Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues.

Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma.

Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing.

Genes are frequently lost or gained in malignant tumors and the analysis of these changes can be informative about the underlying tumor biology. Retinoblastoma is a pediatric intraocular malignancy, and since deletions in chromosome 13 have been described in this tumor, we performed genome wide sequencing with the Illumina platform to test whether recurrent losses could be detected in low coverage data from DNA pools of Rb cases. An in silico reference profile for each pool was created from the human genome sequence GRCh37p5; a chromosome integrity score and a graphics 40 Kb window analysis approach, allowed us to identify with high resolution previously reported non random recurrent losses in all chromosomes of these tumors. We also found a pattern of gains and losses associated to clear and dark cytogenetic bands respectively. We further analyze a pool of medulloblastoma and found a more stable genomic profile and previously reported losses in this tumor. This approach facilitates identification of recurrent deletions from many patients that may be biological relevant for tumor development.

Characterization of a persistent respiratory syncytial virus showing a low-fusogenic activity associated to an impaired F protein.

In this work we have characterized the virus (RSV(48)) present in passage 48 of a respiratory syncytial virus persistently infected murine macrophage-like cell culture. This virus was noncytopathic in macrophages and had a low-fusogenic activity in RSV-permissive cell lines, although the level of this activity varied among the different cell lines tested. The fusogenic activity of RSV(48) in Vero cells, as evaluated by the number and size (nuclei per syncytium) of syncytia, was lower than that shown in cells H358. However, the syncytia formed by RSV(48) in Vero cells increased significantly when the virus was treated with trypsin previous to cell infection and the protease was left in the medium during the development of polykarions. Moreover, the fusogenic activity of RSV(48) was increased by a brief acidic pH treatment of infected cells. These results imply that the RSV(48) F protein was inefficiently activated by intracellular proteases in Vero cells and exposure to low pH favours membrane fusion. Analysis of the nucleotide and the deduced amino acid sequences of the RSV(48) F protein showed nine amino acid residue differences with respect to the RSV(wt) sequence, some of which mapped to positions that suggest they might be responsible for the low-fusogenic activity observed for the RSV(48) F protein.

Origin and differentiation of dendritic cells.

Despite extensive, recent research on the development of dendritic cells (DCs), their origin is a controversial issue in immunology, with important implications regarding their use in cancer immunotherapy. Although, under defined experimental conditions, DCs can be generated from myeloid or lymphoid precursors, the differentiation pathways that generate DCs in vivo remain unknown largely. Indeed, experimental results suggest that the in vivo differentiation of a particular DC subpopulation could be unrelated to its possible experimental generation. Nevertheless, the analysis of DC differentiation by in vivo and in vitro experimental systems could provide important insights into the control of the physiological development of DCs and constitutes the basis of a model of common DC differentiation that we propose.

Early events of rotavirus infection: the search for the receptor(s).

The entry of rotaviruses into epithelial cells seems to be a multistep process. Infection competition experiments have suggested that at least three different interactions between the virus and cell surface molecules take place during the early events of infection, and glycolipids as well as glycoproteins have been suggested to be primary attachment receptors for rotaviruses. The infectivity of some rotavirus strains depends on the presence of sialic acid on the cell surface, however, it has been shown that this interaction is not essential, and it has been suggested that there exists a neuraminidase-resistant cell surface molecule with which most rotaviruses interact. The comparative characterization of the sialic acid-dependent rotavirus strain RRV (G3P5[3]), its neuraminidase-resistant variant nar3, and the human rotavirus strain Wa (G1P1A[8]) has allowed us to show that alpha 2 beta 1 integrin is used by nar3 as its primary cell attachment site, and by RRV in a second interaction, subsequent to its initial contact with a sialic acid-containing cell receptor. We have also shown that integrin alpha V beta 3 is used by all three rotavirus strains as a co-receptor, subsequent to their initial attachment to the cell. We propose that the functional rotavirus receptor is a complex of several cell molecules most likely immersed in glycosphingolipid-enriched plasma membrane microdomains.

Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha(-) and CD8alpha(+) dendritic cells are generated from CD4(low) lymphoid-committed precursors.

