Structural Glycoprotein E2 of Classical Swine Fever Virus Critically Interacts with Host Protein Torsin-1A during the Virus Infectious Cycle.

The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.

A Cell Culture-Adapted Vaccine Virus against the Current African Swine Fever Virus Pandemic Strain.

African swine fever virus (ASFV) causes a virulent, deadly infection in wild and domestic swine and is currently causing a pandemic covering a contiguous geographical area from Central and Eastern Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in devastating economic losses to the swine industry. The most advanced vaccine candidates are live attenuated strains developed using a genetically modified virulent parental virus. Recently, we developed a vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV pandemic strain Georgia (ASFV-G). ASFV-G-ΔI177L is safe and highly efficacious in challenge studies using parental ASFV-G. Large-scale production of ASFV-G-ΔI177L has been limited because it can replicate efficiently only in primary swine macrophages. Here, we present the development of an ASFV-G-ΔI177L derivative strain, ASFV-G-ΔI177L/ΔLVR, that replicates efficiently in a stable porcine cell line. In challenge studies, ASFV-G-ΔI177L/ΔLVR maintained the same level of attenuation, immunogenic characteristics, and protective efficacy as ASFV-G-ΔI177L. ASFV-G-ΔI177L/ΔLVR is the first rationally designed ASF vaccine candidate that can be used for large-scale commercial vaccine manufacture. IMPORTANCE African swine fever is currently causing a pandemic resulting in devastating losses to the swine industry. Experimental ASF vaccines rely on the production of vaccine in primary swine macrophages, which are difficult to use for the production of a vaccine on a commercial level. Here, we report a vaccine for ASFV with a deletion in the left variable region (LVR). This deletion allows for growth in stable cell cultures while maintaining the potency and efficacy of the parental vaccine strain. This discovery will allow for the production of an ASF vaccine on a commercial scale.

Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle.

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

Two predicted α-helices within the prion-like domain of TIAR-1 play a crucial role in its association with stress granules in Caenorhabditis elegans.

Stress granules (SGs) are sites for mRNA storage, protection, and translation repression. TIA1 and TIAR1 are two RNA-binding proteins that are key players in SGs formation in mammals. TIA1/TIAR have a prion-like domain (PrD) in their C-terminal that promotes liquid-phase separation. Lack of any TIA1/TIAR has severe consequences in mice. However, it is not clear whether the failure to form proper SGs is the cause of any of these problems. We disrupted two predicted α-helices within the prion-like domain of the Caenohabditis elegans TIA1/TIAR homolog, TIAR-1, to test whether its association with SGs is important for the nematode. We found that tiar-1 PrD mutant animals continued to form TIAR-1 condensates under stress in the C. elegans gonad. Nonetheless, TIAR-1 condensates appeared fragile and disassembled quickly after stress. Apparently, the SGs continued to associate regularly as observed with CGH-1, an SG marker. Like tiar-1-knockout nematodes, tiar-1 PrD mutant animals exhibited fertility problems and a shorter lifespan. Notwithstanding this, tiar-1 PrD mutant nematodes were no sensitive to stress. Our data demonstrate that the predicted prion-like domain of TIAR-1 is important for its association with stress granules. Moreover, this domain may also play a significant role in various TIAR-1 functions unrelated to stress, such as fertility, embryogenesis and lifespan.

[Comment to the article «Frecuency of diagnosis of bone lesions in cases of anterior glenohumeral dislocation¼]. / Carta al editor sobre el artículo «Frecuencia de diagnóstico de lesiones óseas en luxación glenohumeral anterior¼.

No Abstract available.

Dosimetric properties of KMgF3:Tb + PTFE.

This work presents the results obtained from the dosimetric properties of the new radiation detectors of KMgF3:Tb + PTFE. The thermoluminescent material was obtained by microwave technique. The polycrystalline powder obtained was mixed with polytetrafluoroethylene resin powder in the ratio 2:3 to make dosimeters in pellet form. The thermoluminescent response of these new detectors presented a linear behavior, in the dose range between 1 and 1000 Gy 60Co gamma radiation, the repeatability test in the measurements, during ten cycles of heat treatment, irradiation and readouts, showed ±â€¯3.7% DS, the stability test of thermoluminescent signal, during two months showed that the fading is practically null. For the results obtained, this new detector could be very useful for radiation dosimetry, in clinical applications.

