Central autonomic control of the bone marrow: multisynaptic tract tracing by recombinant pseudorabies virus.

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

Identification of neurones of the brain and spinal cord involved in the innervation of the ductus deferens using the viral tracing method.

Using the viral transneuronal tracing technique cell groups of the spinal cord and brain transsynaptically connected with the ductus deferens were identified. Neurotropic (pseudorabies) virus was injected into the muscular coat of the ductus deferens and after survival times of 3, 4 and 5 days the spinal cord and brain were processed immunocytochemically. Virus-labelled neurones could be detected in the preganglionic sympathetic neurones and the dorsal commissural nucleus (upper lumbar segments) and in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei including the gigantocellular and paragigantocellular nucleus, the lateral reticular nucleus, the nucleus of the solitary tract, the caudal raphe nuclei, the A1/C1, A2, A5 and A7 noradrenergic cell groups and the locus coeruleus. In the hypothalamus significant numbers of virus-infected neurones could be detected in the paraventricular nucleus. In most cases moderate numbers of virus-labelled cells were present in the lateral hypothalamic area, in the retrochiasmatic area, in the periventricular region and in the median preoptic area. Double-labelling immunofluorescence detection of virus-infected neurones and thyrosine hydroxylase (TH) showed colocalization of virus protein and TH in portion of neurones of the A1/C1, A2, A5 and A7 noradrenergic cell groups, in the locus coeruleus and in the hypothalamic paraventricular nucleus. The present results provide the first morphological data on the multisynaptic circuit of neurones innervating the ductus deferens.

Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique.

Using the viral transneuronal tracing technique, the cell groups in the CNS transneuronally connected with the female mammary gland were detected. Lactating and non-lactating female rats were infected with pseudorabies virus injected into the mammary gland. The other group of animals was subjected to virus injection into the skin of the back. Four days after virus injection, infected neurons detected by immunocytochemistry, were present in the dorsal root ganglia ipsilateral to inoculation and in the intermediolateral cell column of the spinal cord. In addition, a few labelled cells could be detected in the dorsal horn and in the central autonomic nucleus (lamina X) of the spinal cord. At this survival time several brain stem nuclei including the A5 noradrenergic cell group, the caudal raphe nuclei (raphe obscurus, raphe pallidus, raphe magnus), the A1/C1 noradrenergic and adrenergic cell group, the nucleus of the solitary tract, the area postrema, the gigantocellular reticular nucleus, and the locus coeruleus contained virus-infected neurons. In some animals, additional cell groups, among others the periaqueductal gray and the red nucleus displayed labelling. In the diencephalon, a significant number of virus-infected neurons could be detected in the hypothalamic paraventricular nucleus. In most cases, virus-labelled neurons were present also in the lateral hypothalamus, in the retrochiasmatic area, and in the anterior hypothalamus. In the telencephalon, in some animals a few virus-infected neurons could be found in the preoptic area, in the bed nucleus of the stria terminalis, in the central amygdala, and in the somatosensory cortex. At the longer (5 days) survival time each cell group mentioned displayed immunopositive neurons, and the number of infected cells increased. The pattern of labelling was similar in animals subjected to virus inoculation into the mammary gland and into the skin. The distribution and density of labelling was similar in lactating and non-lactating rats. The present findings provide the first morphological data on the localization of CNS structures connected with the preganglionic neurons of the sympathetic motor system innervating the mammary gland. It may be assumed that the structures found virus-infected belong to the neuronal circuitry involved in the control of the sympathetic motor innervation of the mammary gland.

Identification of CNS neurons involved in the innervation of the epididymis: a viral transneuronal tracing study.

Cell groups of the spinal cord and the brain transsynaptically connected with the epididymis (caput, cauda) were identified by means of the viral transneuronal tracing technique. Pseudorabies virus was injected into the caput or the cauda epididymidis, and after survival times 4 and 5 days, the spinal cord and brain were processed immunocytochemically. Virus-labeled neurons could be detected in the preganglionic sympathetic neurons (lower thoracic and upper lumbar segments) and following virus injection into the cauda epididymidis, also in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei (lateral reticular nucleus, gigantocellular and paragigantocellular nucleus, A5 noradrenergic cell group, caudal raphe nuclei, locus coeruleus, Barrington's nucleus, nucleus of the solitary tract, periaqueductal gray) and in the diencephalon (hypothalamic paraventricular nucleus, lateral hypothalamus). At the longer survival time, some telencephalic structures also exhibited virus-labeled neurons. The distribution of infected neurons in the brain was similar after virus injection into the caput or cauda epididymidis; however, earlier onset of infection was observed after inoculation into the cauda. The present findings provide the first morphological data on a multisynaptic circuit of neurons innervating the epididymis and presumably involved in the control of epididymal functions. reserved.

