Discovery of a unique novel clade of mosquito-associated bunyaviruses.

Bunyaviruses are the largest known family of RNA viruses, infecting vertebrates, insects, and plants. Here we isolated three novel bunyaviruses from mosquitoes sampled in Côte d'Ivoire, Ghana, and Uganda. The viruses define a highly diversified monophyletic sister clade to all members of the genus Orthobunyavirus and are virtually equidistant to orthobunyaviruses and tospoviruses. Maximal amino acid identities between homologous putative proteins of the novel group and orthobunyaviruses ranged between 12 and 25%. The type isolates, tentatively named Herbert virus (HEBV), Taï virus (TAIV), and Kibale virus (KIBV), comprised genomes with L, M, and S segments of about 7.4 kb, 2.7 kb, and 1.1 kb, respectively. HEBV, TAIV, and KIBV encode the shortest bunyavirus M segments known and did not seem to encode NSs and NSm proteins but contained an elongated L segment with an ∼500-nucleotide (nt) insertion that shows no identity to other bunyaviruses. The viruses replicated to high titers in insect cells but did not replicate in vertebrate cells. The enveloped virions were 90 to 110 nm in diameter and budded at cellular membranes with morphological features typical of the Golgi complex. Viral RNA recovered from infected cells showed 5'-terminal nontemplated sequences of 9 to 22 nt, suggestive of cap snatching during mRNA synthesis, as described for other bunyaviruses. Northern blotting identified RNA species of full and reduced lengths, suggested upon analogy with other bunyaviruses to constitute antigenomic-sense cRNA and transcript mRNAs, respectively. Functional studies will be necessary to determine if this group of viruses constitutes a novel genus in the bunyavirus family.

Cathepsin D deficiency induces cytoskeletal changes and affects cell migration pathways in the brain.

Cathepsin D deficiency is a fatal neurodegenerative disease characterized by extreme loss of neurons and myelin. Our previous studies have demonstrated that structural and functional alterations in synapses are central to the disease pathogenesis. Therefore, we took a systematic approach to examine the synaptic proteome in cathepsin D knock-out mice, where the synaptic pathology resembles that of human patients. We applied quantitative mass spectrometry analysis on synaptosomal fractions isolated from cathepsin D knock-out and control mice at the age of 24 days. From the approximately 600 identified proteins, 43 were present in different amounts (P<0.05, measured in triple biological replicates) in cathepsin D knock-out mice compared to controls. We connected and bridged these 43 proteins using protein interaction data, and overlaid the network with brain specific gene expression information. Subsequently, we superimposed the network with Gene Ontology, pathway, phenotype and disease involvement, allowing construction of a dynamic, disease-protein centered network and prediction of functional modules. The measured changes in the protein levels, as well as some of the bioinformatically predicted ones, were confirmed by quantitative Western blotting or qualitative immunohistochemistry. This combined approach indicated alterations in distinct cellular entities, previously not associated with the disease, and including microtubule associated cytoskeleton and cell projection organization. Cell spreading and wound healing assays confirmed strongly compromised spatial orientation, associated with changes in distribution of focal adhesions and integrin assembly, in cathepsin D deficient cells. These changes might contribute to commencement of synaptic alterations and neuronal degeneration observed in cathepsin D deficiency.