Project description:Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS).
Project description:Transcriptional analysis of global gene expression changes in naïve subjects in response to smallpox vaccination with either DryVax or the replication-competent, attenuated LC16m8 vaccinia virus.
Project description:Smallpox is a highly communicable, often fatal diseae. There is currently no licensed treatment for smallpox and vaccinia virus (VV) is currently used for immunization. While immunization with VV can provide good protection against exposure to the smallpox virus, the current vaccine is far from optimal. Complications occur in 1/1,000-1/10,000 vaccinees, with at least one death per million vaccinees. We have constructed recombinant VV strains which are less pathogenic, yet provide a protective immune response. These viruses contain various mutations in the E3L which is known to block the host antiviral response. Identifying the host genes involved in producing a strong protective immunological response to these attenuated viruses would not only increase our understanding of the proteins and pathways involved in effective smallpox vaccination, but aid in the development of alternative vaccine strains which enhance these specific immune responses. We will determine gene expression patterns in HeLa cells at various times following infection with wtVV and several VV constructs containing mutations in the E3L gene. The VV E3L gene product blocks the host antiviral response by sequestering viral danger signals, including double-stranded RNA and Z-DNA. VV constructs containing mutations in E3L which allow host cell recognition of either of these danger signals leads to a decrease in viral pathogensis. In this project we will dissect the cellular inflammatory response to infection with wtVV in comparison to VV containing mutations in the E3L gene. By understanding why certain strains of VV are non-pathogenic, yet highly immunogenic, it is possible to gain a better understanding on the mechanisms of poxvirus pathogenesis and the host response. We will examine three times points following infection with VV: 2 HPI, 6 HPI and 9 HPI. These times points represent keys points in the virus replication cycle. Several VV constructs will be used which contain mutations in the E3L gene. These constructs alter the ability of E3L to sequester double-stranded RNA and/or Z-DNA and therefore have a direct effect on viral pathogenesis. Fourteen constructs will be used including: mock, wtVV, VVdelE3L, VVE3Ldel83N, VVE3Ldel37N, VVE3Ldel26C, VVE3Ldel7C, VVE3L Y48A, VVE3L P63A, VVE3L K167T, VV-ATV, VV-ADAR/E3L, VVdelK3L, VVdelK3L-E3Ldel37N. Cells will be infected at an MOI of 5 to allow infection of all cells. At each time point, cells will be harvested by scraping. RNA will be isolated using a Trizol RNA extraction protocol (Invitrogen) followed by RNA purification using the RNeasy cleanup kit available from Qiagen.
Project description:Successful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a ‘take’. Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia would reveal differences between takes and no-takes that may help better understand the phenomenon. Using vaccinia proteome microarrays and recombinant protein ELISAs we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication competent smallpox vaccines, DryVax® and APSV. Most that received diluted vaccine failed to respond, whereas 4 other no-takes receiving diluted vaccine, and 4 receiving undiluted vaccine, mounted an antibody response. Interestingly, their antibody profiles were not significantly different from controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax® that was significantly different from takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with pre-existing immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination. Antibody profiling was peformed on sera from vaccinees receiving smallpox vaccines, either Wyeth DryVax or Aventis Pasteur Vaccine ('WetVax') in NIH-sponsored clinical trials. These samples comprise 23 vaccinia naïve no-takes, and 50 vaccinia naïve takes and 25 previoulsy vaccinated takes as controls. Samples from d0 (day of vaccination) and at the peak of the antibody repsonse (d28) were taken from each donor and probed in triplicate against the proteome arrays.
Project description:Fur is known from contemporary written sources to have been a key commodity in the Viking Age. Nevertheless, the fur trade has been notoriously difficult to study archaeologically as fur rarely survives in the archaeological record. In Denmark, fur finds are rare and fur in clothing has been limited to a few reports and not recorded systematically. We were therefore given access to fur from six Danish high status graves dated to the Viking Age. The fur was analysed by aDNA and palaeoproteomics methods to identify the species of origin in order to explore the Viking Age fur trade. Endogenous aDNA was not recovered, but fur proteins (keratins) were analysed by MALDI-TOF and LC-MS/MS. We show that Viking Age skin clothing were often composites of several species, showing highly developed manufacturing and material knowledge. For example, fur was produced from wild animals while leather was made of domesticates. Several examples of beaver fur were identified, a species which is not native to Denmark, and therefore indicative of trade. We argue that beaver fur was a luxury commodity, limited to the elite and worn as an easily recognisable indicator of social status.