Developmental changes in myelin-induced proliferation of cultured Schwann cells.

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells.

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.

Non-specific antiviral response detected in RNA-treated cultured cells of the sandfly, Lutzomyia longipalpis.

Lutzomyia longipalpis is the principal vector of visceral leishmaniasis in the Americas, and can also transmit some viruses. To help develop a gene-silencing system for this sandfly, we transfected cultured embryonic cells with various double-stranded RNAs using West Nile virus (WNV) virus-like particles (VLPs) expressing luciferase as the target RNA to demonstrate effective gene knock-down. When luciferase dsRNA was introduced into these cells, they produced the expected reduction in VLP-encoded luciferase, suggesting specific silencing of the luciferase gene. Surprisingly, we found that unrelated dsRNAs, which included those specific for several L. longipalpis gene sequences and Escherichia coli beta-galactosidase, diminished replication of the VLP-encoded genome. These results are the first indication for a nucleic acid-induced, non-specific antiviral response in this important insect vector.

Out on the farm with DNA vaccines.

DNA vaccination is a rapidly developing technology that offers new approaches for the prevention of disease. This technology may permit the production of new vaccines against diseases that have no current vaccine, as well as allowing the development of improved vaccines to replace existing products. We describe how DNA vaccination is being developed for use in commercial animal production, with an emphasis on viral diseases, and discuss the existing hurdles to its development and use.

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate.

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.

Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus.

Over the last few years we have utilized a system to genetically engineer foot-and-mouth disease virus (FMDV) to produce live-attenuated vaccine candidates. These candidates have been generated in the genetic background of a tissue culture-adapted strain of serotype A12 virus. Based on this A12 system, we created a virus lacking the sequence encoding the leader (L) proteinase (Piccone et al., 1995), and demonstrated that this leaderless virus, A12-LLV2 was avirulent in bovine and swine, and could be used as an attenuated vaccine (Mason et al., 1997; Chinsangaram et al., 1998). The current study shows that a similar leader-deleted chimeric virus containing the genome of the type A12 virus with a substituted type O1 capsid coding region from a bovine-virulent virus can be constructed, and that the virus has low, but detectable virulence in swine. A second chimera specifying a tissue culture-adapted type O1 capsid lacking the RGD cell binding site, was avirulent in swine, but was not sufficiently immunogenic to provide protection from challenge. These results are described with respect to mechanisms of attenuation and antigen formation in live-attenuated virus-inoculated animals.

Flavivirus type-specific antigens produced from fusions of a portion of the E protein gene with the Escherichia coli trpE gene.

Based on previous data showing that JEV and DEN-1 fusion proteins can be used as antigens, we engineered analogous fusion proteins for the same regions of the E proteins of DEN-2, DEN-3, and DEN-4, using the polymerase chain reaction. The resulting fusion proteins were tested in a modified ELISA for their reactivity with mouse immune ascitic fluids (MIAF) generated against 10 different flaviviruses. The results showed that these recombinant antigens could be used as type-specific immunological reagents, and suggest that these antigens could serve as the basis for rapid, safe, and inexpensive flavivirus serosurveys.

Transformation of an insect symbiont and expression of a foreign gene in the Chagas' disease vector Rhodnius prolixus.

A shuttle plasmid was developed that is capable of replicating both in Escherichia coli and in Rhodococcus rhodnii, a bacterial symbiont of the Chagas' disease vector Rhodnius prolixus. We have been able to transform R. rhodnii with this plasmid, infect aposymbiotic R. prolixus with the transformed symbionts, select with the antibiotic thiostrepton, and re-isolate genetically altered symbionts from the insects following successive molts. Symbiotic bacteria are potentially valuable as vehicles for the stable introduction of foreign genes into insects with the goal of eventually altering the ability of the insect to transmit a pathogenic agent.

Japanese encephalitis virus-specific proliferative responses of human peripheral blood T lymphocytes.

