Kenneth B. Platt

A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections.

Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labeling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T4-T13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactive phenylethanolamine-N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

Porcine reproductive and respiratory syndrome virus: a persistent infection.

Persistent infection with porcine reproductive and respiratory syndrome virus (PRRSV) was shown in experimentally infected pigs by isolation of virus from oropharyngeal samples for up to 157 days after challenge. Four 4 week old, conventional, PRRSV antibody-negative pigs were intranasally inoculated with PRRSV (ATCC VR-2402). Serum samples were collected every 2 to 3 days until day 42 post inoculation (PI), then approximately every 14 days until day 213 PI. Fecal samples were collected at the time of serum collection through day 35 PI. Oropharyngeal samples were collected at the time of serum collection from 56 to 213 days PI by scraping the oropharyngeal area with a sterile spoon, especially targeting the palatine tonsil. Turbinate, tonsil, lung, parotid salivary gland, spleen, lymph nodes and serum were collected postmortem on day 220 PI. Virus isolation (VI) on porcine alveolar macrophage cultures was attempted on all serum, fecal and oropharyngeal samples, as well as tissues collected postmortem. Postmortem tonsil tissues and selected fecal samples were also assayed for the presence of PRRSV RNA by the polymerase chain reaction (PCR). Serum antibody titers were determined by IFA, ELISA and SVN. Virus was isolated from all serum samples collected on days 2 to 11 PI and intermittently for up to 23 days in two pigs. No PRRSV was isolated from fecal samples, but 3 of 24 samples were PCR positive, suggesting the presence of inactivated virus. Oropharyngeal samples from each pig were VI positive 1 or more times between 56 and 157 days PI. Oropharyngeal samples from 3 of 4 pigs were VI positive on days 56, 70 and 84 PI. Virus was isolated from one pig on day 157 PI, 134 days after the last isolation of virus from serum from this animal. Virus was isolated from oropharyngeal samples for several weeks after the maximum serum antibody response, as measured by IFA, ELISA and SVN tests. All tissues collected postmortem were VI negative and postmortem tonsil samples were also negative by PCR. An important element in the transmission of PRRSV is the duration of virus shedding. The results of this study provided direct evidence of persistent PRRSV infection and explain field observations of long-term herd infection and transmission via purchase of clinically normal, but PRRSV infected, animals. Effective prevention and control strategies will need to be developed in the context of these results.

Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period. These results clearly indicate that the 15-kD protein is the most immunogenic of the 4 viral proteins identified and may provide the antigenic basis for the development of improved diagnostic tests for the detection of PRRS virus antibodies.

Peripheral and central pathways regulating the kidney: a study using pseudorabies virus

We used the retrograde transneuronal transport of a neurotropic virus, pseudorabies virus (PRV), to identify the neurons in sympathetic ganglia, spinal cord and brain which regulate renal function and renal circulation. PRV was microinjected into the left kidney of 70, pentobarbital-anesthetized, male rats. After an incubation period of 1-4 days, rats were anesthetized and sacrificed. PRV-infected neurons were located immunocytochemically in pre- and paravertebral sympathetic ganglia, the intermediolateral cell column of the T10-T13 segments and several brainstem cell groups: the medullary raphe nuclei, rostral ventrolateral medulla, rostral ventromedial medulla, A5 cell group, and the paraventricular hypothalamic nucleus. In more heavily infected rats, additional labeling was found in the locus coeruleus, periaqueductal gray matter, lateral hypothalamic area, zona incerta, and anterior hypothalamic area. No infected propriospinal neurons were observed in the lateral spinal nucleus or gray matter of the caudal cervical, lumbosacral or thoracic spinal segments not containing infected putative sympathetic preganglionic neurons. The paucity of infected propriospinal neurons in the presence of infected brainstem neurons, even in lightly infected rats, is discussed in reference to the relative importance of descending vs spinal regulation of the sympathetic outflow to the kidney.

CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study

The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains wer e processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluoresence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus. A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decarboxylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled.

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.

Categorization of North American porcine reproductive and respiratory syndrome viruses: epitopic profiles of the N, M, GP5 and GP3 proteins and susceptibility to neutralization.

Summary.  Eleven epitopes were identified by murine monoclonal antibodies (MAbs) that represented the N, M, GP5 and GP3 proteins of the North American (NA) porcine reproductive and respiratory syndrome (PRRS) virus, KY 35 (NVSL 46907). Three discontinuous epitopes of the N and M proteins were designated EpORF7-Fd through Hd and EpORF6-Ad through Cd. Five continuous epitopes of the GP5 and GP3 proteins were designated EpORF5-A through C and EpORF3-A and B. The MAbs representing EpORF5-C and EpORF6-A and B had neutralizing activity. The MAbs representing the above epitopes, except EpORF7-Gd and Hd, expanded the virus marker system described in a previous study in which a panel of 69 NA viruses and the Lelystad virus were categorized into 5 antigenic groups, I15 through V15 based on the presence or absence of 5 continuous epitopes of the N protein. Antigenic groups I15 and II15, which represented 84.7 and 11.6% of all viruses tested, were categorized further into 9 and 4 subgroups, respectively. The remaining NA viruses and the Lelystad virus were distributed among 4 groups, one of which was represented by 2 subgroups. Significant (P<0.05) differences in sensitivity to neutralization of 28 viruses representing 6 antigenic groups by the 3 neutralizing MAbs suggested that sensitivity to neutralization may also be of value in categorizing PRRS viruses.

Porcine reproductive and respiratory syndrome virus: routes of excretion.

Abstract This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post inoculation (PI), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 PI at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may play an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV prolonged recovery of virus from tonsils of swine.

Field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) vary in their susceptibility to antibody dependent enhancement (ADE) of infection.

Seventeen porcine reproductive and respiratory syndrome virus (PRRSV) field isolates, including isolate ISU-P, were evaluated for their susceptibility to antibody dependent enhancement (ADE) of infection mediated by antibodies raised against PRRSV isolate ISU-P. Progeny virus yields of ISU-P and 4 of 16 field isolates in porcine alveolar macrophages (PAM) were reduced following treatment with a concentration of antibody that neutralized ISU-P (p < 0.01). In contrast, the yields of 12 of 17 field isolates were enhanced (p < 0.01). Treatment of all isolates with a 10-fold lower concentration of this antibody significantly (p < 0.01) increased virus yields of all isolates in PAM. However, the degree of enhancement varied among the isolates when compared to the enhancement of the yield of ISU-P. While no differences in enhancement were observed among ISU-P and 9 field isolates, yield enhancement of 6 and 1 isolates were less than and more than the yield enhancement of ISU-P, respectively (p < 0.05). The degree of enhancement mediated by a high concentration of antibody raised against ISU-P was inversely proportional to the ability of the antibody to neutralize the isolates (r = 0.92). In contrast, no direct correlation (r = 0.32) was observed between the degree of enhancement mediated by a low concentration of antibody and the ability of the antibody to neutralize the isolates. These data suggest that the variability in the susceptibility of PRRSV isolates to ADE arise from quantitative and/or qualitative differences in the antigenic determinants associated with virus neutralization and/or ADE. The antigenic diversity and the wide range in the susceptibility to ADE that exists among field isolates indicate that ADE should be taken into consideration in the development of effective immunization strategies for PRRS.