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Ischemic myelomalacia secondary to fibrocartilaginous emboli (FCE) is an idiopathic disease in humans and animals. On the other hand, congenital spinal cord malformations result from neural tube defects in fetal development (ie, spinal dysraphism), with structural anomalies referred to collectively as myelodysplasia. Spinal dysraphisms are frequently accompanied by skin and vertebral abnormalities because of the embryogenic relationship. In this observational case study, we report the pathologic findings of 13, 18- to 24-weeks-old pigs from a large conventional operation that presented with acute paraparesis. Ischemic myelomalacia secondary to FCE was observed in 5 of 13 examined pigs. Congenital spinal cord malformations located between the caudal thoracic and sacral spinal cord were identified in 7 pigs, with structural abnormalities that ranged from diplomyelia/split cord malformation to segmental spinal dysgenesis (myelodysplasia) to caudal agenesis. Concurrent myelomalacia and congenital spinal cord malformations in the same or different sites were noted in 2 pigs. No spinal lesion was observed in 3 pigs. Although gross vertebral abnormalities were not observed herein, intervertebral instability due to minor defects in the articular facets, as well as other unidentified factors, is suspected to contribute high incidence of FCE. It is likely that these congenital malformations were previously underdiagnosed or are possibly new conditions associated with continuous inbreeding and genetic improvement in the modern swine industry.

SARS-CoV-2 (CoV2) infected, asymptomatic individuals are an important contributor to COVID transmission. CoV2-specific immunoglobulin (Ig)—as generated by the immune system following infection or vaccination—has helped limit CoV2 transmission from asymptomatic individuals to susceptible populations (e.g. elderly). Here, we describe the relationships between COVID incidence and CoV2 lineage, viral load, saliva Ig levels (CoV2-specific IgM, IgA and IgG) and inhibitory capacity in asymptomatic individuals between Jan 2021 and May 2022. These data were generated as part of a large university COVID monitoring program and demonstrate that COVID incidence among asymptomatic individuals occurred in waves which mirrored those in surrounding regions, with saliva CoV2 viral loads becoming progressively higher in our community until vaccine mandates were established. Among the unvaccinated, infection with each CoV2 lineage (pre-Omicron) resulted in saliva Spike-specific IgM, IgA and IgG responses, the latter increasing significantly post-infection and being more pronounced than N-specific IgG responses. Vaccination resulted in significantly higher Spike-specific IgG levels compared to unvaccinated infected individuals, and uninfected vaccinees’ saliva was more capable of inhibiting Spike function. Vaccinees with breakthrough Delta infections had Spike-specific IgG levels comparable to those of uninfected vaccinees; however, their ability to inhibit Spike binding was diminished. These data demonstrate that COVID vaccines achieved hoped-for effects in our community, including the generation of mucosal antibodies that inhibit Spike and lower community viral loads, and suggest breakthrough Delta infections were not due to an absence of vaccine-elicited Ig, but instead limited Spike binding activity in the face of high community viral loads.

rQNestin34.5v.2 is an oncolytic herpes simplex virus 1 (oHSV) that retains expression of the neurovirulent ICP34.5 gene under glioma-selective transcriptional regulation. To prepare an investigational new drug (IND) application, we performed toxicology and efficacy studies of rQNestin34.5v.2 in mice in the presence or absence of the immunomodulating drug cyclophosphamide (CPA). ICP34.5 allows HSV1 to survive interferon and improves viral replication by dephosphorylation of the eIF-2α translation factor. rQNestin34.5v.2 dephosphorylated eIF-2α in human glioma cells, but not in human normal cells, resulting in significantly higher cytotoxicity and viral replication in the former compared to the latter. In vivo toxicity of rQNestin34.5v.2 was compared with that of wild-type F strain in immunocompetent BALB/c mice and athymic mice by multiple routes of administration in the presence or absence of CPA. A likely no observed adverse effect level (NOAEL) dose for intracranial rQNestin34.5v.2 was estimated, justifying a phase 1 clinical trial in recurrent glioma patients (ClinicalTrials.gov: NCT03152318), after successful submission of an IND., Graphical Abstract

Wastewater-based epidemiology is an effective tool for monitoring infectious disease spread or illicit drug use within communities. At the Ohio State University, we conducted a SARS-CoV-2 wastewater surveillance program in the 2020-2021 academic year and compared results with the university-required weekly COVID-19 saliva testing to monitor COVID-19 infection prevalence in the on-campus residential communities. The objectives of the study were to rapidly track trends in the wastewater SARS-CoV-2 gene concentrations, analyze the relationship between case numbers and wastewater signals when adjusted using human fecal viral indicator concentrations (PMMoV, crAssphage) in wastewater, and investigate the relationship of the SARS-CoV-2 gene concentrations with wastewater parameters. SARS-CoV-2 nucleocapsid and envelope (N1, N2, and E) gene concentrations, determined with reverse transcription droplet digital PCR, were used to track SARS-CoV-2 viral loads in dormitory wastewater once a week at 6 sampling sites across the campus during the fall semester in 2020. During the following spring semester, research was focused on SARS-CoV2 N2 gene concentrations at 5 sites sampled twice a week. Spearman correlations both with and without adjusting using human fecal viral indicators showed a significant correlation (p 0.05) between human COVID-19 positive case counts and wastewater SARS-CoV-2 gene concentrations. Spearman correlations showed significant relationships between N1 gene concentrations and both TSS and turbidity, and between E gene concentrations and both pH and turbidity. These results suggest that wastewater signal increases with the census of infected individuals, in which the majority are asymptomatic, with a statistically significant (p-value0.05) temporal correlation. The study design can be utilized as a platform for rapid trend tracking of SARS-CoV-2 variants and other diseases circulating in various communities.

