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Chronic Wasting Disease

June 14, 2002

Presolicitation Notice: Q-Lab Services for Transmissible Spongiform Encephalopathy Testing

Solicitation Number: APVSNVXX-0106-2

Wisconsin Viral Research Group (WVRG)
Business Size: 4 full time employees including the two owners

Prior or Current Collaborations with APHIS: None

Consulting Veterinary Pathologist: To be named if BPA is issued (WVRG is a human medical diagnostic laboratory with an M.D. clinical consultant as required by CLIA regulations. A veterinary pathologist will be identified and recruited when the need arises.)

Laboratory Capacity: With current personnel, facilities and other commitments, it can be estimated that WVRG could perform approximately 50 tests per day or 13,000 tests per year.

Price per test: The cost of each test will be $98.

Description of the Laboratory:
WVRG is a Clinical Laboratory Improvement Amendments (CLIA) certified (Identification Number: 52D0921062) clinical diagnostic laboratory with specialty certification for Virology and General Immunology.

With this certification comes assurance that WVRG will be able to:

Follow the QA policies and procedures established by the NVSL
Establish and follow procedures for the tracking of specimens
Establish procedures for the detection and correction of clerical and other errors that may occur
Establish procedures for the timely reporting of testing results including a monthly summary
Establish and maintain a system for the archiving of samples that will include immediate retrieval of individual, specific samples

The owners and co-directors of WVRG are Drs. Konstance Knox and Donald Carrigan (see attached biographical sketches).

Expertise in Immunohistochemical (IHC) Staining Procedures:
Drs. Knox and Carrigan have over twenty years of experience in the use of IHC staining for the diagnosis of disease in various tissues. Documentation of this expertise can be found in the partial list of relevant publications listed below.

  1. Carrigan DR and Johnson KP. (1980). Chronic relapsing myelitis in hamsters associated with experimental measles virus infection. Proc Natl Acad Sci 77:4297-4300.
  2. Toorkey CB and Carrigan DR. (1989). Immunohistochemical detection of an immediate early antigen of human cytomegalovirus in normal tissues. J Infect Dis 160:741-751.
  3. Finkle C, Tapper MA, Knox KK, and Carrigan DR. (1991). Coinfection of cells with human immunodeficiency virus and cytomegalovirus in lung tissue of patients with AIDS. J Acquired Immunodeficiency Syndromes 4:735-737.
  4. Russler SK, Tapper MA, Knox KK, Liepins A, and Carrigan DR. (1991). Pneumonitis associated with coinfection by human herpesvirus six and legionella in an immunocompetent adult. Amer J Pathol 138:1405-1411.
  5. Drobyski WR, Knox KK, Majewski D, and Carrigan DR. (1994). Fatal encephalitis due to variant B human herpesvirus 6 infection in a bone marrow transplant recipient. N Engl J Med 330:1356-1360.
  6. Knox KK and Carrigan DR. (1994). Disseminated active HHV-6 infections in patients with AIDS. Lancet 343:577-578.
  7. Knox KK, Pietryga D, Franciosi R, and Carrigan DR. (1995). Progressive immunodeficiency and fatal pneumonitis associated with HHV-6 infection in an infant. Clin Infect Dis, 20:406-413.
  8. Knox KK and Carrigan DR. (1995). Active human herpesvirus six (HHV-6) infection of the central nervous system in patients with AIDS. J Immune Defic Syndr and Hum Retrovir 9:69-73.
  9. Knox KK, Harrington D and Carrigan DR. (1995). Fulminant human herpesvirus six (HHV-6) encephalitis in an HIV infected infant. J Med Virol, 45:288-292.
  10. Rosenfeld CS, Weinbaum D, Carrigan DR, Knox KK, Andrews DF, and Shadduck RK. (1995). Late graft failure due to dual bone marrow infection with variants A and B of human herpesvirus 6. Exper Hematol, 23:626-629.
  11. MacKenzie I, Carrigan DR, and Wiley CA. (1995). Chronic myelopathy associated with human herpesvirus six. Neurology, 45:2015-2017.
  12. Knox KK and Carrigan DR. (1996). Chronic marrow suppression in a bone marrow transplant patient associated with persistent HHV-6 marrow infection. Clin Infect Dis, 22:174-175.
  13. Knox KK and Carrigan DR. (1996). Active HHV-6 infection in the lymph nodes of HIV infected patients: In vitro evidence that HHV-6 can break HIV latency. J Acquired Immune Defic Syndr and Hum Retrovirol, 11:370-378.
  14. Carrigan DR, Harrington D and Knox KK. (1996). Subacute leukoencephalitis caused by CNS infection with human herpesvirus six manifesting as acute multiple sclerosis. Neurology, 47:145-148.
  15. Knox KK, Brewer JH, Harrington DJ, Henry JM, and Carrigan DR. (2000). Human herpesvirus six and multiple sclerosis: systemic active infections in patients with early disease. Clin Infect Dis, 31:894-903.