Coding in the noncoding DNA strand: A novel mechanism of gene evolution?

The question whether the noncoding DNA strand had or still has the capability for encoding functional polypeptides has been addressed in several articles. The theoretical background of the views advocating this idea arose from two groups of findings. One of them was based on various observations implying that the genetic code was adapted for double-strand coding. The other group of theories arose from the observation of gene-length overlapping open reading frames (O-ORFs) on the antisense DNA strand in a number of genes. In fact, the above theories, which I term selectionist, conceive a novel conception of gene evolution, proposing that new genes can be created by the utilization of antisense DNA strand. In contrast, neutralist theory claims that the O-ORFs are mere by-products of evolutionary processes acting to create special codon usage and base distribution patterns in the coding sequences.

Central nervous system structures labelled from the testis using the transsynaptic viral tracing technique.

In the present study, the transneuronal transport of neurotrophic virus technique was used to identify cell groups of the spinal cord and the brain that are transsynaptically connected with the testis. Pseudorabies virus was injected into the testis and after survival times of 3-6 days, the spinal cord and brain were processed immunocytochemically using a polyclonal antibody against the virus. Virus-infected perikarya were detected in the preganglionic neurones of the spinal cord (T10-L1, L5-S1) and in certain cell groups and areas of the brain stem, the hypothalamus and the telencephalon. In the brain stem, the cell groups and areas in which labelled neurones were present included, among others, the nucleus of the solitary tract, the caudal raphe nuclei, the locus coeruleus and the periaqueductal grey of the mesencephalon. In the hypothalamus, virus infected perikarya were observed in the paraventricular nucleus and in certain other cell groups. Telencephalic structures containing labelled neurones included the preoptic area, the bed nucleus of the stria terminalis, the central amygdala and the insular cortex. These data identify a multisynaptic circuit of neurones in the spinal cord and in the brain which may be involved in the control of testicular functions.

Replication and virulence of early protein 0 and long latency transcript deficient mutants of the Aujeszky's disease (pseudorabies) virus.

Early protein 0 (EP0)-deficient recombinant Aujeszky's disease viruses, Ka-ep0lac and Ba-ep0lac derived from strains Kaplan and Bartha, respectively, were constructed to explore the impact of the mutation on replication, virulence and latency of the virus. Inactivation of the EP0 gene resulted in a mutation of long latency transcript (Cheung et al., 1991) that is located on the complementary DNA strand of EP0 and immediate early protein (IE)175 genes. In infection of immortalized porcine kidney cells, the growth rate and yield of both EP0(-) mutant strains were significantly smaller than that of wild-type virus. Ka-ep0lac was found to be highly virulent, while Ba-ep0lac showed an attenuated phenotype in mice. PCR assay and immunohistochemistry showed that the Ba-ep0lac virus was able to establish latency in the mouse trigeminal ganglia. However, latent virus was not able to reactivate in explant reactivation assays. Accordingly, latent Ba-ep0lac has the potential to be exploited as vectors for the delivery of foreign genes to the nervous system.

CNS structures presumably involved in vagal control of ovarian function.

The contribution of the vagus nerve to viral transneuronal labeling of brain structures from the ovaries demonstrated recently by us was investigated. Unilateral vagotomy was performed prior to ipsilateral intraovarian virus injection. Virus-infected neurons were visualized by immunostaining. In vagotomized rats such neurons were detected only in certain cell groups of the brain (parapyramidal nucleus, A(1), A(5) cell group, caudal raphe nuclei, hypothalamic paraventricular nucleus, lateral hypothalamus). Vagotomy interfered with labeling of several structures that were labeled in controls, including area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, A(7) cell group, Barrington's nucleus, locus coeruleus, periaqueductal gray, dorsal hypothalamus. Findings provide a morphological basis to study the functional significance of brain structures presumably involved in the control of ovarian function and acting via the vagus or the sympathetic nerves.

A putative latency promoter/enhancer (P(LAT2)) region of pseudorabies virus contains a virulence determinant.

Contradictory data have recently been reported on the role of the unique long-internal repeat junction area of pseudorabies (Aujeszky's disease) virus (PrV) genome in the virulence of the virus. To investigate the basis of the difference, four recombinant PrVs mutated at the outer region of inverted repeats that involved a putative latency promoter (P(LAT2)) were constructed in this study. Propagation characteristics of mutant viruses in cultured cells were similar to those of the wild-type virus. However, a 757 bp deletion at this location caused significant reduction in the virulence of PrV after intraperitoneal inoculation of mice and a moderate decrease in the virulence after intracranial inoculation. These results indicate that the P(LAT2) region is an important virulence determinant that may be implicated in the neuroinvasive capability of the virus.

