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Twenty-eight faecal specimens from Sri Lankan buffalo calves shown to be positive for rotavirus group A antigen were subgrouped by an enzyme-linked immunosorbent assay, by using monoclonal antibodies prepared against subgroup I and II antigens. The 13 of the 28 specimens which were classified as strongly positive belonged to subgroup I. Three of five faecal specimens inoculated on to roller cultures of MA104 cells yielded group A subgroup I rotavirus. As with other group A rotaviruses isolated from human beings and young animals, the buffalo isolates required pre-treatment with trypsin and to have trypsin incorporated in the maintenance medium, and the inoculated cell cultures had to be rolled; at least six serial passages were required before distinct rotavirus cytopathic effects were produced in the MA104 cells.

Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted alpha-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.

vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.

Isolates of Vibrio cholerae O1 El Tor from two well-defined cholera outbreaks in Malaysia were analyzed by using pulsed-field gel electrophoresis (PFGE). Isolates from sporadic cases occurring during the same time period were also studied. Digestion of chromosomal DNA from these isolates of V. cholerae O1 with restriction endonucleases NotI (5'-GCGGCCGC-3') and SfiI (5'-GGCCNNNN-3'), followed by PFGE, produced restriction endonuclease analysis (REA) patterns consisting of 13 to 24 bands (ranging in size from 46 to 398 kbp). Analysis of the REA patterns generated by PFGE after digestion with NotI and SfiI suggested the clonal nature and close genetic identity of the isolates obtained during each of the two outbreaks (Dice coefficient, 0.93 to 1.0). Although they had very similar REA patterns, the two outbreak clones were not identical. Isolates of V. cholerae O1 from sporadic cases, on the other hand, appeared to be much more heterogeneous (five different REA patterns detected in the five isolates tested; Dice coefficient, 0.31 to 0.81) than those obtained during the two outbreaks. We conclude that PFGE of V. cholerae O1 chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for molecular typing of V. cholerae isolates for epidemiological purposes.

Faecal samples from 150 buffalo calves, one to 150 days old, located in various districts of Sri Lanka, were examined for group A rotavirus antigen by a screening enzyme linked immunosorbent assay (ELISA). Positive samples were confirmed by the blocking ELISA. In the calves studied 27.3 per cent were diarrhoeic, and the rest were non-diarrhoeic but were in contact with the animals showing diarrhoea. Antigen was detected in 36.6 per cent of the diarrhoeic animals and in 11.9 per cent of the non-diarrhoeic animals. There was a strong association between the presence of antigen in faeces and diarrhoea in these animals (chi 2 = 46.98; P < 0.001). Of the 146 serum samples examined for antirotaviral antibodies, by the blocking ELISA at a single serum dilution (1:20) against a constant dose of antigen (8 units), 68.5 per cent were positive indicating a widespread infection with the virus in the population studied. This is the first record of the detection of rotavirus and its association with diarrhoea in buffalo calves in Sri Lanka.