While WVRG has had no direct experience with IHC staining of tissues from subjects with transmissible spongiform encephalopathies (TSE), this extensive experience will allow the immediate implementation of such staining. It should be noted that several of the publications listed above (numbers 1,5,6,8,9,11,14 and 15) describe studies in which IHC staining of central nervous system (CNS) tissues was used to diagnose viral associated disease. Further, one of the owners of WVRG (DRC) did a post-doctoral fellowship in Neurovirology in the laboratory of Dr. Richard Barringer at the University of California, San Francisco (see biographical sketch). Thus, WVRG has available to it sufficient neurological experience and expertise to successfully implement IHC staining for TSE disease in CNS tissues.

Laboratory Facilities:
The laboratory of WVRG consists of:

Private offices for Drs. Knox and Carrigan

A general office space (160 square feet) for use of employees and for specimen tracking and results reporting. This space includes FAX machines, computers with internet access, telephones, etc.

A large laboratory (520 square feet) containing:

a large sink with adjacent open bench space
a six foot, biosafety level two laminar flow hood
two table top refrigerated centrifuges
2 CO2 incubators
2 non-CO2 incubators
4 foot PCR sample preparation hood
2 microcentrifuges
2 -70oC freezers
2 -20o freezers
standard refrigerator
various water baths, heating blocks, etc.
an AO microscope and a Zeiss photomicroscope
an inverted microscope
a pH meter
a scale for weighing chemicals for buffers
18 square feet (2 ft by 9ft) of available lab bench area
14 square feet (2 ft by 7ft) of available lab bench area

A smaller laboratory (200 square feet) containing:

a sink with adjacent open bench space
a four foot, biosafety level 2 laminar flow hood
2 Hybaid thermocyclers
a Stratagene Robocycler
2 agarose gel apparatuses
an UV light box with photo documentation system
a microplate washer
an EIA reader
1 microcentrifuge
a 37oC incubator
a -20o upright freezer
an upright refrigerator/freezer
a Zeiss fluorescent microscope
a PCR work station with UV light
14 square feet (2 ft by 7ft) of available lab bench area

A 150 square foot storage room containing floor to ceiling storage shelves. Approximately 100 square feet of this room would be available for storage of brain specimens and slides.

Currently, we are not prepared for the dehydration, paraffin embedding and sectioning of formalin fixed tissues since our histological work is performed under subcontract with a nearby hospital laboratory. If we receive a BPA, we will purchase:

a Labconco Paramount Filtered Enclosure for working with formalin fixed tissues and the various solvents that will be required
a Leica EG 1160 Histoembedder Paraffin Embedding System
a Richard-Allan Scientific HM325 Autoretracting Rotary Microtome

These pieces of equipment will be dedicated solely to this project and will be located as a unit on the available open bench space in our larger laboratory.

With respect to expertise in the preparation of slides from formalin fixed, tissues, both Dr. Knox and Dr. Carrigan have extensive experience in histological sample preparation from their early work with measles virus infections of the CNS of golden Syrian hamsters. documentation of this experience can be found in the following papers:

  • Carrigan DR and Johnson KP. (1980). Chronic relapsing myelitis in hamsters associated with experimental measles virus infection. Proc Natl Acad Sci 77:4297-4300.
  • Johnson KP, Swoveland P, Norrby E, and Carrigan DR. (1981). Experimental subacute sclerosing panencephalitis: Measles matrix (M) protein selectively disappears during acute infection. J Infect Dis 144:161-169.
  • Carrigan DR. (1986). Round cell variant of measles virus: Neurovirulence and pathogenesis of acute encephalitis in newborn hamsters. Virology 148:349-359.
  • Carrigan DR. (1986). Round cell variant of measles virus: Mechanisms involved in the establishment of defective viral infection of the central nervous system. Virology 155:614-624.
  • Carrigan DR. (1987). Chronic, relapsing encephalomyelitis associated with experimental measles virus infection. J Med Virol 21:223-230.
  • Carrigan DR and Knox KK. (1990). Identification of interferon resistant subpopulations in several strains of measles virus: Positive selection by growth of the virus in brain tissue. J Virol 64:1606-1613.

Working with formalin fixed, prion infected tissues poses special concerns since the fixation does not inactivate the infectious prions. Dr. Carrigan gained experience in working with CNS and other tissues from patients with Creutzfeldt-Jacob Disease during his graduate work and post-doctoral fellowship at the University of California, San Francisco. Therefore, adequate expertise and experience exists to allow WVRG to safely and efficiently process the brain specimens and to perform the IHC staining assays.