Novel gammaherpesvirus functions encoded by bovine herpesvirus 6 (bovine lymphotropic virus).

The genus Macavirus of the subfamily Gammaherpesvirinae includes viruses that infect lymphoid cells of domestic and wild ruminants and swine, causing asymptomatic latent infections in reservoir hosts. Here, we describe the genome of bovine herpesvirus 6 (BoHV-6), a macavirus ubiquitous in healthy cattle populations. The BoHV-6 genome exhibited architecture conserved in macaviruses, including a repetitive H-DNA region and unique 141 kbp L-DNA region predicted to encode 77 genes. BoHV-6 encoded, in variable genomic regions, a novel complement of genes relative to other characterized macaviruses, probably contributing to distinctive aspects of BoHV-6 infection biology and host range. Most notably, BoHV-6 encoded the first herpesviral protein (Bov2.b2) similar to cellular ornithine decarboxylase, an enzyme that catalyses the first and rate-limiting step in the biosynthesis of polyamines. Bov2.b2 conceivably mediates a novel mechanism by which BoHV-6 promotes cell-cycle-dependent viral replication.

Synthesis and structural studies on (E)-3-(2,6-difluorophenyl)-1-(4-fluorophenyl)prop-2-en-1-one: a promising nonlinear optical material.

A new fluorinated chalcone (E)-3-(2,6-difluorophenyl)-1-(4-fluorophenyl)prop-2-en-1-one was synthesized in 90% yield and crystallized by a slow evaporation technique. Its full structural characterization and purity were determined by scanning electron microscopy, infrared spectroscopy, gas chromatography-mass spectrometry, 1H, 13C and 19F nuclear magnetic resonance, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Raman microspectroscopy, UV-Vis absorption spectroscopy, single crystal X-ray diffraction (XRD) and Hirshfeld surface (HS) analysis. The fluorinated chalcone crystallized in centrosymmetric space group P21/c stabilized by the C-H⋯O and C-H⋯F interactions and the π⋯π contact. The crystalline environment was simulated through the supermolecule approach where a bulk with 378 000 atoms was built. The electric parameters were calculated at the DFT/CAM-B3LYP/6-311++G(d,p) level as function of the electric field frequency. The macroscopic parameters such as linear refractive index and third-order nonlinear susceptibility (χ (3)) were calculated, and the results were compared with experimental data obtained from the literature. The χ (3)-value for the chalcone crystal is 369.294 × 10-22 m2 V-2, higher than those obtained from a few similar types of molecule, showing that the chalcone crystal can be considered as a nonlinear optical material. Also, molecular theoretical calculations such as infrared spectrum assignments, frontier molecular orbital analysis and MEP were implemented, revealing that the most positive region is around the hydrogen atoms of the aromatic rings, and electrophilic attack occurs on the carbonyl group.

A novel dihydrocoumarin under experimental and theoretical characterization.

Coumarins are natural and synthetic active ingredients widely applied in diverse types of medicinal treatments, such as cancer, inflammation, infection, and enzyme inhibition (monoamine oxidase B). Dihydrocoumarin compounds are of great interest in organic chemistry due to their structural versatilities and, as part of our investigations concerning the structural characterization of small molecules, this work focuses on crystal structure and spectroscopic characterization of the synthesized and crystallized compound 4-(4-methoxyphenyl)-3,4-dihydro-chromen-2-one (C16H14O3). Additionally, a theoretical calculation was performed using density functional theory to analyze the sites where nucleophilic or electrophilic attack took place and to examine the molecular electrostatic potential surface. Throughout all of these calculations, both density functional theory and Car-Parrinello molecular dynamics were performed by fully optimized geometry. The spectroscopic analysis indicated the presence of aromatic carbons and hydrogen atoms, and also the carbonyl and methoxy groups that were confirmed by the crystallographic structure. The C16H14O3 compound has a non-classical intermolecular interaction of type C-H⋅⋅⋅O that drives the molecular arrangement and the crystal packing. Moreover, the main absorbent groups were characterized throughout calculated harmonic vibrational frequencies. Also, natural bond orbital analysis successfully locates the molecular orbital with π-bonding symmetry and the molecular orbital with π* antibonding symmetry. Finally, the gap between highest occupied and lowest unoccupied molecular orbitals implies in a high kinetic stability and low chemical reactivity of title molecule.

Induction of neutralizing antibodies to transmissible gastroenteritis virus by anti-idiotypic antibodies.

