In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

Distinctive intrahepatic characteristics of paediatric and adult pathogenesis of chronic hepatitis C infection.

Mechanisms leading to liver damage in chronic hepatitis C (CHC) are being discussed, but both the immune system and the virus are involved. The aim of this study was to evaluate intrahepatic viral infection, apoptosis and portal and periportal/interface infiltrate in paediatric and adult patients to elucidate the pathogenesis of chronic hepatitis C. HCV-infected, activated caspase-3(+) and TUNEL(+) hepatocytes, as well as total, CD4(+), CD8(+), Foxp3(+) and CD20(+) lymphocytes infiltrating portal and periportal/interface tracts were evaluated in 27 paediatric and 32 adult liver samples by immunohistochemistry or immunofluorescence. The number of infected hepatocytes was higher in paediatric than in adult samples (p 0.0078). In children, they correlated with apoptotic hepatocytes (activated caspase-3(+) r = 0.74, p < 0.0001; TUNEL(+) r = 0.606, p 0.0017). Also, infected (p = 0.026) and apoptotic hepatocytes (p = 0.03) were associated with the severity of fibrosis. In adults, activated caspase-3(+) cell count was increased in severe hepatitis (p = 0.009). Total, CD4(+), CD8(+) and Foxp3(+) lymphocyte count was higher in adult samples (p < 0.05). Paediatric CD8(+) cells correlated with infected (r = 0.495, p 0.04) and TUNEL(+) hepatocytes (r = 0.474, p = 0.047), while adult ones correlated with activated caspase-3(+) hepatocytes (r = 0.387, p 0.04). In adults, CD8(+) was associated with hepatitis severity (p < 0.0001) and correlated with inflammatory activity (CD8(+) r = 0.639, p 0.0003). HCV, apoptosis and immune response proved to be involved in CHC pathogenesis of both paediatric and adult patients. However, liver injury in paediatric CHC would be largely associated with a viral cytopathic effect mediated by apoptosis, while in adults it would be mainly associated with an exacerbated immune response.

Evolution of hepatitis C virus hypervariable region 1 in immunocompetent children born to HCV-infected mothers.

Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d(N)/d(S) rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S))] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.

[Genotype characterization of hepatitis C virus infection in children]. / Caracterización genotipica de la infección por el virus de hepatitis C en niños.

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5'untranslated region (5'UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36%) were always positive for HCV RNA, in 5 (20%) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36%) showed alternating RT-nested PCR results and in 2 (8%) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the host's response in the initial stages of it.