scholarly journals Spatiotemporal expression pattern of alpha, beta, and gamma genes during BoHV-5 infection

2019 ◽  
Vol 56 (1) ◽  
pp. e150972
Author(s):  
Didier Quevedo Cagnini ◽  
Peres Ramos Badial ◽  
José Paes de Oliveira-Filho ◽  
Alexandre Secorun Borges ◽  
João Pessoa de Araújo Junior
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Expression Pattern ◽  
Host Cells ◽  
Gapdh Gene ◽  
Bovine Cell ◽  
Viral Genes ◽  
Alpha Beta ◽  
Over Time

Bovine herpesvirus 5 is an alphaherpesvirus that causes nonsuppurative meningoencephalitis in cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Although previous studies elucidated crucial aspects involved in the pathogenesis of the disease, there is a paucity of information regarding the molecular events contributing to infection and replication of BoHV-5. The objective of the present study was to determine the in vitro gene expression pattern of BoHV-5 (e.g., alpha, beta, and gamma genes) and host cells genes (GAPDH and 18S) over time utilizing different quantities of inoculated virus. Three BoHV-5 genes (bICP0, UL9, US4) and one structural bovine cell gene had their expression accessed by real-time PCR. While the expression of BoHV-5 genes increased during the course of infection, GAPDH gene expression decreased in the host cells, evidencing the effect of viral infection on the expression of bovine cell genes. The 18S ribosomal RNA (rRNA) gene was constitutively expressed throughout BoHV-5 infection. Our data clearly demonstrates that GAPDH gene should not be used as a reference gene in studies of BoHV-5 infection because it was influenced by viral infection. However, 18S rRNA was constitutively expressed and, therefore, is recommended for normalization of BoHV-5 infection studies in bovine cells. The expression of viral genes transcripts was not altered by increasing number of viral particles added to the culture. All viral genes included here demonstrated the same expression pattern over time and there was no difference in the expression of viral genes among the various time points. Our data show important differences comparing to classical studies regarding herpesvirus alpha, beta, and gamma genes expression. More research is necessary to improve our understanding about the BoHV-5 biology during infection. Studies employing next-generation sequencing (i.e., RNA-seq), using both in vitro and in vivo models, would be the next logical step to grasp the virus and host cell’s transcriptome changes over the course of infection.

The Use of Lectins as Tools to Combat SARS-CoV-2

2021 ◽  
Vol 27 ◽  
Author(s):  
Daniela Martinez ◽  
Diego Amaral ◽  
David Markovitz ◽  
Luciano Pinto
Keyword(s):  
Viral Infection ◽  
Host Cells ◽  
Viral Fusion ◽  
Cell Receptors ◽  
First Case ◽  
Mannose Binding ◽  
New Treatments ◽  
Viral Surface

Background: in december 2019, china announced the first case of an infection caused by an, until then, unknown virus: sars-cov-2. since then, researchers have been looking for viable alternatives for the treatment and/or cure of viral infection. among the possible complementary solutions are lectins, and proteins that are reversibly bound to different carbohydrates. the spike protein, present on the viral surface, can interact with different cell receptors: ace2, cd147, and dc-signr. since lectins have an affinity for different carbohydrates, the binding with the glycosylated cell receptors represents a possibility of preventing the virus from binding to the receptors of host cells. Objective: in this review we discuss the main lectins that are possible candidates for use in the treatment of covid-19, highlighting those that have already demonstrated antiviral activity in vivo and in vitro, including mannose-binding lectin, griffithsin, banlec, and others. we also aim to discuss the possible mechanism of action of lectins, which appears to occur through the mediation of viral fusion in host cells, by binding of lectins to glycosylated receptors found in human cells and/or binding of these proteins with the spike glycoprotein, present in virus surface.moreover, we also discuss the use of lectins in clinical practice. Conclusion: Even with the development of effective vaccines, new cases of viral infection with the same virus, or new outbreaks with different viruses can occur; so, the development of new treatments should not be discarded. moreover, the discussions made in this work are relevant regarding the anti-viral properties of lectins.

scholarly journals African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-θ-Mediated p300 Transactivation

2008 ◽  
Vol 83 (2) ◽  
pp. 969-980 ◽  
Author(s):  
Aitor G. Granja ◽  
Elena G. Sánchez ◽  
Prado Sabina ◽  
Manuel Fresno ◽  
Yolanda Revilla
Keyword(s):  
Gene Expression ◽  
Inflammatory Response ◽  
Viral Infection ◽  
Host Cell ◽  
Expression Pattern ◽  
African Swine Fever Virus ◽  
Gene Expression Pattern ◽  
African Swine Fever ◽  
Fever Virus ◽  
Amino Terminal

