Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among travelers and residents of developing countries. ETEC produces either a heat-stable toxin or a heat-labile toxin, or both, encoded by plasmid-borne ST and LT genes, respectively. Diagnosis of infection with this subclass of E. coli can be performed with oligonucleotide hybridization probes; however, the sensitivity and specificity of this method are insufficient. A nonradioactive multiplex PCR assay that provides a sensitive and specific method for detecting the presence of either or both toxin genes has been developed. A simple procedure that removed inhibitors of the PCR while efficiently releasing ETEC DNA from stool specimens for subsequent amplification was used. The results for samples from a human volunteer study of ETEC infection indicated that this method of sample preparation results in greater clinical sensitivity than conventional total nucleic acid extraction and ethanol precipitation. Detection of ETEC by a multiplex PCR assay in stool specimens directly processed with a glass matrix and chaotropic solution had greater sensitivity than culture.

The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus-and minus-strand RNA and doublestranded RNA in simian rotavirus SAil-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plusand minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plusto minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labeling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.

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We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp7O and pl5E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp7O proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.