The purpose of this study was to optimize and evaluate the purification techniques, isolation and... more The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Sobre Nicolosi, Alejandra Pia (comp.). La television en la decada kirchnerista. Democracia audiov... more Sobre Nicolosi, Alejandra Pia (comp.). La television en la decada kirchnerista. Democracia audiovisual y batalla cultura l. Buenos Aires: Editorial Universidad Nacional de Quilmes, 280 pp., 2015, ISBN 978-987558-297-2.
Arenaviruses are enveloped viruses that have a bi-segmented negative-stranded RNA genome. The gen... more Arenaviruses are enveloped viruses that have a bi-segmented negative-stranded RNA genome. The genomic RNA segments, large (L) and small (S), use an ambisense coding strategy to encode two open reading frames in opposite orientation, separated by a noncoding intergenic region. Several arenaviruses are etiological agents of emerging diseases. At present, they are responsible for up to 500,000 zoonotic infections per year in endemic areas of Africa and South America and can lead to severe and lethal hemorrhagic fever as well as neurological symptoms. Arenaviridae represents the largest group of hemorrhagic fever (HF)-causing viruses: five of the South American arenaviruses (CHPV, GTOV, JUNV, MACV, and SABV) are associated with HF in humans.
Spectrophotometers are usually at hand at University teaching laboratories, but this is not alway... more Spectrophotometers are usually at hand at University teaching laboratories, but this is not always the case for high school institutions. The same is true of substances that are commonly available in biochemical teaching experiments. In this work, our aim is to show that basic biochemical colorimetric experiments and demonstrations can be done with a homemade spectrophotometer that is easy and inexpensive to build. In the same spirit, we provide examples of experiments that can be performed with ordinary and fairly inexpensive substances. A spectrophotometer is an apparatus intended to measure the degree of absorbance of light in specific wavelength ranges. It consists of three essential elements, a light source, a monochromator, and a detector. The light source and detector define the limits of wavelength and sensitivity of the apparatus, whereas the monochromator separates the light produced by the light source into different small ranges, usually in the nanometer scale. The sampl...
With the arrival of the pandemic in Argentina in March 2020, a working group of scientists from t... more With the arrival of the pandemic in Argentina in March 2020, a working group of scientists from two institutes belonging to the Faculty of Exact and Natural Sciences of the University of Buenos Aires and CONICET, together with colleagues from different academic institutions in the country, decided to put forth our experience and knowledge in data science and associated disciplines, towards helping with decision-making in the context of COVID-19. Data analysis within Argentina and other countries, scenario simulation, as well as rapid response projects- mainly in the province of Buenos Aires- were all within the scope of our aim. This review article outlines some of the activities carried out by our team throughout these pandemic months.
St. Louis encephalitis virus (SLEV) is a mosquito-borne reemerging flavivirus in Argentina. It is... more St. Louis encephalitis virus (SLEV) is a mosquito-borne reemerging flavivirus in Argentina. It is currently necessary to develop specific serological tests that can efficiently discriminate the flaviviruses that circulate in our country. The immunoassays to diagnose SLEV lack specificity because they are based on the detection of structural viral proteins and the human immunoglobulins produced during infection against these proteins cross-react with other flaviviruses. Here, we describe an enzyme-immunoassay designed to detect human IgG antibodies specific to the viral nonstructural protein NS5. The results indicate that NS5 is a promising antigen useful to discriminate SLEV from other circulating flaviviruses.
Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense... more Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.
Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numero... more Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3′ end of viral mRNA. Regarding to this, in this review we provide an update about th...
Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This repor... more Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actual...
Vaccination has been one of the most successful and the most significant scientific advances in h... more Vaccination has been one of the most successful and the most significant scientific advances in human health and life expectancy all around the globe. The World Health Organization considers that immunization should be recognized as the main component of human health right, due to the fact that vaccination prevents 2.5 million deaths annually (World Health Organization, 2011). The most successful vaccines have been developed using conventional methods that follow the paradigm established by Pasteur: "to isolate, inactivate and inject" the pathogen microorganism and mimic a natural infection. Recently, metagenomics have played an important role in the discovery of new immunogens for vaccine design and the selection of antigens based on genomic information. The main approach that has used this strategy has been called "reverse vaccinology". This promising and arising field allows the screening of the entire potential antigenic repertoire of an organism using predic...
Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of ar... more Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further pu...
The Argentine Hemorrhagic Fever, an endemic disease present in a much of Argentina, is caused by ... more The Argentine Hemorrhagic Fever, an endemic disease present in a much of Argentina, is caused by the Junín virus (JUNV). Currently, there are sequences available from several strains of this virus, like those belonging to the vaccine lineage (XJ13, XJ#44 and Candid#1), as well as MC2 (rodent isolate) and IV4454 (human isolate). In this article, we report sequence information on two fragments of genomic segment S of viral isolates from the endemic area. A Nested-RT-PCR was used to amplify discrete genomic regions of 13 isolates of rodent and human origin. The bioinformatics studies revealed a great homogeneity of sequences among the JUNV isolates. The phylogenetic classification showed greater evolutionary distance between the old world arenaviruses (Lassa and LCM virus) than between the new world arenaviruses (JUNV and Machupo virus).► We report sequence information on two fragments of genomic segment S of viral isolates from the endemic area. ► We designed a Nested-RT-PCR to amplify discrete genomic regions of 13 isolates of rodent and human origin. ► Bioinformatics studies revealed a great homogeneity of sequences among the JUNV isolates. ► The phylogenetic classification showed differences in the evolutionary distance between the old world and new world arenaviruses.
The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function dur... more The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.
