Characteristics of the major structural proteins of African swine fever virus: Role as antigens in the induction of neutralizing antibodies. A review.

African swine fever virus (ASFV) has been traditionally a major animal health problem worldwide, with important economic impact for the pig industry. Recently the disease has re-emerged in large areas of the world, including China and Eastern Europe. Therefore, it seems timely to summarize our current understanding of ASFV proteins and their antigenic properties, in connection with potential vaccine formulations. Here we review the main characteristics of the major structural proteins p150, p72 and p17 of ASFV and their antigenic properties. In particular, we emphasize that p17 was detected as a specific antigen in the immunoreaction of pig sera with neutralizing antibodies. In addition, specific immunoreactions against IP97, IP27, IP25.5, IP23 and IP13 viral infection proteins were also detected in these sera. The viral structural proteins have been studied with intracellular and extracellular viruses and, therefore the differences between both classes of viruses were also reviewed.

Partial complementation between the immediate early proteins ICP4 of herpes simplex virus type 1 and IE180 of pseudorabies virus.

We previously described that the immediate early (IE) IE180 protein of PRV can down-regulate the transactivation of the ICP4 promoter of HSV-1, and that the d120 virus (an ICP4-deficient HSV-1 strain) can partially replicate its viral DNA in the presence of the IE180 protein. Herein, we demonstrate that this partial complementation of d120 by IE180 is sufficient for transcription of ß, γ1 and γ2 products such as DNA pol, VP16 and gC, respectively. However, expression levels are low for VP16 and even lower for the gC, such that IE180 is unable to fully substitute for ICP4 functionally. Viral progeny was not detected in PK15 cells expressing PRV IE180.

Dry sliding wear behaviour of ß-type Ti-Nb and Ti-Mo surfaces designed by diffusion treatments for biomedical applications.

The dry sliding wear behaviour of different Ti-Nb and Ti-Mo surfaces was investigated in order to evaluate the role of Nb and Mo ß-stabilizing elements in titanium wear resistance to consider them for biomedical applications. Dry sliding wear tests were performed under unlubricated conditions using a ball-on-plate tribometer (UMT) with reciprocating lineal movement of 1 Hz frequency at different loads (2 and 5 N) and against two counterface materials (alumina and stainless steel) to assess the effect of these parameters on wear. The results indicated an improvement in wear resistance for all the modified Ti surfaces. Metal-on-metal surfaces exhibited higher wear rate than ceramic-on-metal, and higher wear was observed for the more severe conditions. Wear rate values on modified surfaces were between 53% and 96% lower compared to pure Ti tested at 2 N, and up to 79% lower than Ti at 5 N. In both cases the highest wear reduction was observed for Ti-MoNH4Cl surface.

Construction of recombinant pseudorabies viruses by using PRV BACs deficient in IE180 or pac sequences: Application of vBAC90D recombinant virus to production of PRV amplicons.

We describe a simple and efficient method to obtain recombinant pseudorabies virus (PRV) in mammalian cells by using the PRV BACs, PBAC80 deficient in pac sequences and PBAC90 deficient in the IE180 gene. These essential viral sequences were used as targets to obtain viable recombinant viruses. PBAC80 was constructed, confirmed to encode a copy of the IE180 gene regulated by the inducible Ptet promoter, and used to obtain recombinant attenuated PRV viruses that express the EGFP protein (PRV-BT80GF virus). PBAC90 was used to obtain the vBAC90D virus, deficient in IE180 and free of replication-competent revertants, and which can be used as a helper in the production of PRV amplicons.

Inhibition of herpes virus infection in oligodendrocyte cultured cells by valproic acid.

Valproic acid (VPA) is a small fatty acid used for treatment of different neurologic diseases such as epilepsy, migraines or bipolar disorders. VPA modulates different processes of cell metabolism that can lead to alterations in susceptibility of several cell types to the infection of Human Immunodeficiency Virus (HIV), Epstein-Barr virus (EBV), as well as to exert an inhibitory effect on the replication of different enveloped viruses in cultured cells. Taken these data into account and the fact that HSV-1 has been involved in some neuropathies, we have characterized the effect of VPA on this herpesvirus infection of the differentiation/maturation-inducible human oligodendrocyte cell line HOG, which resulted more susceptible to VPA inhibition of virus growth after cell differentiation. In these cells, the role of VPA in virus entry was tackled. Incubation with VPA induced a slight but reproducible inhibition in the virus particles uptake mainly observed when the drug was added in the adsorption or early upon infection. In addition, transcription and expression of viral proteins were significantly downregulated in the presence of VPA. Remarkably, when the infective viral production was assessed, VPA dramatically blocked the detection of infectious HSV-1 particles. Herein, our results indicate that VPA treatment of HOG cells significantly reduces the effect of HSV-1 infection, virus entry and productivity without affecting cellular viability.