"CLUPS": A New Culture Medium for the Axenic Growth of Entamoeba histolytica.

Amebiasis remains a major health problem in Mexico. Therefore, the search for better culture media and low-cost diagnostic and therapeutic tools is fundamental. We present a new culture medium for Entamoeba histolytica which allows the microbe to preserve its virulence factors and ability to induce hepatic abscesses in animal models. The novel CLUPS medium is an improved version of the PEHPS medium, previously designed in our laboratory. The main difference is the substitution of raw beef liver in PEHPS by raw beef lung in the CLUPS medium. To compare the performance of three-culture media (traditional TYI-S-33, PEHPS, and CLUPS), E. histolytica trophozoites were cultured in quintuplicate, followed by the evaluation of phospholipase activity and the induction of liver abscesses in golden hamsters. E. histolytica trophozoites grew significantly better in CLUPS medium than in TYI-S-33. Likewise, CLUPS-cultured trophozoites produced significantly more phospholipases than TYI-S-33-cultured trophozoites. Finally, trophozoites grown in any of the three tested media had similar potential to induce liver abscesses.

Stress-induced germ cell apoptosis by a p53 independent pathway in Caenorhabditis elegans.

In Caenorhabditis elegans, several distinct apoptosis pathways have been characterized in the germline. The physiological pathway is though to eliminate excess germ cells during oogenesis to maintain gonad homeostasis and it is activated by unknown mechanisms. The DNA damage-induced germ cell apoptosis occurs in response to genotoxic agents and involves the proteins EGL-1 and CED-13, and the DNA damage response protein p53. Germ cell apoptosis can also be induced in response to pathogen infection through an EGL-1 dependent pathway. To gain insight into the mechanism and functions of germ cell apoptosis, we investigated whether and how other forms of stress induce this cell death. We found that oxidative, osmotic, heat shock and starvation stresses induce germ cell apoptosis through a p53 and EGL-1 independent pathway. We also learned that the MAPK kinases MEK-1 and SEK-1, and the p53 antagonist protein ABL-1, are essential for stress-induced germ cell apoptosis. We conclude that in C. elegans responses to various stresses that do not involve genotoxicity include an increase in germ cell apoptosis through the physiological pathway.

[Recurrent oral ulcer: clinical characteristic and differential diagnosis]. / Ulceras orales recurrentes: Características clínicas y diagnóstico diferencial.

Recurrent aphthous stomatitis (RAS), are common inflammatory lesions of the oral mucous, usually round or ovoid, circumscribed by erythematous haloes with a yellow-grey floor and mostly painful. The RAS has reached an incidence about 20% in general population, present on any aged group, especially adolescents and young adults. Etiopathogenesis of RAS is not entirely understood. Some factors involved include immune system anomalies, infections, nutritional deficiency, mucous traumatism, food or contact allergy, autoimmunity illness and cancer; together with psychiatric, genetic and environment agents. In this article, main clinical features, etiology related factors, differential diagnosis and initial study of patients consulting for RAS are presented.

Lambs are Susceptible to Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist.

Caenorhabditis elegans ATPase inhibitor factor 1 (IF1) MAI-2 preserves the mitochondrial membrane potential (Δψm) and is important to induce germ cell apoptosis.

