This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To provide expert support to AIDS research conducted at the Primate Center. 1. PROGRESS AND CONCERNS The Virology Services Unit (VS) performed almost 4,000 SIV virus load determinations in FY2008. This is fewer than in FY2007, but we anticipate increased demand in FY2009 as established users plan more and larger experiments. In anticipation of this increased demand, we began a transition to the high-throughput Roche LightCycler 480 quantitative PCR platform in December 2008. This will facilitate an expansion of our services and will simplify batch processing of samples as we transition to charging all users (intra- and extramural) by the end of 2009. Our chargeback system for extramural investigators had 3 users and generated revenues of $5,691. VS also produced over 3,500 vials of high-titer SIVmac239 stock and 3 custom SIV mutants for WNPRC investigators. In response to investigators interested in evaluating the function of Nef protein variants, we developed a """"""""nef cassette"""""""" system that allows us to produce SIVmac239 variants that express nef alleles derived from viruses circulating in infected animals'plasma. Investigators performing vaccine studies are becoming increasingly interested in heterologous challenge viruses. To provide a heterologous challenge for vaccines based on SIVmac239, we produced a large-scale stock (over 450 vials) of the biological isolate SIVsmE660 on rhesus macaque PBMC. The Immunology Services Unit produced approximately 40,000 tetramer tests in 2008, 20,000 of which were shipped to outside investigators at a total of $59,755 in chargebacks. In 2009, we will develop MHC class II tetramers For extramural investigators 253 blood and 198 mucosal samples were processed and 554 sample vials were shipped out. IS also provides expert support to Primate Center and extramural investigators wishing to use our flow cytometry facilities. More than 2,500 hours were used for flow data acquisition during this year. These activities resulted in an additional $41,954 in chargebacks. In 2008 we purchased a new FACSCalibur machine to replace one of our old desktop analyzers. We are in the process of acquiring an additional custom-made BD-LSR II machine to expand our services. We have established or supported the development of several multicolor staining protocols. These include staining panels to identify antigen specific cell populations that recirculate into the lymph node, and show markers of exhaustion;panels that separate different antigen presenting populations, and panels that identify SIV infected cells ex vivo. To promote more objective data analysis in flow cytometry we have introduced and educated users for the use of compensation beads in multicolor staining. We have replaced our existing FACSDiva software with a more updated version, and also equipped our BD-LSR II with a system performance tracking software. 2. ALLOCATION OF RESOURCE ACCESS The central mission of IVS is to provide expert support to AIDS and infectious disease related research conducted at the Primate Center by WNPRC or outside investigators. In fiscal 2008 IS served 8 on-campus and 13 off-campus laboratories supported by both federal and non-federally funded grants. VS supported 4 on-campus and 3 off-campus users in FY2008. 3. DISSEMINATION We regularly update our website to facilitate the attraction of users. We request that projects utilizing Virology and Immunology Services acknowledge the service in manuscripts and presentations. Drs. Friedrich, Wilson and Rakasz, the PIs of VS and IS units, consult closely with users of the service, helping to design experiments and interpret results. 4. TRAINING Dr. Friedrich consults regularly with recognized leaders in SIV virology and molecular biology to develop and refine our techniques. For example, custom SIV mutagenesis methods were developed in collaboration with Dr. Ronald Desrosiers at the New England Primate Center. Quantitative RT-PCR techniques were developed in consultation with Dr. Jeffrey Lifson at the National Cancer Institute. Similarly, Dr. Rakasz has developed IS protocols and technologies in collaboration with Dr. Louis Picker at the Oregon Primate Center. IS staff have been trained in flow cytometry techniques at Beckton Dickinson.
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