Vector competence of Aedes vexans (Diptera: Culicidae) for West Nile virus and potential as an enzootic vector.

Vector competence of Aedes vexans (Meigen) and Culex pipiens pipiens L. (Diptera: Culicidae) for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was compared. Infection rates of both species were similar 14 d after feeding on chickens, with WNV titers ranging from 10(4.2) to 10(8.7) plaque-forming units (PFU)/ml. Median infectious doses and 95% confidence intervals (CI) were 10(6.0(5.8, 63)) and 10(5.7(5.4, 5.9)) PFU for Ae. vexans and Cx. p. pipiens, respectively. WNV transmission was not observed in Ae. vexans that fed on chickens with WNV titers < 10(5.0) PFU/ml, in contrast to a mean (95% CI) transmission rate of 7(2,18)% for Cx. p. pipiens. Mean WNV transmission rates for Ae. vexans and Cx. p. pipiens were 13(7,21)% and 10(5,19)%, respectively, after feeding on chickens with WNV titers of 10(5.3 +/- 0.1) and 10(5.7 +/- 0.1) PFU/ml, and 31(25,37)% and 41(30,53)% after feeding on chickens with WNV titers > or = 10(6.1 +/- 0.1) PFU/ml. Time postinfection (p.i.) significantly influenced WNV transmission by Ae. vexans as indicated by a nearly 10-fold increase in transmission rate between days 7 and 14 p.i. Mean WNV load expectorated with saliva ofAe. vexans was 10(2.4(2.1, 2.7)) PFU, and it was not significantly affected by the titer of chickens on which they originally fed or time p.i. These data indicate that vector competence of the primarily mammalophilic Ae. vexans, which also feeds on birds, approaches that of Cx. p. pipiens for WNV. Because peridomestic mammals, such as cottontail rabbits, squirrels, and chipmunks, develop WNV titers infective for Ae. vexans, this species may play a significant role in WNV enzootic cycles.

The potential of Aedes triseriatus (Diptera: Culicidae) as an enzootic vector of West Nile virus.

The susceptibility of Aedes triseriatus (Say) (Diptera: Culicidae) to low levels of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was determined and compared with that of Culex pipiens L. to assess the likelihood of its participation in an enzootic cycle involving mammals. Ae. triseriatus and Cx. pipiens were exposed to WNV by feeding on baby chickens with WNV serum titers ranging from 10(4.1 +/- 0.1) to 10(8.6 +/- 0.1) plaque-forming units (PFU)/ml and from 10(4.1 +/- 0.1) to 10(7.0) PFU/ml, respectively. Infection rates and 95% confidence intervals (CIs) of 8% (4, 14) and 25% (15, 38) occurred in Ae. triseriatus and Cx. pipiens after feeding on chickens with WNV titers of 10(4.1 +/- 0.1) PFU/ml and increased to 65% (49, 79) and 100% (72, 100) in Ae. triseriatus and Cx. pipiens after feeding on chickens with titers of 10(7.1 +/- 0.1) PFU/ml. The mean infection rate of Ae. triseriatus ranged from 97% (84, 100) to 100% (79, 100) after feeding on chickens with WNV titers of > or = 10(8.2) PFU/ml. The infectious dose (ID)50 values for Ae. triseriatus and Cx. pipiens were 10(6.5) (6.4, 6.7) and 10(4.9) (4.6, 5.1) PFU/ml, respectively. The combined estimated transmission rate of Ae. triseriatus at 14 and 18 d after feeding on chickens with a mean WNV titer of 10(8.6 +/- 0.1) PFU/ml was 55%. Although Ae. triseriatus is significantly less susceptible to WNV than Cx. pipiens, the susceptibility of Ae. triseriatus to WNV titers < 10(5.0) PFU/ml and its ability to transmit WNV suggest that Ae. triseriatus has the potential to be an enzootic vector among mammalian populations.

Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Production and characterization of monoclonal antibodies directed against pseudorabies virus.

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.

Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs.

