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The ISAC facility (Isotope Separation and Acceleration) at TRIUMF has recently started to provide isotopes for pre-clinical nuclear medicine studies. By irradiating ISOL (Isotope Separation OnLine) targets with a 480 MeV proton beam from the TRIUMF H- cyclotron, the facility can deliver a large variety of radioactive isotope beams (RIB) for research in the fields of nuclear astrophysics, nuclear structure and material science with half-lives down to a few milliseconds via an electrostatic beamline network. For the collection of medical isotopes, typically with half-lives in the range of hours or days, we have developed a compact apparatus for the implantation of mass-separated RIB on a target disc at energies between 20-55 keV. In this paper, we also discuss two different retrieval methods of the implanted activity from the implantation target: by chemical etching of the target surface and by recoil collection of implanted alpha emitters.

Abstract Background The shape of the dose-response curve at low doses differs from the linear quadratic model. The effect of a radio-adaptive response is the centre of many studies and well known inspite that the clinical applications are still rarely considered. Methods We studied the effect of a low-dose pre-irradiation (0.03 Gy – 0.1 Gy) alone or followed by a 2.0 Gy challenging dose 4 h later on the survival of the HT29 cell line (human colorectal cancer cells) and on the GM637 cell line (human fibroblasts). Results 0.03 Gy given alone did not have a significant effect on both cell lines, the other low doses alone significantly reduced the cell survival. Applied 4 h before the 2.0 Gy fraction, 0.03 Gy led to a significant induced radioresistance in GM637 cells, but not in HT29 cells, and 0.05 Gy led to a significant hyperradiosensitivity in HT29 cells, but not in GM637 cells. Conclusion A pre-irradiation with 0.03 Gy can protect normal fibroblasts, but not colorectal cancer cells, from damage induced by an irradiation of 2.0 Gy and the application of 0.05 Gy prior to the 2.0 Gy fraction can enhance the cell killing of colorectal cancer cells while not additionally damaging normal fibroblasts. If these findings prove to be true in vivo as well this may optimize the balance between local tumour control and injury to normal tissue in modern radiotherapy.

Natural oak toxicity, a phenomenon sporadically reported in the United States, is due to consumption of any part of most oak trees ( Quercus spp.). Ruminants, mainly cattle, are disproportionately susceptible to oak toxicity. Toxicity is attributed to degradation of the oak plant hydrolysable tannins by rumen microbes and enzymes into absorbable low-molecular-weight metabolites, which are postulated to bind and damage endothelial cells by unknown mechanisms. The clinical manifestations of acute toxicosis are nonspecific or broadly suggestive of renal disease due to acute tubular injury. Here we document the clinical, gross, histopathologic, and novel ultrastructural features of natural acute oak nephrotoxicity in 3 beef calves on 2 farms in Colorado, USA. Gross postmortem findings included perirenal edema with renomegaly and hemorrhagic gastroenteritis. Histologically, renal tubular epithelial necrosis was severe, with hemorrhage and intratubular hyaline casts. Transmission electron microscopy revealed extensive involvement of proximal and distal convoluted tubules, with predominantly intact basement membranes, and glomerular and interstitial endothelial injury and necrosis. The ultrastructural details of toxic nephropathy and vasculopathy induced by oak metabolites in natural cases of bovine oak toxicosis have not been described previously, to our knowledge., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas. We sought to determine if LA meeting a size criterion (> 700 cells) in canine sarcomas met the criteria of TLS and the overall prevalence of LA of any size. RNA expression in large LAs versus aggregate-adjacent sarcoma tissue (AAS) was measured in laser capture microdissected tissue and compared to curl-derived RNA from aggregate-free sarcomas and lymph nodes. CD3, CD20, MUM-1 and PNAd expressions were measured using immunohistochemistry. CD20 and CD3 mRNA were more highly expressed in LA versus AAS (13.8 fold, p = 0.0003 and 2.3 fold, p = 0.043). This was supported by the IHC findings. The large LAs were also enriched in chemokine RNA expression characteristic of TLS (CXCR5 5.8 fold, p < 00001, CCL19 3.68 fold, p = 0.0209, CCL21 6.87 fold, p = 0.0209 and CXCL13 2.68 fold, p = 0.0924). Plasma cells and high endothelial venules were identified in LA containing tumours but not in control tissue. Large LAs were present in 12% of tumours, and LA of any size in 30%. We conclude that large LAs in canine STS are consistent with TLS., (© 2024 The Author(s). Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Objective: Ultraviolet light (UV) is a risk factor for the development of cutaneous neoplasia in many mammalian species. This study evaluated UV exposure as a risk factor of concern for the development of cutaneous neoplasia in equine species due to the significant UV exposure that may accrue over their lifetimes., Animals and Samples: Neoplastic biopsy specimens from 3272 horses that were submitted over a 10-year period to the Colorado State University Diagnostic Medicine Center and to the University of Saskatchewan Western College of Veterinary Medicine and Prairie Diagnostic Services were evaluated., Procedure: This retrospective study assessed the spatial relationships between altitude, latitude, longitude, and UV maximum value and the probability of UV-induced cutaneous neoplasia., Results: Cases from areas at high altitude proved to have a higher prevalence of UV-induced cutaneous neoplasia than those from areas at lower elevations. A multivariable regression analysis demonstrated that altitude was the only factor significantly and positively associated with the diagnosis of UV-induced neoplasia., Conclusion and Clinical Relevance: Evidence of cutaneous neoplasia in horses and environmental factors that influence the degree of UV exposure in a geographic location may aid in diagnosis and suggest preventive measures from UV overexposure., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

