Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice.

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.

PKU is a reversible neurodegenerative process within the nigrostriatum that begins as early as 4 weeks of age in Pah(enu2) mice.

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.

Pathologic and immunohistochemical findings in hypothalamic and mesencephalic regions in the pah(enu2) mouse model for phenylketonuria.

The Pah(enu2) mouse, created through ethylnitrosurea mutagenesis, is a model for phenylketonuria. These mice have elevated serum phenylalanine levels, hypopigmentation, and behavior and movement abnormalities, and female mice exhibit a maternal phenylketonuria syndrome. We evaluated the brains of adult and juvenile Pah(enu2) mice for consistent, demonstrable lesions to elucidate various neuropathologic processes and to assess the efficacy of various treatment modalities such as AAV-mediated gene therapy. One aspect of the disease may involve the effect of hyperphenylalanemia on catecholamine function. High levels of phenylalanine inhibit enzymes that are important in the conversion of tyrosine and tryptophan to their respective neurotransmitter derivatives, including dopamine. Therefore, assessment of dopaminergic regions was of interest in this study. Histologic evaluation of juvenile and adult brains revealed an increased cellular density as early as 4 wk of age in the middle to posterior hypothalamus and substantia nigra. The infiltrating cells showed immunoreactivity for CD11b and had morphologic characteristics of macrophages. There was marked expression of inducible nitric oxide synthase in these dopaminergic regions that co-localized to CD11b-positive cells. The CD11b-positive cells and increased inducible nitric oxide synthase expression in these regions may function in a neuroregulatory manner to compensate for alterations in dopamine metabolism.

Cytologic diagnosis of Lawsonia intracellularis proliferative ileitis in a Japanese snow macaque (Macaca fuscata).

Diffuse ileal thickening and ileocecocolic lymphadenomegaly were observed during exploratory laparotomy in a 2-year-old male Japanese snow macaque (Macaca fuscata) that had flu-like signs and diarrhea. Cytologic examination of ileal biopsy imprints revealed many mature, mildly karyolytic neutrophils and fewer well-differentiated lymphocytes, eosinophils, macrophages, and plasma cells in a background containing amorphous, necrotic material. Tightly cohesive sheets of moderately pleomorphic epithelial cells also were seen. The cytologic diagnosis was chronic, active, mixed inflammation with atypical epithelial cells and necrosis. Histologically, the mucosal and crypt epithelium was moderately hyperplastic with a loss of goblet cells, increased mitoses, and frequent crypt abscesses. Within the lamina propria and extending into the submucosa was a marked neutrophilic infiltrate, with low numbers of lymphocytes, histiocytes, plasma cells, and eosinophils. The histologic diagnosis was chronic, diffuse, marked suppurative and lymphocytic ileitis. Warthin-Starry silver staining of the ileal biopsy and imprint specimens demonstrated numerous pleomorphic, curved bacilli consistent with Lawsonia intracellularis. Polymerase chain reaction (PCR) and immunohistochemistry confirmed the identity of the infectious agent. L intracellularis infection may be underdiagnosed because silver stain is required to visualize the organism with light microscopy and because the pathognomonic crypt hyperplasia may be complicated by secondary pathologic changes. Application of silver stain to cytologic specimens should be considered when distal intestinal lesions associated with hyperplastic epithelium, with or without inflammation, hemorrhage, or necrosis, are identified in animals with clinical signs of enteritis, especially in frequently affected species or in stressed or young animals.

Synovial fluid from an African spur-thighed tortoise (Geochelone sulcata).

A 4.5-year old, male African spur-thighed tortoise (Geochelone sulcata) was presented to the University of Florida Veterinary Teaching Hospital with a 2-week history of lethargy, anorexia, constipation, dyspnea, and coughing up fluid or vomiting. Laboratory results included an inflammatory leukogram and a marked increase in plasma uric acid concentration. Synovial fluid from multiple joints was thick, chalky white, and opaque, with a grainy consistency. Microscopically, the fluid contained numerous brown, needle-like crystals consistent with urates (gout). Gross necropsy findings and histopathology confirmed a diagnosis of systemic gout, with urate deposition, gout tophi, and underlying necrosis in multiple organs, including kidneys, lung, and liver. Dehydration with concurrent renal insufficiency may have impaired urate excretion and led to a build-up of urates in the blood and tissues of this tortoise. A high protein diet also may have contributed to the development of gout. Cytologic evaluation of synovial fluid can be used as a quick and definitive tool to diagnose gout in tortoises.