Clinical laboratories, the select agent program, and biological surety (biosurety).

The threat of bioterrorism has led to increased concerns over the availability of biological select agents and toxins (BSAT). Congress has implemented several public laws that have led to the development of federal regulations by the Centers for Disease Control and Prevention (CDC) and the US Department of Agriculture. The CDC regulation 42 CFR 73 has a direct impact on all clinical laboratories that may at some time identify BSAT in a clinical specimen. The Department of Defense has imposed a more stringent layer of regulation called biological surety (biosurety) on top of the requirements of 42 CFR 73 for military laboratories that possess BSAT. However,42 CFR 73 falls into the framework of biosurety. Both sets of regulations have four pillars (safety, physical security, agent account-ability, and personnel reliability) that are built on a foundation of training and covered by a roof of management (operations and plans).

Filovirus-like particles as vaccines and discovery tools.

Ebola and Marburg viruses are members of the family Filoviridae, which cause severe hemorrhagic fevers in humans. Filovirus outbreaks have been sporadic, with mortality rates currently ranging from 30 to 90%. Unfortunately, there is no efficacious human therapy or vaccine available to treat disease caused by either Ebola or Marburg virus infection. Expression of the filovirus matrix protein, VP40, is sufficient to drive spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. The addition of other filovirus proteins, including virion proteins (VP)24, 30 and 35 and glycoprotein, increases the efficiency of VLP production and results in particles containing multiple filovirus antigens. Vaccination with Ebola or Marburg VLPs containing glycoprotein and VP40 completely protects rodents from lethal challenge with the homologous virus. These candidate vaccines are currently being tested for immunogenicity and efficacy in nonhuman primates. Furthermore, the Ebola and Marburg VLPs are being used as a surrogate model to further understand the filovirus life cycle, with the goal of developing rationally designed vaccines and therapeutics. Thus, in addition to their use as a vaccine, VLPs are currently being used as tools to learn lessons about filovirus pathogenesis, immunology, replication and assembly requirements.

Association of ebola virus matrix protein VP40 with microtubules.

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization.

Professional antigen-presenting dendritic cells (DCs) are critical in regulating T cell immune responses at both systemic and mucosal sites. Many Lactobacillus species are normal members of the human gut microflora and most are regarded as safe when administered as probiotics. Because DCs can naturally or therapeutically encounter lactobacilli, we investigated the effects of several well defined strains, representing three species of Lactobacillus on human myeloid DCs (MDCs) and found that they modulated the phenotype and functions of human MDCs. Lactobacillus-exposed MDCs up-regulated HLA-DR, CD83, CD40, CD80, and CD86 and secreted high levels of IL-12 and IL-18, but not IL-10. IL-12 was sustained in MDCs exposed to all three Lactobacillus species in the presence of LPS from Escherichia coli, whereas LPS-induced IL-10 was greatly inhibited. MDCs activated with lactobacilli clearly skewed CD4(+) and CD8(+) T cells to T helper 1 and Tc1 polarization, as evidenced by secretion of IFN-gamma, but not IL-4 or IL-13. These results emphasize a potentially important role for lactobacilli in modulating immunological functions of DCs and suggest that certain strains could be particularly advantageous as vaccine adjuvants, by promoting DCs to regulate T cell responses toward T helper 1 and Tc1 pathways.

The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22.

The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.