The potential of anti-idiotypic antibodies (anti-ids) as immunogens against transmissible gastroenteritis virus (TGEV) was tested in a heterologous system. A month-old pig was immunized with a neutralizing murine monoclonal antibody (MAb, 5A5) of the IgG2a isotype, specific for the E2 glycoprotein of TGEV. The anti-ids were isolated from the serum of the immunized pig by affinity chromatography, initially on a 5A5-Sepharose column, followed by repeated adsorption on a mouse IgG2a column. The swine anti-ids thus obtained bound to the MAb 5A5 (the idiotype), but not to MAbs of the same isotype IgG2a but of different idiotypes. The anti-ids also inhibited the binding of 5A5 to TGEV in a concentration-dependent manner. Mice immunized with the anti-ids produced antibodies to TGEV. These antibodies, neutralized TGEV in vitro and inhibited the binding of 5A5 to TGEV.

Characterization of foot-and-mouth disease virus by monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against foot-and-mouth disease (FMD) virus types O1 Campos Br1/58, A24 Cruzeiro Br1/55, and C3 Indaial Br1/71, which are the strains used for production of FMD vaccines in the majority of South American countries. Within the library of MAbs produced, a group was selected on the basis of their neutralizing titer in cell culture, protective titer in suckling mice, sensitivity to trypsin, and specificity for virus structural proteins. The MAbs were utilized in an ELISA test format to compare European and South American representative field isolates with vaccine production strains in their r1 relationship as obtained by 50% complement fixation (CF50) with polyclonal antibodies (PAb) and their virus neutralization (VN) relationship obtained with sera from one-time-vaccinated and from revaccinated cattle, respectively. The MAbs selected varied in their reactivity against the different strains and, therefore, enabled us to compare field FMDV strains to those against which the MAbs were produced, with definite advantages over the r1 and VN ratios. Thus, panels of MAb produced with the vaccine strains and appropriately selected are significantly useful for the FMD-control programs because they serve to provide guidance on the immunological coverage provided by the vaccines against FMDV strains circulating in the field. The MAbs are also useful for the differentiation of FMD virus strains.

Quantitation of latency established by attenuated strains of Pseudorabies (Aujeszky's disease) virus.

A quantitative and differential polymerase chain reaction (PCR) was developed that measures the ability of Pseudorabies virus (PRV) to colonize tissues that are targets for latency. This PCR is based on the co-amplification of viral target sequences and that of a gene of the host species: the porcine Nuclear Factor 1 gene, which serves as an internal standard. The technique was found to be sensitive and reproducible. Using this technique, individual variabilities were detected in the level of colonization of trigeminal ganglia established by a PRV strain among different animals. At a population level, it was established that two different PRV attenuated strains establish latency with different efficiencies, when applied by the same route and at similar inoculation dose.

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.

Study of neutralizing monoclonal antibodies to bovine herpes virus type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy.

Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.

Gene specific assay to differentiate strains of pseudorabies virus.

The need for compatibility between Pseudorabies vaccination and disease eradication measures has caused the production and release of diverse Pseudorabies virus (PRV) vaccine strains with altered genetic makeups due to the deletion of specific genes. These genes code for antigens used as differential serologic markers. By use of polymerase chain reaction (PCR), it is possible to determine, in a rapid and sensitive way, if a given PRV strain has a "wildtype" genotype, or if instead it carries a deletion for a specific gene. A sequence of 217 bp was selected as an amplification target within the gene of the essential glycoprotein 50 (gp50). Another sequence of 173 bp was selected in the joint area of glycoprotein 63 (gp63) and glycoprotein I (gI) genes. Under optimal amplification conditions, the simultaneous use of both PCR tests allowed us to differentiate specifically gI negative strains from several other wild type PRV strains, utilizing cell culture-propagated virus, acutely and latently infected neural tissue of mice and pigs as source of DNA targets. This kind of test will be useful for the rapid identification of PRV strains detected in tissues from individual animals, especially in cases of single reactors occurring in vaccinated herds. At the same time, the gene-defined PCR test will be useful for the evaluation of vaccines in their ability to prevent latency, by permitting unequivocal differentiation between vaccine and challenge virus strains.

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection.

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.

Detection of latent pseudorabies virus in swine using in situ hybridization.

We have examined methods for detection of pseudorabies virus (PRV) latency in three groups of swine; naturally infected animals obtained from a field case; animals which have been experimentally infected with Becker or Iowa strains of PRV; and single reactors (single seropositive animals within PRV-free herds). In situ hybridization was shown to be more sensitive than explanation/co-cultivation for the detection of latent virus. Nervous tissues, in particular the trigeminal ganglia, were found to be the most reliable source for detecting latent PRV. The presence of latent PRV was not detected in lymphoid tissues examined.

Effect of maternally acquired Aujeszky's disease (pseudorabies) virus-specific antibody in pigs on establishment of latency and seroconversion to differential glycoproteins after low dose challenge.