The T lymphocytes play an important role in prevention and recovery from viral infections. To characterize T lymphocyte responses to Japanese encephalitis (JE) virus infections, we analyzed JE virus-specific T lymphocytes in peripheral blood mononuclear cells (PBMC) obtained from seven JE patients and 10 vaccinees who had received a formalin-inactivated, purified JE virus vaccine (Biken vaccine). These PBMC were examined for proliferative responses against live JE virus, a glutaraldehyde-fixed lysate of cells infected with JE virus, and extracellular particles (EPs; subviral membrane vesicles released from cells infected with recombinant vaccinia viruses encoding the JE virus premembrane and envelope proteins). Japanese encephalitis virus-specific T cell proliferation was demonstrated with PBMC from both patients and vaccinees after stimulation with infectious JE virus or the lysate of JE virus-infected cells. Proliferating PBMC included CD4+ T lymphocytes and CD8+ T lymphocytes in responses to either form of JE viral antigens. Responses to EPs were observed only with PBMC from some American vaccinees whose PBMC also responded to the virus and lysate. These results indicate that JE virus infection and immunization with an inactivated JE vaccine induce JE virus-specific CD4+ and CD8+ T memory lymphocytes that can be induced to proliferate by infectious JE virus and noninfectious JE antigens.

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.

Strategy for producing new foot-and-mouth disease vaccines that display complex epitopes.

Widely used inactivated vaccines for foot-and-mouth disease (FMD) induce protective immunity, but vaccine production plants and residual virus in the vaccine itself have been implicated in disease outbreaks. The structure of the FMD virion has been determined, and although much of the surface of the viral particle is produced by complex folding of the three surface-exposed capsid proteins (VP1-3), some surface regions representing important linear epitopes can be mimicked by recombinant proteins or synthetic peptides. Vaccine candidates based on these products stimulate immune responses to foot-and-mouth virus (FMDV), but do not always protect livestock from disease. The basis of protective immunity to FMDV has been explored using genetic engineering to produce antigenic chimeras of the virus. Studies with these chimeras have shown that a strong and protective immune response can be generated in livestock to epitopes outside the sequential epitopes incorporated into previous subunit vaccine candidates. Genetic engineering of the virus has also been used to demonstrate that changes within the sequence encoding an arginine-glycine-aspartic acid (RGD) sequence in VP1 abrogate virus binding to cells in culture, confirming the role of RGD as the receptor binding site. Based on this information, genetically stable viruses which cannot bind to cells have been created by deleting the nucleotides coding the RGD sequence. The receptor binding site-deleted viruses have been shown to be non-infectious in tissue culture, mice, and swine. Cattle vaccinated with these viruses are protected from disease when challenged with virulent FMDV, demonstrating that they could serve as the basis for safer FMD vaccines.

Engineering better vaccines for foot-and-mouth disease.

Although efficacious and safe, current vaccines for FMD suffer from drawbacks. Among these are that the immune response to the vaccine interferes with the ability to detect vaccinated animals that have subsequently become infected and could carry and shed the virus, creating an obstacle to re-instating disease-free status to countries/regions that vaccinate to control outbreaks. Multiple diagnostic tests are available to identify animals that have been infected with FMDV by detection of antibodies to viral non-structural proteins (NSP) that are present in low concentration in traditional vaccines and are poorly immunogenic in vaccine preparations. However, these tests are not 100% reliable. To circumvent this problem, we have developed a new generation of vaccines that express the "empty capsid" subunit of the virus, in the absence of one of the most immunogenic NSPs, 3Dpol. Here we describe delivery of the empty capsid subunits by recombinant replication-defective human adenovirus type 5 (Ad5). These Ad5-vectored empty capsid vaccines can protect pigs from FMDV challenge as early as 7 days post-vaccination. A second problem with current FMD vaccines is that they do not induce protective immunity quickly, a drawback that is likely to be shared by our Ad5-vectored empty capsid vaccine. To overcome this problem, we have developed a prophylactic antiviral treatment consisting of an Ad5 encoding porcine interferon alpha (pIFNalpha). Administration of Ad5-pIFNalpha protects swine from FMD as early as one day post-administration. The combination of this antiviral treatment and the empty capsid subunit vaccine should induce rapid and complete protection from FMD, and could overcome current diagnostic problems.

Prospects, including time-frames, for improved foot and mouth disease vaccines.

