Serological detection of infection with diverse human and simian immunodeficiency viruses using consensus env peptides

Cross-species transmission has been shown to play an important role in the emergence of human retroviruses. We developed a generic enzyme immunoassay using synthetic peptides from gp41 and C2V3 consensus sequences (human immunodeficiency virus [HIV] type 1 [HIV-1] groups M, O, and N and the homologous region of simian immunodeficiency virus [SIV] strains from chimpanzees [SIVcpz], SIVcpzGAB1 and SIVcpzANT) to detect divergent HIV and SIV. A cocktail of peptides from gp41 and C2V3 (M-O) detected all HIV-1 group M and O sera and showed cross-reactivity with SIVcpz sera. Further, a mixture of C2V3 peptides (GAB1-ANT) failed to detect HIV-1 infections but reacted with all SIVcpz sera, allowing discrimination of SIVcpz from HIV-1 infections. Since most SIVcpz sera cross-reacted with HIV-1 peptides, we next evaluated SIVcpz serum reactivity with rapid tests for HIV-1/2. SIVcpzANT and SIVcpzUS sera reacted with the Sero-strip and Multispot assays. Both tests are sensitive in detecting group M (97 100%, respectively), although Multispot has lower sensitivity for group O detection (67%) than does Sero-strip (100%). The limited volume and time required to perform these assays make them a generic tool for field screening. The env peptide-based assay and rapid tests should allow for the identification of emerging variants of HIV and SIV.     

External quality control assessment in PCR diagnostics of dengue virus infections

BACKGROUND: Increased travelling to countries endemic for dengue fever (DF) demands efficient laboratory diagnostics. Nucleic acid amplification techniques (NAT) are now frequently used for rapid diagnosis of imported viral diseases. Different PCR systems are available. OBJECTIVES: In order to assess the quality of molecular diagnostics of dengue virus infections, an external quality assurance (EQA) in PCR diagnostics was conducted. Study design: A panel of 10 human plasma samples was prepared and spiked with dengue virus types DEN-1 to DEN-4. In addition, a 10-fold dilution series (1:10-1:10(4) ) of DEN-3 virus was included. The panel was pre-tested by nested RT-PCR, in-house real-time PCR, and a commercial real-time PCR kit. The samples were inactivated by gamma irradiation and shipped in freeze dried state. Thirteen laboratories, within the European network for the diagnostics of imported viral diseases (ENIVD) took part using either single-round, nested, or real-time RT-PCR methods. Two laboratories used two methods in parallel, summarising up to 15 comparable results. RESULTS: 33-100% correct results were achieved. All laboratories detected DEN-2 correctly, followed by DEN-1 (14 positive results of 15), DEN-3 (12/15) and DEN-4 (11/15). Testing of the serial dilution revealed low sensitivity in many labs, with results ranging from 33 to 80% of correctly tested samples. CONCLUSION: The EQA gives a feedback of the quality of the RT-PCR system used by each respective laboratory. The different test systems and amplification conditions demonstrate the importance of external quality control measures.     

Magnetic resonance fast Fourier imaging

A new method for fast magnetic resonance imaging is presented. It provides a more rapid data acquisition than two-dimensional Fourier imaging (2DFI) by a factor which may be chosen depending on the required signal-to-noise ratio of the image. In addition to the readout gradient of 2DFI, the present method employs an oscillating modulation gradient. In this way, a curved alternating trajectory in k space is sampled after each spin excitation. For a p-times accelerated data acquisition, the trajectory consists of p periods, where p is of the order of 2 to 8 for low-frequency gradient modulation but can be chosen higher if certain hardware requirements are met. Adequate sampling density in k space is obtained by scanning shifted trajectories after subsequent spin excitations. The method can be combined with volume imaging (3DFI) and multiple slice 2DFI. It was implemented on a standard Philips Gyroscan system without any hardware modifications. Results obtained for an acceleration factor p = 4 are shown.     