Two dendritic cell (DC) subsets have been identified in the murine system on the basis of their differential CD8alpha expression. CD8alpha(+) DCs and CD8alpha(-) DCs are considered as lymphoid- and myeloid-derived, respectively, because CD8alpha(+) but not CD8alpha(-) splenic DCs were generated from lymphoid CD4(low) precursors, devoid of myeloid reconstitution potential. Although CD8alpha(-) DCs were first described as negative for CD4, our results demonstrate that approximately 70% of them are CD4(+). Besides CD4(-) CD8alpha(-) and CD4(+) CD8alpha(-) DCs displayed a similar phenotype and T-cell stimulatory potential in mixed lymphocyte reaction (MLR), although among CD8alpha(-) DCs, the CD4(+) subset appears to have a higher endocytic capacity. Finally, experiments of DC reconstitution after irradiation in which, in contrast to previous studies, donor-type DCs were analyzed without depleting CD4(+) cells, revealed that both CD8alpha(+) DCs and CD8alpha(-) DCs were generated after transfer of CD4(low) precursors. These data suggest that both CD8alpha(+) and CD8alpha(-) DCs derive from a common precursor and, hence, do not support the concept of the CD8alpha(+) lymphoid-derived and CD8alpha(-) myeloid-derived DC lineages. However, because this hypothesis has to be confirmed at the clonal level, it remains possible that CD8alpha(-) DCs arise from a myeloid precursor within the CD4(low) precursor population or, alternatively, that both CD8alpha(+) and CD8alpha(-) DCs derive from an independent nonlymphoid, nonmyeloid DC precursor. In conclusion, although we favor the hypothesis that both CD8alpha(+) and CD8alpha(-) DCs derive from a lymphoid-committed precursor, a precise study of the differentiation process of CD8alpha(+) and CD8alpha(-) DCs is required to define conclusively their origin.

A new cysteine in rotavirus VP4 participates in the formation of an alternate disulfide bond.

Most animal rotaviruses bind to a cell surface molecule that contains sialic acid (SA). We have recently isolated variants from simian rotavirus RRV which show an SA-independent phenotype. The VP4 protein of these variants was shown to have three amino acid changes with respect to the wt protein, one of them being Tyr-267 --> Cys. In this work, we have investigated whether the new cysteine could interfere with the disulfide bond (Cys-318/Cys-380) present in the VP5* subunit of the wt protein. Cysteine residues 318 and 380 were mutagenized in gpr8 and RRV VP4 genes, and the wt and mutant genes were transcribed and translated in vitro. The protein products were analysed by electrophoresis under reducing and non-reducing conditions. This approach showed that, in the VP4 protein synthesized in vitro, Cys-267 is capable of forming an alternate disulfide bond with Cys-318. This alternate bond also seems to occur in the VP4 protein present in the variant gpr8 virus particles.

Serotype specificity of the neutralizing-antibody response induced by the individual surface proteins of rotavirus in natural infections of young children.

The relative contribution of the rotavirus surface proteins, VP4 and VP7, to the induction of homotypic as well as heterotypic neutralizing antibodies (NtAbs) in natural infections was studied. The NtAb titers of paired sera from 70 infants with serologically defined primary rotavirus infections were determined with a panel of rotavirus reassortants having one surface protein from a human rotavirus (serotypes G1 to G4 for VP7 and P1A and P1B for VP4) and the other surface protein from a heterologous animal rotavirus strain. A subset of 37 children were evaluated for epitope-specific antibodies to the two proteins by an epitope-blocking assay. The infants were found to seroconvert more frequently to VP4 than to VP7 by both methods, although the titers of the seroconverters were higher to VP7 than to VP4. Both proteins induced homotypic as well as heterotypic NtAbs. G1 VP7 frequently induced a response to both G1 and G3 VP7s, while G3 VP7 and P1A VP4 induced mostly homotypic responses.

Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle.

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle.

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.

Identification of a T-helper cell epitope on the rotavirus VP6 protein.

In this work, we have studied the T-helper (Th)-cell response against rotavirus, in a mouse model. Adult BALB/c mice were inoculated parenterally with porcine rotavirus YM, and the Th-cell response from spleen cells against the virus and two overlapping fragments of the major capsid protein VP6 (VP6(1-192) and VP6(171-397)) were evaluated in vitro. The Th cells recognized the YM virus and the two protein fragments, suggesting that there are at least two Th-cell epitopes on the VP6 molecule. To study the specificity of Th cells against VP6 at the clonal level, we established two Th-cell hybridomas cross-reactive for the VP6 protein of rotavirus strains YM and SA11. Both hybridomas recognized the VP6(171-397) polypeptide, and a synthetic peptide comprising the amino acids 289 to 302 (RLSFQLVRPPNMTP) of YM VP6 in the context of the major histocompatibility complex class II IEd molecule. The Th-cell hybridomas recognized rotavirus VP6 in a highly cross-reactive fashion, since they could be stimulated by eight different strains of rotavirus, including the murine rotavirus EDIM, that represent five G serotypes and at least two subgroups. The amino acid sequence of the VP6 epitope is highly conserved in most group A rotavirus strains sequenced so far. On the other hand, it was found that Th cells specific for the VP6 epitope may constitute an important proportion of the total polyclonal Th-cell response against rotavirus YM in spleen cells. These results demonstrate that VP6 can be a target for highly cross-reactive Th cells.

[Workshop on Molecular Epidemiology of Viral Diseases]. / Taller de Epidemiología Molecular sobre Enfermedades Virales.