When the electrochemical proton gradient is disrupted in the mitochondria, IF1 (Inhibitor Factor-1) inhibits the reverse hydrolytic activity of the F1Fo-ATP synthase, thereby allowing cells to conserve ATP at the expense of losing the mitochondrial membrane potential (Δψm). The function of IF1 has been studied mainly in different cell lines, but these studies have generated contrasting results, which have not been helpful to understand the real role of this protein in a whole organism. In this work, we studied IF1 function in Caenorhabditis elegans to understand IF1´s role in vivo. C. elegans has two inhibitor proteins of the F1Fo-ATPase, MAI-1 and MAI-2. To determine their protein localization in C. elegans, we generated translational reporters and found that MAI-2 is expressed ubiquitously in the mitochondria; conversely, MAI-1 was found in the cytoplasm and nuclei of certain tissues. By CRISPR/Cas9 genome editing, we generated mai-2 mutant alleles. Here, we showed that mai-2 mutant animals have normal progeny, embryonic development and lifespan. Contrasting with the results previously obtained in cell lines, we found no evident defects in the mitochondrial network, dimer/monomer ATP synthase ratio, ATP concentration or respiration. Our results suggest that some of the roles previously attributed to IF1 in cell lines could not reflect the function of this protein in a whole organism and could be attributed to specific cell lines or methods used to silence, knockout or overexpress this protein. However, we did observe that animals lacking IF1 had an enhanced Δψm and lower physiological germ cell apoptosis. Importantly, we found that mai-2 mutant animals must be under stress to observe the role of IF1. Accordingly, we observed that mai-2 mutant animals were more sensitive to heat shock, oxidative stress and electron transport chain blockade. Furthermore, we observed that IF1 is important to induce germ cell apoptosis under certain types of stress. Here, we propose that MAI-2 might play a role in apoptosis by regulating Δψm. Additionally, we suggest that IF1 function is mainly observed under stress and that, under physiological conditions, this protein does not play an essential role.

Vaccination against Louping Ill Virus Protects Goats from Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus.

Treatment with interferon-alpha delays disease in swine infected with a highly virulent CSFV strain.

Interferon-alpha (IFNα) can effectively inhibit or abort a viral infection within the host. It has been reported that IFN induction and production is hindered during classical swine fever virus (CSFV) infection. Most of those studies have been performed in vitro, making it difficult to elucidate the actual role of IFNs during CSFV infection in swine. Here, we report the effect of IFNα treatment (delivered by a replication defective recombinant human adenovirus type 5, Ad5) in swine experimentally infected with highly virulent CSFV strain Brescia. Treatment with two different subtypes of IFNα delayed the appearance of CSF-related clinical signs and virus replication although it did not prevent lethal disease. This is the first report describing the effect of IFNα treatment during CSFV infection in swine.

Treatment of presumed bacterial pneumonia in ambulatory children.

In a prospective, randomized, open study, a combination of trimethoprim and rifampin (TMP/R) 20 mg/kg/day was compared with ampicillin (AMP) 150 mg/kg/day, both given orally twice daily for 10 days, for the treatment of 60 children who had mild community-acquired pneumonia. The control group comprised 112 healthy children. The overall duration of the disease was 8.5 +/- 3.6 days in the TMP/R group vs 6.0 +/- 1.1 days in the AMP group. Fever persisted for 7.0 +/- 1.8 days in the TMP/R-treated patients vs 5.2 +/- 1.0 days in the AMP-treated patients. At the end of the 10 days, nasopharyngeal cultures were negative in all patients in the AMP group and in 25 of the 30 patients in the TMP/R group. These five patients were clinical and microbiologic failures. We conclude that in infants and children with mild community-acquired pneumonia, treatment with AMP for 10 days is more effective than treatment with a combination of TMP/R for clinical cure and eradication of bacterial pathogens.

Pharmacologic modification of the time course of ovum transport in guinea pigs.

Various agents were examined for their effects on ovum transport in the guinea pig. Estrogen significantly accelerated ovum transport in this species. The experiments further demonstrated that estrogen did not act by inducing prostaglandin synthesis, nor by altering plasma progesterone levels. The estrogen-induced acceleration was significantly antagonized by tamoxifen, an antiestrogen that acts by interfering with estrogen receptor synthesis. Cycloheximide also antagonized the effects of estrogen on ovum transport. These data suggest that the modification of ovum transport by estrogen is due to the entrance of estrogen into the nuclei of target cells, and subsequent protein synthesis. Although we assume that this action occurs at the level of the oviduct, our experiments do not prove this assumption.

