2004 West Nile Bird Testing

1. 2004 West Nile Bird Testing

2. Oral Swab Collection • Collect specimens on birds from the family Corvidae (American Crows, Blue Jays) and birds of prey. • Collect only specimens on birds that are no more than 24 hours old with out signs of decay. • If the carcass is soft and mushy, has an obvious odor, skin discoloration, feathers or skin that rub off easily, and has insects or maggots DO NOT submit for testing. It is too old.

3. Oral Swab Collection Cont. • Submit specimens to OLS in the supplied boxes or a clean or unmarked box properly labeled with the shipping address and what kind of specimen it contains (diagnostic specimen oral swab). • Fill out the submission form COMPLETELY.

4. What To Send OLS • After you swab in oral cavity of the bird, place the swab into a vial of transport media. If needed break off the wooden shaft. Tightly cap the media tube, and label the tube with the unique ID number that matches the submission form. Unique ID should be obtained by calling IDEP at 1-800-423-1271 or 304-558-5358 • Place the tube of media into a biohazard bag that is in the kit. One tube per bag. • Place the bag (s) into the shipping box and mail to OLS. Please mail on the day of collection or refrigerate and mail the following day. Do not mail on Fridays.

5. What You Will Receive In A Kit From OLS When you order a kit from OLS you will receive in it the following: • A mailing box for up to 3 specimens • 3 tubes of media • 6 swabs • 3 parts of gloves • 3 bags for collection • 2 biohazard bags • 1 mask

6. Instructions for Sending Oral Swabs to WVDHHR/OLS for West Nile Virus Testing Test request form: REFRIGERATE the MEDIA UPON ARRIVAL until ready to use Please include a test request/submission form for each specimen. Fill out the form as completely as possible. Mailing container: Please package the specimen as directed below. DO NOT CARRY IT ON YOUR PERSON! After swabbing the oral cavity place the cotton tip of the swab into the provided transport media (one swab per tube of media). Break off the wooden end if needed. Tightly cap the media. Place the tube into the provided biohazard bag. Place this bag into the provided bubble wrap container and place up to 6 tubes in each box for mailing. Mail or return the box to OLS the same day as collection. If you can not mail the day of collection wait no longer then the next day refrigerate the specimen until shipped. Please DO NOT mail specimens on Fridays.

7. Instruction Forms OLS Containers: Our laboratory will supply containers and forms upon request by written requisition. Contact the West Nile section to receive supplies. Reporting of testing results: Test results will normally be available 4 days to 2 weeks after receipt of specimen. During periods of heavy submission, turn‑around times may be longer. Receipt of a hard copy of the results will be mailed within one day after completion of testing. Shipping address: West Virginia Department of Health & Human Resources Office of Laboratory Services/Arbovirus Testing 167 11th Avenue South Charleston, WV 25303 Please address questions to Wendy Hess at the Serology/Arbovirus Section at 304‑558‑3530 Ext. 2402.

8. Human Sample Submission Form WEST VIRGINIA DEPARTMENT OF HEALTH AND HUMAN RESOURCES Office of Laboratory Services (OLS) 167 11th Avenue South Charleston, WV 25303 Phone: (304) 558‑3530 FAX: (304) 558‑2006 ARBOVIRUS TEST SUBMISSION FORM Patient Name: Date of birth______/______/____AddressCity: County:State:______ Zip:____________ Date symptoms began ______/______/______ Recent Travel? Specimen Information Date serum collected ______/______/______Date CSF collected ______/______/______ Indicate the clinical syndrome(s): Was a lumbar puncture performed? Headache and fever Yes Total WBC count __________ Aseptic meningitis Protein __________________ Encephalitis No To whom should the results be forwarded: Name ___________________________________________ Address _________________________________________ __________________________________________ Fax _____________________ Phone __________________ (Hard copies of all results will be mailed to the above person.)