The major inducible 70 kDa heat shock protein (hsp70) is induced by and supports intracellular replication of viruses belonging to diverse families. Paradoxically, this virus-hsp70 interaction is protective in mouse models of viral neurovirulence, enhancing T cell mediated immune clearance in an interferon β (IFN-β)-dependent manner. Protection reflects early release of hsp70 from viable infected neurons and induction of strong innate immune responses in uninfected brain macrophages, including the induction of IFN-β through Toll-like receptor 4. Potency of the response is inherent in the fact that hsp70 is released at a time when pathogen-associated molecular patterns (PAMPs) are in low abundance, and that the innate response is driven by uninfected cells, free from viral interference. Release of hsp70 from viable cells is primarily exosomal, and infection enhances total exosome release and hsp70 content on the surface of exosomes. Exosome content of hsp70 reflects levels of hsp70 in the infected cell. Findings have broad virological relevance and support a protective role for fever, a potent stimulus for hsp70 induction. While protective in the context of microbial infection, recent findings support potential untoward effects of inappropriate extracellular hsp70 release in non-infectious neuroinflammatory conditions.

A 4 yr old border collie presenting for mydriasis and decreased mentation and a 7 yr old Boston terrier presenting for obtundation, head tilt, and paraparesis were both evaluated using MRI. Findings in both included mass lesions of the thalamus and brainstem that were hypo- to isointense on T1-weighted images and hyperintense on T2-weighted images with regions of hypointensity, and robust contrast enhancement and displacement of adjacent structures. Postmortem histopathology findings, tumor location, and a mixed pattern of epithelial cell differentiation were consistent with germ cell tumor in both cases. Germ cell tumor of the suprasellar region is an infrequently reported neoplasm of dogs and imaging findings in this species have not been well described in the prior literature.

A 28-year-old, female umbrella cockatoo (Cacatua alba) was evaluated because of lethargy, anorexia, regurgitation, and coelomic swelling of 6 month's duration, which corresponded to cessation of egg laying. Radiographs and ultrasound examination demonstrated extensive deposits of coelomic fat and an enlarged oviduct. Exploratory celiotomy demonstrated copious amounts of firm, nodular fat completely surrounding the gastrointestinal tract, resulting in extensive chronic adhesions between intestinal loops. Free yolk was present in the cranial left coelom, yolk coelomitis was diagnosed, and a salpingohysterectomy was performed. Two days after surgery, the bird stopped passing feces and began regurgitating after eating, and a colonic obstruction was demonstrated via contrast radiography. Euthanasia was elected, and necropsy revealed an obstruction of the distal colon caused by extraluminal compression by adhesions of firm, nodular fat. Histologic examination demonstrated extensive fat necrosis with granulomatous inflammation, characterized by cords of necrotic fat surrounded by multinucleated giant cells and epithelioid macrophages with scattered lymphocytes and plasma cells and rare heterophils. The clinical signs, gross lesions, and histologic lesions are characteristic of massive fat necrosis (lipogranulomatosis) in ruminants and mesenteric panniculitis in humans and companion mammals. This is the first report, to our knowledge, of this disease in psittacine birds.

Male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice are frequently used in prostate cancer research because their prostates consistently develop a series of preneoplastic and neoplastic lesions. Disease progression in TRAMP mouse prostates culminates in metastatic, poorly differentiated carcinomas with neuroendocrine features. The androgen dependence of the rat probasin promoter largely limits transgene expression to the prostatic epithelium. However, extra-prostatic transgene-positive lesions have been described in TRAMP mice, including renal tubuloacinar carcinomas, neuroendocrine carcinomas of the urethra, and phyllodes-like tumors of the seminal vesicle. Here, we describe the histologic and immunohistochemical features of 2 novel extra-prostatic lesions in TRAMP mice: primary anaplastic tumors of uncertain cell origin in the midbrain and poorly differentiated adenocarcinomas of the submandibular salivary gland. These newly characterized tumors apparently result from transgene expression in extra-prostatic locations rather than representing metastatic prostate neoplasms because lesions were identified in both male and female mice and in male TRAMP mice without histologically apparent prostate tumors. In this article, we also calculate the incidences of the urethral carcinomas and renal tubuloacinar carcinomas, further elucidate the biological behavior of the urethral carcinomas, and demonstrate the critical importance of complete necropsies even when evaluating presumably well characterized phenotypes in genetically engineered mice.

Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 10 6 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 10 6 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo , thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies.

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro , hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.

Two Boxer dogs developed progressive ataxia in association with a neoplastic infiltration of the spinal leptomeninges. In the first dog, the leptomeningeal neoplasm encompassed the entire cord and the ventral aspect of the brainstem and extended bilaterally into the piriform lobes. In the second, the neoplasm surrounded the C1–C3 segments of the spinal cord and the brainstem without involvement of the brain or spinal cord parenchyma. In both dogs, the neoplastic cells had variably distinct cell borders, clear to eosinophilic cytoplasm, and a round to ovoid hyperchromatic nucleus. Neoplastic cells were immunopositive for Olig2 and doublecortin in both dogs and for vimentin in one dog but were immunonegative for glial fibrillary acidic protein, S-100, CD34, E-cadherin, cytokeratin, CD3, and CD20. The morphological and immunohistochemical features of the neoplastic cells were consistent with an oligodendrocyte lineage. This hitherto poorly recognized neoplasm in dogs is analogous to human leptomeningeal oligodendrogliomatosis.

The intrinsically disordered C-terminal domain (NTAIL) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The NTAIL region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489–506. In the previous studies published in this journal, we obtained experimental evidence supporting a KD for the NTAIL–XD binding reaction in the nM range and also showed that an additional NTAIL region (Box3, aa 517–525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (KD in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in NTAIL–XD binding. Since our previous studies relied on NTAIL-truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an NTAIL devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482–525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these NTAIL forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point.

The features of gliomatosis cerebri involving the brainstem and cerebellum in a 3-year-old dog are described. In magnetic resonance (MR) images, there was diffuse loss of the cerebellar folia and cerebellar gray and white matter contrast. Multiple illdefined T2-hyperintensities were present in the cerebellar parenchyma. A poorly defined, T2-hyperintense mass effect was present ventral to the pons and rostral medulla. No contrast enhancement was noted. Cerebrospinal fluid (CSF) was normal. Postmortem examination was consistent with gliomatosis cerebri, based on compatible histopathology and immunohistochemical findings. Although rare, gliomatosis cerebri should be included as a differential for diffuse infiltrative central nervous system (CNS) lesions.

Dog 1 was a 3-week-old male Border Terrier puppy referred to the Neurology and Neurosurgery Service of the Veterinary Medical Center, The Ohio State University, for ataxia and tremors. The signs were first noticed 5 days before presentation when the puppy first attempted to walk on its own. The owners reported that the tremors affected mainly the hindquarters. No improvement or worsening of the tremors had been noted over this period. An injectable corticosteroid (unknown type and dose) had been administered 4 days before referral, with no changes in the clinical signs. The tremors were reported to disappear when the puppy was sleeping. There were 3 other puppies from the same litter, which were reported to be normal, as were the parents. On presentation, the puppy showed severe generalized coarse body tremors (with low frequency and high amplitude), most severe in the hindquarters, which showed a characteristic swinging side-to-side or “rocking horse” movement (as illustrated in the supplementary Video S1). The tremors also involved the head and thoracic limbs, but to a lesser degree, and disappeared when the dog was asleep or at rest. Cerebellar ataxia was noted when the dog was trying to walk. The neurological examination was difficult because of the severity of the tremors, but it showed an absent menace bilaterally (considered normal for a 3-week-old puppy), delayed proprioceptive positioning, and hopping that was slightly hypermetric on all limbs. The remaining cranial nerves, spinal reflexes, cutaneous trunci reflex, and vertebral column palpation were unremarkable. Physical examination was within normal limits. Because the characteristics of the tremors, which were coarse and affected mainly the hindquarters, a diffuse or multifocal CNS disorder such as a congenital disorder of myelination was considered the main differential diagnosis. A cerebellar lesion (infectious or inflammatory cerebellar disease, cerebellar hypoplasia, neonatal cerebellar ataxia) also was considered possible, although less likely, based on the type of tremors noted. A complete blood count (CBC) and biochemical profile showed changes considered normal for a puppy. The owners, who also were the breeders, chose humane euthanasia. A complete necropsy was performed. A cerebrospinal fluid (CSF) sample was collected from the cerebellomedullary cistern immediately after euthanasia. The fluid was colorless and clear, with a total protein of 55 mg/ dL (reference range

The role of extracellular 70 kDa heat shock protein 70 (ehsp70) in central nervous system inflammation is vastly understudied, despite evidence supporting the ability to drive a pro-inflammatory state. We investigated the presence of ehsp70 in cerebrospinal fluid (CSF) and serum of dogs with Steroid Responsive Meningitis-Arteritis (SRMA), with the hypothesis that an ehsp70 response would occur, and might play a role in the pathogenesis of this disease. Samples from 30 dogs acutely affected with SRMA, and 30 dogs treated with corticosteroids and currently in clinical remission from SRMA were compared with normal dogs. Serum and CSF concentrations of ehsp70 were quantified using an enzyme-linked immunosorbent assay. An ehsp70 response occurred in the CSF of dogs with SRMA and this response was attenuated by corticosteroid treatment. There was no correlation between serum and CSF concentrations of ehsp70, supporting local production and release of ehsp70 and not simply leakage from serum. Dogs with SRMA thus represent a powerful spontaneous model by which to study the role of ehsp70 in CNS inflammation.