In humans, rounded atelectasis is defined as focal lung collapse that radiologically appears as a round mass-like lesion in the periphery of the lung. In general, human patients with rounded atelectasis have a history of pleural effusion and abnormal pleura and characteristic CT findings help to distinguish rounded atelectasis from pulmonary neoplasia without the need for invasive surgical biopsy. This retrospective multi-center case series describes rounded atelectasis in four cats and one dog. Chylothorax was seen in four patients and an eosinophilic and lymphoplasmacytic effusion was seen in one patient. All patients had solitary or multifocal subpleural pulmonary masses (26 masses total in 5 patients) with diffuse, multifocal, or focal visceral and parietal pleural thickening. All the masses but one were broad-based towards the visceral pleura. Masses were most common in the ventral or lateral aspect of the lungs. Indistinctness at the hilar aspect of the lesion was seen in all masses; a "comet tail" sign was seen in 14 of 26 masses. On postcontrast images, the lesions were homogeneously enhanced in 24 of 26 masses and heterogeneous in two of 26 masses. Other findings include ground glass opacities (n = 5), parenchymal bands (n = 4), mild to moderate lymphadenopathy (n = 4), and compensatory hyperinflation of the lung lobes not affected by atelectasis (n = 2). Histopathology of four cases revealed atelectasis with fixed pleural folds, chronic pleuritis, and mild to moderate pleural fibrosis. Awareness of rounded atelectasis in veterinary species will enable inclusion of a benign etiology into the differential diagnosis for subpleural masses in cases with pleural abnormalities., (© 2022 American College of Veterinary Radiology.)

River water temperatures are increasing globally, particularly in urban systems. In winter, wastewater treatment plant (WWTP) effluent inputs are of particular concern because they increase water temperatures from near freezing to ~7-15 °C. Recent laboratory studies suggest that warm overwinter temperatures impact the reproductive timing of some fishes. To evaluate winter water temperature's influence in the wild, we sampled Johnny Darter Etheostoma nigrum from three urban South Platte River tributaries in Colorado upstream and downstream of WWTP effluent discharge sites. Fish were collected weekly during the spring spawning season of 2021 and reproductive development was determined from histological analysis of the gonads. Winter water temperatures were approximately 5-10 °C greater ~300 m downstream of the WWTP effluent compared to upstream sites, and approximately 3°C warmer at sampling sites ~5000 m downstream of the effluent discharge. Females collected downstream of WWTP effluent experienced accelerated reproductive development compared to upstream by 1-2 weeks. Water quality, including total estrogenicity, and spring water temperatures did not appear to explain varying reproductive development. It appears that small increases in winter water temperature influence the reproductive timing in E. nigrum . Further investigations into how shifts in reproductive timing influence other population dynamics are warranted., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

A 15-y-old castrated male Maine Coon cat was evaluated for an ulcerated soft tissue mass on the right hindlimb that had been observed for 4 mo and had grown rapidly. A 3 × 3 cm soft, raised, amorphous, and ulcerated subcutaneous mass was observed on the lateral right metatarsus. In-house cytology via fine-needle aspiration was nondiagnostic. Incisional biopsy of the mass and further staging was declined, and amputation was elected. The amputated limb was submitted for histopathology, which revealed severe chronic nodular granulomatous dermatitis and multifocal granulomatous popliteal lymphadenitis with large numbers of intralesional fungal hyphae. Fungal PCR and sequencing on formalin-fixed, paraffin-embedded tissue identified Chalastospora gossypii . No adjunctive therapy was elected at the time. The patient has done well clinically 1 y post-operatively. C. gossypii is a rare microfungus found worldwide and is considered a minor pathogen of several plants. To our knowledge, infection by this fungus has not been reported previously in veterinary species. Features in our case are comparable to other mycotic infections. Nodular granulomatous mycotic dermatitis and cellulitis, although uncommon, should be a differential for soft tissue masses in veterinary species; C. gossypii is a novel isolate.

Recent studies have sought to optimize the fecal flotation procedure to improve the detection of helminth eggs in terrestrial domestic species. It is unclear, however, whether these efforts in optimization are applicable to parasite species of marine environments, and verification of veterinary diagnostic procedures is clinically important. It was hypothesized that the eggs belonging to the parasites of pinnipeds would have different specific gravities (SpG) than those belonging to terrestrial hosts. Fecal samples were collected from each of 25 stranded pinnipeds representing three species ( Zalophus californianus , Phoca vitulina , Mirounga angustirostris ), and modified double centrifugal flotations were performed on 1-g samples. Among the 22 California sea lions sampled, trematode, ascarid, and cestode eggs were detected in 17/22 (77%), 10/22 (45%), and 4/22 (18%) individuals, respectively. Sugar-gradient modified centrifugation flotations were then conducted on a subset of 10 samples from California sea lions to evaluate the distribution of eggs in fractions representing varying SpG. Higher numbers of ascarid eggs were found in fractions representing a lower SpG (1.00-1.15), whereas trematode eggs belonging to the genus Zalophotrema were found in significantly higher numbers in the fraction representing 1.25 ( P < 0.05). In conclusion, the SpG of trematode, ascarid, and cestode eggs from pinnipeds appears to be similar to those from terrestrial hosts, but numerous factors may affect their ability to be detected using traditional diagnostic approaches. Further exploration into the nature of the variability noted may lead to improved diagnostics in marine parasitology.

