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A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.

Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE.We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy.Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects.17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13.The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.

Black Creek Canal (BCC) virus is a newly identified hantavirus from Florida which is carried by the cotton rat (Sigmodon hispidus) and is associated with hantavirus pulmonary syndrome (HPS). We have investigated the interaction of BCC virus with polarized epithelial cells to examine whether entry and release of this virus occur at specific plasma membrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluorescence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC virus infection. In contrast, Vero C1008 cells infected basolaterally exhibited a barely detectable level of BCC virus-synthesized proteins. Titration of virus from apical and basolateral media of infected cells has demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the basolateral media. The site of BCC virus release from polarized cells is, therefore, different from that previously described for release of other members of the family Bunyaviridae and may reflect one of the determinants of hantavirus pathogenesis. In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the apical surface of polarized cells. Electron microscopy studies of the infected cells revealed evidence of BCC virus budding at the plasma membrane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Since the same site of virus assembly was recently described for Sin Nombre virus, it is likely that all of the new American hantaviruses associated with HPS utilize this same type of virus maturation.

SummaryRequirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for a lymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.

Analysis of a limited number of laboratory strains of rabies virus had demonstrated the presence of a genome region bounded by two transcription termination and polyadenylation-like (TTP) signals (approximately 400 to 450 nucleotides apart) which was located between the end of the glycoprotein (G) coding sequence and the beginning of the L polymerase coding sequence. Although this region had been suggested to represent a remnant or pseudogene (~), no detailed analysis had been carried out to examine this possibility. Here we present the nucleotide sequence analysis of this genome region for several laboratory rabies virus strains and a large number of diverse rabies viruses detected directly in brain tissue of naturally infected animals, Only one distinct lineage of the laboratory strains and none of the wild-type rabies viruses contained the upstream -I-IP-like signal, indicating that only the downstream -I-IP motif is the authentic G mRNA transcription termination and polyadenylation and signal. Phylogenetic analysis of sequence differences provided no evidence of laboratory strains containing the two -I-IP-like signals being ancestral to any of the viruses possessing only the downstream -I-IP sequence motif. These data indicate that this region of the rabies virus genome encodes a G mRNA with a long 3 ' noncoding region with no evidence of a pseudogene. Rabies virus is a member of the Lyssavirus genus of the Rhabdoviridae family, a diverse group of animal, plant, and insect viruses, including vesicular stomatitis virus (VSV), rabies and related viruses, infectious hematopoietic necrosis virus (IHNV), and bovine ephemeral fever virus (BEFV) (1, 2). These viruses all possess nonsegmented, negative-sense, single-stranded RNA genomes of approximately 10 to 15 kb in length. The virus genomes commonly encode at least five mRNAs, which are located and also sequentially transcribed in the general pattern 3'-nucleocapsid protein (N), polymerase-associated phosphoprotein (P or M1), matrix protein (M or M2), glycoprotein(s) (G), and large polymerase protein (L)-5'. These genomes usually contain very little noncoding nucleotide sequence and gene junctions are small. The most common exception to this overall genetic structure is the genome region located between the G mRNA termination codon and the L mRNA initiation signal. In IHNV, an additional gene encoding a nonstructural protein (NV) is located in between the G and L genes (3). BEFV contains an additional nonstructural glycoprotein (GNs) in this genome area that has been proposed to have evolved by duplication of the original giycoprotein gene (4). Further, other small open reading frames