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.

Measles virus is highly neuroinvasive, yet host immune responses are highly effective at limiting neurovirulence in humans. We know that neurons are an important target of infection and that both IFN-γ and -β expression are observed in the measles virus-infected human brain. Rodent models can be used to understand how this response is orchestrated. Constitutive expression of the major inducible 70-kDa heat-shock protein is a feature of primate tissues that is lacking in mice. This article examines the importance of addressing this difference when modeling outcomes of brain infection in mice, particularly in terms of understanding how infected neurons may activate uninfected brain macrophages to produce IFN-β and support T-cell production of IFN-γ, a mediator of noncytolytic viral clearance. New and historical data suggest that the virus heat-shock protein 70 relationship is key to a protective host immune response and has potential broad relevance.

An immunocompetent animal disease model based on infection with Ehrlichia chaffeensis would facilitate research toward understanding mechanisms responsible for the broad range of clinical signs associated with human monocytic ehrlichiosis (HME). The adaptability of this model for the experimental feeding of tick species and stages and for testing therapies comparable to those for human diseases are additional advantages of large animal models. Herein, we summarize pathology reports for calves that developed fatal disease after experimental inoculation with E. chaffeensis. Elevated liver enzyme levels and lung pathology among these calves corroborated earlier reports of severe HME. Thus, an experimental disease model based on infection of outbred immunocompetent hosts with E. chaffeensis could be within our grasp for the first time.

Human monocytic ehrlichiosis (HME) is a zoonotic emerging tick-borne disease with clinical signs that range from mild symptoms to multiple organ failure and death.Ehrlichia chaffeensis, the aetiologic agent of HME, is reported to infect a divergent range of mammals. Although cattle are common hosts of the primary vector of this pathogen, the susceptibility of this host toE. chaffeensishas not been reported to date. This study was undertaken to determine if cattle could provide a useful infection model ofE. chaffeensis. Dairy calves were injected with DH82 cells infected with the Arkansas, St Vincent or 91HE17 strain ofE. chaffeensis, and monitored for signs of clinical ehrlichiosis and for infection of peripheral blood and ticks by PCR assay. Splenectomized and spleen-intact calves were injected with cryopreserved stabilates ofE. chaffeensis-infected DH82 cells for the first experiment. Mild clinical signs were occasionally observed among these calves, and only two blood samples were PCR-positive, while several ticks fed on each calf tested PCR-positive. The second experiment involved injection of normal calves with active cultures of the sameE. chaffeensisstrains. Interestingly, three of six calves inoculated with active cultures became recumbent and died or had to be euthanized. All of the surviving calves in this experiment tested PCR-positive on multiple dates, but fewer ticks fed on these calves were PCR-positive. These results suggest that a bovine disease model could facilitate the understanding of factors that affect the severity of HME.

Recent reports project a deficiency of veterinary pathologists, indicating a need to train highly qualified veterinary pathologists, particularly in academic veterinary medicine. The need to provide high-quality research training for veterinary pathologists has been recognized by the veterinary pathology training program of the Ohio State University (OSU) since its inception. The OSU program incorporates elements of both residency training and graduate education into a unified program. This review illustrates the components and structure of the training program and reflects on future challenges in training veterinary pathologists. Key elements of the OSU program include an experienced faculty, dedicated staff, and high-quality students who have a sense of common mission. The program is supported through cultural and infrastructure support. Financial compensation, limited research funding, and attractive work environments, including work–life balance, will undoubtedly continue to be forces in the marketplace for veterinary pathologists. To remain competitive and to expand the ability to train veterinary pathologists with research skills, programs must support strong faculty members, provide appropriate infrastructure support, and seek active partnerships with private industry to expand program opportunities. Shortages of trained faculty may be partially resolved by regional cooperation to share faculty expertise or through the use of communications technology to bridge distances between programs. To foster continued interest in academic careers, training programs will need to continue to evolve and respond to trainees’ needs while maintaining strong allegiances to high-quality pathology training. Work–life balance, collegial environments that foster a culture of respect for veterinary pathology, and continued efforts to reach out to veterinary students to provide opportunities to learn about the diverse careers offered in veterinary pathology will pay long-term dividends for the future of the profession.

Highlights • No cross-neutralization was detected between PEDV and PdCV. • A two-way cross-reactivity was detected between PEDV and PdCV. • Conserved epitope(s) in viral N proteins may contribute to antigenic cross-reactivity. • Prevention of PEDV and PdCV will require the development of separate virus-specific vaccine products., Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PdCV) cause indistinguishable clinical signs and pathological changes in swine. Here we investigated the antigenic relationship between PEDV and PdCV. We provide the first evidence that conserved epitope(s) on the respective viral nucleocapsid proteins cross-react with each other although virus neutralization cross-reactivity was not observed. As a practical matter, prevention of these two very similar diseases of swine will require the development of separate virus-specific vaccine products.

Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.

A neuronal storage disease affecting 5 captive Humboldt penguins is described. One bird died after 3 days of lethargy and anorexia. The 4 remaining birds died after a slowly progressing course of disease with signs that included lethargy, weakness, and neurologic dysfunction. Neurologic signs included dysphagia and ataxia. Gross lesions in the first animal to die consisted of hepatosplenomegaly indicative of avian malaria, which was confirmed histologically. The 4 remaining animals were mildly to moderately emaciated. Moderate to marked vacuolation of the neuronal perikarya was observed in Purkinje cells, neurons of the brainstem nuclei, and motorneurons of the spinal cord in all birds. By electron microscopy the vacuoles represented multilayered concentric lamellar structures. These findings were indicative of sphingolipidosis. All animals had been prophylactically treated for avian malaria, aspergillosis, and possible bacterial infections with chloroquine, itraconazole, and enrofloxacin. circumstantial evidence implicates chloroquine therapy as the possible cause of the storage disease.

The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.

The major inducible 70-kDa heat shock protein (hsp72) binds measles virus (MV) nucleocapsids and increases MV gene expression. The cytoplasmic tail of the MV N protein (N TAIL ) contains three hydrophobic domains (Box-1–3) that are potential targets of hsp72 interaction. Low affinity binding to Box-3 is correlated to hsp72-dependent stimulation of MV minireplicon reporter gene expression whereas interactions between hsp72 and Box-1 and/or -2 have not been documented. The present work showed that virus deficient in Box-3/hsp72 interaction retains the ability to form nucleocapsid/hsp72 complexes, identifying Box-2 but not Box-1 as a mediator of high affinity hsp72 binding. Box-2 is the binding site for the viral P protein X domain (XD), where P tethers the viral polymerase to nucleocapsid in support of transcription and genome replication, and competitive inhibition of XD binding to N TAIL by hsp72 was shown. Recognition of a common binding site by P and hsp72 represents a potential mechanism for host cell modulation of viral gene expression.

Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. All ponies remained asymptomatic until the termination of the experiment, day 79 postinoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just before inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples, except that from pony no. 4 on day 28; this pony had received 1 X 10(7) sporocysts. Using indirect immunofluorescence antibody tests (IFATs), 7 serum samples were found to be positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies, with heaviest infections in the tongue. All sarcocysts examined histologically appeared to contain only microcytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites, whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as being S. neurona infected using IFAT, and further research is needed on the serologic diagnosis of S. neurona infections.

Nervous tissue subjected to hyperthermic pre-conditioning is resistance to numerous insults although in vitro, the same treatment can increase gene expression and cytopathic effect of neurotropic paramyxoviruses, including measles virus (MV). The present work determined whether the in vivo relationship between hyperthermic pre-conditioning and MV infection would be to increase neuropathogenicity or, conversely, to promote clearance. Balb/c mice 36 h of age were exposed to a 41 degrees C hyperthermic treatment for 30 min. Intracranial inoculation of mice with Edmonston MV was performed at 6 h following the heat treatment, a time point exhibiting elevated levels of the major inducible 70-kDa heat shock protein in brain, a hallmark of pre-conditioning. Forty-seven percent of the non-heated animals supported a persistent cytopathic infection at 21-day post infection (PI) based upon the quantitative detection of viral RNA in brain using real time RT-PCR. Cytopathic effect in the infected brains was proportionate to viral RNA burden. In contrast, infected stress conditioned mice lacked significant cytopathic effect and clearance was demonstrated in 95% of the animals. Analysis of shorter post-infection intervals showed that levels of viral RNA in brain were equivalent between stress conditioned and non-conditioned mice at 2 and 7 days PI, with clearance being first evident in both groups at 14 days. The temporal onset and progression of clearance was correlated to splenocyte blastogenic responsiveness to purified MV antigen but not the production of MV-specific antibody. Collectively, these results support the hypothesis that stress conditioning enhances the efficacy of cell-mediated immune responses known to mediate viral clearance from brain.

The 72 kDa heat shock protein (HSP72) is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV) nucleocapsid protein (N), a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins.

