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.

Surveillance of adamantane resistance among influenza A H3 viruses isolated in Argentina between 2001 and 2007.

A dramatic rise in the frequency of resistance to adamantane drugs by influenza A H3 viruses, associated with a single amino acid replacement in the viral matrix M2 protein, has occurred in multiple countries worldwide in recent years. We investigated the frequency of adamantane-resistant influenza A H3 viruses in Argentina during the period 2001-2007. We used reverse transcription followed by polymerase chain reaction. The obtained products were sequenced for the detection of mutations of the M2 gere relevant to the resistance phenotypes. The HA1 sequences of the sensitive and resistant strains were also analyzed to clarify whether they had any relevance to the resistant mutations. Twenty out of 55 (36%) strains were identified with the resistance-conferring substitution at amino acid 31 (Serine 31 Asparagine). No resistant viruses were detected between 2001 and 2005. All strains isolated in 2006 and four out of five isolates from 2007 were resistant. None of the patients had received previous treatment with amantadine and/or rimantadine. The HA1 analysis showed that there were only two changes (Serine193 Phenylalanine and Aspartic acid 225 Asparagine) present in the strains with the M2 substitution at position 31. Our data indicate that since 2006 there has been a significant increase of adamantane-resistant influenza A H3 viruses, which raises concern over the spread of these viruses in Argentina.

Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.

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.

Influenza circulating strains in Argentina exhibit differential induction of cytotoxicity and caspase-3 in vitro.

BACKGROUND: Human influenza infections are a significant cause of morbidity worldwide. Though damage to the respiratory epithelium and has been related to apoptosis, which occurs subsequent to influenza virus infection, little information is available regarding cell cytotoxicity of human strains. OBJECTIVE: To study cytotoxicity performed in vitro by various circulating strains in Argentina. The study sample consisted of three vaccine strains (H1N1, H3N2, and B) administered during 1999-2000 in South America and three strains isolated from clinical samples, one, NAC (H1N1) obtained from an adult inpatient with human pneumonia; and the other two (T) and (T2) (H3N2) with influenza syndrome. Viral antigen was detected by an immunofluorescence test, conducted prior to viral isolation in MDCK cells. Strains were subtyped by the hemmaglutination inhibition test. Cytotoxic properties were determined by lactate dehydrogenase reaction (LDH), crystal violet staining and Hoechst staining. Caspase-3 activity, morphological changes of apoptosis, and viral yields were measured in MDCK infected cells. RESULTS AND CONCLUSIONS: Cells infected by each of the strains exhibited apoptosis morphology by Hoechst staining and caspase-3 activity was high for both H1N1 strains. Further, high levels of LDH activity were detected for NAC and H3N2 strains tested, indicating the possible role of different viral proteins or functions on cell cytotoxicity. The NAC strain, isolated from human pneumonia and antigenically related to A/New Caledonia /20/99 (H1N1), was the highest cytotoxic strain and an excellent inducer of caspase-3 activity. In turn, no parameter was related to different viral yields. We conclude that human strains studied in this paper may be useful tools in the characterization of molecular determinants involved in viral cytopathogenicity.

[Glycogen synthase activity in parodontal disease (author's transl)]. / Activite glycogene-synthase dans la maladie paradontale.

A biochemical study of an enzyme participating in the synthesis of glycogen is presented, with particular regard to the fluctuations in the amounts of this polysaccharide in human gingival epithelium, during inflammation. The increase in the activity of UDPglucose : glycogen glucosyltransferase can be related to the accumulation of glycogen. Some kinetic parameters of this enzyme are described.

Genetic characterization of wild-type measles viruses isolated during the 1998 measles epidemic in Argentina.

This study reports on genomic characterization of six measles virus (MV) isolates obtained from a measles epidemic in Argentina in 1998. Reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of the carboxyl-terminal region of the nucleoprotein (N) gene of these isolates classified all of them as wild type MV of D6 genotype. MVs of D6 genotype with identical nucleotide sequences in the region analyzed were also identified during the 1997 measles epidemic in Brazil and the 1999 measles outbreak in Uruguai. These results suggest that the MVs associated with the 1998 measles epidemic in Argentina might have originated from Brazil. As the D6 genotype is also widely distributed in Europe, it is possible that this genotype was brought to South America from Europe.

[Detection of a new genomic variation of Adenovirus 7 associated to severe infections of the lower respiratory tract]. / Detección de una nueva variación genómica de Adenovirus 7 asociada a infecciones severas del tracto respiratorio inferior.

We report a new genomic variation of Adenovirus 7, associated to severe infections of the lower respiratory tract isolated during September 1990, from children under 3 years of age and living in Buenos Aires city. The restriction analysis with the BamHI, BglI, BglII and SmaI restriction endonucleases demonstrated that the new variation is highly related to the recently described Adenovirus 7h.