Expression of the immediate early IE180 protein under the control of the hTERT and CEA tumor-specific promoters in recombinant pseudorabies viruses: Effects of IE180 protein on promoter activity and apoptosis induction.

Since the pseudorabies virus (PRV) genome encodes for a single immediate-early protein, IE180, we reasoned that this strong transactivating protein could represent a key regulatory switch that could be genetically manipulated in order to alter its tropism towards cancer cells. We therefore initiated studies to test whether the human telomerase reverse transcriptase (hTERT) and carcinoembryonic antigen (CEA) tumor promoters could functionally replace the IE180 promoter. We show that both promoters can functionally substitute the IE180 promoter in plasmid constructs and recombinant viruses, and observed that IE180 differentially auto-regulated each promoter tested, with PRV IE180 negatively regulating the hTERT promoter but positively hyper-activating the CEA promoter. Interestingly, we also observed that the recombinant PRV-TER and PRV-CEA viruses preferentially replicated in diverse cancer cell lines compared to control non-cancer cells, and the PRV-CEA was capable of additionally inducing a profound apoptotic phenotype which we correlated to the overexpression of IE180.

Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53.

The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3 weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2 weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described.

The IE180 protein of pseudorabies virus suppresses phosphorylation of translation initiation factor eIF2α.

We have previously shown that the porcine alphaherpesvirus pseudorabies virus (PRV) efficiently interferes with phosphorylation of the eukaryotic translation initiation factor eIF2α. Inhibition of phosphorylation of eIF2α has been reported earlier for the closely related alphaherpesvirus herpes simplex virus 1 (HSV-1) through its ICP34.5 and US11 proteins. PRV, however, does not encode an ICP34.5 or US11 orthologue. Assays using cycloheximide, UV-inactivated PRV, or phosphonoacetic acid (PAA) showed that de novo expression of one or more (immediate) early viral protein(s) is required for interference with eIF2α phosphorylation. In line with this, a time course assay showed that eIF2α phosphorylation was abolished within 2 h after PRV inoculation. PRV encodes only one immediate-early protein, IE180, the orthologue of HSV-1 ICP4. As reported earlier, a combinational treatment of cells with cycloheximide and actinomycin D allowed expression of IE180 without detectable expression of the US3 early protein in PRV-infected cells. This led to a substantial reduction in eIF2α phosphorylation levels, indicative for an involvement of IE180. In support of this, transfection of IE180 also potently reduced eIF2α phosphorylation. IE180-mediated interference with eIF2α phosphorylation was not cell type dependent, as it occurred both in rat neuronal 50B11 cells and in swine testicle cells. Inhibition of the cellular phosphatase PP1 impaired PRV-mediated interference with eIF2α phosphorylation, indicating that PP1 is involved in this process. In conclusion, the immediate-early IE180 protein of PRV has the previously uncharacterized ability to suppress phosphorylation levels of the eukaryotic translation initiation factor eIF2α.

Construction and properties of a recombinant pseudorabies virus with tetracycline-regulated control of immediate-early gene expression.

A study was carried out to determine whether altering the control of expression of the IE180 gene of pseudorabies virus (PRV), by replacing the IE180 promoter with the tetracycline-responsive promoter (Ptet), affects virus replication and virulence. This PRV-BT90 mutant virus was constructed by complementation and recombination in Hela Tet-Off cells. The virus yield produced by infection of Hela Tet-Off cells with PRV-BT90 was similar to that of the parental virus vBecker2. Viral replication of PRV-BT90 was reduced in Vero cells as reflected by a reduction of virus yield and plating efficiency compared to vBecker2. PRV-BT90 plaque formation in Hela Tet-Off cells was inhibited in the presence of doxycycline, whereas vBecker2 plaque formation was not affected. Subcutaneous infection of mice with the two viruses revealed a LD(50) higher than 10(6) TCID(50) for the PRV-BT90 mutant virus while the LD(50) was 178 TCID(50) for the vBecker2 parental virus.

Regulation of pseudorabies virus gG glycoprotein gene promoter independently of pseudorabies immediate early IE180 protein.

