Viral cysteine proteinases

Summary Dozens of novel cysteine proteinases have been identified in positive single-stranded RNA viruses and, for the first time, in large double-stranded DNA viruses. The majority of these proteins are distantly related to papain or chymotrypsin and may be direct descendants of primordial proteolytic enzymes. Virus genome synthesis and expression, virion formation, virion entry into the host cell, as well as cellular architecture and functioning can be under the control of viral cysteine proteinases during infection. RNA virus proteinases mediate their liberation from giant multidomain precursors in which they tend to occupy conserved positions. These proteinases possess a narrow substrate specificity, can cleave in cis and in trans, and may also have additional, nonproteolytic functions. The mechanisms of catalysis, substrate recognition and RNA binding were highlighted by the recent analysis of the three-dimensional structure of the chymotrypsin-like cysteine proteinases of two RNA viruses.     

cDNA probes for the diagnosis of bovine torovirus (Breda virus) infection.

A genomic cDNA library of RNA from Breda virus (BRV), a bovine torovirus, was prepared. The nucleotide sequence of the 3' end of the genome was found to be highly conserved (93% identical) between BRV and Berne virus, the torovirus prototype. Cross-hybridization experiments were performed to select Berne virus cDNA clones for use as probes in a dot hybridization assay; the objective was to detect heterologous torovirus RNA in fecal material. A rapid RNA extraction method was employed to make the test applicable for routine diagnosis. Samples from calves after experimental and natural infection with BRV were assayed to establish the sensitivity and specificity of the test and to compare the test with the enzyme-linked immunosorbent assay (ELISA) for antigen detection. For this purpose, 53 samples from seven infected calves were tested with both methods. In the ELISA, BRV was detected in six fecal samples from three inoculated calves. By use of the hybridization test, 16 samples from seven calves reacted positively. With one exception, only postinoculation samples were found positive in hybridization. No signal was seen in feces from uninoculated calves or from calves infected with rotavirus or coronavirus.     

Perforated colorectal neoplasms: correlation of clinical, contrast enema, and CT examinations

Results of clinical, contrast enema (CE), and computed tomographic (CT) examinations in 39 patients with perforated colorectal neoplasms were retrospectively reviewed. Twenty patients were toxemic at initial presentation, but in only four patients was the diagnosis of perforated colorectal neoplasm initially suspected clinically. CE study was performed in 22 patients and enabled the diagnosis of perforated neoplasm in 11 cases, neoplasm alone in eight, and neither neoplasm nor perforation in three. CT was performed in 38 patients and enabled the diagnosis of perforated neoplasm in 36; pericolic phlegmon but no mass lesion was evident in two. In 16 patients, CT also demonstrated metastatic disease. Because of its reliability in establishing the diagnosis and staging the extent of the inflammatory and neoplastic disease, CT is indicated in cases of suspected or proved perforated colorectal neoplasm and in cases in which CE study findings are indeterminate or suggestive of perforated neoplasm.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.