[Antigenic relationship between influenza A (H3N2) strains circulating in Argentina and vaccine strains]. / Estudio antigenico de cepas de influenza A (H3N2) circulantes en la Argentina y su relacion con las cepas vacunales.

Influenza epidemic season occurs usually from May to September in Argentina, so that the vaccine produced in the northern hemisphere to be administered during October-November may be out of phase for Argentina. In order to determine if the locally circulating strains in Argentina are antigenically close by related to the vaccine strains administered, they were compared with the influenza viruses isolated from May 1994 to December 1997. Clinical samples (9866) were nasopharyngeal aspirates from children hospitalized for acute lower respiratory tract infection and nasal-pharyngeal swabs from adults with influenza syndrome. Initial laboratory diagnosis was performed by immunofluorescence assay, followed by isolation in MDCK cells. Influenza A viruses (242) were detected and subtyped by hemagglutination inhibition (HAI) with WHO FLU Reagent Kit. A subset of the isolated viruses was antigenically analyzed by the WHO Collaborating Center at CDC, Atlanta, USA. Influenza A (H3N2) viruses characterized as circulating in Argentina during the last four years matched partially with the antigens present in the vaccines administered during 1994-97 period. These antigenic variants sometimes circulate late in the year (October 1994 and 1997) initiating the following influenza season and becoming prevalent. They were present 2 years later in the vaccine formula administered in the southern hemisphere. The HAI results of our isolates show that they are highly specific with the homologous antiserum and much less specific with antibodies against vaccine strains. The difference is 16 to 64 fold different. These results demonstrate the need to intensify influenza laboratory surveillance in order to obtain the best possible vaccine.

[Etiological and clinical evaluation of low acute respiratory infections in children]. / Evaluación etiológica y clínica de infecciones respiratorias agudas bajas en una población infantil.

Viral laboratory diagnosis was correlated with clinical and epidemiological data from 80 hospitalized children with acute lower respiratory infection (ALRI). They all were less than 5 years-old and were studied from May to September 1993. Fifteen percent of them were malnourished and 75% had some unsatisfied basic necessity. Nasopharingeal aspirates were obtained the first day of hospitalization, and diagnosis for respiratory viruses was performed by the immunofluorescence test with monoclonal antibodies. Routine laboratory determinations, x-ray studies, and clinical data were not conclusive to determine viral etiology. Forty-one percent of the children had a positive viral diagnosis: the most important agent was Respiratory Syncytial Virus (78.7%) followed by Adenovirus (9.1%), Influenza A (6.1%) and Parainfluenza (3%). The peak of incidence was observed in June and the majority of the patients remained hospitalized less than 10 days. Six children died: two of them had viral pneumonia and could not receive mechanical respiratory assistance. The percentage of children who received antibiotics was high, 61.2%, in spite of the fact that 34.7% of these patients had a laboratory confirmed viral etiology. The availability of rapid laboratory viral diagnosis may contribute to decrease the use of antibiotics and improve the management of patients.

[Rabies vaccine inactivated with ethylenimine. Duration of the immunity in dogs]. / Vacuna antirrábica inactivada con etilenimina. Duración de immunidad en perros.

The duration-of-immunity afforded by the ethylenimine inactivated rabies vaccine produced in BHK cells with the PV strain, was studied in dogs. One hundred per cent of the dogs became serologically positive 7 days after vaccination; the same percentage was still positive 3 years after vaccination with one dose of the vaccine. A good correlation was observed between the antibody profiles as determined by the titers obtained with the mouse neutralization and fluorescent field inhibition techniques. The correlation was not as good when the number of international units per ml was determined by both tests. The best correspondence between serological response and resistance to challenge was observed when the antibodies were determined by the number of international units per ml, using the neutralization test in mice. All the dogs challenged 12 and 25 months after vaccination resisted challenge; 89% (8/9) were protected 36 months after vaccination. Inactivated vaccines can be as effective to control rabies as those prepared with modified live virus; moreover, the inactivated vaccines are more stable and safer than the latter.

[Comparison of 2 techniques for the laboratory diagnosis of influenza virus infections]. / Comparación de dos técnicas para el diagnóstico de laboratorio de infecciones por el virus influenza.

