Multilocus sequence analysis of 'Candidatus Phytoplasma asteris' strain and the genome analysis of Turnip mosaic virus co-infecting oilseed rape.

AIM: Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes. METHODS AND RESULTS: Phytoplasma ('Candidatus Phytoplasma') presence was confirmed by PCR-RFLP and 16S rRNA gene sequencing. Phylogenetic analyses of phytoplasma amp, tufB, secY, groEL and ribosomal protein genes confirmed its affiliation to the 'Ca. P. asteris' species. However, in the amp gene encoding a specific protein crucial for insect transmission specificity, significant SNPs were found. Biological and serological tests revealed the co-infection with Turnip mosaic virus (TuMV). The phylogenetic analysis of full TuMV genome sequence, the first reported from the Balkans, classified it into the world-B phylogenetic lineage. CONCLUSIONS: A pathogenic complex consisting of 'Ca. P. asteris' and TuMV found to co-infect oilseed rape plants for the first time was molecularly characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Rape phyllody is a serious problem in rapeseed production. The molecular information from this first multi-gene analysis of 'Ca. P. asteris' strain associated with rape phyllody as well as the first report of the complete sequence of TuMV isolate from the Balkans is a starting point for understanding the disease complexity and management.

Clinical features and etiology of pneumonia in acid-fast bacillus sputum smear-negative HIV-infected patients hospitalized in Asia and Africa.

OBJECTIVES: To determine the main causes of acid-fast bacillus sputum smear-negative pneumonia in Asian and African HIV-infected patients DESIGN AND SETTING: A prospective multicenter study (ANRS 1260) of consecutive hospitalized patients in tertiary hospitals in Phnom Penh, Ho Chi Minh City, Bangui and Dakar. INTERVENTION: Use of the same clinical, radiological and biological methods at the four sites; regular quality controls of participating laboratories; final review of medical records by experts. Similar criteria used to establish diagnoses. RESULTS: In all 462 patients were enrolled, 291 in Asia and 171 in Africa. The median CD4 cell count was 25 cells/microl. Radiological opacities were diffuse in 42% of patients and localized in 45%. Fiberoptic bronchoscopy was performed in 354 patients, at similar rates in the four sites. A definite and/or probable diagnosis was obtained in 375 patients (81%). Pneumocystis jiroveci pneumonia, bacterial pneumonia, AFB sputum smear-negative tuberculosis and other infections (fungi, parasites, atypical mycobacteria) were diagnosed in respectively 47, 30, 17 and 12% of Asian patients and 3, 48, 26 and 5% of African patients. CONCLUSION: In South-east Asia, acid-fast bacillus smear-negative pneumonia is caused by a wide variety of pathogens. When possible, fiberoptic bronchoscopy must be performed rapidly if clinical data are not highly suggestive of bacterial pneumonia, Pneumocystis jiroveci pneumonia or tuberculosis. In contrast, in Africa, bacterial pneumonia and tuberculosis are responsible for the large majority of cases. Fiberoptic bronchoscopy should be restricted to patients with clinical and/or radiological findings not suggestive of bacterial pneumonia or tuberculosis, antibiotic failure, and three consecutive negative sputum smears.

Predictors of pneumocystosis or tuberculosis in HIV-infected Asian patients with AFB smear-negative sputum pneumonia.

