A Novel Live Attenuated Respiratory Syncytial Virus Vaccine Candidate with Mutations in the L Protein SAM Binding Site and the G Protein Cleavage Site Is Protective in Cotton Rats and a Rhesus Macaque.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children of <5 years of age worldwide, infecting the majority of infants in their first year of life. Despite the widespread impact of this virus, no vaccine is currently available. For more than 50 years, live attenuated vaccines (LAVs) have been shown to protect against other childhood viral infections, offering the advantage of presenting all viral proteins to the immune system for stimulation of both B and T cell responses and memory. The RSV LAV candidate described here, rgRSV-L(G1857A)-G(L208A), contains two modifications: an attenuating mutation in the S-adenosylmethionine (SAM) binding site of the viral mRNA cap methyltransferase (MTase) within the large (L) polymerase protein and a mutation in the attachment (G) glycoprotein that inhibits its cleavage during production in Vero cells, resulting in virus with a "noncleaved G" (ncG). RSV virions containing the ncG have an increased ability to infect primary well-differentiated human bronchial epithelial (HBE) cultures which model the in vivo site of immunization, the ciliated airway epithelium. This RSV LAV candidate is produced efficiently in Vero cells, is highly attenuated in HBE cultures, efficiently induces neutralizing antibodies that are long lasting, and provides protection against an RSV challenge in the cotton rat, without causing enhanced disease. Similar results were obtained in a rhesus macaque.IMPORTANCE Globally, respiratory syncytial virus (RSV) is a major cause of death in children under 1 year of age, yet no vaccine is available. We have generated a novel RSV live attenuated vaccine candidate containing mutations in the L and G proteins. The L polymerase mutation does not inhibit virus yield in Vero cells, the cell type required for vaccine production, but greatly reduces virus spread in human bronchial epithelial (HBE) cultures, a logical in vitro predictor of in vivo attenuation. The G attachment protein mutation reduces its cleavage in Vero cells, thereby increasing vaccine virus yield, making vaccine production more economical. In cotton rats, this RSV vaccine candidate is highly attenuated at a dose of 105 PFU and completely protective following immunization with 500 PFU, 200-fold less than the dose usually used in such studies. It also induced long-lasting antibodies in cotton rats and protected a rhesus macaque from RSV challenge. This mutant virus is an excellent RSV live attenuated vaccine candidate.

Fibroblastic osteosarcoma with epithelioid and squamous differentiation in a dog.

A fibroblastic osteosarcoma with epithelioid and squamous differentiation in the distal femur of a 9-y-old spayed female Greyhound dog is described. Grossly, the tumor consisted of a pale-white, firm-to-hard mass that replaced the medullary and cortical areas of the distal end of the right femur. Histologically, the mass was composed predominantly of spindle cells admixed with areas of mineralized and non-mineralized osteoid matrix that were surrounded by stellate osteoblasts and scattered multinucleate giant cells, consistent with the diagnosis of a fibroblastic osteosarcoma. In addition, well-demarcated clusters of neoplastic epithelioid cells and foci of squamous differentiation with keratin pearls were present throughout the neoplasm. The spindle cells, epithelioid cells, and areas of squamous differentiation expressed cytoplasmic immunostaining for osteocalcin and osteonectin. The spindle cells and epithelioid cells were also immunopositive for vimentin. Epithelioid cells also expressed occasional cytoplasmic immunostaining for pancytokeratin (PCK) Lu-5, and areas of squamous differentiation were immunoreactive for PCK Lu-5 and high molecular weight CK; these areas were inconsistently immunoreactive for CK 5-6 and immunonegative for low molecular weight CK. Foci of squamous differentiation were not located within blood or lymphatic vessels, given that no immunoreactivity for factor VIII-related antigen was observed around these areas. A thorough autopsy and an evaluation of the medical history excluded a primary carcinoma or other neoplasm elsewhere in the dog. The findings were consistent with a diagnosis of fibroblastic osteosarcoma with epithelioid and squamous differentiation.

Double outlet right ventricle with subpulmonary ventricular septal defect (Taussig-Bing anomaly) and other complex congenital cardiac malformations in an American Quarter Horse foal.