Branches of the thoracic sympathetic trunk in the human fetus

Summary The segmental organization of the thoracic sympathetic trunk and all its ramifications was studied in 6 human fetuses (16–22 weeks) by means of the acetylcholinesterase in toto staining method. Each trunk was divided into 12 sympathetic segments. A segment is defined as that part of the sympathetic trunk which is connected via its rami communicates with one spinal nerve, without discriminating between grey and white rami. The diameter of the rami communicantes and their direction towards the spinal nerves are variable. The number of peripheral segmental ramifications of the trunk is much larger than assumed previously. Each thoracic sympathetic segment gives off at least 4–5 nerves. Three categories of nerves are discerned: (1) large splanchnic rootlets confined to the greater, lesser and least thoracic splanchnic nerves, (2) medium-sized splanchnic nerves directed towards thoracic viscera, some of which give off branches towards costovertebral joint plexuses and, described for the first time in man, (3) small nerves which ramify extensively and form nerve plexuses in the capsule of the costovertebral joints. The majority of the ramifications is formed by the nerves of the third category. The existence of Kuntz's nerve, connecting the 2nd intercostal nerve and 1st thoracic spinal nerve, is confirmed in four specimens. The nerve plexuses of the costovertebral joints receive a segmentally organized innervation: they receive their input from the neighbouring sympathetic segment and the one cranial to it.It is concluded that the thoracic sympathetic branches in man show a complex, segmentally organized pattern and may have a considerable component of somatosensory nerve fibers. The complex relationships must be taken into account in surgical sympathectomies.     

The psychosomatic theory of bronchial asthma.

The author discusses the development of the psychosomatic asthma theory as a paradigm of theory formation in psychomatic medicine. The first formulation of the theory was based on clinical and psychiatric observations. It was tested by psychological, physiological and experimental methods and as a result was reformulated and extended. In its present form it regards asthmatic breathing as a reaction of a predisposed personality structure (partly hereditary, partly acquired during a youth situation in which overprotection by a domineering parent played a large role), to an ambivalent conflict with a key figure. The resulting frustration is not acted out by aggressive, flight, or depressive behaviour, but inhibited; thereby the motoric and verbal discharges are displaced into (substituted by) a respiratory behaviour pattern, which is characterised by an abnormally forceful contraction of the abdominal muscles during the expiration. The resulting high intraabdominal pressure is transmitted into the thorax where it pushes the posterior membranaceous wall of the trachea and large bronchi forward into the lumen and thus produces a long stretched obstruction of the large airways. The passage of the air through the compressed large air passages under high pressure and low velocity is the mechanism which causes the typical wheeze and the other manifestations of the asthmatic airway obstruction. A hypothesis is suggested for the ways in which this psychoneurogenic respiratory behaviour contributes to the so-called bronchial hyper-reactivity and the secondary development of allergies.     

Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.      

Characterisation of a recently isolated lyssavirus in frugivorous zoo bats

Summary.  In July 1997 a lyssavirus was isolated in Denmark from a colony of Egyptian flying foxes (Rousettus aegyptiacus) originating from a Dutch zoo. Sequencing of a 400 nucleotides coding region of the nucleoprotein and of a major part of the G-protein ectodomain encoding region of the newly isolated virus, revealed a very high similarity with European Bat Lyssavirus subtype 1a (EBL-1a). For characterisation of the recently isolated lyssavirus in frugivorous zoo bats, 16 frugivorous bats (Rousettus aegyptiacus) of the same colony and 80 mice were experimentally infected with the Rousettus isolate or with a well defined EBL-1a strain isolated from a Dutch insectivorous bat (Eptesicus serotinus). Inoculation viruses were titrated in mice to determine LD₅₀‘s of both isolates. Clinical signs of inoculated bats were recorded during 6 weeks. After showing neurological signs or at the end of the experimental infection all animals were euthanized. During the experimental infection sera and various tissues of inoculated bats were collected. Immunoassays, mouse inoculation tests (MIT) and polymerase chain reaction (PCR) were employed for detection of lyssavirus specific antibodies, antigen or RNA. Five bats inoculated with the Rousettus isolate and 2 bats inoculated with the Eptesicus isolate showed neurological signs. The remaining 9 bats survived and cleared the virus; at least under the detection limit of the used assays. Despite a much higher pathogenicity of the Rousettus isolate observed in mice, LD₂₅’s in bats were quite the same for the 2 isolates. The pathogenicity of both isolates suggested that like many other mammals, Rousettus aegyptiacus bats could be victims of lyssavirus infection besides reservoir hosts of infectious EBL1a. There was no significant difference in detecting the different lyssavirus isolates in Rousettus aegyptiacus bats. An employed immunoperoxidase staining (IP) method was very useful for sensitive detection and localization of lyssavirus antigen in histologic preparates. Received July 26, 1999/Accepted February 18, 2000     

Partial characterization of a Hantavirus isolated from a Clethrionomys glareolus captured in Belgium

A Hantavirus was isolated in Vero-E6 cells from lungs of a free living bank vole (Clethrionomys glareolus) captured in Turnhout, Province of Antwerp--Northern part of Belgium. With help of monoclonal antibodies the Belgian Hantavirus isolate could be clearly differentiated from Hantaan virus strain 76-118, Prospect Hill virus strain PH1 and SR11, a Hantavirus isolated from laboratory Wistar rat in Japan, but not from the nephropathia epidemica virus strain H?lln?s.