Joel Rovnak

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.

Seroprevalence of bovine immunodeficiency-like virus and bovine leukemia virus in a dairy cattle herd

To determine the prevalence of single vs. dual infection with bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV), sera (n = 95) from a dairy cattle herd were analyzed for anti-BIV and anti-BLV antibodies by an enzyme linked immunosorbent assay. Twenty-one percent (20/95) of samples were BIV-seropositive, while 52% (49/95) of the same samples were BLV-seropositive. A significantly greater percentage of BIV-seronegative samples were BLV-seropositive, 57% (43/75), than were BIV-seropositive samples, 30% (6/20). There was no significant correlation between data ranked from least to greatest amount of anti-viral antibody. Five cattle had persistent lymphocytosis (PL); all five were BLV-seropositive and two were BIV-positive. The mean anti-BLV titer was significantly greater in PL cattle, as compared at non-PL cattle, whereas there was no significant difference between the mean anti-BIV titer in PL cattle, as compared with non-PL cattle. These results provide additional information on the seroprevalence of naturally occurring BIV infection, and indicate that BIV can exist independent of other common infectious agents, such as BLV. Further, the results suggest that infection with BIV is not associated with an increased rate of infection with other infectious agents such as BLV.

Novel gammaherpesviruses in North American domestic cats, bobcats and pumas: identification, prevalence and risk factors

ABSTRACT Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus), and puma (Puma concolor) blood cell DNA samples from California, Colorado, and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4-kb region of each putative virus species, including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology. IMPORTANCE Gammaherpesviruses (GHVs) establish lifelong infection in many animal species and can cause cancer and other diseases in humans and animals. In this study, we identified the DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus), and pumas (Puma concolor; also known as mountain lions, cougars, and panthers). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified to be native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the United States. In contrast, puma virus was found only in a specific region of Southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to the route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.

The correlation between the direct and indirect detection of bovine leukemia virus infection in cattle.

Ninety-three cattle from a herd naturally infected with bovine leukemia virus (BLV) were tested for the presence of BLV infection by two indirect indicators, anti-BLV antibodies and lymphocytosis, and two direct indicators, BLV provirus and BLV gp51 antigen expression in peripheral blood mononuclear cells (PBMC). Forty-eight percent (45/93) of the cattle were seropositive, and of these, 53% (24/45) were provirus-positive. Freshly isolated PBMC were negative for gp51 antigen expression, but 11 cattle were positive following short-term culture of their PBMC; 10 of these were seropositive/provirus-positive cattle, and one was a seropositive/provirus-negative cow. Lymphocytosis was present in eight cattle, all of which were seropositive/provirus-positive/gp51-positive. Four cattle were provirus-positive, but negative for all other indicators of BLV infection; a second blood sample was collected from three of these cattle at a later date, at which time two of the three had seroconverted. These results suggest that depending on the stage of the infection, the pathogenesis of BLV in cattle may involve fundamental differences in the host-viral relationship, including the number of cells infected or the number of copies of integrated provirus per cell, regulation of expression of viral antigens, induction of the anti-viral immune response, and the polyclonal or monoclonal proliferation of lymphocytes.

Walleye Dermal Sarcoma Virus Cyclin Interacts with Components of the Mediator Complex and the RNA Polymerase II Holoenzyme

ABSTRACT Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.

SWAN-1, a Caenorhabditis elegans WD Repeat Protein of the AN11 Family, Is a Negative Regulator of Rac GTPase Function

Rac GTPases are key regulators of cell shape and cytoskeletal organization. While some regulators of Rac activity are known, such as GTPase-activating proteins (GAPs) that repress Rac activity, other Rac regulators remain to be identified. The novel Caenorhabditis elegans WD-repeat protein SWAN-1 was identified in a yeast two-hybrid screen with the LIM domains of the Rac effector UNC-115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases. The swan-1(ok267) loss-of-function mutation suppressed defects caused by the hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and filopodia formation induced by constitutively active Rac in C. elegans neurons. Furthermore, SWAN-1(+) transgenic expression suppressed the effects of overactive Rac, including ectopic lamellipodia and filopodia formation in C. elegans neurons, ectopic lamellipodia formation in cultured mammalian fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular morphogenesis and cytoskeletal organization. While broadly conserved across species, SWAN-1 family members show no sequence similarity to previously known Rac inhibitors.

Walleye Dermal Sarcoma Virus: Molecular Biology and Oncogenesis

Retroviruses have been detected in most vertebrate species and are etiologic agents of a variety of neoplastic diseases. The study of retroviruses has been instrumental in uncovering the molecular mechanisms responsible for oncogenesis. Retroviruses have been isolated from three neoplastic diseases in fish, two of which affect the dermis and regress naturally coincident with spawning. This feature provides a unique model to study mechanisms of tumor development and regression. Three complex retroviruses, isolated from walleye (Sander vitreus) with dermal sarcoma and epidermal hyperplasia, are the members of the newest retroviral genus, Epsilonretrovirus. Three accessory proteins, encoded by walleye dermal sarcoma virus (WDSV), function in the regulation of host and viral gene expression and cell cycle, alter cell-signaling pathways to promote cell proliferation and block apoptosis, and, finally, induce apoptosis through dissipation of the mitochondrial membrane potential.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

A rapid, specific, sensitive, and inexpensive method has been developed that detects RNA from a Zika virus strain associated with the current outbreak. LAMP shines light on Zika virus Rapid and simple assays to detect infectious agents are key to tracking emerging epidemics. Chotiwan et al. describe a loop-mediated amplification (LAMP) assay that detects Zika virus RNA in human biofluids such as serum and semen as well as in mosquitoes, the insect vector that transmits the disease. This approach successfully distinguished the Asian-lineage Zika virus, associated with the current outbreak in the Americas, from the African-lineage Zika virus. This LAMP assay should enable tracking of the Asian-lineage strain as it moves into new geographical locations. A key advantage of this approach is detection without the need for RNA purification or copying RNA into DNA. Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.

Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assays and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKCalpha, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.

Walleye Dermal Sarcoma Virus Retroviral Cyclin Directly Contacts TAF9

ABSTRACT Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. A WDSV accessory gene encodes a cyclin homolog or retroviral cyclin (rv-cyclin). WDSV rv-cyclin was found to be associated with transcription complexes and to affect transcription in a cell-type and promoter-dependent manner. It inhibited the WDSV promoter in walleye fibroblasts and activated transcription from GAL4 promoters when fused to the GAL4 DNA binding domain, and an activation domain (AD) has been localized to 30 amino acids in the carboxyl region. rv-cyclin can block the pulldown of transcription coactivators by the AD of VP16, and the isolated rv-cyclin AD interferes specifically with the interaction between the carboxyl halves of the VP16 AD, VP16C, and TATA-binding protein-associated factor 9 (TAF9). The carboxyl region and isolated AD can bind TAF9 directly in assays of protein-protein interaction in vitro. Furthermore, rv-cyclin and the isolated rv-cyclin AD interfere specifically with the function of VP16C in transcription assays. A previously identified motif within the VP16C sequence mediates TAF9 binding, and this motif is present in the activation domains of a variety of TAF9-binding transcriptional activators. A similar motif is present in the rv-cyclin AD, and point mutations within this motif affect rv-cyclin function and protein-protein interactions. The results support a model of transcription regulation by direct interaction with TAF9.