Infection of porcine alveolar macrophages by the porcine reproductive and respiratory syndrome virus (PRRSV) was significantly enhanced in vitro by antibody raised against the PRRSV isolate ISU-P (p < 0.01). Increased yields and infection rates were highly correlated (r = 0.95) and the ratio of yield to infection rate was greater than 1.4, suggesting that more than one mechanism was responsible for enhanced infection. Antibody-dependent enhancement (ADE) of infection was also demonstrated in vivo using a completely randomized block design (n = 16). The mean level and duration of viremia were greater (p < 0.05) in pigs injected with subneutralizing amounts of PRRSV-specific IgG prior to virus challenge than in control pigs injected with normal IgG. In contrast, virus replication was significantly (p < 0.01) inhibited in pigs with neutralizing antibody titers of 4 log2. The period of time that subneutralizing levels of antibody can persist and contribute to ADE of PRRSV infection was estimated in 4 pigs injected with PRRSV-specific IgG to yield an initial neutralizing antibody titer of 3.8 log2. Neutralizing activity declined to undetectable levels by day 37 postinjection (PI). ADE activity was first detected in undiluted sera on day 20 PI and persisted through day 62 PI. Western immunoblot analysis of sera collected between days 37 and 62 PI detected antibodies specific for the 15-kDa nucleocapsid and 26-kDa glycosylated envelope proteins. These results strongly suggest that ADE has the potential to contribute to the pathogenesis of PRRSV infection and is mediated by antibody specific for the 26-kDa envelope protein.

Studies on the biology of Ixodes dammini in the upper midwest of the United States.

The seasonal activity pattern of Ixodes dammini was monitored in south-central Wisconsin in 1989 and 1990. Immature tick activity and abundance were assessed by small mammal trapping (732 Peromyscus leucopus examined) and supplemented with flagging. Tick survival and molting times were evaluated by maintaining ticks in environmental chambers at field sites. Results indicate the biology of I. dammini in the upper midwest is similar to that previously reported from the eastern United States. One notable difference was that seasonal larval activity appeared broader (late April through October) and peaked earlier (on 9 July 1989 mean was 7.7 larvae/mouse; on 9 June 1990 mean was 7.3 larvae/mouse). Nymphal activity on P. leucopus peaked in May of both years (mean = 2.5 in 1989; mean = 1.8 in 1990). Bloodfed females placed in the field in early May 1990 oviposited in early June, and larvae emerged by early August. All bloodfed stages successfully overwintered at field sites.

Impact of dengue virus infection on feeding behavior of Aedes aegypti.

In addition to heavily infecting the salivary glands of Aedes aegypti (L.) mosquitoes, dengue viruses produce a significant infection of the nervous system, involving the brain, Johnston's organ, compound eye, and thoracic and abdominal ganglion. To determine if dengue infection affects feeding behavior of Ae. aegypti we measured feeding times, counted the number of feeding delays or interruptions, and by in situ immunocytochemistry techniques determined the spatial and temporal distribution of dengue infections in females parenterally infected with dengue 3 virus. The mean of the total time required for feeding by infected mosquitoes was significantly longer than the time required by uninfected mosquitoes. Similarly, the mean of the time spent probing was significantly longer in infected mosquitoes than in uninfected mosquitoes when day after inoculation was considered. Significant increases in the length of feeding activity in infected mosquitoes corresponded to virus infection in organs that are known to control or influence activities associated with blood feeding. Sequential infections of the salivary glands (five days postinoculation [PI]), brain and compound eye (eight days PI), and Johnston's organ and midgut and abdominal ganglion (11 days PI) of most mosquitoes were observed. The increased time required by infected Ae. aegypti mosquitoes to acquire a blood meal may contribute to the efficiency of Ae. aegypti as a vector of dengue virus. Longer feeding periods are more likely to be interrupted by the host, which increases the chance that an infected mosquito will probe or feed on additional hosts.

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.

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.

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 labeling 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.

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.

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens.

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.

The evaluation of a lectin-agarose based subunit vaccine and complementary diagnostic antigen for Aujeszky's disease (pseudorabies) in the pig.

Eleven out of 25 pigs were immunized with a lectin--agarose based subunit vaccine for Aujeszky's disease (AD). The vaccine was prepared by extracting protective antigens from a non-ionic detergent (Triton-X-100) extract of AD virus-infected PK-la cells with Lens culinaris agglutinin immobilized on agarose beads. Two groups of 3 and 4 pigs received 2 doses of vaccine each containing 426 micrograms of adsorbed protein. Two groups of 2 pigs each received 2 vaccine doses containing either 23 or 33 micrograms of adsorbed protein. All vaccinated pigs survived a nasal challenge of 10(8.5) PFU of virulent AD virus while 13 out of 14 (93%) uninoculated controls died between Days 5 and 9 post challenge. This immunizing preparation qualified as a practical subunit vaccine because pigs were protected with relatively small amounts of protective antigen while at the same time remained free of detectable antibody to a complementary diagnostic antigen. This antigen was obtained in relatively pure form from the maintenance medium of virus-infected cells 4 h post-inoculation. In addition both high and low dose vaccinates failed to produce detectable antibody to at least one other antigen complex. The composition of Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin (RCA)-purified AD viral antigen preparations were also compared by crossed immunoelectrophoretic techniques. Both preparations contained two antigen complexes and two individual antigens in common. Each preparation also contained its own unique antigen complex. The RCA purified antigen preparation also contained small quantities of a single antigen that was not detectable in the LCA antigen preparation.