This study investigated whether (1) passively immune pigs could become latently infected after challenge with low doses of wild type pseudorabies virus (PRV) and (2) if seroconversion to PRV could be consistently detected using two commercially available differential diagnostic ELISAs. Three litters of piglets with passively acquired PRV serum neutralizing (SN) antibody (geometric mean titers 47.03 to 95.10) were challenged at 6 to 12 days of age with 236 to 500 TCID50 of Shope strain virus; pigs were vaccinated at 11 weeks of age with a commercially available genetically engineered vaccine (TK- gE- gG- Iowa S62 strain PRV). Vaccination was intended to reduce the risk of reactivation of latent infection resulting in spread of virulent PRV infection to previously uninfected pigs during the experiment. Vaccination at this age also approximated common field practices in infected herds. After 15 weeks, all challenged pigs were seropositive on the PRV glycoprotein (g or gp) E differential ELISA but were seronegative on the gG differential ELISA. All three challenge groups had pigs that were latently infected as evidenced by the detection of PRV DNA by polymerase chain reaction (PCR) assay of their trigeminal ganglia (TG). There was a significant inverse relationship observed for age at challenge and the proportion of PCR positive pigs in the group 15 weeks postchallenge (p = 0.0004). This trend was independent of the passively acquired PRV SN antibody titers at challenge. In this study, passively acquired antibody did not provide protection against establishment of latent infection in piglets after exposure to low doses of virulent PRV. These latent infections were detected serologically by only one of two available differential diagnostic ELISA.

Experimental infection of sheep with bovine herpesvirus type-5 (BHV-5): acute and latent infection.

We demonstrated that sheep are susceptible to acute and latent infection by bovine herpesvirus type-5 (BHV-5). Lambs inoculated intranasally with two South American BHV-5 isolates replicated the virus with titers up to 10(7.1) TCID50/ml for up to 15 days and showed mild signs of rhinitis. Four lambs in contact with the inoculated animals acquired the infection and excreted virus for up to seven days. One lamb developed progressive signs of neurological disease and was euthanized in extremis. Clinical signs consisted of tremors of the face, bruxism, ptyalism, incoordination, lateral flexion of the neck and head, circling, walking backwards, recumbency and paddling. The virus was detected in the anterior and posterior cerebrum, dorso- and ventro-lateral cortex, cerebellum, pons, midbrain and olfactory bulb. Viral nucleic acids were demonstrated in neurons and astrocytes of the anterior and ventro-lateral cortex by in situ hybridization. Histological changes consisting of non-suppurative meningitis, perivascular mononuclear cuffing, focal gliosis, neuronal necrosis and intranuclear inclusions were observed in the anterior cerebrum, ventro-lateral cortex and midbrain. Dexamethasone treatment at Day 50 pi resulted in reactivation of the latent infection and virus shedding in 13/16 (81%) of the lambs. Together with previous reports of BHV-5 antibodies in sheep, these findings show that sheep are fully susceptible to BHV-5 suggesting that infection by BHV-5 in sheep may occur naturally.

Role of neutralizing antibodies in PRRSV protective immunity.

Little has been known about the components of the immune system that are effective in the protection of a pig against PRRSV infection. Although antibodies were initially perceived as a deleterious, ineffective component of the PRRSV-specific immune response, neutralizing antibodies (NA) are now considered to be an important correlate of protective immunity against PRRSV. This paper reviews the current knowledge on arterivirus-specific NA, the role that NA have in protection against infection with PRRSV, as well as the viral molecular structures that are responsible for the production of this type of antibodies by the pig. This information should prove central to the design of new generation vaccines against PRRSV.

A capture-enzyme immunoassay for rapid diagnosis of transmissible gastroenteritis virus.

Two enzyme immunoassays (EIA) were developed for the detection of swine transmissible gastroenteritis virus (TGEV) antigens. The 2 EIAs used the same detecting system, a monoclonal antibody conjugated to horseradish peroxidase, but used different capture systems including a monoclonal antibody (m-EIA) or a polyclonal antibody (p-EIA). The EIAs were compared with the fluorescent antibody test (FAT) and electron microscopy (EM) for the detection of TGEV in intestinal samples of experimentally inoculated gnotobiotic piglets and of conventional diarrheic pigs submitted for diagnosis. In the gnotobiotic piglets experimentally inoculated with TGEV, 81.8% (9/11) were positive for TGEV by p-EIA, and 72.7% (8/11) were positive by m-EIA. In comparison, 81.8% (9/11) were positive by FAT and 27.2% (3/11) were positive by EM. Three noninfected controls were negative by all tests. In the diagnostic samples, 86.0% (43/50) were positive by p-EIA, 68.2% (30/44) were positive by m-EIA, 28.6% (14/49) were positive by IFA, and 38.0% (19/50) were positive by EM. The m-EIA had a higher agreement with FAT and EM than did p-EIA.