A 3-year-old, female spayed, Pug-cross dog presented with a 2-year history of nasal congestion and focal facial deformity. Complete surgical excision was elected following nasopharyngoscopy, endoscopic biopsy and subsequent computed tomography (CT) scan. Nasopharyngoscopy revealed a friable, polypoid mass of the right nasal conchae occluding the right nasal passage. Biopsies of the mass showed expansion of the submucosa by dense bundles of well-differentiated smooth muscle cells that were strongly immunoreactive for ⍺-smooth muscle actin and desmin. Post-procedure CT revealed a large space-occupying mass in the nasal cavity with minimal lysis of the adjacent calvarium. Histology following complete excision confirmed the results obtained on the initial endoscopic biopsy. The final diagnosis was smooth muscle hamartoma., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Antilipidemic drugs are routinely detected in effluent and surface waters downstream of wastewater treatment plants. A mixture exposure study with nine environmentally relevant antilipidemic drugs was performed with zebrafish (Danio rerio, ZF) and fathead minnow (Pimephales promelas, FHM) embryos to investigate the effects on sensitive embryologic stages. Zebrafish embryos were exposed nominally to: (a) 0.005 μM, (b) 0.05 μM, or (c) 0.5 μM of each drug in the mixture. Fathead minnow embryos were exposed nominally to: (a) 0.0005 μM, (b) 0.005 μM, or (c) 0.05 μM of each drug in the mixture. Several of the individual drug concentrations were within ranges previously found in the environment. Multiple metrics demonstrate that (a) exposure of ZF and FHM embryos to antilipidemic drugs during embryonic development results in lethal and sublethal effects, (b) ZF were more sensitive than FHM based on median lethal concentration (LC 50 0.02 μM and 0.05 μM, respectively), but FHM exhibited more severe abnormal sublethal morphologies than zebrafish embryos, and (c) the sublethal effects differed between the two species. This model identified novel specific endpoints for assessing sensitive, sublethal effects of pharmaceuticals in the environment. Abnormal myofiber birefringence pattern, hemorrhage, and heart rate are not included in standard evaluations but each of these metrics demonstrated a dose-dependent response in this study. Results demonstrate risk to fish development with potential repercussions at the population level, especially if environmental concentrations increase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A 12-y-old spayed female Schipperke dog with a previous diagnosis of inflammatory bowel disease was presented with a 2-mo history of severe colitis. The patient's condition progressed to hepatopathy, pneumonia, and dermatitis following management with prednisolone and dexamethasone sodium phosphate. Colonic biopsies identified severe necrosuppurative colitis with free and intracellular parasitic zoites. Postmortem examination confirmed extensive chronic-active ulcerative colitis, severe acute necrotizing hepatitis and splenitis, interstitial pneumonia, ulcerative dermatitis, myelitis (bone marrow), and mild meningoencephalitis with variable numbers of intracellular and extracellular protozoal zoites. PCR on samples of fresh colon was positive for Neospora caninum . Immunohistochemistry identified N. caninum tachyzoites in sections of colon, and a single tissue cyst in sections of brain. Administration of immunosuppressive drugs may have allowed systemic dissemination of Neospora from the intestinal tract.

Background: Plague caused by Yersinia pestis is a highly infectious and potentially fatal zoonotic disease that can be spread by wild and domestic animals. In endemic areas of the northern hemisphere plague typically cycles from March to October, when flea vectors are active. Clinical forms of disease include bubonic, septicemic, and pneumonic plague. All clinical forms are uncommon in dogs and the pneumonic form is exceedingly rare., Case Presentation: Two mixed breed young-adult male domestic dogs presented to Colorado veterinarians with fever and vague signs that progressed to hemoptysis within 24 h. Case 1 presented in June 2014, while Case 2 occurred in December 2017. Thoracic radiography of Case 1 and 2 revealed right dorsal and right accessory lobe consolidation, respectively. In Case 1 initial differential diagnoses included pulmonary contusion due to trauma or diphacinone toxicosis. Case 1 was euthanized ~ 24 h post presentation due to progressive dyspnea and hemoptysis. Plague was confirmed 9 days later, after the dog's owner was hospitalized with pneumonia. Case 2 was treated as foreign body/aspiration pneumonia and underwent lung lobectomy at a veterinary teaching hospital. Case 2 was euthanized after 5 days of hospitalization when bacterial culture of the excised lobe yielded Yersinia pestis. Both dogs had severe diffuse necrohemorrhagic and suppurative pneumonia at post mortem examination., Conclusions: Both dogs were misdiagnosed due to the atypical lobar presentation of an extremely rare form of plague in a species that infrequently succumbs to clinical disease. Presentation outside of the typical transmission period of plague was also a factor leading to delayed diagnosis in Case 2. Erroneous identification by automated bacterial identification systems was problematic in both cases. In endemic areas, plague should be ruled out early in febrile dogs with acute respiratory signs, hemoptysis, lobar or diffuse pathology, and potential for exposure, regardless of season. Seasonal and geographic distributions of plague may shift with climate change, so vigilance by primary care veterinarians is warranted. Timely submission of samples to a veterinary diagnostic laboratory could expedite accurate diagnosis and reduce potential for human and domestic animal exposure.

Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In December 2017, a dog that had pneumonic plague was brought to a veterinary teaching hospital in northern Colorado, USA. Several factors, including signalment, season, imaging, and laboratory findings, contributed to delayed diagnosis and resulted in potential exposure of >116 persons and 46 concurrently hospitalized animals to Yersinia pestis.

Background: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography., Materials and Methods: Relatively low-resolution WSI scans (0.50 μm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography., Results: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation ( R 2 = 0.9916) and by agreement with a pathologist ( R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) ( R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all ( n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286)., Conclusions: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy., Competing Interests: There are no conflicts of interest.

Background: Perianal (hepatoid) gland tumors are common in dogs, and the distinction between the benign and malignant forms is clinically important. Cytology of these tumors typically contains many large hepatoid cells and fewer small basal cells., Objectives: The objective of this study was to determine whether the proportion of the smaller basaloid reserve cells in cytologic samples from perianal tumors correlates with malignancy., Methods: Eighty-three cases of cytologically diagnosed perianal gland tumors with corresponding histopathologic sections were identified from two separate institutions and included six (7.2%) malignant tumors and 77 (92.8%) benign tumors. The proportion of basal cells from each sample was evaluated., Results: No difference between these groups was found, although the study was sufficiently powered to detect an approximately 1.5-fold change in basal cell proportion., Conclusions: This report found no evidence that the proportion of basal cells in canine perianal tumor cytology is an indication of the potential for malignancy. We, therefore, do not recommend citing this feature in cytologic reports or when communicating with clinicians., (© 2018 American Society for Veterinary Clinical Pathology.)

