Development of methodology based on commercialized SERS-active substrates for rapid discrimination of Poxviridae virions.

Surface-enhanced Raman spectroscopy (SERS) can be made an attractive approach for identification of Raman-active compounds and biological materials (i.e., toxins, viruses, or intact bacterial cells/spores) through development of reproducible, spatially uniform SERS-active substrates. Recently, reproducible (from substrate-to-substrate), spatially homogeneous (over large areas) SERS-active substrates have been commercialized and are now available in the marketplace. We have utilized these patterned surfaces to acquire SERS spectral signatures of intact bovine papular stomatitis, pseudocowpox, and Yaba monkey tumor viruses. Salient spectral signature features make it possible to discriminate among these genetically distinct Poxviridae-Chordopoxvirinae virions. In addition, partial least-squares, a multivariate calibration method, has been used to develop personal computer-borne algorithms useful for classification of unknown Parapoxvirus (e.g., bovine papular stomatitis virus and pseudocowpox virus) samples based solely on SERS spectral signatures. To our knowledge, this is the first report detailing application of these commercial-off-the-shelf (COTS) SERS-active substrates to identification of intact poxviruses.

Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus.

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqManchemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens (n=29) associated with rash-causing illnesses were not detected using this assay.

What is your diagnosis?: Multifocal subcutaneous tumors in a young male baboon.

A juvenile male baboon (Papio cynocephalus anubis), while being held in quarantine prior to research assignment at the Washington National Primate Research Center (WaNPRC), presented with three soft tissue masses on the right elbow, wrist, and knee on routine physical examination. The masses were subcutaneous and did not appear to cause any discomfort to the animal. An excisional biopsy of the knee mass was submitted for histopathologic analysis and found to be poorly defined, rapidly growing, and of undetermined histogenesis. Euthanasia was elected, and a full necropsy done with samples collected for histopathologic analysis. By immunohistochemistry, the "large" cells in the masses were suggestive of a mesenchymal, nonmacrophage origin because of their being positive for vimentin and negative for cytokeratin and the macrophage maturation marker CD68. Eosinophilic intracytoplasmic inclusions were seen on HE-stained sections and corresponded to aggregates of ovoid to brick-shaped viral particles on transmission electron microscopy (TEM), which proved to be the key diagnostic tool for this case. The masses were determined to be Yaba monkey tumor virus (YMTV)-induced "benign histiocytomas" by TEM.

A Yaba-like condition in a young baboon (Papio anubis).

A rapidly developing proliferation of subcutaneous nodules on the leg of a young baboon is described. Histologically the nodules consisted of plump round cells, many of which contained intracytoplasmic eosinophilic viral-like inclusions.

Burkitt's lymphoma.

Clinical and epidemiologic features of Burkitt's lymphoma are reviewed. Epidemiologic studies suggest that simultaneous infection with Epstein-Barr (E-B) virus and malaria may be involved as etiologic agents. On the other hand we have found that in the Amazon region of Brazil and Peru both malaria and E-B virus infection is common among children, yet Burkitt's lymphoma is rare. The possibility exists that other concomitant etiologic agents and genetic factors are also involved. Several investigators suggested the possible involvement of Reo 3 virus. We have found antibodies against Yaba virus. A laboratory worker who accidentally inoculated himself with Yaba virus developed a histiocytoma which when inoculated into Asiatic monkeys produced typical Yaba tumors. This was the first case that Koch's postulates were fulfilled in a virus induced neoplasm in man. Therapeutically, the best clinical results were obtained in those patients who were treated with small doses of cyclophosphamide. On the basis of somewhat inadequate follow-up studies, it is estimated that "five year cures" were obtained in about 10% of the patients.

Experimental infection of pheasants with Lednice (Yaba 1) virus.

Young pheasants (24-hour- and 7-day-old) are capable of producing viraemia and antibodies after experimental infection with Lednice virus even after low doses of virus (0.7-0.9 log mouse LD50/g). Viraemia lasts approximately 3 days, but the titres of virus in the blood are low. The possible role of young pheasants in the circulation of virus in nature is discussed.

Experimental infection of Macaca mulatta monkeys with Lednice (Yaba 1) virus.

Macaca mulatta monkeys were inoculated with Lednice (Yaba 1) virus strain 6118. The animals developed no clinical signs and the body temperature and values of complete haematological examination remained within normal limits. In spite of positive immunofluorescence in regional lymph nodes, no virus could be recovered from them by passages in mice. Viraemia was not demonstrated. The antibody response was directly proportional to the amount of virus inoculated, i.e. it was significant after a higher virus dose or after repeated inoculation. The results obtained are discussed in respect of and in comparison with serological findings in man.

Studies on viremia and antibody formation in ducklings and goslings after experimental infection with Lednice (Yaba 1) virus.

A very low virus level was found for a short time in the blood of ducklings and goslings after experimental Lednice (Yaba 1) virus infection by subcutaneous (scut) and intranasal (inas) route and in blood of ducklings also after infection by intracerebral (icer) route. Subsequently virus neutralizing antibody formation was examined. Antibody formation was found in most of animals. In goslings onset of antibody formation seems to be slower than in ducklings after the same virus infection dose; vice versa, in ducklings antibody titer decreased after 6 weeks. Using low infection virus dose, neutralizing antibody level in ducklings was lower than in the former case. On examining the blood and the organs, the virus was detected in a very low amount and for a very short time in blood and spleen of goslings and only in blood of ducklings.

Immunodiffusion analysis of Yaba poxvirus structural and associated antigens.

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.

Some physiochemical properties of Yaba poxvirus deoxyribonucleic acid.

Yaba tumor poxvirus deoxyribonucleic acid (DNA) has a density of 1.6905 in CsCl and its T(m) value in 0.015 m citrate in saline is 82.3. The guanine plus cytosine content estimated from these properties was taken to be 32.5 +/- 0.5%, a value 2 to 3% less than for DNA from other poxviruses reported to date.