The 8th American College of Veterinary Pathologists (ACVP) symposium, held at the 2012 Experimental Biology meeting in San Diego, California (http://experimentalbiology.org/ content/AboutEP.aspx), was organized by the ACVP Intersociety Experimental Pathology Committee in conjunction with the American Society for Investigative Pathology (ASIP; http://www.asip.org/). The symposium facilitates interactions and cross-disciplinary collaborations between veterinary and physician pathologists, scientists, and basic researchers. The American Society for Nutrition cosponsored this year’s symposium. The symposium topic was evolutionary aspects of animal models. An evolutionary perspective is increasingly being embraced as essential for a comprehensive understanding of disease. The symposium highlighted the importance of an evolutionary perspective in the development and use of animal models of human disease. Speakers emphasized how evolution has differentially molded cardiovascular physiology, response to infectious agents, and aging in both man and animals. In addition, the evolutionary trade-offs between phenotypic selection and disease, which has provided many spontaneous animal models of human diseases, were discussed. Dr Robert Hamlin, The Ohio State University, opened with the challenges for studying cardiovascular disease in a talk entitled: ‘‘Animals as models of human cardiovascular disease: the search to overcome outdated evolutionary homeostatic mechanisms.’’ He addressed the fact that animals used to model human diseases often have very different cardiovascular physiology from humans and how these differences affect the predictive value of safety studies. As an example, cardiovascular drug safety testing was expanded significantly upon discovering that the antihistamine terfenadine is cardiotoxic when not completely metabolized. Its toxic effect is induced by the inhibition of the cytochrome P450 3A4 (CYP 3A4) isoform, a circumstance that caused death in a small percentage of people due to an unexpected interaction with other medications. CYP 3A4 is inhibited by a variety of commonly prescribed drugs as well as grapefruit. In its toxic form, terfenadine induces a prolonged QT interval and thus can trigger a fatal arrhythmia; however, this effect could not be modeled in rats and mice since they do not have the hERG ion channel that caused the human arrhythmia. Dr Hamlin further emphasized the importance of matching characteristics of the experimental animal with those of the human phenotype. In listing several animals that should not be used to model specific human diseases, he included guinea pigs and dogs as poor subjects for studies of coronary collateralization as their coronary arteries have a much more extensive collateral circulation than is present in human beings. An interesting theme was how many cardiovascular diseases arise or worsen due to outmoded ‘‘protective’’ homeostatic mechanisms. For example, volume depletion was the primary mode of death for prehistoric man; however, in the developed world, the mechanisms that evolved to assist survival in conditions of volume depletion (thirst, decreased urine production, and vasoconstriction) now complicate more modern maladies such as diabetes, congestive heart failure, and pulmonary disease. Such examples emphasize the need for careful consideration of the evolutionary similarities and differences between man and the animals used in research. To summarize, Dr Hamlin questioned how we can expect to model a heterogeneous population with a homogeneous animal model when disease characteristics may be determined by population heterogeneity. He concluded that selecting surrogates to study human pathophysiology should

The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

Hyperthermia above a critical threshold results in multisystemic changes that include neurological manifestations of heat stroke. It is unknown if the latter represents an intrinsic thermal sensitivity of the CNS or whether injury is secondary to physiological responses of non-CNS origin. To address this issue, the present work examined functional, structural, and biochemical changes in the CNS of dogs subjected to a thermal dosage immediately below that which induces disseminated intravascular coagulation with secondary multiple organ injury. The experimental approach is previously reported, inducing a 42.5°C, 90 min, whole body hyperthermia while preventing other physiological responses to treatment, including respiratory alkalosis and significant reductions in mean arterial pressure. Functional analyses included neurologic examinations and brainstem auditory evoked potential recordings in the post-treatment interval in both hyperthermic and euthermic control populations. Biochemical and structural analyses examined the expression of 70-kDa heat shock proteins, cytokines, markers of astroglial and microglial injury/activation, evidence of vascular endothelial damage, and evidence of neuronal and axonal injury in brain between 0.5 h and 8 days from the end of the treatment. The only significant change associated with treatment was induction of the major inducible 70-kDa heat shock protein, this being most prominent in the cerebellum with maximal expression at 6 h and a return to baseline by 8 days. Collectively, from these results we suggest that the canine brain is intrinsically resistant to sublethal hyperthermia such that when CNS lesions occur, they do so in the presence of other physiological derangements.

The spinal cord ependymal layer (SEL) is a recent focus in spinal cord injury (SCI) research because of its potential to serve as a source of endogenous neural stem cells. Dogs are an important spontaneous model of SCI; however, there is a paucity of information available in the literature regarding the canine SEL. Here we describe the histologic appearance and immunohistochemical staining patterns of the SEL in normal dogs ( n = 4) and dogs with acute SCI caused by intervertebral disk extrusion ( n = 7). Immunohistochemical staining for PCNA, Ki-67, caspase 3, E-cadherin, GFAP, and vimentin was employed in both groups. Staining for Ki-67 was absent in the SEL of normal and SCI-affected dogs, indicating possible restricted proliferative capacity of the canine SEL acutely after SCI. GFAP-positive cells were increased after SCI at both at the lesion epicenter and at proximal spinal cord sites ( P = .001 and P = .006, respectively), supporting the possibility of astrocytic differentiation within the SEL after SCI. Total E-cadherin staining did not differ between normal and SCI-affected dogs ( P = .42 for lesion epicenter, P = .09 at proximal sites) and was restricted to the apical cell surface in normal dogs. After SCI, E-cadherin staining was membrane-circumferential and cytosolic in nature, indicating possible loss of cellular polarity after injury that could drive cell migration from the SEL to injury sites. Enhanced GFAP expression and changes in E-cadherin expression patterns support additional studies to evaluate the canine SEL as a source of endogenous neural precursors that may be modulated for future clinical interventions after SCI.