OBJECTIVES: To identify predictors of Pneumocystis jiroveci pneumonia (PCP) or pulmonary tuberculosis (TB) in acid-fast bacillus smear-negative HIV-infected patients and to develop clinical prediction rules. DESIGN: A cohort study conducted in consecutive hospitalized Asian patients. METHODS: Multivariate analyses were performed on the Cambodian sample to determine clinical, radiological, and biological predictors of PCP or TB at hospital admission. The Vietnamese sample was kept for independent validation. RESULTS: In Cambodia, the gold standard technique for TB and PCP were fulfilled in 172 (27 cases) and 160 (84 cases) patients, respectively. For TB, independent predictors included the following: headache [odds ratio (OR) 3.0; 95% confidence interval (CI) 1.04 to 8.6], localized radiological opacity (OR 5.8; 95% CI 1.9-17.9), and mediastinal adenopathy (OR 10.1; 95% CI 3.5 to 29.0); and for PCP: resting oxygen saturation <90% (OR 3.3; 95% CI 1.3 to 8.5 for resting arterial oxygen saturation >or=80%; and OR 9.1; 95% CI 1.8 to 44.5 for resting arterial oxygen saturation <80%), trimethoprim-sulphamethoxazole prophylaxis (OR 0.1; 95% CI 0.04 to 0.6), and diffuse radiological shadowing (OR 7.0; 95% CI 2.7 to 18.6). PCP risk predicted by a score based on these 3 factors ranged from 3% to 92% (Cambodia). When tested on Vietnamese patients (n = 69, 38 with PCP), the score maintained correct predictive ability (c-index = 0.72) but with poor calibration. CONCLUSIONS: The PCP score could provide a useful clinical tool to identify PCP among acid-fast bacillus smear-negative pneumonia and start specific therapy.

Hemadsorption of mumps virus examined by light and electron microscopy.

The hemadsorption (HAD) reaction of chick embryo cells infected with mumps virus was studied by means of light and electron microscopy, with special reference to the plasma membrane of the infected cell. The concomitant observation of membrane-free aggregates of viral nucleocapsid in the cytoplasm and attached red blood cells on the surface of the same cell indicated that only infected cells hemadsorbed and that hemagglutinin is confined within the infected cell. The attachment of red blood cells to morphologically intact cell membrane prior to its differentiation into viral envelope suggested that the HAD phenomenon, dependent on the presence of hemagglutinin, was independent of the viral maturation process. The gap of low electron density normally separating the morphologically intact membrane of the tissue culture cell and that of the red blood cell at the binding site was replaced by newly formed surface projections in HAD involving a segment of differentiated plasma membrane.

Immuno-electron microscopy of the morphogenesis of mumps virus.

The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy.

Replication of mumps virus in human leukocyte cultures.

Duc-Nguyen, Huu (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and Werner Henle. Replication of mumps virus in human leukocyte cultures. J. Bacteriol. 92:258-265. 1966.-Human peripheral leukocyte cultures maintained in the presence of phytohemagglutinin (PHA) were found to support to some extent the replication of mumps virus. When such cultures were exposed, within 24 hr after their initiation, to a high input multiplicity of virus, successful infection, as determined by immunofluorescence and plaque assays, did not become evident before the 3rd or 4th day. On exposure of cultures 4 to 5 days old, viral replication was detectable within 2 days. In both instances, peak immunofluorescence and virus titers were reached when the cultures were 7 to 9 days old and composed mainly of blast forms. With decreasing input multiplicities of infection, cells containing viral antigen and production of infectious viral progeny became detectable with increasing delay. No significant viral replication was noted in surviving cells maintained in the absence of PHA. These results indicate that mainly, if not solely, the PHA-stimulated cells of the lymphocytic series support viral multiplication. The extent of the infectious process was limited, however, because the life span of the cultures was not significantly shortened, the yields of infectious virus per immunofluorescent cell were at all times low, and most infected cells contained only a few well-delineated small masses of antigen, suggestive of an abortive infection. Only fresh cultures were capable of synthesizing interferon on stimulation by mumps, Newcastle disease, or Sendai viruses. When the cultures were set up in the presence of PHA, this capacity was lost within 24 hr. PHA per sefailed to induce detectable production of an interferon under the conditions used. The implications of these findings are discussed.

An electron microscopic study of changes at the surface of influenza-infected cells as revealed by ferritin-conjugated antibodies.

The sequence of events associated with liberation of the PR8 strain of influenza virus at the surface of chorioallantoic membranes (CAM) was studied by means of ferritin-conjugated antibodies, one of which was specific for the V antigen of the virus and the other for host cell antigen. With an input multiplicity of approximately 0.001 per cell, viral progeny were first detected by infectivity titrations and electron-microscopy after 12 and 13 hours, respectively. As infection proceeded, viral antigen progressively accumulated at the cell surface, while host cell antigen diminished in amount. It was concluded that normal host cell protein did not constitute an integral part of the surface structure of the virus.