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay.

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.

Efficacy of a deletion mutant bovine herpesvirus-1 (BHV-1) vaccine that allows serologic differentiation of vaccinated from naturally infected animals.

Fifteen bovine herpesvirus-1 (BHV-1)-negative calves were vaccinated intramuscularly with 10(7.4) plaque-forming units of a double-deletion BHV-1 mutant (IBRV(NG)dltkdlgIII), and 6 remained as nonvaccinated controls. Thirty days after vaccination, the animals were challenged by nasal instillation of 10(8.2) CCID50 of a virulent BHV-1 strain (Cooper). The vaccinated calves were protected against wildtype virus challenge as demonstrated by clinical evaluation. Most of the vaccinates developed only a mild rhinitis (lasting an average of 6.5 days) with almost no systemic symptoms, whereas the controls developed a serious illness characterized by rhinitis (mean = 11.5 days), conjunctivitis, hyperthermia, apathy, loss of appetite, and dyspnea. The vaccinates also shed significantly less virus and for a shorter period of time (mean = 5.5 days) than the controls (mean = 9 days). Thirty days after vaccination, the vaccinates were negative in an anti-gIII specific blocking enzyme-linked immunosorbent assay (ELISA), despite the fact that most of them had developed neutralizing antibodies (serum neutralization titers ranging from 1:2 to 1:16). Seroconversion to gIII was detected as early as 7 days postinfection (dpi). Fourteen days after the challenge, all the animals exposed to wildtype BHV-1 had developed anti-gIII antibodies and were positive in this differential serologic test. Six controls plus 8 vaccinates kept in isolation were still positive to gIII when tested at 75 dpi. The use of the IBRV(NG)dltkdlgIII strain in conjunction with an anti-gIII specific blocking ELISA kit represents a powerful tool for BHV-1 control/eradication programs.

Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates.

Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV). Conventional virus isolation procedures showed the specimens contained BVDV. The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification. The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P. One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates. An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers. The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting.

Characterization of antibody response to porcine reproductive and respiratory syndrome virus ORF5 product following infection and evaluation of its diagnostic use in pigs.

The sensitivity and specificity of recombinant open reading frame 5 products used in the Western blotting assay for confirmation of porcine reproductive and respiratory syndrome virus (PRRSV) serologic status were evaluated. The recombinant antigen-based assays were specifically compared with a commercial enzyme-linked immunosorbent assay (ELISA) for PRRSV antibodies using 1) PRRSV antibody-negative reference sera (n = 30), 2) naturally infected pig sera (n = 40), 3) sequential sera obtained from 24 experimentally infected pigs, and 4) sera submitted to 3 state diagnostic laboratories (n = 200). The recombinant antigen assay yielded an average increased sensitivity of 10% over the commercial PRRSV ELISA. The negative controls (group 1 sera) showed no difference between the 2 assays. This comparison confirmed that the recombinant antigen-specific assay was more sensitive than the commercial ELISA and is well suited for routine confirmation of the presence of PRRSV antibodies.

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model.

The suitability of a rabbit seizure model for studying the neuropathogenesis of bovine herpesvirus type 5 (BHV-5) encephalitis was evaluated. Intranasal administration of BHV-5 (strain TX89) together with intramuscular administration of dexamethasone produced seizures in 70% of rabbits tested and meningo-encephalitis in 100%. Infectious BHV-5 was consistently isolated from the following sites: olfactory bulb; anterior cortex, containing the frontal cortex, olfactory tract and anterior portion of the olfactory cortex; posterior cortex, containing the temporal, parietal, piriform, entorhinal and occipital cortices; amygdala; hippocampus. Less frequently, BHV-5 was isolated from the midbrain and diencephalon, the pons and medulla, the cerebellum, and the trigeminal ganglia. Rabbits similarly infected with the Cooper strain of bovine herpesvirus type 1 showed no neurological signs or meningo-encephalitis, and virus was not recovered from the brain. The brains of BHV-5-infected rabbits showed neuronal degeneration, leptomeningitis, gliosis and perivascular cuffing, predominantly in the olfactory cortex (piriform and entorhinal cortices), amygdala and hippocampus. Mild lymphocytic meningitis was seen in the olfactory bulb and focal lymphocytic infiltration was sometimes present in the medulla and cerebellum. BHV-5, specific antigens and nucleic acids were detected in the olfactory cortex, amygdala and hippocampus by immunohistochemical methods and in-situ hybridization. The results suggested that, after intranasal BHV-5 inoculation, the virus spread to the central nervous system via the olfactory and trigeminal pathways. The olfactory pathway was more susceptible than the trigeminal pathway to neuropathogenic effects.