Detection of hepatitis A virus proteins in infected BS-C-1 cells.

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its tropism for the liver in infected hosts, a nonlytic infection in hepatocytes, and a slow and nonlytic growth cycle in cultured cells. Although the genome structure and organization of HAV appear to be similar to those of the other picornaviruses, the viral proteins synthesized in infected cells have not been previously characterized. We have utilized specific antisera raised in rabbits to recombinant HAV proteins expressed in Escherichia coli in an effort to identify both structural and nonstructural proteins in BS-C-1 cells throughout the course of a viral replication cycle. Replication was monitored by dot blot hybridization of viral genomes. Structural proteins VP0, VP1, VP2, and VP3 were found to accumulate during the infection cycle as did viral RNA. Nonstructural proteins 2C and 3D were not detected on immunoblots, although a minor amount of 2C could be detected by immunoprecipitation of lysates of radiolabeled, infected cells. The relative sensitivities of the various antisera were determined, and the failure to observe nonstructural proteins was shown not to be due to decreased sensitivity of the detection reagents. Thus, it appears that HAV nonstructural proteins do not accumulate in infected cells to levels comparable to those of capsid proteins.

The use of neutrophils, macrophages and organ cultures to assess the penetration of human cells by antimicrobials.

Recent studies of pathogenic mechanisms of bacteria have revealed that in addition to traditional intracellular parasites such as Brucella spp. and Listeria, many other human pathogens reside inside cells during disease processes. Thus, studies of how well antimicrobials are delivered to and perform within phagocytic or endocytic vacuoles have become of increasing importance. Whereas studies of penetration of PMN neutrophils and macrophages have indicated whether antimicrobials penetrate the cytoplasm of human cells, studies in organ cultures can reveal whether antimicrobials enter phagosomes or endocytic vacuoles where the bacteria actually reside. Such information is probably more predictive of antimicrobial efficacy in naturally occurring intracellular infections than are data from studies with PMN's or macrophages.

Attachment factors amont enterotoxigenic Escherichia coli from patients with acute diarrhea from diverse geographic areas.

To cause diarrhea, enterotoxigenic Escherichia coli (ETEC) must initially colonize the small bowel. Different surface structures have been implicated in this initial attachment. Recognized attachment factors include colonization factor antigens I and II (CFA/I and CFA/II) and type I pili. Several methods of detection for each of these factors have been reported. In this study, we screened for the presence of these attachment factors among enterotoxigenic E. coli isolated from 40 patients with acute diarrhea and 40 asymptomatic control individuals and examined their ability to attach to ATCC 407 human intestinal cells in vitro. Of 40 patients with diarrhea, 16 (40%) had enterotoxigenic E. coli isolates which exhibited an attachment trait. Fourteen (35%) of these isolates demonstrated the ability to attach to ATCC 407 cells, whereas only four isolates from asymptomatic controls attached (P < 0.02). A total of 20% of the patient isolates and 7.5% of the control isolates possessed CFA/I. Only one patient isolate demonstrated CFA/II. Evidence for type I pili was found on 10% of the patient isolates and 12.5% of the control isolates. Attachment to ATCC 407 cells allowed the detection of 87.5% (14 of 16) of enterotoxigenic E. coli isolates with any type of attachment trait. Of the 14 cases demonstrating attachment ability to ATCC 407 cells, 7 did not attach in the presence of mannose. Three of these showed evidence for both CFA/I and type I pili, one showed only CFA/I, and one showed only type I pili. Two of those mannose-sensitive attaching isolates showed no other demonstrable trait. Seven patient isolates showed mannose-resistant attachment. Of these, two were classified as possessing CFA/I, and one was classified as possessing CFA/II. The four remaining isolates could not be classified into any recognized attachment factor category, suggesting that other attachment factors remain to be identified.