9. Human Specimen Collection Instructions Instructions for Sending Specimens to WVDHHR/OLS for West Nile Virus Testing Test request form: Please include a test request form for each specimen. Fill out the form as completely as possible. Testing will not be initiated without inclusion of the date of onset of symptoms and date each specimen was collected. Name of patient is required also. Mailing container: Please put your specimen in a substantial container. Wrap your specimen in absorbent material and pack it in 2 different plastic containers to insure that any leakage is contained. If you are requesting serology, there is no need to keep it frozen. If you are hand carrying specimens, please observe the packing instructions above to secure the specimens in transit. DO NOT CARRY IT ON YOUR PERSON! OLS Containers: Our laboratory will supply containers and forms upon request by written requisition. Contact the Container Room to receive supplies. Specimen types and amounts: Acute and convalescent specimens should be sent together whenever available. If one of these specimens is unavailable, we will accept single specimens for testing. Ideal timing of specimens is as follows: acute specimens should be taken 10 days after onset of symptoms. Convalescent specimens should be taken 2‑3 weeks after the acute sample. Keep in mind if the acute sample is taken prior to 10 days post‑onset of symptoms, antibodies may not have had time to develop and a false negative test may result. Testing for serology is done on serum and cerebrospinal fluid (CSF) specimens. Please send at least 0.5 ml of serum and at least 1 ml of CSF for proper testing to proceed. The larger amount of CSF specimen is required because these samples are routinely tested undiluted. Do not send whole blood. Reporting of testing results: Test results will normally be available 4 days to 2 weeks after receipt of specimen. During periods of heavy submission, turn‑around times may be longer. Receipt of a hard copy of the results will be mailed within one day after completion of testing. Initial serologic testing will be performed using IgM capture ELISA and IgG ELISA. If the initial results are positive, further confirmatory testing may delay the reporting of final results. Shipping address: West Virginia Department of Health & Human Resources Office of Laboratory Services/Arbovirus Testing 167 11th Avenue South Charleston, WV 25303 Contacts: Wendy Hess or Kirsten Snowden at the Serology/Arbovirus Section at 304‑558‑3530

10. LABORATORY TESTING

11. Laboratory TestingWest Nile Virus • Serological based diagnosis • Sample Type: Human clinical samples • Detection target: Antibody / Antigen • Molecular based diagnosis • Sample Type: Mosquitoes and Birds (Oral Swabs) • Detection Target: Nucleic Acid (RNA) *The most sensitive WNV detection assay for field-collected specimens is RNA detection

12. Molecular Diagnostics • Making copies of a specific portion of the target’s (ex. WNV) genome i.e. DNA or RNA • Different Methods • PCR (Usually DNA) • NASBA (Usually RNA) • NASBA • Nucleic Acid Sequence Based Amplification • Method (to be) used at OLS to diagnose WNV in birds and mosquitoes

13. How NASBA Works • Sample • Grind mosquitoes into a “mosquito milkshake” • Transport media containing oral swab (birds) • Add sample to lysis buffer • Inactivates the virus • Releases nucleic acids (RNA) • Denatures nucleases that destroy RNA • Stabilizes the RNA for processing or storage

14. How NASBA Works • Extraction and Isolation of the RNA • The NucliSens Extractor makes this an automated procedure • Some sample prep required • Addition of silica to bind RNA • Extractor performs a series of washes to “clean” and isolate the RNA • 45 minutes for 10 samples NucliSens Extractor

15. How NASBA Works • Amplification of the RNA • Make millions of copies of the RNA for detection • The more you have the easier it is to see (detect) • Requires primers to initiate the amplification • Short pieces of commercially made RNA that bind specifically to your target RNA (ex. WNV) • Just like priming a pump with a small amount of water

16. How NASBA Works • Detection of the RNA • Is it there? • NucliSens EasyQ Reader • Incubator and Analyzer • Automated • Real Time • Simultaneous amplification and detection • Molecular beacons • Similar to primers • Fluorescent attachment • 90 minutes for 96 WNV samples NucliSens EasyQ reader

17. Questions?