Intraluminal proteolytic activation plays an important role in replication of type 1 reovirus in the intestines of neonatal mice.

Oral inoculation of suckling mice with reovirus serotype 1 (strain Lang) results in the conversion of intact virions to intermediate subviral particles (ISVPs) in the intestinal lumen. Digestion of virus in vitro with chymotrypsin or trypsin reveals two distinct forms of ISVPs, while the predominant species of ISVPs found in the small intestinal lumen appears to be identical to the chymotrypsin product. The in vivo conversion of virions to ISVPs was blocked by pretreatment of mice with protease inhibitors, resulting in inefficient replication of reovirus in intestinal tissue. The early inhibition of viral replication in suckling mice pretreated with protease inhibitors was not observed when suckling mice were inoculated with ISVPs generated by in vitro digestion with either chymotrypsin or trypsin. However, replication was decreased during secondary rounds of replication in mice receiving repeated doses of protease inhibitors, suggesting that luminal proteolytic digestion is important in rendering progeny virions infectious in the gut.     

Sequence comparisons of developmentally regulated collagen genes of Caenorhabditis elegans

Collagen genes col-6, col-7 (partial), col-8, col-14 and col-19 from the nematode Caenorhabditis elegans were sequenced, and compared to the previously sequenced genes col-1 and col-2. The genes are between 1.0 and 1.2 kb in length, and each includes one or two short introns. The presumptive promoter regions contain sequences similar to the eukaryotic TATA promoter element. Two distinct, conserved sequences were found in the presumptive promoter regions of, respectively, the dauer larva-specific genes col-2 and col-6, and the primarily adult-specific genes col-7 and col-19. The domain structures of the collagen polypeptides are similar: each polypeptide contains two triple-helix forming (Gly-X-Y)n domains, one of 30-33 amino acids (aa), and the other of 127-132 aa. The latter domain is interrupted by one to three short (2-8 aa) non-(Gly-X-Y)n segments that occur at relatively conserved locations in each polypeptide. Sets of cysteine residues flank the (Gly-X-Y)n domains in all of the polypeptides. The genes can be placed into three families based upon amino acid sequence similarities. Genes within a family do not always exhibit similar developmental expression programs, suggesting that structural and regulatory regions of the genes have evolved separately. The codon usage in the genes is highly asymmetrical, with adenine appearing in the third position of 85% of the glycine codons, and 93% of the proline codons.     

Growth and survival of reovirus in intestinal tissue: role of the L2 and S1 genes.

Reovirus serotype 1 Lang can be recovered in high titer from the intestines of neonatal mice up to day 8 after peroral inoculation. By contrast, reovirus serotype 3 Dearing cannot be recovered from intestinal tissue past day 4 after peroral inoculation. This difference between the two reoviruses was mapped by using reassortants generated from nonmutagenized laboratory stocks. When the L2 and S1 genes of reovirus serotype 3 Dearing were present in reassortants, the reassortants behaved like serotype 3 Dearing in exhibiting a decreased capacity to be recovered from intestinal tissue. Likewise, viruses which contained the L2 and S2 genes from serotype 1 Lang exhibited an enhanced capacity to grow and survive, which is characteristic of serotype 1 Lang. Thus, the capacity of reovirus to survive in intestinal tissue was determined by the L2 and S1 genes.     

Derivation and characterization of an efficiently myocarditic reovirus variant.

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.     

gm: a practical tool for automating DNA sequence analysis

The gm (gene modeler) program automates the identification ofcandidate genes in anonymous, genomic DNA sequence data, gmaccepts sequence data, organism-specific consensus matricesand codon asymmetry tables, and a set of parameters as input;it returns a set of models describing the structures of candidategenes in the sequence and a corresponding set of predicted aminoacid sequences as output, gm is implemented in C, and has beentested on Sun, VAX, Sequent, MIPS and Cray computers. It iscapable of analyzing sequences of several kilobases containingmulti-exon genes in >1 min execution time on a Sun 4/60. Received on December 4, 1989; accepted on February 28, 1990     

A METHOD FOR ISOLATION OF CAMPYLOBACTER SPP. (JEJUNI, COLI AND LARI) FROM SHELLFISH AND THE MARINE ENVIRONMENT

A method has been developed to detect thermophilic species of Campylobacter in shellfish, marine and tributary waters, sediment and farm runoff by-products such as manure and silage. The method consists of a 48 h enrichment incubation and subcultured to selective agars. Presumptive colonies confirmed with a latex agglutination (antibodies) to common flagellar antigens of C. jejuni, C. coli and C. lardi. Over an 8 year period, West Coast estuaries (Washington, Oregon, and California) were sampled, resulting in analysis of a total of 512 samples. Results suggest that Campylobacter spp. are well distributed in the marine environment. Two enrichment broths were compared for the recovery of campylobacters from environmental samples. The method described in the Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) (1984), was compared to a modified method. Use of the modified method described here resulted in higher recovery rates of Campylobacter spp. Recoveries of campylobacters from sediment, shellfish, and water were 10,13, and 28% higher for the modified method, respectively.     

Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2.

We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae. This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2. Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution. Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts. Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2. Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding. Thus, we implicate specific residues of p53 in different DNA and protein interactions.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

Interference following mixed infection of reovirus isolates is linked to the M2 gene.

Following infection by pairs of reovirus isolates consisting of combinations of reovirus T1 Lang, T2 Jones, or T3 Dearing, we found that one of the isolates interfered with the yield of progeny RNA derived from the other parents. The most significant interference was produced by T2 Jones or T3 Dearing, when mixed with T1 Lang. Genetic analysis revealed that the presence of the M2 gene in the interfering parent (in the T1 Lang x T3 Dearing pair) was linked to interference. Studies on interference in infected cells indicated that interference occurs after adsorption and penetration.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.