Production of 22 nm HBsAG particles by human lymphocytes infected with a recombinant vaccinia virus containing the coding sequence for hepatitis B virus surface antigen.

To examine the possible role of lymphocytes in the course of hepatitis B virus infection, we studied peripheral blood lymphocytes and Epstein-Barr virus transformed B-cells for their capability to produce hepatitis B virus gene products. Infection of these cells with a recombinant vaccinia virus containing the hepatitis B virus surface antigen resulted in the production and secretion of hepatitis B virus surface antigen shortly after infection reaching a peak after three days. When the supernatants of the cells were analyzed on density gradients, a peak of reactivity for hepatitis B virus surface antigen was reached at 1.21 g/cm3. Electron microscopy revealed the presence of spherical and filamentous HBsAg particles. These findings show that human lymphocytes are capable of producing hepatitis B virus surface antigen.     

Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90.

A DNA probe (D4S95) that detects a variable number of tandem repeats and a single-site-variation polymorphism after digestion with a single restriction enzyme, AccI, has previously been described. The order of this probe relative to the gene for Huntington disease (HD) and other previously described markers has not been established. Analysis of 24 affected families with HD has shown that D4S95 is in tight linkage with the gene causing HD, with a maximal Lod score of 12.489 at a theta of .03. D4S90 is a probe which maps to 4p16.3, telomeric to D4S95, and detects polymorphisms with HincII and other enzymes. In one affected person, recombination has occurred between D4S10 and HD, between D4S95 and HD, and in all likelihood also between D4S90 and HD, which strongly suggests that the gene for HD is telomeric to all these DNA probes. This suggests that the gene causing HD is located in the most distal region of the short arm of chromosome 4, flanked by D4S90 and the telomere, and supports the locus order D4S10-D4S95-D4S90-HD-telomere. D4S95 is a most useful DNA marker for predictive testing programs, while D4S90 will serve as a useful starting point for identifying DNA fragments closer to the gene for HD.     

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.     

Serial Selection for Resistance to a Wild-Type and to a Genetically Modified Nucleopolyhedrovirus in Trichoplusia ni

In previous work, cabbage loopers (Trichoplusia ni) evolved 22-fold resistance to the single nucleocapsid nucleopolyhedrovirus of T. ni (TnSNPV) after 26 generations of selection with the virus. The goal of the present study was to determine if T. ni could evolve resistance to the recombinant Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV-AaIT) that expresses an insect specific neurotoxin and to determine if it was influenced by prior development of resistance to TnSNPV. To answer these questions, the T. ni line that had been exposed to TnSNPV was divided into two sublines at generation 27. One of them was serially selected for resistance to AcMNPV-AaIT (subline TnSNPV/AcMNPV-AaIT), while the other one was mock infected with distilled water (subline TnSNPV/H₂O). The same was done with the line that was used as a control from generations 1 to 26 (subline H₂O/AcMNPV-AaIT and subline H₂O/H₂O). After 17 generations of selection with AcMNPV-AaIT, T. ni that had not been previously exposed to TnSNPV evolved only twofold resistance to AcMNPV-AaIT. However, those that had been selected with TnSNPV evolved fourfold resistance to AcMNPV-AaIT. Exposure to AcMNPV-AaIT conferred cross-resistance to TnSNPV in only one subline, subline H₂O/AcMNPV-AaIT. Resistance to AcMNPV-AaIT did not affect the developmental time, pupal weight, egg production, or percentage of egg hatch of T. ni.     

Defining the cost of the Egyptian lymphatic filariasis elimination programme

BACKGROUND: Ultrasonography (USG) is known to be a suitable tool for diagnosis in lymphatic filariasis as the adult filarial nematode Wuchereria bancrofti in scrotal lymphatic vessels of infected men can be detected by the characteristic pattern of movement, the Filaria Dance Sign. In onchocerciasis, moving adult worms have not yet been demonstrated by USG. In addition the verification of drug effects on living adult Onchocerca volvulus filariae in trials is hampered by the lack of tools for longitudinal observation of alterations induced by potentially macrofilaricidal drugs in vivo. The present study was carried out to determine the frequency of detection of moving adult filariae of O. volvulus by USG. METHODS: In an endemic region for onchocerciasis in Ghana, 61 patients infected with onchocerciasis were recruited by palpation and onchocercomas examined by USG using an ultrasound system equipped with a 7.5 - 10 MHz linear transducer. Onchocercomas were recorded on videotape and evaluated with regard to location, number and size, as well as to movements of adult filariae. RESULTS: In the 61 patients 303 onchocercomas were found by palpation and 401 onchocercomas were detected by USG. In 18 out of 61 patients (29.5%), altogether 22 nodules with moving adult O. volvulus filariae were detected and are presented in animated ultrasound images as mp-4 videos. CONCLUSION: Ultrasonographical examinations of onchocercomas where living adult filariae can be displayed may serve as a new tool for the longitudinal observation in vivo of patients with onchocerciasis undergoing treatment and as an adjunct to histological evaluation.     

Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004); however, the function or mechanism by which EXON0 affects BV production is unknown. In this study, we further elucidated the function of EXON0 by investigating the localization of EXON0 in infected Sf9 cells and in virions and by identifying interactions between EXON0 and other viral proteins. In addition, electron microscopy was used to study the cellular localization of nucleocapsids in cells transfected with an exon0 knockout (KO) virus. The results showed that EXON0 was localized to both the cytoplasm and the nuclei of infected Sf9 cells throughout the infection. Western blotting results also showed that EXON0 was purified along with BV and occlusion-derived virus (ODV). The fractionation of BV into the nucleocapsid and envelope components showed that EXON0 localized to the BV nucleocapsid. Yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy revealed that it interacted with nucleocapsid proteins FP25 and BV/ODV-C42. Cells transfected with the exon0 KO virus exhibited normally appearing nucleocapsids in the nuclei in numbers equal to those in the nuclei of cells transfected with the EXON0 repaired virus. In contrast, the numbers of nucleocapsids in the cytoplasm of cells transfected with the exon0 KO virus were significantly lower than those in the cytoplasm of cells transfected with the repaired virus. These results support the conclusion that EXON0 is required in the BV pathway for the efficient egress of nucleocapsids from the nucleus to the cytoplasm.