ABSTRACT During a viral infection, reprogramming of the host cell gene expression pattern is required to establish an adequate antiviral response. The transcriptional coactivators p300 and CREB binding protein (CBP) play a central role in this regulation by promoting the assembly of transcription enhancer complexes to specific promoters of immune and proinflammatory genes. Here we show that the protein A238L encoded by African swine fever virus counteracts the host cell inflammatory response through the control of p300 transactivation during the viral infection. We demonstrate that A238L inhibits the expression of the inflammatory regulators cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) by preventing the recruitment of p300 to the enhanceosomes formed on their promoters. Furthermore, we report that A238L inhibits p300 activity during the viral infection and that its amino-terminal transactivation domain is essential in the A238L-mediated inhibition of the inflammatory response. Importantly, we found that the residue serine 384 of p300 is required for the viral protein to accomplish its inhibitory function and that ectopically expressed PKC-θ completely reverts this inhibition, thus indicating that this signaling pathway is disrupted by A238L during the viral infection. Furthermore, we show here that A238L does not affect PKC-θ enzymatic activity, but the molecular mechanism of this viral inhibition relies on the lack of interaction between PKC-θ and p300. These findings shed new light on how viruses alter the host cell antiviral gene expression pattern through the blockade of the p300 activity, which represents a new and sophisticated viral mechanism to evade the inflammatory and immune defense responses.

229 EXPRESSION OF mRNA ENCODING GLYCOLYTIC ENZYMES IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FSH

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
E. S. Caixeta ◽  
P. Ripamonte ◽  
M. F. Machado ◽  
R. B. da Silva ◽  
C. Price ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
In Vitro Maturation ◽  
Housekeeping Gene ◽  
Cumulus Cells ◽  
Glycolytic Enzymes ◽  
Mrna Abundance ◽  
Relative Expression ◽  
Significant Difference

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.

Patients who failed to conceive following an in vitro fertilization cycle can be clustered into different failure causes using gene expression hierarchical analysis†

2020 ◽  
Vol 103 (3) ◽  
pp. 599-607
Author(s):  
Chloé S Fortin ◽  
Scot Hamilton ◽  
Martin Laforest ◽  
Marie-Claude Léveillé ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Clustering Analysis ◽  
Gene Expression Pattern ◽  
Hierarchical Analysis ◽  
Stimulation Protocol ◽  
Follicular Cells ◽  
Vitro Fertilization ◽  
Different Response

Abstract The patient’s response to an IVF stimulation protocol is highly variable and thus difficult to predict. When a cycle fails, there are often no apparent or obvious reasons to explain the failure. Having clues on what went wrong during stimulation could serve as a basis to improve and personalize the next protocol. This exploratory study aimed to investigate if it is possible to distinguish different failure causes or different follicular responses in a population of nonpregnant IVF patients. Using qRT-PCR, we analyzed a panel of genes indicative of different failure causes in patients who did not achieve pregnancy following an IVF cycle. For each patient, a pool of follicular cells from all aspirated follicles was used as a sample which gives a global picture of the patient’s ovary and not a specific picture of each follicle. We performed hierarchical clustering analysis to split the patients according to the gene expression pattern. Hierarchical analysis showed that the population of nonpregnant IVF patients could be divided into three clusters. Gene expression was significantly different, and each cluster displayed a particular gene expression pattern. Follicular cells from patients in clusters 1, 2 and 3 displayed respectively a pattern of gene expression related to large incompetent follicles with a higher apoptosis (over matured), to follicles not ready to ovulate (under mature) and to an excess of inflammation with no visible symptoms. This study reinforces the idea that women often have different response to the same protocol and would benefit from more personalized treatments.

scholarly journals In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells

2007 ◽  
Vol 88 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Daniel Doucet ◽  
Anic Levasseur ◽  
Catherine Béliveau ◽  
Renée Lapointe ◽  
Don Stoltz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Genomic Dna ◽  
Cultured Cells ◽  
Germ Line ◽  
Choristoneura Fumiferana ◽  
Genome Segment ◽  
Host Cells ◽  
Dsdna Viruses

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
R. Cancian ◽  
M. Macelai ◽  
G. Tavares ◽  
R. S. Valente ◽  
E. S. Caixeta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Cellular Response ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Canonical Pathways

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

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