Inactivated foot and mouth disease (FMD) vaccines have been used successfully as part of eradication programmes. However, there are a number of concerns with the use of such vaccines and the recent outbreaks of FMD in disease-free countries have increased the need for improved FMD control strategies. To address this requirement, new generation FMD vaccines are being developed. Currently, one of the most promising of these vaccine candidates utilises an empty viral capsid subunit delivered to animals by a live virus vector. This candidate, a replication-defective recombinant human adenovirus containing the capsid and 3C proteinase coding regions of FMD virus (FMDV), induces an FMDV-specific neutralising antibody response in inoculated animals. Upon challenge with a virulent animal-passaged homologous virus, swine and cattle vaccinated with this recombinant adenovirus are protected from clinical signs of FMD as well as from FMDV replication. One inoculation of a high dose of this vaccine candidate protected swine from challenge as early as seven days after vaccination.

Procedures for preventing the transmission of foot-and-mouth disease virus to pigs and sheep by personnel in contact with infected pigs.

The most effective method of containing an outbreak of foot-and-mouth disease (FMD) is by the culling of livestock. However, qualified people must diagnose the disease before the culling can begin, and they must avoid susceptible animals after having been in contact with infected premises, to prevent them from transmitting the virus. To test the effectiveness of biosecurity procedures in preventing the transmission of FMD virus (O/UK/35/2001) investigators contacted and sampled pigs inoculated with FMD virus for approximately 45 minutes and then contacted and sampled sentinel pigs and sheep after either using no biosecurity procedures, or washing their hands and donning clean outerwear, or showering and donning clean outerwear. The virus was detected in the nasal secretions of one investigator immediately after the postmortem investigation of the inoculated pigs but was not detected in samples collected between approximately 12 and 84 hours later. After the contaminated personnel had showered and changed into clean outerwear they did not transmit the strain of FMD virus to susceptible pigs and sheep.

One hundred pregnancies after treatment with pulsatile luteinising hormone releasing hormone to induce ovulation.

OBJECTIVE: To review treatment with pulsatile luteinising hormone releasing hormone in infertile women who do not ovulate and are resistant to clomiphene after 100 pregnancies achieved with this treatment. DESIGN: Retrospective analysis of 146 courses of treatment over 434 cycles. SETTING: Infertility clinic. PATIENTS: 118 Women whose failure to ovulate was due to idiopathic hypogonadotrophic hypogonadism (n = 39), amenorrhoea related to low weight (n = 17), organic pituitary disease (n = 15), or polycystic ovaries (n = 47). INTERVENTIONS: Dose of 15 micrograms luteinising hormone releasing hormone/pulse subcutaneously every 90 minutes given with a miniaturised pump throughout cycle monitored by ultrasound. Women with hypogonadotrophic hypogonadism had 48 courses, women with amenorrhoea related to low weight 23, women with organic pituitary disease 18, and women with polycystic ovaries 57. END POINT: Follow up of 100 pregnancies achieved in 77 women during six years after introducing treatment. MEASUREMENTS and main results--One hundred pregnancies (seven multiple, 28 miscarriages). Cumulative rates of pregnancy were 93-100% at six months in women with idiopathic hypogonadotrophic hypogonadism, amenorrhoea related to low weight, and organic pituitary disease. In women with polycystic ovaries (cumulative rate of pregnancy 74%) adverse prognostic factors were obesity, hyperandrogenism, and high luteinising hormone concentrations, which were also associated with a high rate of early pregnancy loss. CONCLUSIONS: Treatment with pulsatile luteinising hormone releasing hormone is safe, simple, and effective, and the preferred method of inducing ovulation in appropriately selected patients. Compared with exogenous gonadotrophin treatment there is little need for monitoring, no danger of hyperstimulation, and a low rate of multiple pregnancies.

Evaluation of infectivity and transmission of different Asian foot-and-mouth disease viruses in swine.