A 4-week-old American Quarter Horse colt presented with a recent history of diarrhea and decreased activity level. On initial physical examination, the animal was bright and alert and major findings were limited to a loud systolic heart murmur radiating widely over both sides of the thorax. While in the hospital, the clinical condition of the foal warranted further imaging to determine the cause and extent of cardiac disease. A variety of congenital cardiac malformations were identified during echocardiographic examination and autopsy, including a double outlet right ventricle and a subpulmonary interventricular septal defect (Taussig-Bing anomaly), ventricular inversion with atrioventricular discordance, tricuspid valve atresia, a septum primum interatrial septal defect, mitral valve dysplasia with a cleft in the septal mitral valve cusp, aortic, and subaortic stenosis, tubular hypoplasia of the ascending aorta and the aortic arch, a patent ductus arteriosus, an aberrant circumflex coronary artery, and aberrant left and right subclavian arteries. Echocardiographic and postmortem findings of the cardiac defects in this foal are presented and discussed.

Cellulitis caused by the Burkholderia cepacia complex associated with contaminated chlorhexidine 2% scrub in five domestic cats.

Isolates of the Burkholderia cepacia complex (BCC) are known as plant and human pathogens. We describe herein BCC infections as the cause of subcutaneous abscesses and purulent cellulitis in 5 cats. All cats were presented with an open wound, and 4 received standard wound care and empiric antibiotic therapy. Despite treatment, clinical signs worsened in 4 cats. Isolates of the BCC were obtained from all 5 cases. Two cats were submitted for postmortem examination. Subcutaneous abscesses with draining fistulas were observed. Histopathology revealed severe, pyogranulomatous cellulitis with intralesional gram-negative bacilli. Based on susceptibility results, the other 3 cats were administered effective antibiotics and recovered without complications. The BCC was cultured from the 2% chlorhexidine surgical scrub solution used in the clinic, suggesting the source of infection for 4 of 5 cats. Given the ability to grow in antiseptic solutions, the extra steps required to culture from antiseptics, and innate multidrug resistance, the BCC poses a challenge to both detect and treat. Although the BCC causes disease almost exclusively in humans with cystic fibrosis or immunodeficiency, the bacteria should also be a differential for nosocomial infections in veterinary patients.

Pulmonary capillary hemangiomatosis and hypertrophic cardiomyopathy in a Persian cat.

Pulmonary capillary hemangiomatosis (PCH) and pulmonary veno-occlusive disease (PVOD) are rare causes of primary pulmonary hypertension in humans, and, in 2016, were reported in dogs. A 1-y-old, neutered male Persian cat was presented for autopsy after sudden death several hours after grooming. Grossly, the lungs were mottled red-to-pink, contained rubbery-to-firm nodular foci, and there was moderate-to-marked left-sided cardiomegaly and left atrial dilation, consistent with hypertrophic cardiomyopathy. Microscopically, there was multifocal to regionally extensive capillary proliferation within pulmonary alveolar septa and around respiratory bronchioles, with nodular aggregates of densely arranged capillaries that replaced pulmonary alveolar spaces. Rare occlusive venous remodeling was identified in Verhoeff-van Gieson-stained sections. The gross and microscopic changes were consistent with PCH with rare features of PVOD. Hypertrophic cardiomyopathy was interpreted as potentially contributing to the cause of death, but unrelated to the pulmonary vascular proliferation.

Attenuation of the pressor response to exogenous angiotensin by angiotensin receptor blockers and benazepril hydrochloride in clinically normal cats.