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic malignancy in dogs. Recently, MYC and BCL2 expression levels determined with immunohistochemistry (IHC) were found to be prognostic in people with DLBCL. We hypothesized that canine DLBCL can be similarly subdivided into prognostic subtypes based on expression of MYC and BCL2. Cases of canine DLBCL treated with CHOP chemotherapy were retrospectively collected and 43 dogs had available histologic tissue and complete clinical follow-up. Median values of percent immunoreactive versus immunonegative cells were used to determine positive or negative expression status. Completion of CHOP was significantly associated with a positive outcome. Compared with human patients, our canine DLBCL patients had high IHC expression of both MYC and BCL2, and relative expression levels of one or both markers were not associated with clinical outcome., (© 2016 John Wiley & Sons Ltd.)

Acanthomatous ameloblastoma is a common, locally invasive, nonmetastasizing tumor of the canine oral cavity. The long-term prognosis for canine acanthomatous ameloblastoma is good if complete excision can be achieved, usually by maxillectomy or mandibulectomy. A variant of acanthomatous ameloblastoma with atypical foci was noted in 5 dogs. There was no age, breed, or sex predisposition. Atypical cells were pleomorphic with a high mitotic rate. They were immunohistochemically negative for cytokeratin, vimentin, melan A, PNL2, CD3, Pax5, CD18, chromogranin A, and synaptophysin. Ultrastructurally, the atypical cells had modest amounts of electron-lucent cytoplasm, abundant rough endoplasmic reticulum, zonula adherens junctions, cleaved or irregular nuclei, and occasional cytoplasmic structures consistent with secretory granules or lysosomes. Complete excision was achieved by maxillectomy or mandibulectomy in 3 dogs; the lesion was incompletely excised in 2 dogs. No ancillary therapy was elected in any patient. No local recurrence or distant metastasis was reported in any case. One patient died of heart failure 20 mo following complete excision; all other patients were alive at last follow-up (average follow-up: 18.8 mo, range: 6-30 mo). The histogenesis of the atypical foci is unclear, but atypical foci within acanthomatous ameloblastoma do not appear to be associated with metastasis or with a poor prognosis relative to acanthomatous ameloblastoma with typical histologic morphology.

A diagnosis of neoplasia was noted in 125 of 357 donkeys (35%) in our review of medical records from 5 veterinary schools in the United States and Canada. Equine sarcoid was the most common tumor in our study, accounting for 72% of all tumors and 82% of cutaneous tumors. Soft-tissue sarcomas were the second most common skin tumors. All other types of neoplasia were rare. Important differences in the occurrence of neoplasia in donkeys compared to horses included the rarity or absence of squamous cell carcinoma in any organ system and gray horse melanoma. Lymphosarcoma, the most common malignant tumor in horses, appears to be very rare in donkeys. We report several tumors in donkeys including melanocytoma, peripheral nerve sheath tumor, and gastrointestinal stromal tumor. Our data demonstrate commonalities as well as differences in neoplastic diseases of donkeys and horses. Understanding differences in carcinogenesis among these 2 closely related species can inform researchers pursuing pathogenic mechanisms of equine disease and inform veterinary diagnosticians regarding tumor prevalence., (© 2016 The Author(s).)

A 7-month-old female domestic shorthair cat was diagnosed with chronic regenerative hemolytic anemia characterized by increased osmotic fragility of unknown etiology. At 13 months of age, the cat was evaluated for acute collapse. The cat was icteric with severe hyperbilirubinemia but no hematocrit changes. Severe obtundation and lateral recumbency progressed to tetraparesis and loss of proprioception in all 4 limbs, and a cerebellar or brainstem lesion was suspected. Postmortem examination revealed suppurative cholangiohepatitis and acute neuronal necrosis in the nuclei of the brainstem and cerebellum, consistent with bilirubin encephalopathy. This is the first known occurrence of cholangiohepatitis and bilirubin encephalopathy in an adult cat with chronic hemolytic anemia. Although rare, bilirubin encephalopathy should be considered a possible sequela to hyperbilirubinemia in adult patients. It remains unknown whether increased osmotic fragility was related to the cholangiohepatopathy., (© The Author(s) 2015.)

Infections by the digenetic trematode, Ribeiroia ondatrae, cause severe limb malformations in many North American amphibians. Ribeiroia ondatrae also infects fishes as second intermediate hosts, but less is known about the pathology and immune responses initiated in infected fish, even though reports of infected fish date back to early 1900s. To this end, we experimentally exposed juvenile Bluegills Lepomis macrochirus to three doses of R. ondatrae cercariae and monitored the pathology, parasite infection success, and humoral responses over 648 h. All exposed fish became infected with metacercariae, and the average infection load increased with exposure dose. Histologically, infection was associated with acute hemorrhages in the lateral line and local dermis at 36 h, followed by progressive granulomatous inflammation that led to the destruction of encysted metacercariae. Correspondingly, over the course of 648 h we observed an 85% decline in average infection load among hosts, reflecting the host's clearance of the parasite. Infection was not associated with changes in fish growth or survival, but did correlate with leukocytosis and neutrophilia in circulating host blood. Understanding the physiological responses of R. ondatrae in Bluegill will help to clarify the ecological effects of this parasite and provide a foundation for subsequent comparisons into its effects on behavior, individual health, and population dynamics of Bluegill.