Most isolates of foot-and-mouth disease virus (FMDV) display a broad host range. Since the late 1990s, the genetic lineage of PanAsia topotype FMDV serotype O has caused epidemics in the Far East, Africa, the United Kingdom, France, the Netherlands, and numerous other countries throughout Europe and Asia. In contrast, there are several FMDV isolates that exhibit a more restricted host range. A Cathay topotype isolate of FMDV serotype O from the 1997 epizootic in Taiwan (O/TAW/97) demonstrated restricted host specificity, only infecting swine. Methods used to evaluate infectivity and pathogenicity of FMDV isolates in cattle are well-documented, but there has been less progress studying transmission and pathogenicity of FMDV isolates in pigs. In previous studies designed to examine pathogenicity, various chimeric viruses derived from O/TAW/97 were intradermally inoculated in the heel bulb of pigs. Subsequent quantitative scoring of disease and evaluation of virus released into nasal secretions and blood was assessed. Here we prove the usefulness of this method in direct and contact inoculated pigs to evaluate infectivity, pathogenicity and transmission of different Asian FMDV isolates. Virus strains within the Cathay topotype were highly virulent in swine producing a synchronous disease in inoculated animals and were efficiently spread to in-contact naïve pigs, while virus strains from the PanAsia topotype displayed more heterogeneous properties.

Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells.

The Japanese encephalitis virus (JE) structural glycoprotein (E) and two nonstructural glycoproteins (NS1 and NS1') were processed differently by JE-infected vertebrate and invertebrate cell lines. All three proteins were released slowly (t1/2 greater than 6 hr) from JE-infected monkey cells (Vero cells). Mosquito cell lines released E at a similar rate (t1/2 greater than 8 hr), while NS1 and NS1' were retained in an undegraded form in the cell layer. The proteolytic processing of the three proteins appeared identical in both cell types, but some differences in N-linked glycosylation were observed. E, NS1, and NS1' found within the infected cells of both types contained high-mannose oligosaccharide groups for more than 8 hr after synthesis. Additional sugar residues were added to the single E protein oligosaccharide group prior to release from Vero cells, while sugar residues were trimmed from the E protein oligosaccharide group prior to release from mosquito cells. The forms of NS1 and NS1' found in the culture fluid of infected Vero cells contained one complex and one high-mannose oligosaccharide. All three glycoproteins released from JE-infected Vero cells were associated with extracellular particles, the virion in the case of E and a low density particle in the case of and NS1' exhibited amphipathic properties in Triton X-114 extraction experiments. Taken together, these results suggest that both the structural (E) and nonstructural (NS1 and NS1') glycoproteins were pathway of the infected Vero cells, assembled into particles, and then released into the extracellular fluid.

Foot-and-mouth disease virus undergoes restricted replication in macrophage cell cultures following Fc receptor-mediated adsorption.

We have previously reported that foot-and-mouth disease virus (FMDV) can enter an Fc receptor (FcR)-expressing cell line by antibody-dependent enhancement. Since FMDV can establish a persistent infection in animals in the presence of high levels of neutralizing antibodies (carrier state), we examined macrophages for their ability to be infected by the virus in the presence of antibody. The murine macrophage cell line P388D1 or porcine macrophage-monocytes isolated from peripheral blood were incubated with antibody-complexed virus. Under these conditions, host protein synthesis was rapidly inhibited in both cell types, but not in cells incubated either with virus alone or with imine-inactivated antibody-complexed virus. Virus-specific structural and nonstructural proteins were synthesized in antibody-complexed virus-infected P388D1 cells, while only nonstructural proteins were detected in porcine macrophage cultures. Negative-strand RNAs were detected in both cell types, indicating that RNA replication had taken place. Cultures of P388D1 cells transfected with viral RNA produced very low levels of infectious virus, and infection with virus-antibody complexes, followed by a brief wash with pH 6.0 buffer to remove residual input virus, allowed the detection of low levels of productive replication. Thus, macrophages can be infected with FMDV via FcR-mediated adsorption, and infection of these cells could contribute to pathology or provide a reservoir of infectious virus in carrier animals.

Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein.

The role of the Japanese encephalitis virus (JEV) premembrane (prM) protein in maturation of the envelope (E) glycoprotein was evaluated by using recombinant vaccinia viruses encoding E in the presence (vP829) or absence (vP658) of prM. Immunofluorescence analyses showed that E appeared to be localized in the endoplasmic reticulum of cells infected with JEV, vP829, or vP658. However, reactivity with monoclonal antibodies and behavior in Triton X-114 indicated that E produced in the absence of prM behaved abnormally. Furthermore, E produced in the presence of prM by recombinant vaccinia viruses could be incorporated into flavivirus pseudotypes, whereas E synthesized in the absence of prM could not. These results demonstrate that cosynthesis of prM is required for proper folding, membrane association, and assembly of the flavivirus E protein.