OBJECTIVE: To compare the attenuation of the angiotensin I-induced blood pressure response by once-daily oral administration of various doses of angiotensin receptor blockers (irbesartan, telmisartan, and losartan), benazepril hydrochloride, or lactose monohydrate (placebo) for 8 days in clinically normal cats. ANIMALS: 6 healthy cats (approx 17 months old) with surgically implanted arterial telemetric blood pressure-measuring catheters. PROCEDURES: Cats were administered orally the placebo or each of the drug treatments (benazepril [2.5 mg/cat], irbesartan [6 and 10 mg/kg], telmisartan [0.5, 1, and 3 mg/kg], and losartan [2.5 mg/kg]) once daily for 8 days in a crossover study. Approximately 90 minutes after capsule administration on day 8, each cat was anesthetized and arterial blood pressure measurements were recorded before and after IV administration of each of 4 boluses of angiotensin I (20, 100, 500, and 1,000 ng/kg). This protocol was repeated 24 hours after benazepril treatment and telmisartan (3 mg/kg) treatment. Differences in the angiotensin I-induced change in systolic arterial blood pressure (ΔSBP) among treatments were determined. RESULTS: At 90 minutes after capsule administration, only losartan did not significantly reduce ΔSBP in response to the 3 higher angiotensin doses, compared with placebo. Among drug treatments, telmisartan (3 mg/kg dosage) attenuated ΔSBP to a significantly greater degree than benazepril and all other treatments. At 24 hours, telmisartan was more effective than benazepril (mean ± SEM ΔSBP, 15.7 ± 1.9 mm Hg vs 55.9 ± 12.42 mm Hg, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that telmisartan administration may have advantages over benazepril administration for cats with renal or cardiovascular disease.

Evaluation of a rapid pressor response test in healthy cats.

OBJECTIVE: To evaluate angiotensin I and angiotensin II rapid pressor response tests in healthy cats. ANIMALS: 6 purpose-bred sexually intact male cats. PROCEDURES: Telemetric blood pressure (BP) implants were placed in all cats. After 2 weeks, cats were anesthetized for challenge with exogenous angiotensin I or angiotensin II. Continuous direct arterial BP was recorded during and immediately after IV administration of boluses of angiotensin I or angiotensin II at increasing doses. Blood pressure responses were evaluated for change in systolic BP (SBP), change in diastolic BP (DBP), and rate of increase of SBP by 4 observers. RESULTS: Following IV angiotensin I and angiotensin II administration, transient, dose-dependent increases in BP (mean ± SEM change in SBP, 25.7 ± 5.2 and 45.0 ± 9.1; change in DBP, 23.4 ± 4.7 mm Hg and 36.4 ± 7.8 mm Hg; for 100 ng of angiotensin I/kg and angiotensin II/kg, respectively) and rate of increase of SBP were detected. At angiotensin I and II doses < 2.0 ng/kg, minimal responses were detected, with greater responses at doses ranging from 20 to 1,000 ng/kg. A significant effect of observer was not found. No adverse effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The rapid pressor response test elicited dose-dependent, transient increases in SBP and DBP. The test has potential as a means of objectively evaluating the efficacy of various modifiers of the renin-angiotensin-aldosterone system in cats. Ranges of response values are provided for reference in future studies.

Altered immune phenotype in peripheral blood cells of patients with scleroderma-associated pulmonary hypertension.

Pulmonary arterial hypertension is a common and fatal complication of scleroderma that may involve inflammatory and autoimmune mechanisms. Alterations in the gene expression of peripheral blood mononuclear cells have been previously described in patients with pulmonary arterial hypertension. Our goal is to identify differentially expressed genes in peripheral blood mononuclear cells in scleroderma patients with and without pulmonary hypertension as biomarkers of disease. Gene expression analysis was performed on a Microarray Cohort of scleroderma patients with (n = 10) and without (n = 10) pulmonary hypertension. Differentially expressed genes were confirmed in the Microarray Cohort and validated in a Validation Cohort of scleroderma patients with (n = 15) and without (n = 19) pulmonary hypertension by RT-qPCR. We identified inflammatory and immune-related genes including interleukin-7 receptor (IL-7R) and chemokine receptor 7 as differentially expressed in patients with scleroderma-associated pulmonary hypertension. Flow cytometry confirmed decreased expression of IL-7R on circulating CD4+ T-cells from scleroderma patients with pulmonary hypertension. Differences exist in the expression of inflammatory and immune-related genes in peripheral blood cells from patients with scleroderma-related pulmonary hypertension compared to those with normal pulmonary artery pressures. These findings may have implications as biomarkers to screen at-risk populations for early diagnosis and provide insight into mechanisms of scleroderma-related pulmonary hypertension.