Equine cutaneous lymphoma is an uncommon disease that can present with variable clinical signs, immunosuppression, and rapid systemic disease progression. Various subtypes of equine lymphoma have been described and classified according to a veterinary adaptation of the World Health Organization classification system, but little data is available regarding the association between lymphoma subtypes and epidemiological criteria and/or clinical outcome. The objective of the current study was to classify previously diagnosed cases of equine cutaneous lymphoma and correlate subtypes with clinical data to investigate epidemiological trends and prognostic implications. Results from the study confirm T-cell-rich, large B-cell lymphoma (TCRLBCL) as the predominant subtype in equine cutaneous lymphoma. Cutaneous T-cell lymphoma (CTCL) was the second most common phenotype in the current study. To the authors' knowledge, a description of the morphological and immunohistochemical features of CTCL in horses, an epitheliotropic neoplasm similar to the cutaneous epitheliotropic lymphoma variant (mycosis fungoides) observed in human beings and canids, has not been previously described. Less common lymphoma subtypes identified in the study include diffuse large B-cell lymphoma and anaplastic large T-cell lymphoma. Quarter Horses almost exclusively developed TCRLBCL, while the frequency of CTCL was increased significantly in Thoroughbreds compared with other breeds. TCRLBCLs more frequently presented as multiple masses while CTCLs were more often solitary nodules. When multiple regions of the body were affected, 100% of cases were TCRBCL. Finally, there was an observable trend for longer survival in cases of TCRLBCL without local recurrence, suggesting adequate surgical excision may be an effective treatment modality., (© 2014 The Author(s).)

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects.

A 3-year-old neutered male Australian Shepherd mix dog presented with metastatic soft tissue sarcoma arising from a primary tumor in the left lumbar fascia. Two separate metastases to the lungs were characterized by neoplasia within bronchiolar walls, which caused obstruction of the bronchiolar lumina and atelectasis of adjacent alveoli, a characteristic feature of endobronchial metastasis. Neoplastic cells of the primary lumbar neoplasm, metastatic pulmonary lesions, and additional widespread metastatic masses identified postmortem were similarly immunoreactive for vimentin, but non-immunoreactive for cytokeratin, cluster of differentiation 18, synaptophysin, chromogranin, and desmin. The present report describes a naturally occurring case of endobronchial metastasis in a dog.

Juvenile salmon sharks beach yearly along the California coast, primarily during late summer and early fall. Fresh, frozen, and formalin-fixed tissues from 19 stranded salmon sharks were collected for examination. Histopathology revealed meningitis or meningoencephalitis in 18 of 19 shark brains with intralesional bacteria observed in 6 of the affected brains. Bacterial culture of fresh or frozen brain, liver, and/or heart blood from 13 sharks yielded pure cultures characterized molecularly and/or biochemically as belonging to the genus Carnobacterium. The 16s ribosomal DNA sequence of 7 tissue isolates from 7 separate sharks was 99% homologous to C. maltaromaticum (GenBank FJ656722.1). Sequence of the large ribosomal DNA intergenic spacer region (ISR) was 97% homologous to C. maltaromaticum (AF374295.1). This is the first report of Carnobacterium infection in any shark species, and the authors posit that brain infection caused by Carnobacterium is a significant cause of morbidity and mortality in juvenile salmon sharks found stranded along the Pacific coast of California.

A retrospective study evaluated the clinical data and histologic features of non-neoplastic dermatologic lesions in skin biopsies from horses, donkeys, and mules submitted over a 10-year period to the Colorado State University Diagnostic Medicine Center and to the University of Saskatchewan Western College of Veterinary Medicine and Prairie Diagnostic Services. A total of 1793 non-neoplastic biopsies were available for analysis. Based on the histologic description and morphologic diagnosis, each submission was classified as inflammatory, or non-neoplastic/non-inflammatory. Results of this study demonstrate that non-neoplastic equine cutaneous biopsies are predominantly inflammatory, but histopathology alone may not be sufficient to definitively identify the underlying etiologies driving inflammatory lesions. Clinical correlation, including breed, age, and distribution of the cutaneous lesion, as well as ancillary tests such as bacterial or fungal culture are required to complete the clinical picture and arrive at the appropriate clinical diagnosis.

An approximately 4-year-old castrated male, Saanen cross goat presented to the Colorado State University Veterinary Teaching Hospital for evaluation and removal of a 22 cm × 22 cm, dark red, thickened, and crusted cutaneous lesion along the left ventrolateral thorax. An initial incisional biopsy performed approximately 8 weeks earlier was suspicious for cutaneous hemangiosarcoma. Surgical excision was deemed to be the most appropriate treatment option for this goat. A complete physical exam, complete blood count, and chemistry profile were performed and results were within normal limits. Thoracic radiographs and abdominal ultrasound were performed to rule out metastatic disease and comorbid conditions; no metastatic lesions or other abnormalities were observed. En bloc surgical excision of the affected skin was performed and the entire tissue was submitted for histopathology. A final diagnosis of cutaneous hemangiolipoma was reached upon extensive sectioning and histologic examination of the larger tissue specimen. The goat recovered well from surgery and has had no further complications up to 9 months postoperatively. To our knowledge, this is the first case report of a hemangiolipoma in a goat and surgical excision for such lesions appears to be a viable treatment method.