Objective—To determine analgesic effects of intraneural injection of ethyl alcohol or formaldehyde in the palmar digital nerves of horses. Animals—6 horses. Procedures—Ethyl alcohol was injected in the medial palmar digital nerve of 1 forelimb, and formaldehyde was injected in the contralateral nerve. The lateral palmar digital nerve in 1 forelimb was surgically exposed, but not injected, and the contralateral lateral palmar digital nerve was not treated. For each heel, severity of lameness in response to experimentally induced heel pain (lameness score and peak vertical force), thermal reaction time, and heel skin sensitivity scores were recorded. Heel pain was experimentally induced by advancing a threaded bolt through a custom-made horseshoe to apply pressure to the palmar horned aspect of the hoof. Horses were followed up for 112 days, when a subset of nerves was sampled for histologic analysis. Results—Alcohol and formaldehyde significantly reduced all measures of heel pain, and analgesia was evident over the 112 days of the study. Pastern circumference was significantly greater for formaldehyde-treated than for alcohol-treated limbs. Histologic evaluation showed preservation of nerve fiber alignment with an intact epineurium, loss of axons, axon degeneration, fibrosis, and inflammation in alcohol-treated and formaldehyde-treated nerves. Conclusions and Clinical Relevance—Results suggested that intraneural injection of either ethyl alcohol or formaldehyde in the palmar digital nerves of horses resulted in substantial, but partial, heel analgesia that persisted for at least 112 days. No advantage of formaldehyde over alcohol was found, and formaldehyde resulted in greater soft tissue inflammation.

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas and Sarcocystis neurona is the most common etiologic agent. The distribution of S. neurona infections follows the geographical distributions of its definitive hosts, opossums (Didelphis virginiana, Didelphis albiventris). Recently, cats and skunks were reported as experimental and armadillos as natural intermediate hosts of S. neurona. In the present report, raccoons (Procyon lotor) were identified as a natural intermediate host of S. neurona. Two laboratory-raised opossums were found to shed S. neurona-like sporocysts after ingesting tongues of naturally-infected raccoons. Interferon-gamma gene knockout (KO) mice fed raccoon-opossum-derived sporocysts developed neurologic signs. S. neurona was identified immunohistochemically in tissues of KO mice fed sporocysts and the parasite was isolated in cell cultures inoculated with infected KO mouse tissues. The DNA obtained from the tongue of a naturally-infected raccoon, brains of KO mice that had neurological signs, and from the organisms recovered in cell cultures inoculated with brains of neurologic KO mice, corresponded to that of S. neurona. Two raccoons fed mature S. neurona sarcocysts did not shed sporocysts in their feces, indicating raccoons are not likely to be its definitive host. Two raccoons fed sporocysts from opossum feces developed clinical illness and S. neurona-associated encephalomyelitis was found in raccoons killed 14 and 22 days after feeding sporocysts; schizonts and merozoites were seen in encephalitic lesions.

Electroencephalography (EEG) is a valuable diagnostic test to identify functional disturbances in brain activity. The purpose of this study was to assess the validity of EEG as a diagnostic indicator of intracranial diseases in horses. The validity of EEG was estimated by comparing clinical, clinicopathologic, and histopathologic findings to EEG findings in 20 horses examined for seizures, collapse, or abnormal behavior between 1984 and 1997. A bipolar left-to-right, back-to-front montage and a bipolar circular montage were recorded from sedated (4) and anesthetized (16) horses. Visual and semiquantitative masked analysis of EEG recording 1st was validated on 10 horses presented for problems other than intracranial diseases. EEG pattern was normal in 7 of the 20 clinically affected horses. Abnormal EEG patterns included high-voltage slow waves and discrete paroxysmal activity with or without generalized activity in 13 horses. Histopathologic diagnoses in 10 horses included meningoencephalitis, neuronal necrosis, congenital anomalies, cerebral edema, and abscess. All of these horses had abnormal EEG patterns (sensitivity, 100%) with a positive neuroanatomic correlation in 7 animals. Localization of histopathologic and EEG abnormalities did not correlate in 15% of the horses (3/20). The cause of neurologic signs could not be explained at postmortem examination in 10 animals and the EEG pattern was normal in 7 of these horses (specificity, 70%). In conclusion, equine EEG was a sensitive tool in the diagnosis of intracranial disorders.

Neurologic disease in horses caused by Sarcocystis neurona is difficult to diagnose, treat, or prevent, due to the lack of knowledge about the pathogenesis of the disease. This in turn is confounded by the lack of a reliable equine model of equine protozoal myeloencephalitis (EPM). Epidemiologic studies have implicated stress as a risk factor for this disease, thus, the role of transport stress was evaluated for incorporation into an equine model for EPM. Sporocysts from feral opossums were bioassayed in interferon-gamma gene knockout (KO) mice to determine minimum number of viable S. neurona sporocysts in the inoculum. A minimum of 80,000 viable S. neurona sporocysts were fed to each of the nine horses. A total of 12 S. neurona antibody negative horses were divided into four groups (1–4). Three horses (group 1) were fed sporocysts on the day of arrival at the study site, three horses were fed sporocysts 14 days after acclimatization (group 2), three horses were given sporocysts and dexamethasone 14 days after acclimatization (group 3) and three horses were controls (group 4). All horses fed sporocysts in the study developed antibodies to S. neurona in serum and cerebrospinal fluid (CSF) and developed clinical signs of neurologic disease. The most severe clinical signs were in horses in group 1 subjected to transport stress. The least severe neurologic signs were in horses treated with dexamethasone (group 3). Clinical signs improved in four horses from two treatment groups by the time of euthanasia (group 1, day 44; group 3, day 47). Post-mortem examinations, and tissues that were collected for light microscopy, immunohistochemistry, tissue cultures, and bioassay in KO mice, revealed no direct evidence of S. neurona infection. However, there were lesions compatible with S. neurona infection in horses. The results of this investigation suggest that stress can play a role in the pathogenesis of EPM. There is also evidence to suggest that horses in nature may clear the organism routinely, which may explain the relatively high number of normal horses with CSF antibodies to S. neurona compared to the prevalence of EPM.

