Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED 01 FEB 2008 N/A - 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Assay for and replication of Karshi (mammalian tick-borne flavivirus 5b. GRANT NUMBER group) virus in mice. American Journal for Tropical Medicine and Hygiene 78:344-347 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Turell, MJ Whitehouse, CA Butler, A Baldwin, C Hottel, H Mores, CN 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION United States Army Medical Research Institute of Infectious Diseases, REPORT NUMBER TR-07-049 Fort Detrick, MD 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Little is known about the replication of Karshi virus, a member of the mammalian tick-borne flavivirus group, in its rodent hosts. Therefore, we developed a novel quantitative real-time RT-PCR assay and measured the amount of viral RNA in selected tissues of infected Swiss Webster mice. Two-day-old mice were highly susceptible, with 100% fatality between days 9 and 12 after infection, while 9-day-old mice were less susceptible with death occurring only rarely. In nearly all cases, mice inoculated when 2-days old contained similar numbers of viral genome equivalents from blood and liver samples from any given mouse, with titers declining after day 7. In contrast, the amount of viral RNA in the brain began to rise rapidly 4 days after exposure, peaked at about 6 days after virus exposure with a titer of >1013 genome equivalents/g, and remained at that level until euthanasia or death. Viral profiles were similar in needle-inoculated or tick-exposed mice 15. SUBJECT TERMS Karshi virus, replication, flavivirus, tick-borne, RT-PCR, laboratory animals, mice 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE SAR 4 unclassified unclassified unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18