The pseudorabies virus (PRV) glycoprotein known as gG is generally regarded as an early protein, and the immediate early IE180 protein regulates its expression during infection. This study, however, provides evidence that although induction by IE180 is observed, the expression of a marker protein (EGFP), or gG itself, under the control of the gG promoter, can also occur independently of the expression of IE180. This result was demonstrated both with transient transfection assays using plasmids and with viral infections. In transient transfections, the expression under control of the gG promoter depends on the cell type and surprisingly, can be 1.3-fold higher than the expression under the control of the IE180 promoter in Hela Tet-Off cells. Recombinant PRV S3 was constructed by replacing gE in the PRV genome with a chimeric transgene, expressing EGFP under the control of the gG promoter. In PK15 cells infected with NIA-3 wild-type virus or with S3 recombinant virus, expression of gG PRV mRNA (or EGFP mRNA) under the control of the gG promoter in the presence of cycloheximide was detected by RT-PCR. This again indicates that some basal expression was produced in infected cells independently of IE180. This expression was augmented by IE180 protein in both plasmid transfections and viral infections.

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E.

Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.

A comparison of enhanced green fluorescent protein expression induced by immediate-early cytomegalovirus (IE-CMV) and gG pseudorabies virus (gG-PRV) promoters, using pseudorabies virus amplicons as vectors.

This study compares the expression efficiencies of the IE-CMV and gG-PRV promoters following their transfection into cultured human and monkey cells, using pseudorabies virus amplicons as vectors and enhanced green fluorescence protein (EGFP) as an expression marker. EGFP expression was similarly strong with both promoters. Pseudorabies virus amplicons appear to be useful vectors in gene expression studies due to their replication in the presence of helpers and their wide range of cellular hosts.

Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus.

Recombinant pseudorabies viruses (PRVs) gIS8 and N1aHTK were constructed by the insertion of a chimeric gene (alpha4-TK) from herpes simplex virus type 1 (HSV-1) into wild-type PRV. HSV-1 TK expression by these recombinant viruses resulted in enhanced sensitivity to ganciclovir, compared to that of the wild-type PRV, and was similar to the sensitivity shown by HSV-1. Infection with gIS8 or N1aHTK recombinant viruses led to expression of HSV-1 TK mRNA as an immediate-early (IE) gene, observed by downregulation of the HSV-1 alpha4 promoter. This negative regulation was due to a PRV IE protein, IE180. IE180, however, does not have all the regulatory functions of the infected-cell protein ICP4, as it does not restore the growth of ICP4-deficient HSV-1 mutants.

Immunological properties of a DNA plasmid encoding a chimeric protein of herpes simplex virus type 2 glycoprotein B and glycoprotein D.

A DNA plasmid containing a chimeric sequence encoding both herpes simplex virus type 2 (HSV-2) glycoprotein B (gB) and glycoprotein D (gD) external domains (pcgDB) was used to immunize BALB/c mice against genital HSV-2 infection. To determine the efficacy of this vaccine, groups of mice immunized with the pcgDB plasmid were compared with animals immunized with plasmids corresponding to the individual proteins (pcgBt or pcgDt), administered separately or in combination (pcgBt + pcgDt). We studied the response of the different mouse groups to viral challenge by analyzing clinical disease (vaginitis), serum antibody levels, as well as lymphoproliferative responses and cytokine production by spleen cells. Increased IFN-gamma levels correlated with prolonged survival in mice immunized with the plasmid pcgDB, relative to mice immunized with plasmids coding for the individual proteins alone or in combination. Our results show that immunization with the plasmid encoding the chimeric protein is advantageous over separate proteins. These findings may have important implications for the development of multivalent DNA vaccines against HSV and other complex pathogens.

Expression and inheritance of hypersensitive resistance to rice hoja blanca virus mediated by the viral nucleocapsid protein gene in transgenic rice.

Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars.

Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule).

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.

[Proximal vein by-pass in the treatment of venous stenosis in expanded polytetrafluoroethylene prosthesis for hemodialysis]. / By-pass a vena proximal para el tratamiento de estenosis venosas en prótesis de politetrafluoroetileno expandido para hemodiálisis.

