Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Blood flow in the oral mucosa of normal and atherosclerotic rhesus monkeys.

Little data is available on regional blood flow in the normal primate oral mucosa and none on that in atherosclerotic animals. Three adult Rhesus monkeys were maintained on a normal diet and 4 on a high fat, high cholesterol diet for 20 months. Radiolabelled microspheres were used to measure blood flow in skin and 16 oral mucosa regions. In normal animals, blood flow ranged from 160.81 to 8.68 ml/min/100 gm tissue. Blood flow in the same regions of atherosclerotic animals showed significantly lower values than in controls, ranging from 65.90 to 1.04 ml/min/100 gms tissue. However, the relative blood flow to the different regions was not significantly different between control and atherosclerotic animals. Histologic examination of tissue from the atherosclerotic animals revealed gross intimal plaques occluding the lumina of the carotids and atherosclerotic lesions in the lingual arteries. It is concluded that the decrease in blood flow in oral mucosa in the atherosclerotic animals is not the result of local changes in the mucosal vasculature but may be related to lesions seen in the major afferent vessels.

Quantitative immunoassay of Treponema denticola serovar C in adult periodontitis.

Murine monoclonal antibodies specific for Treponema denticola serovar C were produced and characterized in this study. An immunoassay was then developed by using these monoclonal antibodies, and the T. denticola serovar C antigen content of subgingival plaque was quantitated for samples taken from patients with periodontitis and healthy volunteers. The human subgingival plaque samples were grouped by severity of disease and pocket depth measurements at the collection site. The T. denticola serovar C content per milligram of subgingival plaque from deep pockets (greater than 6 mm) of patients with severe periodontitis was found to be twice that of samples collected from deep pockets (4 to 6 mm) of patients with moderate periodontitis or samples collected from healthy subjects (pocket depth, less than 4 mm).

Quantitative relationship of Treponema denticola to severity of periodontal disease.

The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.