Communications Project

Document Type:Master's Thesis
Name:Shawn Christopher Walker
Degree:Master of Science
Department:Veterinary Medical Sciences
Committee Chair: Dr. Thomas E. Toth
Committee Members:Dr. Janice Longstreth
Dr. David Moore
Dr. Merle Pierson
Keywords:Virology, Rotavirus, Enzyme, Inactivation
Date of defense:December 9, 1997
Availability:Release the entire work for Virginia Tech access only.
After one year release worldwide only with written permission of the student and the advisory committee chair.


DEVELOPMENT OF A NEW CLASS OF VIRAL DISINFECTANTS: ENZYMATIC INACTIVATION OF SA-11 ROTAVIRUS. Shawn Walker, Thomas E. Toth. Virginia-Maryland Regional College of Veterinary Medicine, Department of Biomedical Sciences and Pathobiology. The non-enveloped, pH- and heat resistant rotavirus (RV), which is cross species-infective among cattle, swine and humans may cause dehydration and high mortality in the young. Rotaviruses are inactivated only by corrosive and toxic disinfectants. In this study, the effects of bacterial proteases as a new type of disinfectants on simian rotavirus (SA-11) were analyzed. SA-11 rotavirus replicates in cells causing cytopathic effect (CPE) and is similar in protein composition to cattle and swine RV. Preliminary experiments tested the temperature and pH sensitivity of SA-11 rotavirus. At pH 8.5, 45C was the highest temperature at which no loss in viral titer was seen, and the virus was still infective following treatment at 65C for 2 hrs. pH sensitivity tests were then conducted for two hours at 45C, with pH 5 being the lowest and pH 8.5 being the highest at which no loss in titer was observed. Four proteases were then tested for effectiveness at inactivating SA-11 rotavirus at their pH optimal at 45C. Alcalase was selected as the most efficient protease. Alcalase was found to inactivate SA-11 at 25C, and pH 8.5 in 3 days, indicating that enzymes were relatively effective at lower temperatures. SA-11 rotavirus virus was then tested for sensitivity to pH at 25C and 15C in absence of enzyme. At pH 2, 25C a ~4 log reduction was seen following 15 min of treatment, with viable virus still remaining after 8 hrs, at 15C a ~1.75 log reduction was seen following 2 hrs, and a ~4 log reduction following 8 hrs of treatment. At pH 4 and 6, at 25C and 15C no effect on SA-11 titer was seen after 120 hrs treatment. The enzyme was then tested at 1.0% and 0.1% enzyme concentration, at 15C and 25C, and pH 6 to determine efficacy of enzyme at sub-optimal conditions. Following treatment with 1.0% Alcalase at 25C a ~3.25 log reduction, and at 15C, 1.0% Alcalase, a ~1.75 log reduction was seen at 120 hrs. At 15C, 1.0% Alcalase a ~1.75 log reduction was seen at 120 hrs. Treatment with 0.1% Alcalase at 25C, pH 6 resulted in ~2.25 log reduction after 120 hrs. At 15C, 0.1% Alcalase a ~1.25 log reduction was seen following 120 hrs. The results showed that proteases, used as viral disinfectants, were not as effective at inactivating rotaviruses under simulated field conditions as originally hoped, nevertheless the ease of application and moderate but definite efficacy against rotaviruses may help reduce rotaviral infections and severity of clinical signs in young animals.

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