PIP: The effects of various agents on the transport of ovum was tested in 39 guinea pigs. The administration of estrogens significantly accelerated this process. Estrogens did not act through the induction of prostaglandin synthesis or by altering plasma progesterone levels. Estrogen-induced ovum acceleration was significantly antagonized by tamoxifen, an antiestrogen that acts by interfering with estrogen receptor synthesis. Cycloheximide also antagonized the effects of estrogen on ovum transport. A modification of ovum transport by estrogens is thought to be due to the entrance of estrogen into the nuclei of the target cells, and through subsequent protein synthesis. It is assumed that this process takes place at the oviduct level, although the experiment did not prove this.

Time course of ovum transport in guinea pigs.

The time course of ovum transport was determined in cycling guinea pigs. The occurrence of ovulation was determined in assessing changes in vaginal cytology. The day of the postovulatory leukocyte influx was considered day 1 of the cycle. Ovum transport in guinea pigs is characterized by a relatively long sojourn in the ampulla, followed by rapid transport through the isthmus. This pattern is similar to that seen in women and subhuman primates and different from the pattern in rabbits.

Uptake of (188)Re-beta-naphthyl-peptide in cervical carcinoma tumours in athymic mice.

Radiolabelled somatostatin analogues have been used in diagnostic and therapeutic nuclear medicine to treat cancerous tumours. Lanreotide, a cyclic octapeptide, beta-naphthyl-peptide, with antiproliferative action on human small cell lung carcinoma was (188)Re labelled and characterised, and its biodistribution was studied in mice. Molecular modelling indicates that the lipophilic radiopharmaceutical might be an oxo-rhenium (V) penta-coordinated complex. The implanted human cervical tumour of epidermoid origin was positive for cytokeratins and Vimentin. Uptake of (188)Re-labelled peptide in the implanted tumour in athymic mice was 6.2+/-2.9% and was rapidly cleared via the hepatobiliary system. (188)Re-beta-naphthyl-peptide might be a potential therapeutic agent.

Labeling peptides with rhenium-188.

A direct labeling technique via EHDP for the preparation of 188Re-somatostatin analogue peptide beta-(2-naphthyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-amide complex was developed. The influence of reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were investigated. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in the radiopharmaceutical solution. Results showed that under the procedure reported herein 188Re-peptide complex can be prepared with a radiochemical purity of 90% and a specific activity up to 1.8 GBq mg-1 without radiolytic degradation of the product.

Tuberculosis: an increasing problem among minorities in the United States.

Although the number of tuberculosis cases reported annually in the United States has decreased markedly during the past three and a half decades, the decrease among whites has been considerably greater than among nonwhites. As a result of this widening gap, nearly two-thirds of the cases reported in 1987 occurred in minority populations and, for the first time in history, the number of cases among blacks exceeded the number of cases among non-Hispanic whites. From 1985 to 1987, tuberculosis among blacks increased 6.3 percent and among Hispanics, by 12.7 percent, but it decreased 4.8 percent among non-Hispanic whites. Much of the increase appears attributable to tuberculosis occurring among persons infected with the human immunodeficiency virus (HIV). Although there are many obstacles to the elimination of the disease in minority populations, numerous strategies have been developed and are being implemented to address this situation.

Uptake of the 188Re(V)-DMSA complex by cervical carcinoma cells in nude mice: pharmacokinetics and dosimetry.

The uptake of the rhenium-188 (188Re(V)-DMSA) complex of dimercaptosuccinic acid by cervical carcinoma cells in nude mice was evaluated. The pharmacokinetics and dosimetry calculations in normal rats were also evaluated. The images obtained in mice did not show significant accumulation in metabolic organs and the biodistribution studies showed that 3.52 +/- 0.76% of the injected activity per gram (n = 4) was taken up by the tumor. This percentage produces a cumulated activity of 35.63 +/- 8.40 MBq h and an equivalent dose per injected activity of 260 +/- 8.91 mSv/MBq. Pharmacokinetics and dosimetry of the 1887e(V)-DMSA complex indicate that this radiopharmaceutical could be evaluated in patients with soft tissue tumors, since the risk of radiation damage to the kidney or red bone marrow could not be an obstacle for its application in therapeutic nuclear medicine.