OBJECTIVE: To show the long-term results of 97 politetraflouroethylene dialysis grafts submitted to a graft by-pass to treat graft-vein stenosis. MATERIALS AND METHODS: Venous stenoses were studied and diagnosed by means of fistulography in cases with fistula dysfunction or during surgery for graft thrombectomy. Both early and late complication rates were studied, as well as primary and secondary patency rates. RESULTS: Number of cases, 97. Mean age, 58 years (7-79). Diabetic nephropathy: 19.5%. Types of grafts in which stenoses developed: straight forearms 13; loop forearm 9; 6 mm upper arm 36; 6-8 mm upper arm 34; brachio-jugular 4; femoro-femoral 1. Overall follow-up time: 2,427 graft-months. Mean follow-up time: 21 +/- 5 months. Late complication rate: 0.30 episodes per graft-year of follow-up. Re-stenosis rate: 0.12 graft-year of follow-up. Primary cumulative patency rate: 70%, 62%, 51%, 45% at one, two, three and four years, respectively. Secondary cumulative patency rate: 87%, 79%, 74% and 71% at one, two, three and four years, respectively (p < 0.0016). No differences were observed between secondary patency observed after by-pass to treat dysfunction or thrombosis (p = 0.09259). DISCUSSION: In our experience, by-pass to proximal vein is associated with good results both at short and long term, probably because the intimal hyperplasia area is excluded and because by-pass is performed on an already dilated vein. The procedure can be performed under local anesthesia and in an outpatient basis between dialysis, with little discomfort for the patient.

By-pass a vena proximal para el tratamiento de estenosis venosas en prótesis de politetrafluoroetileno expandido para hemodiálisis / By-pass to proximal vein for treatment of venous stenoses in politetrafluorethylene grafts for haemodialysis

Objetivo. Mostrar los resultados a largo plazo de 97 by-pass a vena proximal para tratar estenosis protésis-vena periféricas de prótesis de politetrafluoroetileno (PTFE) para hemodiálisis.Material y métodos. Las estenosis venosas fueron estudiadas y diagnosticadas con fistulografía en casos de disfunción de la fístula o durante el procedimiento de trombectomía de las prótesis. Se estudiaron la tasa de complicaciones precoces y tardías, así como las curvas de permeabilidad primaria y secundaria.Resultados. Número de casos: 97. Edad media: 58 (7-79). Nefropatía diabética: 19,5 por ciento. Tipos de prótesis en las que se desarrollaron las estenosis: 13 rectas de antebrazo, 9 antebrazo curvas, 36 brazo 6 mm, 34 brazo 6-8 mm, 4 humeroyugulares y 1 femorofemoral. Tiempo global de seguimiento: 2.427 meses. Tiempo medio de seguimiento: 21ñ 5 meses. Tasa de complicaciones totales: 0,30 episodios prótesis/año de seguimiento. Tasa de reestenosis: 0,12 prótesis/año de seguimiento. Curva actuarial de permeabilidad primaria: 70 por ciento, 62 por ciento, 51 por ciento, 45 por ciento al primer, segundo, tercer y cuarto año, respectivamente. Curva actuarial de permeabilidad secundaria: 87 por ciento, 79 por ciento, 74 por ciento y 71 por ciento al primer, segundo, tercer y cuarto año, respectivamente (p < 0,0016). No hubo diferencia en la curva de función secundaria si el by-pass fue realizado por malfunción o trombosis. Conclusiones. En nuestra experiencia el by-pass a vena proximal tiene buenos resultados a corto y largo plazo probablemente por excluir la zona de hiperplasia intimal y por realizar el by-pass sobre vena dilatada. El tratamiento puede ser realizado bajo anestesia local, en régimen ambulatorio y en período interdiálisis con mínimas molestias para los pacientes (AU)

No disponible

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain.

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.

Molecular phylogeny and morphological homoplasy in fruitbats.

The present study evaluates the evolutionary framework of the Old World fruitbats based on the cytochrome b and 16S rRNA mitochondrial gene sequences from a wide range of taxa. Phylogenetic analyses indicated that morphology-based subfamilies and most suprageneric groups are nonnatural assemblages. They also support the existence of an endemic African clade of fruitbats. The discrepancy between the evolutionary relationships yielded by molecular and morphological data sets may be, at least in part, explained by the recurrent retention of primitive morphology (Rousettus-like) across different lineages. The maintenance of primitive characters in different groups of flying foxes, as well as morphological convergence in nectar-feeding bats and possibly also in short-muzzle bats, may have led to high levels of homoplasy, resulting in misleading taxonomic arrangements. This may be particularly so with respect to high taxonomic levels based on morphological characters.