A new high-pressure high-temperature deformation apparatus to study the brittle to ductile transition in rocks.

Understanding the micro-mechanisms underlying the localized-ductile transition (LDT) as well as the brittle-plastic transition (BPT) has become crucial for our wider understanding of crustal processes and seismicity. Given how difficult in situ observations of these transitions are to perform, laboratory experiments might be our only way to investigate the processes active under these conditions (high T and high P). Here, we present Triaxial AppaRatus for GEoThermal energy, a new gas-based triaxial apparatus located at EPFL in Switzerland that was specifically designed to operate under conditions where both the LDT and BPT can occur in geomaterials. We show that the machine is capable of deforming rock samples at confining pressures of up to 400 MPa, temperatures of up to 800 °C, and pore pressures (liquid or gas) of up to 300 MPa while keeping the temperature gradient along samples of 40 mm in length and 20 mm in diameter minimal (less than 30 at 700 °C). Most importantly, the maximum load is 1000 kN (stresses as high as 2.2 GPa on 24 mm samples and 3 GPa on 20 mm samples), allowing for the deformation of very competent rock samples. Moreover, during deformation, the pair of syringe pore pressure pumps allow for continuous permeability or dilatancy recording. We benchmarked our machine against existing data in the literature and show that it accurately and precisely records stress, strain, permeability, pressure, and temperature.

Monoclonal antibody against Saint Louis encephalitis prM viral protein.

Saint Louis encephalitis virus belongs to Flavivirus genus; Flaviviridae family jointly with other medically important flaviviruses including dengue virus and West Nile virus. The biological properties and functions of prM flavivirus protein are under investigation due to its importance in the generation of infectious virion and host interactions. Monoclonal antibodies have become powerful tools in this approach. Also the use of monoclonal antibodies has been successfully applied for antigenic analysis, clinical diagnosis and treatments. Here, using an immunofluorescence assay we describe a monoclonal antibody (mAb 3D2) that uniquely recognizes native prM Saint Louis encephalitis virus protein expressed in either C6/36-HT or Vero cells. In conclusion, mAb3D2 has significant potential for use in (a) the diagnosis of infections caused by this virus and (b) therapeutic use to treat patients infected by this virus and fundamental research to understand the role of the prM in the Saint Louis encephalitis virus infectious process.

Isolation of Mycoplasma genitalium from patients with urogenital infections: first report from the Latin-American region.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe a modified culture system based on Vero cells grown in medium 199 with 2% foetal bovine serum (FBS). The culture system was evaluated using early passage M. genitalium strains M6271 and M6311 with growth monitoring by quantitative TaqMan PCR. Eleven endocervical swabs and one male urethral swab positive by 16S rRNA and MgPa1-3 PCRs were quantified and inoculated into Vero cell suspensions in medium 199 supplemented with 2% FBS and antibiotics. Cultures were incubated for 14 days. Cell passages and growth monitoring by TaqMan PCR were performed until the growth of M. genitalium reached ≥10(6) geq/mL. Confirmation of the new M. genitalium strains was performed by sequencing a 281 bp fragment of mgpB. The growth of Mycoplasma genitalium strains was recorded for all urogenital swab specimens in the modified cell-culture system. Growth of M. genitalium was obtained within 2 months and yielded 12 M. genitalium strains with all 11 isolates from females of an identical, but unique genotype. To our knowledge, this is the first successful isolation of M. genitalium in the Latin-American region. The use of Vero cell culture in 199 medium with 2% FBS is a method comparable to the Ultroser G culture system for isolation of M. genitalium. Genotyping of clinical samples and isolates should be performed to document the absence of cross-contamination.

First epidemic of echovirus 16 meningitis in Cuba.

From April to September 2000, an epidemic of aseptic meningitis spread throughout Cuba, with 16,943 reported cases. Virologic studies identified echovirus 16 as the cause of this epidemic. This is the first reported isolate of echovirus 16 from patients with viral meningitis in Cuba.

Study of biologic attributes of Cuban dengue 2 virus after serial passage in primary dog kidney cells.

