A, and six variant Orf A transcripts have been identified. Northern analysis indicated that a 3.3-kb transcript, encoding full-length Orf A, is the predominant transcript in developing, but not regressing, tumors. Three Orf A proteins, one full-length and two amino-truncated forms, were expressed in mammalian and piscine cells, and their intracellular locations were determined. The full-length form was nuclear and concentrated in interchromatin granule clusters, defined by colocalization with SC-35. The amino-truncated forms were cytoplasmic. Fusion of amino-terminal portions of Orf A to a heterologous protein demonstrated that residues 1-112 were necessary for nuclear localization. Mutation of aa K80 and/or E110 disrupted nuclear localization, suggesting a mechanism similar to that of cellular A- and D-type cyclins for its nuclear import.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

Sequence and transcriptional analyses of the fish retroviruses walleye epidermal hyperplasia virus types 1 and 2: evidence for a gene duplication.

Walleye epidermal hyperplasia virus types 1 and 2 (WEHV1 and WEHV2, respectively) are associated with a hyperproliferative skin lesion on walleyes that appears and regresses seasonally. We have determined the complete nucleotide sequences and transcriptional profiles of these viruses. WEHV1 and WEHV2 are large, complex retroviruses of 12,999 and 13,125 kb in length, respectively, that are closely related to one another and to walleye dermal sarcoma virus (WDSV). These walleye retroviruses contain three open reading frames, orfA, orfB, and orfC, in addition to gag, pol, and env. orfA and orfB are adjacent to one another and located downstream of env. The OrfA proteins were previously identified as cyclin D homologs that may contribute to the induction of cell proliferation leading to epidermal hyperplasia and dermal sarcoma. The sequence analysis of WEHV1 and WEHV2 revealed that the OrfB proteins are distantly related to the OrfA proteins, suggesting that orfB arose by gene duplication. Presuming that the precursor of orfA and orfB was derived from a cellular cyclin, these genes are the first accessory genes of complex retroviruses that can be traced to a cellular origin. WEHV1, WEHV2, and WDSV are the only retroviruses that have an open reading frame, orfC, of considerable size (ca. 130 amino acids) in the leader region preceding gag. While we were unable to predict a function for the OrfC proteins, they are more conserved than OrfA and OrfB, suggesting that they may be biologically important to the viruses. The transcriptional profiles of WEHV1 and WEHV2 were also similar to that of WDSV; Northern blot analyses detected only low levels of the orfA transcripts in developing lesions, whereas abundant levels of genomic, env, orfA, and orfB transcripts were detected in regressing lesions. The splice donors and acceptors of individual transcripts were identified by reverse transcriptase PCR. The similarities of WEHV1, WEHV2, and WDSV suggest that these viruses use similar strategies of viral replication and induce cell proliferation by a similar mechanism.

Assessment of bovine leukemia virus transcripts in vivo.

Reverse transcriptase PCR (RT-PCR) consistently detected bovine leukemia virus transcripts in fresh cells, and competitive RT-PCR enumerated these transcripts. The detection of transcripts in limited numbers of tumor cells indicated that expression occurs in a minority of cells. The data suggest that individual cells contain hundreds of copies of the tax/rex transcript in vivo.

Walleye retroviruses associated with skin tumors and hyperplasias encode cyclin D homologs.

Walleye dermal sarcoma (WDS) and walleye epidermal hyperplasia (WEH) are skin diseases of walleye fish that appear and regress on a seasonal basis. We report here that the complex retroviruses etiologically associated with WDS (WDS virus [WDSV]) and WEH (WEH viruses 1 and 2 [WEHV1 and WEHV2, respectively]) encode D-type cyclin homologs. The retroviral cyclins (rv-cyclins) are distantly related to one another and to known cyclins and are not closely related to any walleye cellular gene based on low-stringency Southern blotting. Since aberrant expression of D-type cyclins occurs in many human tumors, we suggest that expression of the rv-cyclins may contribute to the development of WDS or WEH. In support of this hypothesis, we show that rv-cyclin transcripts are made in developing WDS and WEH and that the rv-cyclin of WDSV induces cell cycle progression in yeast (Saccharomyces cerevisiae). WEHV1, WEHV2, and WDSV are the first examples of retroviruses that encode cyclin homologs. WEH and WDS and their associated retroviruses represent a novel paradigm of retroviral tumor induction and, importantly, tumor regression.

Three closely related herpesviruses are associated with fibropapillomatosis in marine turtles.

Green turtle fibropapillomatosis is a neoplastic disease of increasingly significant threat to the survivability of this species. Degenerate PCR primers that target highly conserved regions of genes encoding herpesvirus DNA polymerases were used to amplify a DNA sequence from fibropapillomas and fibromas from Hawaiian and Florida green turtles. All of the tumors tested (n = 23) were found to harbor viral DNA, whereas no viral DNA was detected in skin biopsies from tumor-negative turtles. The tissue distribution of the green turtle herpesvirus appears to be generally limited to tumors where viral DNA was found to accumulate at approximately two to five copies per cell and is occasionally detected, only by PCR, in some tissues normally associated with tumor development. In addition, herpesviral DNA was detected in fibropapillomas from two loggerhead and four olive ridley turtles. Nucleotide sequencing of a 483-bp fragment of the turtle herpesvirus DNA polymerase gene determined that the Florida green turtle and loggerhead turtle sequences are identical and differ from the Hawaiian green turtle sequence by five nucleotide changes, which results in two amino acid substitutions. The olive ridley sequence differs from the Florida and Hawaiian green turtle sequences by 15 and 16 nucleotide changes, respectively, resulting in four amino acid substitutions, three of which are unique to the olive ridley sequence. Our data suggest that these closely related turtle herpesviruses are intimately involved in the genesis of fibropapillomatosis.

Detection of a novel bovine lymphotropic herpesvirus.

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.

Two closely related but distinct retroviruses are associated with walleye discrete epidermal hyperplasia.

Walleye discrete epidermal hyperplasia (WEH) is a hyperproliferative skin disease that is prevalent on adult walleye fish throughout North America. We have identified two retroviruses associated with WEH, designated here as walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2), that are closely related to one another (77% identity) and to walleye dermal sarcoma virus (64% identity) within the polymerase region. WEHV1 and/or WEHV2 viral DNA was readily detected by PCR in hyperplastic tissue samples, but only low levels of viral DNA were detected in uninvolved skin. Southern blot analysis showed one to three copies of integrated WEHV2 viral DNA in lesions but did not detect WEHV2 viral DNA in uninvolved skin from the same fish. Northern blots detected abundant levels of WEHV1 and/or WEHV2 virion RNA transcripts of approximately 13 kb in hyperplastic tissue, but virion RNA was not observed in uninvolved skin and muscle. These results suggest that WEHV1 and WEHV2 are the causative agents of discrete epidermal hyperplasia.