Objective: To identify epidemiological trends in cutaneous neoplasms affecting equids in central North America and compare them with previously reported trends., Design: Retrospective case series., Sample: 3,351 cutaneous biopsy specimens from 3,272 equids with a neoplastic diagnosis., Procedures: Diagnostic reports from 2 diagnostic laboratories (Colorado State University and Prairie Diagnostic Services Inc) were reviewed for frequency of specific lesions and epidemiological trends. Variables included in analyses (if known) were age, sex, breed, geographic location, date of diagnosis, location of neoplasm on the body, and presence or absence of ulceration., Results: Sarcoid, squamous cell carcinoma (SCC), and melanoma were the 3 most common tumors diagnosed. Tumors associated with UV radiation (SCC, SCC in situ, hemangioma, hemangiosarcoma) were 2.3 (95% confidence interval [CI], 1.8 to 3.0) times as common in biopsy specimens received by Colorado State University than in specimens received by Prairie Diagnostic Services Inc. Appaloosa horses and American Paint horses, respectively, were 7.2 (95% CI, 5.6 to 9.2) and 4.4 (95% CI, 3.6 to 5.4) times as likely as other breeds to have tumors associated with UV radiation. Thoroughbreds were predisposed to cutaneous lymphoma, whereas Arabians were more likely to have melanomas. Draft and pony breeds were 3.1 (95% CI, 1.9 to 5.1) times as likely as other breeds to have benign soft tissue tumors. Morgans and pony breeds more commonly had basal cell tumors. Tumors in the perianal region were significantly more likely to be SCC or melanoma while tumors on the limbs were more likely to be giant cell tumor of soft parts., Conclusions and Clinical Relevance: Signalment, anatomic location of the mass, and geographic location of the horse can be used to help equine practitioners formulate differential diagnoses for cutaneous masses. Further research is necessary to identify the biological basis for the development of many equine cutaneous neoplasms.

Lymphoma in the left femoral nerve of a 10-year-old English Cocker Spaniel caused complete paralysis of the affected limb. Neoplastic cells were immunopositive for CD79a and Pax5 and negative for CD3. Neoplastic cells were in multiple lymph nodes and one kidney but spared bone marrow. The clinical and histologic features in this case resemble those of the rare human condition of neurolymphomatosis.

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.

Herpesviruses are characterized by the ability to establish lifelong latent infections and to reactivate periodically, leading to recurrent disease. The herpes simplex virus type 1 (HSV-1) genome is maintained in a quiescent state in sensory neurons during latency, which is characterized by the absence of detectable viral protein synthesis. Cellular factors induced by stress may act directly on promoters within the latent viral genome to induce the transcription of viral genes and trigger reactivation. In order to identify which viral promoters are induced by stress and elucidate the cellular mechanism responsible for the induction, we generated a panel of HSV-1 promoter-luciferase constructs and measured their response to heat shock. Of the promoters tested, those of ICP0 and ICP22 were the most strongly upregulated after heat shock. Microarray analysis of lytically infected cells supported the upregulation of ICP0 and ICP22 promoters by heat shock. Mutagenic analysis of the ICP0 promoter identified two regions necessary for efficient heat-induced promoter activity, both containing predicted nuclear factor Y (NF-Y) sites, at bases -708 and -75 upstream of the transcriptional start site. While gel shift analysis confirmed NF-Y binding to both sites, only the site at -708 was important for efficient heat-induced activity. Reverse transcription-PCR analysis of selected viral transcripts in the presence of dominant-negative NF-Y confirmed the requirement for NF-Y in the induction of the ICP0 but not the ICP22 promoter by heat shock in lytically infected cells. These findings suggest that the immediate-early ICP0 gene may be among the first genes to be induced during the early events in HSV-1 reactivation, that NF-Y is important for this induction, and that other factors induce the ICP22 promoter.

ICP22, an immediate-early protein of herpes simplex virus type 1 (HSV-1), is required for viral replication in nonpermissive cell types and for expression of a class of late viral proteins which includes glycoprotein C. An understanding of the mechanism of ICP22 function has been complicated by the coexpression of the full-length protein with an in-frame, C-terminus-specific protein, U(S)1.5. In this report, we confirm that the U(S)1.5 protein is a bona fide translation product since it is detected during infections with three laboratory strains and two low-passage clinical isolates of HSV-1. To clarify the expression patterns of the ICP22 and U(S)1.5 proteins, we examined their synthesis from plasmids in transient expression assays. Because previous studies had identified two different U(S)1.5 translational start sites, we attempted to determine which is correct by studying the effects of a series of deletion, nonsense, and methionine substitutions on U(S)1.5 expression. First, amino acids 90 to 420 encoded by the ICP22 open reading frame (ORF) migrated at the mobility of U(S)1.5 in sodium dodecyl sulfate-polyacrylamide gels. Second, introduction of a stop codon downstream of M90 ablated expression of both ICP22 and U(S)1.5. Finally, mutation of M90 to alanine (M90A) allowed expression of full-length ICP22 while dramatically reducing expression of U(S)1.5. Levels of U(S)1.5 but not ICP22 protein expression were also reduced in cells infected with an M90A mutant virus. Thus, we conclude that expression of IC22 and that of U(S)1.5 can occur independently of each other and that U(S)1.5 translation initiates at M90 of the ICP22 ORF.

Of the five herpes simplex virus type 1 immediate early (IE) proteins, the least is known about the function of ICP22 during productive infection and latency. Research characterizing the physical and functional properties of the protein has been limited because ICP22 has proven to be difficult to express in transient assays. In addition, genetic analysis of ICP22 has been complicated by the fact that the C terminus of ICP22 is expressed as a discrete protein product. In order to characterize properties of mutant and wild-type ICP22, we developed a transient expression system. We found that ICP22 can be expressed at detectable levels when placed under the control of the cytomegalovirus IE promoter, confirming recent observations by K. A. Fraser and S. A. Rice (J. Virol. 81:5091-5101, 2007). We extended this analysis to show that ICP22 can also be expressed from its own promoter in the presence of other viral factors, either by coexpression with ICP0 or by infection with an ICP22 null virus. Notably, infection of cells transfected with an ICP22 expression vector yielded ICP22 protein that was modified in a manner similar to that of ICP22 protein detected in wild-type-infected cells. We go on to demonstrate that the failure of ICP22 protein to be expressed in transiently transfected cells was not due to inactivity of the ICP22 promoter, but rather to the ability of ICP22 to inhibit expression of reporter gene activity, including its own, in transient assays. Of special note was the observation that expression of ICP22 was sufficient to prevent transactivation of reporter genes by ICP0. Finally, transient expression of ICP22 was sufficient to complement replication of an ICP22 null virus, demonstrating that this system can be used to study functional properties of ICP22. Collectively, this transient expression system facilitates tests of the physical and functional properties of ICP22 and ICP22 mutants prior to introduction of mutant genes into the viral genome.