Evaluation of a diagnostic antigen for the detection of Aujeszky's disease virus-infected subunit-vaccinated pigs.

An early virus protein complex that is found in the maintenance medium of Aujeszky's disease (AD) virus-infected cells was evaluated as a subunit diagnostic antigen (SUDA) in the enzyme-linked immunosorbent assay (ELISA). This antigen was found in purer form and in larger quantities for up to 12 h post-infection in the maintenance medium of AD virus-infected MDBK cell cultures than in the maintenance medium of virus-infected porcine Fallopian tube (PFT) and PK1a cell cultures. The SUDA was shown to be compatible with a lectin-derived subunit vaccine by the absence of positive ELISA reactions for antibody to this antigen in 25 AD virus-free subunit-vaccinated pigs. Following virus challenge, all fo 24 surviving vaccinated pigs seroconverted to SUDA within 10 days. Compatibility with the vaccine was further demonstrated by the absence of positive ELISA reactions for antibody to SUDA in 12 pigs that received five or six consecutive vaccine doses at 3-wk intervals. The sensitivity of the ELISA with SUDA was demonstrated by the detection of antibody in virus-infected vaccinated and non-vaccinated pigs for at least 15 and 22 weeks, respectively, following exposure to virus. The SUDA was also economical: it was calculated that 8000-14 000 tests could be run with the antigen present in the maintenance medium of one 850 cm2 plastic tissue culture roller bottle of virus-infected MDBK cells.

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.

The effect of a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccine treatment on virus shedding in previously PRRSV infected pigs.

Two experiments were conducted to investigate if virus shedding could be reduced following a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccination (KV) of PRRSV infected pigs. In experiment 1, PRRSV infected pigs were vaccinated with KV on days 14 and 28 following infection. Viremia and serum neutralizing (SN) antibody were compared to infected pigs with no KV. The second experiment was conducted in an identical manner. In addition to viremia and SN antibody, virus in oropharyngeal scrapings and interferon gamma (IFN-gamma) producing cells were monitored. Magnitude and duration of viremia were not different between KV vaccinated and non-vaccinated groups. No virus was detected in oropharyngeal scraping from any pig, nor was there a difference in the detection of viral RNA. In both experiments, however, increases in SN titer and number of IFN-gamma producing cells were observed. The SN titer was significantly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 42-56 following infection in experiments 1 and 2, respectively. The number of IFN-gamma producing cells was slightly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 63. These observations suggest that KV had no effect on virus shedding. However, previously infected pigs responded immunologically to KV, as demonstrated by increases in SN antibody titers and IFN-gamma producing cells.

Porcine reproductive and respiratory syndrome virus: routes of excretion.

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 (p.i.), 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 p.i. 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 plan an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV with prolonged recovery of virus from tonsils of swine.

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.

Longevity and spontaneous flight activity of Culex tarsalis (Diptera: Culicidae) infected with western equine encephalomyelitis virus.

The longevity of an Iowa strain of Culex tarsalis Coquillett fed blood meals containing 2 concentrations of western equine encephalomyelitis virus from Iowa (WEE-7738) was compared with that of Cx. tarsalis fed blood without virus. Females exposed to 4.7-5.0 log TCID50 per mosquito of WEE-7738 did not live as long as mosquitoes exposed to 2.7-3.0 log TCID50 per mosquito or controls. Only 1% of mosquitoes fed blood containing the higher virus concentration survived to day 18 after exposure. However, 13% of mosquitoes fed blood with the lower virus titer and 19.5% of the controls were still alive on day 18 after exposure. Flight activity scores of Cx. tarsalis infected with 4.7-5.0 log TCID50 per mosquito of WEE-7738 were 27.5% lower, and there were 26.1% fewer spontaneous flights than noninfected controls from days 6-11 after infection. After day 8 after infection, infected Cx. tarsalis had 37.1% lower activity scores and 40.0% fewer spontaneous flights than noninfected controls. Virus infection did not affect how long a mosquito flew in a 24-h period (the daily flying time) or the duration of individual flights. The spontaneous flight activity pattern (circadian rhythm) of infected mosquitoes was identical to those of controls. Both infected and noninfected mosquitoes began spontaneous flight activity at 2000-2100 hours (CST) and were active throughout the entire dark phase of the 24-h cycle. Although mosquitoes were active throughout the night, there was a burst or peak of activity between 2200 and 2300 hours when the complete dark cycle began. These results indicate that the adverse effect of WEE infection on longevity and spontaneous flight activity of Cx. tarsalis may decrease vectorial capacity of Cx. tarsalis for WEE.