Causes of canine juvenile hydrocephalus have been well documented. However, the effects of hydrocephalus on periventricular white matter have been only partially described. The present report shows that hydrocephalus-associated lesions of the periventricular white matter, i.e., formation of diverticula, clefts, and tears, are prevalent. Marked hydrocephalus was identified at necropsy in 20 juvenile dogs between 1990 and 1999. The severity grade was based upon a ratio of lateral ventricular dimensions to cortical thickness. All animals exhibited ependymal lesions consisting of attenuation, with or without abortive attempts of ependymal regeneration, and ulceration. In 10 dogs (50%), unilateral or bilateral periventricular diverticula and cleft formation in the region of the caudate nucleus were observed. The diverticula were formed at the caudal pole of the caudate nucleus, communicated with the ventricular lumen, and were associated with ependymal denudation. Loss of the ependymal lining probably contributes to a bulk shift of cerebrospinal fluid from the ventricular lumen to the periventricular white matter, leading to diverticulum formation. Clefts were observed within the parenchyma at the border of the internal capsule and putamen, consistent with an ischemic insult. Occasionally tearing with separation of the caudate nucleus from the subcortical white matter was found, representing unification of expanding clefts and diverticula. In one of the few clinically well-documented cases, tearing was correlated with a sudden decline in neurologic function, culminating in euthanasia. However, tears and clefts may exhibit a chronicity of several days, as indicated by the presence of astroglial scars along the lesion margins.

Use of hyperthermia in the treatment of cancer and viral infection has received renewed interest. However, the in vivo relationship between hyperthermia and direct versus indirect effects upon hemostasis are incompletely defined, although we do know that disseminated intravascular coagulation (DIC) is a common sequel to heat stroke. The purpose of the present study was to more precisely define the relationship between hyperthermia and derangements of hemostasis, thereby providing a guideline for the development of safe hyperthermia treatment regimens. The present investigation examined the in vivo effects of high-grade whole-body hyperthermia (WBH) (42.5 degrees C, 90 min) on hemostasis in a canine model. Induction of hyperthermia via extracorporeal circulation of heated blood (ECC-WBH) caused thrombocytopenia, increased plasma fibrin degradation products (FDPs), prolonged clotting times, increased serum liver enzymes, and evidence of spontaneous bleeding. However, when WBH was induced by peritoneal lavage (PL-WBH), transient thrombocytopenia was the only significant alteration. Temporal correlation between hemostatic alterations and elevations in serum alanine aminotransferase (ALT) levels in the ECC-WBH treatment group suggested that liver injury is responsible, at least in part, for the coagulopathy associated with high-grade hyperthermia and that in the absence of liver injury, identical degrees of hyperthermia cause only incidental decreases in platelet numbers.

Cellular levels of heat shock proteins (HSPs) are elevated in response to physiologic states accompanying acute virus infection (e.g. fever). The objective of the present work was to define the antiviral effect of purified human lymphoblastoid IFN in the presence of HSP over-expression. For this purpose, canine distemper virus (CDV) was used since the response of CDV transcription and persistent infection phenotype to elevated HSP is characterized. First, the effect of elevated HSP on CDV lytic infection phenotype in Vero and CV1 cells was defined, and results extended to the closely related measles virus (MV). Cells expressing elevated levels of the major inducible 70-kDa HSP (hsp72) supported the emergence of large plaque variants of both CDV and MV from small plaque purified inocula. IFN treatment concurrent with infection caused a dosage-dependent reduction in the expression of large plaque variants without affecting hsp72 levels or total plaque number. In contrast to the stress response-induced large plaque variant, small plaques were resistant to the antiviral effects of IFN. These data demonstrate the ability of IFN to selectively abrogate the pro-viral effects of HSP over-expression, inhibiting the formation of a plaque phenotype that is correlated to enhanced virulence in animal models of morbillivirus encephalitis.

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-z) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-z has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-z has only minor activity. Selective inhibition of PKC-z activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-z in the virus replication cycle. The broad tissue expression of PKC-z parallels the pantropic nature of CDV infections, suggesting that PKC-z activity is a determinant of cellular permissiveness to CDV replication. q 1997 Academic Press