In vivo experimentation with simian herpesviruses: assessment of biosafety and molecular contamination.
In vivo studies with highly pathogenic viruses prompt concerns regarding the persistence of infectious virus in pathology specimens. Although formalin fixation of tissues may inactivate infectious virus, fixation may also degrade viral nucleic acid and antigens, thereby limiting detection of virus in tissues by polymerase chain reaction (PCR) amplification or immunohistochemistry (IHC). We sought to: 1) assess the rate of inactivation of infectious virus in tissue specimens during formalin fixation, 2) assess IHC recognition of viral antigens and PCR detection of viral DNA after long-term (14 d) formalin fixation, and 3) investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Infectious baboon herpesvirus HVP2 could be recovered from fresh tissues of infected mice but not those fixed in formalin for >/=24 h. The intensity of IHC staining of viral antigen was unaffected by the duration of formalin fixation. PCR detection of viral DNA was negatively impacted by formalin fixation and/or heat inherent to paraffin processing; however, amplification of very short DNA sequences using real-time PCR was not affected. Lastly, microtome contamination by viral DNA was demonstrated by PCR screening of uninoculated control tissues that were sectioned after sectioning infected tissues. In summary, infectious virus is inactivated after only 24 h of formalin fixation whereas IHC staining remains sensitive in tissues fixed for up to 14 d. Formalin fixation does degrade DNA, but viral DNA can be detected by PCR amplification of very short DNA sequences. In addition, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive PCR amplification.
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA. jerry.ritchey@okstate.edu
Jerry W
JW
Ritchey
Darla H
DH
Black
Kristin M
KM
Rogers
Richard
R
Eberle
D000818:Animals; D000830:Animals, Laboratory; D000956:Antigens, Viral / Q000032:analysis*; D001933:Brain Stem / Q000737:chemistry / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D004866:Equipment Contamination; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006568:Herpesvirus 1, Cercopithecine / Q000235:genetics / Q000276:immunology / Q000502:physiology; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D018139:Simplexvirus / Q000235:genetics / Q000276:immunology / Q000502:physiology; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods / Q000592:standards; D038301:Virus Inactivation
D000956:Antigens, Viral; D004279:DNA, Viral; D005404:Fixatives; D005557:Formaldehyde