This report describes the methodology, diagnostic yield, and adverse events (AE) associated with frame-based stereotactic brain biopsies (FBSB) obtained from 26 dogs with solitary forebrain lesions. Medical records were reviewed from dogs that underwent FBSB using two stereotactic headframes designed for use in small animals and compatible with computed tomographic (CT) and magnetic resonance (MR) imaging. Stereotactic plans were generated from MR and CT images using commercial software, and FBSB performed both with (14/26) and without intraoperative image guidance. Records were reviewed for diagnostic yield, defined as the proportion of biopsies producing a specific neuropathological diagnosis, AE associated with FBSB, and risk factors for the development of AE. Postprocedural AE were evaluated in 19/26 dogs that did not proceed to a therapeutic intervention immediately following biopsy. Biopsy targets included intra-axial telencephalic masses (24/26), one intra-axial diencephalic mass, and one extra-axial parasellar mass. The median target volume was 1.99 cm3. No differences in patient, lesion, or outcome variables were observed between the two headframe systems used or between FBSB performed with or without intraoperative CT guidance. The diagnostic yield of FBSB was 94.6%. Needle placement error was a significant risk factor associated with procurement of non-diagnostic biopsy specimens. Gliomas were diagnosed in 24/26 dogs, and meningioma and granulomatous meningoencephalitis in 1 dog each. AE directly related to FBSB were observed in a total of 7/26 (27%) of dogs. Biopsy-associated clinical morbidity, manifesting as seizures and transient neurological deterioration, occurred in 3/19 (16%) of dogs. The case fatality rate was 5.2% (1/19 dogs), with death attributable to intracranial hemorrhage. FBSB using the described apparatus was relatively safe and effective at providing neuropathological diagnoses in dogs with focal forebrain lesions.

Human noroviruses (NoVs), a major cause of viral gastroenteritis, are difficult to study due to the lack of a cell-culture and a small-animal model. Pigs share with humans the types A and H histo-blood group antigens on the intestinal epithelium and have been suggested as a potential model for studies of NoV pathogenesis, immunity and vaccines. In this study, the effects of age and a cholesterol-lowering drug, simvastatin, on the susceptibility of pigs to NoV infection were evaluated. The median infectious dose (ID50) of a genogroup II, genotype 4 (GII.4) 2006b variant was determined. The ID50 in neonatal (4–5 days of age) pigs was ≤2.74×103 viral RNA copies. In older pigs (33–34 days of age), the ID50 was 6.43×104 but decreased to <2.74×103 in simvastatin-fed older pigs. Evidence of NoV infection was obtained by increased virus load in the intestinal contents, cytopathological changes in the small intestine, including irregular microvilli, necrosis and apoptosis, and detection of viral antigen in the tip of villi in duodenum. This GII.4 variant was isolated in 2008 from a patient from whom a large volume of stool was collected. GII.4 NoVs are continuously subjected to selective pressure by human immunity, and antigenically different GII.4 NoV variants emerge every 1–2 years. The determination of the ID50 of this challenge virus is valuable for evaluation of protection against different GII.4 variants conferred by NoV vaccines in concurrence with other GII.4 variants in the gnotobiotic pig model.

Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.

The lifespan of diabetic patients is 7-8 y shorter than that of the general population because of hyperglycemia-induced vascular complications and damage to other organs such as the liver and skeletal muscle. Here, we investigated the effects of epicatechin, one of the major flavonoids in cocoa, on health-promoting effects in obese diabetic (db/db) mice (0.25% in drinking water for 15 wk) and Drosophila melanogaster (0.01-8 mmol/L in diet). Dietary intake of epicatechin promoted survival in the diabetic mice (50% mortality in diabetic control group vs. 8.4% in epicatechin group after 15 wk of treatment), whereas blood pressure, blood glucose, food intake, and body weight gain were not significantly altered. Pathological analysis showed that epicatechin administration reduced the degeneration of aortic vessels and blunted fat deposition and hydropic degeneration in the liver caused by diabetes. Epicatechin treatment caused changes in diabetic mice that are associated with a healthier and longer lifespan, including improved skeletal muscle stress output, reduced systematic inflammation markers and serum LDL cholesterol, increased hepatic antioxidant glutathione concentration and total superoxide dismutase activity, decreased circulating insulin-like growth factor-1 (from 303 ± 21 mg/L in the diabetic control group to 189 ± 21 mg/L in the epicatechin-treated group), and improved AMP-activated protein kinase-α activity in the liver and skeletal muscle. Consistently, epicatechin (0.1-8 mmol/L) also promoted survival and increased mean lifespan of Drosophila. Therefore, epicatechin may be a novel food-derived, antiaging compound.

Regulatory T cells (Tregs) are a subset of T cells that are responsible for maintaining peripheral immune tolerance and homeostasis. The hallmark of Tregs is the expression of the forkhead box P3 (FoxP3) transcription factor. Natural regulatory T cells (nTregs) are a distinct population of T cells that express CD4 and FoxP3. nTregs develop in the thymus and function in maintaining peripheral immune tolerance. Other CD4+, CD4-CD8-, and CD8+CD28- T cells can be induced to acquire regulatory function by antigenic stimulation, depending on the cytokine milieu. Inducible (or adaptive) Tregs frequently express high levels of the interleukin 2 receptor (CD25). Atypical Tregs express FoxP3 and CD4 but have no surface expression of CD25. Type 1 regulatory T cells (Tr1 cells) produce IL-10, while T helper 3 cells (Th3) produce TGF-β. The function of inducible Tregs is presumably to maintain immune homeostasis, especially in the context of chronic inflammation or infection. Induction of Tregs in coronaviral infections protects against the more severe forms of the disease attributable to the host response. However, arteriviruses have exploited these T cell subsets as a means to dampen the immune response allowing for viral persistence. Treg induction or activation in the pathogenesis of disease has been described in both porcine reproductive and respiratory syndrome virus, lactate dehydrogenase elevating virus, and mouse hepatitis virus. This review discusses the development and biology of regulatory T cells in the context of arteriviral and coronaviral infection.

The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified in the genus Hepevirus of the family Hepeviridae (11,17). It is now known that hepatitis E is a zoonotic disease and that animal reservoirs exist for HEV (13, 26-29, 38, 40). The inefficient replication of HEV in cell cultures has hindered progress in understanding the biology of HEV. The problem has been overcome partially by either characterizing individually expressed proteins from expression vectors or transfecting cells and intrahepatically inoculating animals with capped RNA transcripts generated in vitro from infectious clones (16,17,32,33). More recently, efficient in vitro HEV replication systems have been reported (3,31,35,37), which may aid in future understanding of HEV replication.The 7.2-kb RNA genome of HEV contains three open reading frames (ORFs), namely, ORF1, ORF2, and ORF3, flanked by 5Ј-and 3Ј-nontranslated regions (2). The putative functional domains in the ORF1 protein include methyltransferase, protease, helicase, and RNA-dependent RNA polymerase (RdRp) domains (2). ORF2 encodes the major capsid protein, whereas ORF3 encodes a small multifunctional protein (2,6,18,30,39). The methyltransferase and guanylyltransferase activities in capping of the viral RNA (25), the role of RdRp in viral RNA synthesis...

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ-specific CD4 T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α-specific CD8 T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the UnitedStates. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.