Sequence and expression analyses of the UL37 and UL38 genes of Aujeszky's disease virus.

Previously, we sequenced the HSV-1 Ul39-Ul40 homologue genes of Aujeszky's disease virus (ADV), also designated as pseudorabies virus (Kaliman et al., 1994a, b). Now we report the nucleotide sequence of the adjacent DNA that encodes Ul38, the 5'-region (750 bp) of Ul37, and the promoter regions between these divergently arranged two genes. The ADV Ul38 gene encodes a protein of 368 amino acids. Amino acid sequence comparison of ADV Ul38 with that of other herpesviruses revealed significant structural homology. In a transcription study using RNase protection assay and Northern blot hybridization, we found that the Ul38 gene had one initiation site, but the Ul37 gene was initiated at two transcription sites with two potential initiator AUGs, one of which was dominant. Comparison of ADV Ul37, Ul38 and ribonucleotide reductase gene expression showed that these genes belong to the same temporal class with early kinetics. Data of structural and transcriptional studies suggest that regulation of the expression of these two ADV genes could differ from that of the HSV-1 virus.

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.

Specific amino acid content and codon usage account for the existence of overlapping ORFS.

Here we present a novel hypothesis for the origin of overlapping open reading frames (O-ORFs) observed in the 'non-coding frames' of several genes of yeast chromosome II. By computer analysis it was found that the specific amino acid content and base distribution pattern at certain genomic locations and the presence of O-ORFs were related. This observation prompt us to conclude that these O-ORFs are mere statistical curiosities without any biological function, which is in contrast to the hypotheses proposed by other authors.

Lacrimal preganglionic neurons form a subdivision of the superior salivatory nucleus of rat: transneuronal labelling by pseudorabies virus.

Transneuronal viral tracing was applied to localize preganglionic parasympathetic neurons in the brainstem which innervate the extraorbital lacrimal gland in the rat. The Bartha strain of pseudorabies virus was injected into the lacrimal gland, and after different survival times, the superior cervical and Gasserian ganglia, the upper thoracic spinal cords and the brainstems were immunostained by antiviral antiserum. Virus-labelled neurons appeared in the ganglia and in the ventrolateral part of the ipsilateral brainstem at the pontomedullary junction 45 h after inoculation. The virus-labelled brainstem neurons comprised a subgroup of the superior salivatory nucleus (SSN) located between the root fibers of the facial nerve and the nuclei of the superior olive, and were clearly distinguished from the tyrosine hydroxylase (TH)-immunopositive, A5 catecholaminergic neurons by double immunostaining. The number of infected cells in the ipsilateral SSN was increased by 72 h, and labelled neurons appeared in the intermediolateral cell column (IML) of the ipsilateral thoracic spinal cord. In rats with cervical ganglionectomy prior to the virus injection in the lacrimal gland, virus-infected cells appeared in the SSN, but not in the thoracic spinal cord, indicating that preganglionic SSN cells were infected via parasympathetic axons of the facial nerve. A double-virus tracer labelling technique was applied to determine the topographical relationship between the preganglionic parasympathetic neurons of the lacrimal gland and those of the submandibular gland within the SSN. Simultaneous injection of Bartha strain of pseudorabies virus into the submandibular gland, and a lacZ gene-containing Bartha-derived virus strain into the lacrimal gland (and vice versa) demarcated a ventral lacrimal and a dorsal submandibular subgroup in the SSN.

Construction of a recombinant herpesvirus expressing the jellyfish green fluorescent protein.

Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission.

Analysis of the equalization of inverted repeats and neurovirulence using a pseudorabies virus mutant strain altered at the Ul/Ir junction.

A recombinant pseudorabies (Aujeszky's disease) virus (PrV) designated as vE16lac was constructed by deleting a 3-kbp DNA segment spanning the junction of long and short components of the viral genome, and by replacing the deleted segment with a lacZ-expression cassette. The aim of constructing this mutant was (a) to determine whether the terminal repeat (Tr) can serve as a template for the regeneration of the internal repeat (Ir), and (b) whether this deletion causes a reduction in the neuroinvasiveness of the virus. To analyze the mechanism of equalization, revertant viruses were selected and structurally characterized from vE16lac infection of PK-15 cells, mice and pigs. Because all revertants acquired Ir sequences identical to that of the wild-type virus, the equalization process occurred using the Tr as a template to reconstitute the Ir. We also found that the recombinant virus vE16lac was virulent in both pigs and mice. The data are discussed in view of studies performed with similar PrV mutants by other authors (Rall et al., 1992, Dean and Cheung, 1995 and Dean et al., 1996).