The clinical or epidemiological influenza diagnosis require fast, sensitive and accessible techniques for small laboratories. In order to investigate the sensitivity of the methods currently used in Argentina, the rapid diagnosis by indirect immunofluorescent assay (IF) was compared to the rapid viral culture in MDCK cells. The diagnosis of influenza virus infection was performed on 81 nasal and pharyngeal swabs collected from outpatients with upper respiratory infection, influenza-like syndrome. The samples were collected during 1998 winter season and both techniques were tested. The IF specificity and sensitivity obtained were 91.9% and 59.5%, respectively. In the selection of the assay to be used for influenza diagnosis, the limitations of the simplest techniques such as IF should be considered. Furthermore, it is advisable to set up an optimized culture method in complex laboratories since culture is the only technique which allows the reference centers to perform the full characterization of the isolates.

Regional perspectives on influenza surveillance in South America.

The implementation of influenza vaccination programs emphasize the necessity of an extended influenza surveillance in the countries of the region. As it is based on the activity of National Influenza Centers we intend to make a description of their work, their historical background and further development. Technical capacities in influenza South America laboratories, and networks in Argentina, Brazil and Chile are shown. Examples of different viral circulation in a unique country or in countries with common borders illustrate the importance of the information obtained by means of the virological surveillance. Specific characteristics of the region as long distances and the lack of modern information systems require a suitable fitting of the systems that are working in Northern Hemisphere countries. The contribution of motivated physicians and public health workers must not to be disregarded. The following actions are proposed: optimizing technical capacities of National Influenza Centers in order to improve the quality of data available; improving the communication of the information obtained by surveillance activities to all the participants; increasing the cooperation among the countries; motivate health authorities to become aware of influenza impact in public health.

Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM.

This report is concerned with the application of the enzyme immunoassay to measure the antibodies in humans vaccinated against rabies or presenting symptoms of rabies without having been vaccinated. By the same technique we identify the IgG and IgM classes of antibodies. The antigen (5 microgram/ml), purified virus, is readily adsorbed into polystyrene tube by passive adsorption. The use of only one dilution for each serum assay (1/200) is particularly suitable for epidemiological studies. Antibody response of subjects in the course of rabies vaccination was an obvious application. After 5 inoculations of tissue culture vaccine the IgM response was poor and late; it was even negative in two cases. The IgG response appeared early on the 7th day. In the same way we tried to follow antibody response in three cases of rabies in man. Seroneutralisation (SN) antibody were not detected at the beginning of the illness. In case 1 antibodies were found on the 12th day, in case 2 on the 7th day and in case 3 on the 8th day. When we assayed the serum samples for immunoenzymatic test, we found that the sera became positive some days earlier: on the 5th for case 1, already on the 1st day for the two others. In each of these three cases the positivity of the test corresponded to the presence of IgM class globulins since IgG detection remained negative as did the SN test. Our results could have some clinical interest concerning future rabies treatment and early diagnosis.

Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM results.

Techniques usually employed for the detection of rabies' antibodies are costly, time consuming, and sometimes fail to detect early antibodies. The introduction of immunoenzymatic techniques in the serology of viral disease represents a new and important advance. We therefore adapted this technique to the detection of rabies antibodies. We have found that the sera from rabies patients who had not received antirabies treatment do not show seroneutralizing antibodies until several days after the onset of symptoms. However, antibodies can be detected some days earlier by the immunoenzymatic method in the same samples. Furthermore, the immunoenzymatic test was applied to the detection of both the IgM or the IgG class of antirabies antibodies using an antihuman Ig-or antihuman IgG-peroxydase conjugate.

[Rapid detection of rabies antibodies by immunoenzymatic assay]. / Epreuve immunoenzymatique pour la détection rapide des anticorps antirabiques.

The antigen is a semipurified tissue culture virus, and peroxidase conjugated anti-human immunoglobulines are used. When sera from immunized humans are evaluated by this procedure, the values obtained show a good correlation with seroneutralization titres. The detection of antibodies by this method is earlier than by seroneutralization.

[Inactivation of rabies virus by chemical agents]. / Inactivacion del virus rabico por agentes quimicos

To study the virucidal activity of several chemical agents available locally in Argentina for rabies virus, was considered to be very useful for physicians treating persons bitten by rabid dogs and for those responsible for the sterilization of rabies contaminated areas. CVS fixed rabies virus suspensions were treated for one minute at room temperature with soaps of different quality, anionic (most of them derivates of dodecyl-bencene-sulfonic acid) and cationic (dodecyl, tetradecyl, hexadecyl-trimethyl-ammonium bromide) detergents, lemon juice, vinegar, hydrochloric acid, sodium carbonate, etc. Most of these agents inactivated 4 logarithms of virus (99.99%) which is approximately the amount of virus present in the saliva of experimentally infected dogs. It is noteworthy that a popular treatment for animal bites in several latin America countries is lemon juice, while the scientific community may still recommend the use of nitric acid which has a definitive necrotic and scarring effect on the treated wound. The need to eliminate organic matter prior to sterilization of contaminated areas because of interference with the virucidal activity of the agents was also confirmed.