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0(-)) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0(-) virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0(-) virus.

Two in-frame, C-terminal isoforms of the herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), OBPC-1 and OBPC-2, and a unique C-terminal transcript, UL8.5, are specified by HSV-1 DNA. As the first isoform identified, OBPC-1 was initially assumed to be the product of the UL8.5 transcript. Recent evidence has demonstrated, however, that OBPC-1 is a cathepsin B-mediated cleavage product of OBP, suggesting that OBPC-2 is the product of the UL8.5 transcript. Because both OBPC-1 and -2 contain the majority of the OBP DNA binding domain, we hypothesized that both may be involved in regulating origin-dependent, OBP-mediated viral DNA replication. In this paper, we demonstrate that OBPC-2 is, indeed, the product of the UL8.5 transcript. The translational start site of OBPC-2 was mapped, and a virus (M571A) that does not express this protein efficiently was constructed. Using M571A, we have shown that OBPC-2 is able to bind origin DNA, even though it lacks seven N-terminal amino acid residues of the previously mapped OBP DNA binding domain, resulting in a revision of the limits of the OBP DNA binding domain. Consistent with their proposed roles in regulating viral DNA replication, OBPC-1 and -2 act together to down-regulate viral DNA replication in vitro. During functional studies in vivo, OBPC-2 was identified as a factor that increases mortality in the mouse ocular model of HSV-1 infection.

Although the seven viral proteins required for herpes simplex virus type 1 (HSV-1) DNA replication have been identified, the mechanism by which viral DNA synthesis is regulated is unclear. HSV-1 DNA replication is thought to occur in two stages: origin-dependent DNA replication (stage I) mediated by the origin binding protein (OBP), followed by origin- and OBP-independent DNA replication (stage II). The mechanism that facilitates the switch from stage I to stage II is unknown; however, it must involve the loss of OBP function or OBP itself from the replication initiation complex. Previous studies from this laboratory identified a transcript (UL8.5) and protein (OBPC) that are in frame with and comprise the C terminus of the gene specifying OBP. Because of its DNA binding ability, OBPC has been hypothesized to mediate the switch from stage I to stage II. Here, we identify a second protein (OBPC-2) that is also in frame with the C terminus of OBP but comprises a smaller portion of the protein. We demonstrate that the protein originally identified (OBPC-1) is a cathepsin B-mediated cleavage product of OBP, while OBPC-2 may be the product of the UL8.5 transcript. We further demonstrate that the cleavage of OBP to yield OBPC-1 is dependent upon viral DNA replication. These results suggest that cleavage may be a mechanism by which OBP levels and/or activity are regulated during infection.

A recombinant HSV-1 virus expressing EGFP from the HCMV major immediate early promoter (KOS-CMVGFP) was constructed to monitor viral replication and spread in vitro and in mice. KOS-CMVGFP replicated as efficiently as wild-type virus, strain KOS, in single cycle growth experiments in Vero cells indicating that the recombinant virus has no significant growth defects in vitro. Following ocular inoculation of mice, KOS-CMVGFP exhibited slight but statistically significant reductions in mouse tear film titers relative to wild-type virus. Progression of virus infection of the eyes, periocular tissue, and snout was readily followed by fluorescence microscopy. Insertion of the EGFP expression cassette into the KOS genome had no effect on the efficiency of establishment of latency as determined by quantitative competitive PCR of viral genomes in latently infected TG. KOS-CMVGFP reactivated with wild-type kinetics and efficiency by explant cocultivation, but exhibited a significant delay in the kinetics and a modest reduction in the efficiency of reactivation compared to KOS in the more sensitive TG cell culture model. Notably, EGFP expression preceded the detection of infectious virus by greater than 24 h in both ex vivo models and thus is a useful marker of the early stages in the induction of reactivation.

The host innate response to viral infection includes the production of interferons, which is dependent on the coordinated activity of multiple transcription factors. Herpes simplex virus 1 (HSV-1) has been shown to block efficient interferon expression by multiple mechanisms. We and others have demonstrated that HSV-1 can inhibit the transcription of genes promoted by interferon regulatory factor-3 (IRF-3), including interferon beta (IFN-beta), and that the immediate-early ICP0 protein is sufficient for this function. However, the exact mechanism by which ICP0 blocks IRF-3 activity has yet to be determined. Unlike some other viral proteins that inhibit IRF-3 activity, ICP0 does not appear to affect phosphorylation and dimerization of IRF-3. Here, we show that a portion of activated IRF-3 co-localizes with nuclear foci containing ICP0 at early times after virus infection. Co-localization to ICP0-containing foci is also seen with the IRF-3-binding partners and transcriptional co-activators, CBP and p300. In addition, using immunoprecipitation of infected cell lysates, we can immunoprecipitate a complex containing ICP0, IRF-3, and CBP. Thus we hypothesize that ICP0 recruits activated IRF-3 and CBP/p300 to nuclear structures, away from the host chromatin. This leads to the inactivation and accelerated degradation of IRF-3, resulting in reduced transcription of IFN-beta and an inhibition of the host response. Therefore, ICP0 provides an example of how viruses can block IFN-beta induction by sequestration of important transcription factors essential for the host response.

