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1.  Analysis of HDAC6 and BAG3-Aggresome Pathways in African Swine Fever Viral Factory Formation 
Viruses  2015;7(4):1823-1831.
African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes.
PMCID: PMC4411678  PMID: 25856634
African swine fever virus; aggresome; HDAC6; BAG3; viral factories
2.  New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization 
BioMed Research International  2014;2014:517125.
The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation.
PMCID: PMC4022197  PMID: 24877108
3.  BACs-on-Beads Technology: A Reliable Test for Rapid Detection of Aneuploidies and Microdeletions in Prenatal Diagnosis 
BioMed Research International  2014;2014:590298.
The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.
PMCID: PMC3985206  PMID: 24795887
4.  Antibodies against Marinobacter algicola and Salmonella typhimurium Flagellins Do Not Cross-Neutralize TLR5 Activation 
PLoS ONE  2012;7(11):e48466.
Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA)-derived flagellins (F and FR) as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF) flagellins (FljB and FliC). We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2) and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure, tumors, ionizing radiations and also to improve the outcome of bone marrow transplants).
PMCID: PMC3498291  PMID: 23155384
5.  Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry 
PLoS ONE  2012;7(11):e48853.
Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P2). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.
PMCID: PMC3486801  PMID: 23133661
6.  Small Rho GTPases and Cholesterol Biosynthetic Pathway Intermediates in African Swine Fever Virus Infection 
Journal of Virology  2012;86(3):1758-1767.
The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection.
PMCID: PMC3264358  PMID: 22114329
7.  Dynamics and Predictive Potential of Antibodies against Insect-Derived Recombinant Leishmania infantum Proteins during Chemotherapy of Naturally Infected Dogs 
A predictive marker for the success treatment of canine leishmaniasis is required for the application of a more rational therapy protocol, which must improve the probability of cure and reduce Leishmania resistance to drugs. We investigated the dynamics and predictive value of antibodies against insect-derived recombinant L. infantum proteins rKMPII and rTRYP by using an enzyme-linked immunosorbent assay with retrospective serum samples from 36 dogs during treatment of canine leishmaniasis. In the entire group of dogs, concentrations of antibodies against rKMPII and rTRYP significantly decreased earlier than concentrations of antibodies against crude total Leishmania antigen (one versus six months), which suggested that the dynamics of antibodies against recombinant proteins may be useful for assessing clinical improvement after treatment. Interestingly, decreases in antibody concentrations against rKMPII occurred earlier in disease-free dogs than in dogs that remain clinically ill one year after beginning of treatment, which suggested that these antibodies may be useful for predicting disease-free survival one year after the beginning of therapy against canine leishmaniasis.
PMCID: PMC2861386  PMID: 20439957
8.  Serological Immunoassay for Detection of Hepatitis E Virus on the Basis of Genotype 3 Open Reading Frame 2 Recombinant Proteins Produced in Trichoplusia ni Larvae▿  
Journal of Clinical Microbiology  2009;47(10):3276-3282.
Hepatitis E virus (HEV) is a major cause of acute hepatitis in humans, and strains of genotypes 1 and 2 are endemic in many regions with suboptimal sanitary conditions. In many industrialized countries, HEV strains of genotype 3 are highly endemic in swine, and an increased number of autochthonous infections with HEV genotype 3 strains have been reported lately. Serological studies of HEV infection are often conducted with commercial assays based on peptides and recombinant proteins of HEV genotype 1 and 2 strains. For some patients with proven HEV genotype 3 infections, these assays failed to detect specific antibodies, and they are not applicable or validated for the detection of anti-HEV antibodies in swine. To elucidate the incidence of hepatitis E in regions where HEV genotype 3 infections can be expected, and to study the seroprevalence of HEV in swine, new tools with broad specificity for all genotypes of HEV are needed. We present the expression and partial characterization of recombinant HEV genotype 3 open reading frame 2 (ORF-2) proteins and their usefulness as diagnostic antigens in detecting anti-HEV antibodies in humans and swine with proven HEV genotype 3 infections. The recombinant antigens were produced at relatively high yields and at low cost upon infection of Trichoplusia ni larvae with recombinant baculoviruses expressing recombinant HEV genotype 3 ORF-2 proteins. The enzyme-linked immunosorbent assay based on the recombinant proteins showed good specificity and sensitivity for anti-HEV genotype 3 immunoglobulin G detection in human and swine sera. These recombinant HEV genotype 3 ORF-2 proteins might be added to diagnostic kits containing HEV genotype 1 and 2 antigens in order to develop a broadly sensitive new tool for the diagnosis of hepatitis E.
PMCID: PMC2756903  PMID: 19656986
9.  A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for Bid and Noxa 
Virology  2008;375(2):561-572.
Several large DNA viruses encode Bcl-2 protein homologues involved in the regulation of the cellular apoptosis cascade. This regulation often involves the interaction of these viral proteins with diverse cellular Bcl-2 family members. We have identified the specific interactions of A179L, an African swine fever virus (ASFV) Bcl-2 homologue, with the active forms of the porcine BH3-only Bid protein (truncated Bid p13 and p15). Transient expression of ASFV A179L gene in Vero cells prevented apoptosis induced by these active forms of Bid protein. Interestingly, A179L protein was able to interact, also with the main core Bcl-2 proapoptotic proteins Bax and Bak, and with several BH3-only proteins with selective binding restrictions for full length Bid and Noxa. These results suggest a fine regulation for A179L action in the suppression of apoptosis in infected cells which is essential for efficient virus replication.
PMCID: PMC2572728  PMID: 18329683
vBcl-2, viral Bcl-2; cBcl-2, cellular Bcl-2; ORF, open reading frame; HA, hemagglutinin tag; Bid, BH3 interacting domain death agonist; tBid, truncated Bid; ASFV, African swine fever virus.; Apoptosis; Bid; Virus–cell interaction; African swine fever virus
10.  Intracellular Localization of Vaccinia Virus Extracellular Enveloped Virus Envelope Proteins Individually Expressed Using a Semliki Forest Virus Replicon† 
Journal of Virology  2000;74(22):10535-10550.
The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.
PMCID: PMC110928  PMID: 11044098

Results 1-10 (10)