A workshop on viral epidemiology was held on September 29, 1995 at the Medical School of the Universidad Nacional Autónoma de Mexico. The aim of this workshop was to promote interaction among scientists working in viral epidemiology. Eighteen scientists from ten institutions presented their experiences and work. General aspects of the epidemiology of meaningful viral diseases in the country were discussed, and lectures presented on the rota, polio, respiratory syncytial, dengue, papiloma, rabies, VIH and hepatitis viruses.

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit.

A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB). Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3' end of the DNA that codes for the leader peptide, and the 5' end of the gene encoding mature CTB. The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside. The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide. The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity. Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed.

Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain.

Flavivirus genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. The N-termini of several nonstructural proteins are produced by cleavage at dibasic sites by a two-component viral proteinase consisting of NS2B and NS3. NS3 contains a trypsin-like serine proteinase domain at its N-terminus, whereas the function of NS2B in proteolysis is yet to be determined. We have used an NS3-specific antiserum, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex both in vitro and in vivo. The N-terminal 184 residues of NS3 are sufficient to form the complex with NS2B. The complex forms efficiently when the NS2B and NS3 are translated from two different mRNAs as well as when NS2B and NS3 are translated as a polyprotein from the same mRNA. A chimeric complex can be formed between yellow fever NS2B and a chimeric yellow fever-dengue 2 NS3. Using anti-NS3 antisera, we also found that a 50-kDa fragment of NS3, consisting of the N-terminal approximately 460 residues, is produced in infected mammalian cells. This fragment is not produced in infected mosquito cells, but will form in Triton X-100 lysates of mosquito cells. The cleavage of NS3 to form this fragment is catalyzed by the NS3 proteinase itself and proteolysis requires NS2B. Examination of the amino acid sequence of NS3 reveals a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase; this site occurs within a conserved RNA helicase sequence motif. The importance of this alternatively processed form of NS3 and its role in the replication cycle of dengue virus remain to be determined.

Rotavirus YM gene 4: analysis of its deduced amino acid sequence and prediction of the secondary structure of the VP4 protein.

We have determined the complete nucleotide sequence of the VP4 gene of porcine rotavirus YM. It is 2,362 nucleotides long, with a single open reading frame coding for a protein of 776 amino acids. A phylogenetic tree was derived from the deduced YM VP4 amino acid sequence and 18 other available VP4 sequences of rotavirus strains belonging to different serotypes and isolated from different animal species. In this tree, VP4 proteins were grouped by the hosts that the corresponding viruses infect rather than by the serotypes they belong to, suggesting that this protein is involved in the host specificity of the viruses. In an attempt to predict the secondary structure of the VP4 protein, we selected the more divergent VP4 sequences and made a secondary structure analysis of each protein. In spite of variations within the individual structures predicted, there was a general structural pattern which suggested the existence of at least two different domains. One, comprising the amino-terminal 63% of the protein, is predicted to be a possible globular domain rich in beta-strands alternated with turns and coils. The second domain, represented by the remaining, carboxy-terminal part of VP4, is rich in long stretches of alpha-helix, one of which, 63 amino acids long, has heptad repeats resembling those found in proteins known to form alpha-helical coiled-coils. The predicted secondary structure correlates well with the available data on the protein accessibility delineated by immunological and biochemical findings and with the spike structure of the protein, which has been determined by cryoelectron microscopy.

The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus.

We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. López, J. Gen. Virol. 68:633-642, 1987). In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase. This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus.

Prevalent patterns of serotype-specific seroconversion in Mexican children infected with rotavirus.

The level of neutralizing antibodies to rotaviruses belonging to serotypes 1, 3, and 4 was determined in acute- and convalescent-phase sera from 36 Mexican children with rotaviral diarrhea. Most of the infants who seroconverted fell into one of the following three patterns: single seroconversion to serotype 1; seroconversion to serotypes 1 and 4; or seroconversion to all three serotypes tested. The heterotypic neutralizing antibody responses to rotavirus infections are discussed.

Synthesis in Escherichia coli and immunological characterization of a polypeptide containing the cleavage sites associated with trypsin enhancement of rotavirus SA11 infectivity.

About 45% of the rotavirus SA11 VP3 gene was inserted into a thermoinducible expression plasmid under the control of phage lambda PL promoter. The primary translation product predicted on the basis of the plasmid construction was a hybrid protein in which the 98 amino-terminal amino acids of phage MS2 polymerase were followed by amino acids 42 to 387 of the VP3 protein, which included the region containing the cleavage sites associated with trypsin enhancement of infectivity. On induction, a polypeptide that had the expected mol. wt. and contained VP3-related amino acid sequences as judged by immunological criteria, was synthesized to a level representing about 15% of the total bacterial protein. When a bacterial lysate enriched for the fusion polypeptide was injected into mice, it induced antibodies which inhibited haemagglutination and neutralized SA11 rotavirus infectivity.