Neuronal labeling in the rat brain and spinal cord from the ovary using viral transneuronal tracing technique.

In the present investigations the viral transneuronal labeling method, which is able to reveal hierarchial chains of central nervous system (CNS) neurons, was applied to identify sites in the CNS connected with the ovary and presumably involved in the control of ovarian functions. Pseudorabies virus was injected into the ovaries of rats and a few days later (at various times after the injection) the spinal cord and brain were examined for virus-infected neurons from the ovary. The virus-labeled nerve cells were identified by immunocytochemistry using polyclonal antiviral antibody. Virus-labeled neurons were detected both in the spinal cord and the brain. In the spinal cord such elements were observed in the intermediolateral cell column, in the dorsal horn close to the marginal zone and in the central autonomic nucleus. In the medulla oblongata and pons, neurons of several nuclei and cell groups (area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, paragigantocellular nucleus, parapyramidal nucleus, A1, A5 and A7 cell groups, caudal raphe nuclei, locus ceruleus, subceruleus nucleus, Barrington's nucleus, Kölliker-Fuse nucleus) were found to be transneuronally labeled. In the mesencephalon, the ventrolateral part of the periaqueductal gray matter contained virus-labeled neurons. In the diencephalon, a very intensive cell body labeling was observed in the hypothalamic paraventricular nucleus and a few virus-infected neurons could be detected in the lateral and dorsal hypothalamus, in the arcuate nucleus, zona incerta, perifornical area and in the anterior hypothalamus. Concerning the telencephalic structures, virus-labeled cells were found in the bed nucleus of the stria terminalis and in the central amygdala nucleus. These findings provide the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the ovary and the CNS, and give a detailed account of the structures involved in this pathway.

A restriction cleavage and transfection system for introducing foreign DNA sequences into the genome of a herpesvirus.

This report describes a simple and efficient system for construction of recombinant pseudorabies (Aujeszky's disease) virus (PrV) which is based on the use of a unique restriction site inserted into the viral genome. This system enables the recovery of genetically modified viruses without screening or selection for a specific phenotype, since practically all mature viral particles obtained carry the foreign sequences. To demonstrate, we introduced the tumour suppressor protein-53 (p53) gene into two different intergenic locations of PrV: the ribonucleotide reductase (rr) gene and the promoter of a putative latency gene (PLAT), located at the inverted repeat (IR) region of the viral genome. As a first step, we engineered a unique EcoRI recognition site into the rr gene or into both copies of PLAT with the help of marker transfer using the bacterial lacZ gene. Then, in both cases viral DNAs were cut with the restriction endonuclease EcoRI followed by treatment with calf intestinal phosphatase and used for cotransfection into porcine kidney cells with a plasmid containing the p53 gene flanked by viral DNAs homologous to the target region. As a result of this process, in most of the experiments, we obtained recombinant viruses without the background of parental viruses. Here we show that this method can be used for directional insertion of exogenous sequences into either the unique or the IR region of the PrV chromosome. In principle, this system should be applicable to the construction of recombinant derivatives of any viruses having infectious DNA.

In vivo studies on Aujeszky's disease virus mutants.

We report the construction and in vivo analysis of three recombinant Aujeszky's disease virus (ADV) strains containing mutations at three different loci of the genome. Mutant vE16lac was generated by deleting of 2976 bp DNA fragment which covers 1851 bp of the right arm of UL component, the UL-US junction, the "a" element of the internal repeat (IR) region and a putative LAT promoter. Mutant vRRlac was generated by deletion of a 1805 bp fragment from the coding region of the large and small subunits of ribonucleotide reductase gene (rr). The third mutant, vTKlac, was constructed using insertional mutagenesis of the thymidine kinase gene (tk). In the constructed mutants a lacZ gene expression cassette was either inserted into the target gene (vTKlac) or replaced the deleted DNA segment (vE16lac, vRRlac). Constructed recombinant viruses were analyzed by infecting pigs and monitoring the virus excretion from nasal fluid and disease symptoms. Tissue specimens were collected for virus isolation and pathological examination. Strains vTKlac and vRRlac retained the ability to establish an infection, but showed reduced replication efficiency in the respiratory tract and were unable to attack the central nervous system (CNS) of pigs. Thus, both deletions induce significant attenuation of the virus measured by decrease of virulence in infected pigs. Strain vE16lac showed disease symptoms similar to that of wild type and could be detected in the CNS of pigs.