The immediate-early regulatory protein ICP22 is required for efficient replication of herpes simplex virus type 1 in some cell types (permissive) but not in others (restrictive). In mice infected via the ocular route, the pathogenesis of an ICP22- virus, 22/n199, was altered relative to that of wild-type virus. Specifically, tear film titers of 22/n199-infected mice were significantly reduced at 3 h postinfection relative to those of mice infected with wild-type virus. Further, 22/n199 virus titers were below the level of detection in trigeminal ganglia (TG) during the first 9 days postinfection. On day 30 postinfection, TG from 22/n199-infected mice contained reduced viral genome loads and exhibited reduced expression of latency-associated transcripts and reduced reactivation efficiency relative to TG from wild-type virus-infected mice. Notably, the first detectable alteration in the pathogenesis of 22/n199 in these tests occurred in the eye prior to the onset of nascent virus production. Thus, ICP22- virions appeared to be degraded, cleared, or adsorbed more rapidly than wild-type virions, implying potential differences in the composition of the two virion types. Analysis of the protein composition of purified extracellular virions indicated that ICP22 is not a virion component and that 22/n199 virions sediment at a reduced density relative to wild-type virions. Although similar to wild-type virions morphologically, 22/n199 virions contain reduced amounts of two gamma2 late proteins, US11 and gC, and increased amounts of two immediate-early proteins, ICP0 and ICP4, as well as protein species not detected in wild-type virions. Although ICP22- viruses replicate to near-wild-type levels in permissive cells, the virions produced in these cells are biochemically and physically different from wild-type virions. These virion-specific differences in ICP22- viruses add a new level of complexity to the functional analysis of this immediate-early viral regulatory protein.

This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G1/early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0- viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0- viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0- virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0- viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.

Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.

In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-I(LR) and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-I(LR) in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-I(LR) exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-I(LR) reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-I(LR)-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

Technical challenges associated with mutagenesis of the large oriL palindrome have hindered comparisons of the functional roles of the herpes simplex virus type 1 (HSV-1) origins of DNA replication, oriL and oriS, in viral replication and pathogenesis. To address this problem, we have developed a novel PCR-based strategy to introduce site-specific mutations into oriL and other large palindromes. Using this strategy, we generated three plasmids containing mutant forms of oriL, i.e., pDoriL-I(L), pDoriL-I(R), and pDoriL-I(LR), containing point mutations in the left, right, and both copies, respectively, of the origin binding protein (OBP) binding site (site I) which eliminate OBP binding. In in vitro DNA replication assays, plasmids with mutations in only one arm of the palindrome supported origin-dependent DNA replication, whereas plasmids with symmetrical mutations in both arms of the palindrome were replication incompetent. An analysis of the cloned mutant plasmids used in replication assays revealed that a fraction of each plasmid mutated in only one arm of the palindrome had lost the site I mutation. In contrast, plasmids containing symmetrical mutations in both copies of site I retained both mutations. These observations demonstrate that the single site I mutations in pDoriL-I(L) and pDoriL-I(R) are unstable upon propagation in bacteria and suggest that functional forms of both the left and right copies of site I are required to initiate DNA replication at oriL. To examine the role of oriL and oriS site I in virus replication, we introduced the two site I mutations in pDoriL-I(LR) into HSV-1 DNA to yield the mutant virus DoriL-I(LR) and the same point mutations into the single site I sequence present in both copies of oriS to yield the mutant virus DoriS-I. In Vero cells and primary rat embryonic cortical neurons (PRN) infected with either mutant virus, viral DNA synthesis and viral replication were efficient, confirming that the two origins can substitute functionally for one another in vitro. Measurement of the levels of oriL and oriS flanking gene transcripts revealed a modest alteration in the kinetics of ICP8 transcript accumulation in DoriL-I(LR)-infected PRN, but not in Vero cells, implicating a cell-type-specific role for oriL in regulating ICP8 transcription.

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0(-) mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in 90% of 7134- and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK(cs), a known target of ICP0-directed degradation. The results of the PML and DNA-PK(cs) immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.

In the absence of effective vaccines to control herpesvirus infections, nucleosidic antiviral drugs have been the mainstay of clinical treatment since their development in the late 1970s. However, given the drawbacks of these drugs, including the increasing emergence of drug-resistant clinical isolates, new strategies for treating herpesvirus infections are warranted. A range of promising new drugs with novel molecular targets has been developed, but will they cure latent infections?

Herpes simplex virus (HSV) establishes productive (lytic) infections in nonneuronal cells and nonproductive (latent) infections in neurons. It has been proposed that HSV establishes latency because quiescent neurons lack cellular factors required for productive infection. It has been further proposed that these putative factors are induced following neuronal stress, as a requirement for HSV reactivation. To date, the identity of these putative cellular factors remains unknown. We have demonstrated that cyclin-dependent kinase (cdk) 1, 2, or 7 is required for HSV replication in nonneuronal cells. Interestingly, cdks 1 and 2 are not expressed in quiescent neurons but can be induced in stressed neurons. Thus, cdks may be among the cellular proteins required for HSV reactivation whose neuronal expression is differentially regulated during stress. Herein, we determined that neuronal expression of nuclear cdk2, cdk4, and cyclins E and D2 (which activate cdks 2 and 4, respectively) was induced following explant cultivation, a stressful stimulus that induces HSV reactivation. In contrast, neuronal expression of cdk7 and cytoplasmic cdk4 decreased during explant cultivation, whereas cdk3 was detected in the same small percentage of neurons before and after explant cultivation and cdks 1, 5, and 6 were not detected in neuronal cell bodies. HSV-1 reactivated specifically in neurons expressing nuclear cdk2 and cdk4, and an inhibitor specific for cdk2 inhibited HSV-1 reactivation. We conclude that neuronal levels of cdk2 are among the factors that determine the outcome of HSV infections of neurons.