The purpose of this study was to optimize and evaluate the purification techniques, isolation and... more The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Sobre Nicolosi, Alejandra Pia (comp.). La television en la decada kirchnerista. Democracia audiov... more Sobre Nicolosi, Alejandra Pia (comp.). La television en la decada kirchnerista. Democracia audiovisual y batalla cultura l. Buenos Aires: Editorial Universidad Nacional de Quilmes, 280 pp., 2015, ISBN 978-987558-297-2.
Arenaviruses are enveloped viruses that have a bi-segmented negative-stranded RNA genome. The gen... more Arenaviruses are enveloped viruses that have a bi-segmented negative-stranded RNA genome. The genomic RNA segments, large (L) and small (S), use an ambisense coding strategy to encode two open reading frames in opposite orientation, separated by a noncoding intergenic region. Several arenaviruses are etiological agents of emerging diseases. At present, they are responsible for up to 500,000 zoonotic infections per year in endemic areas of Africa and South America and can lead to severe and lethal hemorrhagic fever as well as neurological symptoms. Arenaviridae represents the largest group of hemorrhagic fever (HF)-causing viruses: five of the South American arenaviruses (CHPV, GTOV, JUNV, MACV, and SABV) are associated with HF in humans.
Spectrophotometers are usually at hand at University teaching laboratories, but this is not alway... more Spectrophotometers are usually at hand at University teaching laboratories, but this is not always the case for high school institutions. The same is true of substances that are commonly available in biochemical teaching experiments. In this work, our aim is to show that basic biochemical colorimetric experiments and demonstrations can be done with a homemade spectrophotometer that is easy and inexpensive to build. In the same spirit, we provide examples of experiments that can be performed with ordinary and fairly inexpensive substances. A spectrophotometer is an apparatus intended to measure the degree of absorbance of light in specific wavelength ranges. It consists of three essential elements, a light source, a monochromator, and a detector. The light source and detector define the limits of wavelength and sensitivity of the apparatus, whereas the monochromator separates the light produced by the light source into different small ranges, usually in the nanometer scale. The sampl...
With the arrival of the pandemic in Argentina in March 2020, a working group of scientists from t... more With the arrival of the pandemic in Argentina in March 2020, a working group of scientists from two institutes belonging to the Faculty of Exact and Natural Sciences of the University of Buenos Aires and CONICET, together with colleagues from different academic institutions in the country, decided to put forth our experience and knowledge in data science and associated disciplines, towards helping with decision-making in the context of COVID-19. Data analysis within Argentina and other countries, scenario simulation, as well as rapid response projects- mainly in the province of Buenos Aires- were all within the scope of our aim. This review article outlines some of the activities carried out by our team throughout these pandemic months.
St. Louis encephalitis virus (SLEV) is a mosquito-borne reemerging flavivirus in Argentina. It is... more St. Louis encephalitis virus (SLEV) is a mosquito-borne reemerging flavivirus in Argentina. It is currently necessary to develop specific serological tests that can efficiently discriminate the flaviviruses that circulate in our country. The immunoassays to diagnose SLEV lack specificity because they are based on the detection of structural viral proteins and the human immunoglobulins produced during infection against these proteins cross-react with other flaviviruses. Here, we describe an enzyme-immunoassay designed to detect human IgG antibodies specific to the viral nonstructural protein NS5. The results indicate that NS5 is a promising antigen useful to discriminate SLEV from other circulating flaviviruses.
Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense... more Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.
Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numero... more Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3′ end of viral mRNA. Regarding to this, in this review we provide an update about th...
Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This repor... more Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actual...
Vaccination has been one of the most successful and the most significant scientific advances in h... more Vaccination has been one of the most successful and the most significant scientific advances in human health and life expectancy all around the globe. The World Health Organization considers that immunization should be recognized as the main component of human health right, due to the fact that vaccination prevents 2.5 million deaths annually (World Health Organization, 2011). The most successful vaccines have been developed using conventional methods that follow the paradigm established by Pasteur: "to isolate, inactivate and inject" the pathogen microorganism and mimic a natural infection. Recently, metagenomics have played an important role in the discovery of new immunogens for vaccine design and the selection of antigens based on genomic information. The main approach that has used this strategy has been called "reverse vaccinology". This promising and arising field allows the screening of the entire potential antigenic repertoire of an organism using predic...
Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of ar... more Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4-8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further pu...
The Argentine Hemorrhagic Fever, an endemic disease present in a much of Argentina, is caused by ... more The Argentine Hemorrhagic Fever, an endemic disease present in a much of Argentina, is caused by the Junín virus (JUNV). Currently, there are sequences available from several strains of this virus, like those belonging to the vaccine lineage (XJ13, XJ#44 and Candid#1), as well as MC2 (rodent isolate) and IV4454 (human isolate). In this article, we report sequence information on two fragments of genomic segment S of viral isolates from the endemic area. A Nested-RT-PCR was used to amplify discrete genomic regions of 13 isolates of rodent and human origin. The bioinformatics studies revealed a great homogeneity of sequences among the JUNV isolates. The phylogenetic classification showed greater evolutionary distance between the old world arenaviruses (Lassa and LCM virus) than between the new world arenaviruses (JUNV and Machupo virus).► We report sequence information on two fragments of genomic segment S of viral isolates from the endemic area. ► We designed a Nested-RT-PCR to amplify discrete genomic regions of 13 isolates of rodent and human origin. ► Bioinformatics studies revealed a great homogeneity of sequences among the JUNV isolates. ► The phylogenetic classification showed differences in the evolutionary distance between the old world and new world arenaviruses.
The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function dur... more The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.
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