OBJECTIVE: The serial passage of dengue viruses in primary dog kidney (PDK) cells has resulted in selection of attenuated viruses. However, the molecular changes responsible for loss of virulence are not well characterized. This article describes the isolation and biologic attributes of one dengue 2 virulent strain as a first step to allow the study of determinants of virulence at the molecular level. METHODS: A15 dengue 2 Cuban strain was isolated from the viremic plasma of a patient with uncomplicated dengue fever during the 1981 epidemic. This was then subjected to serial passage in PDK cells. Viruses resulting from several PDK passages were compared to the parent strain for plaque size and temperature sensitivity, neurovirulence in newborn mice, and cytopathogenic effects on LLC-MK(2) and C6/36-HT cell lines. RESULTS: A15 dengue 2 Cuban strain was successfully propagated in PDK cells. Primary dog kidney 52 to 53 viruses exhibited several biologic attributes, such as small plaques, temperature sensitivity, reduced mouse neurovirulence, and cytopathic effect in permissive cell lines. CONCLUSIONS: These results represent the first step to allow attenuation of this strain of dengue 2 virus.

[Plesiomonas shigelloides, a Vibrionaceae to be taken into account]. / Plesiomonas shigelloides una Vibrionaceae en quien pensar.

The antigenic structure and antimicrobial susceptibility were studied in 99 strains isolated from patients with acute diarrhea (6 strains from an outbreak of digestive transmission disease in Santiago de Cuba) and a strain isolated from a patient who died from infections neurological syndrome (INS, meningitis). Four new serotypes (093, 994, 095, 096), which had not been described in the world classification, were identified from the Cuban isolated strains and were included in the International Serotyping Scheme by the International Reference Center located in Prague, Czech Republic. For the first time in Cuba, the circulation of serotypes 017:H11, 011: H2, 023. H1alc, 057: H3 which show cross reaction to Shiguella species was proved. Those strains from the outbreak of digestive disease belonged to serotype 050: H11 and had a thermostable toxin. The first case of infectious neurologic syndrome with Plesionomas shigelloides etiology reported in Cuba was described; the strain corresponded to serotype 050: H11. The worldwide reported pattern of antimicrobial resistance was demonstrated.

[Hemorrhagic pneumonia caused by influenza virus: virological diagnosis]. / Neumonía hemorrágica por Influenza virus: diagnóstico virológico.

The paper presents the case of a female patient who was admitted to "Calixto García" General Hospital with respiratory distress and hypovolemic or septic shock. She was diagnosed with viral hemorrhagic pneumonia. From the endotracheal secretion taken as a sample, the influenza virus was isolated as etiological agent, which, through the hemaglutination inhibition technique, was characterized as a strain belonging to H3N2 subtype, very similar to strain A/Johannesburg/33/94 from the antigenic viewpoint. The patient recovered satisfactorily after being treated with rivabirin.

Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells.

A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results suggest that the rapid centrifugation assay may be useful for detection of dengue virus in clinical specimens.

[Biological behavior of 3 strains of the dengue-2 virus in 2 cell lines of mosquitoes]. / Comportamiento biológico de 3 cepas del virus dengue-2 en 2 líneas celulares de mosquitos.

Strains A-15 (isolated in Cuba, 1981), Jamaica (isolated in Jamaica, 1981) and Nueva Guinea "C" (standard) from dengue-2 virus were compared according to the time of appearance of the cytopathic effect (CPE), to the time of appearance of specific fluorescence and to the kynetics of viral multiplication on being innoculated in the cell lines AP-61 (Aedes pseudoscutellaris) and C6/36 HT (Aedes albopictus). The results showed that the CPE of highest intensity and earliest appearance was for A-15, followed by Jamaica and Nueva Guinea "C" (NGC). AP-61 seems to favor the CPE of Jamaica with respect to that of the same strain in C6/36 HT. The fluorescence was earlier for Jamaica and A-15 and more intensive for the latter, whereas NGC manifested late. This behaviour was similar in the 2 cellular systems. The greatest titres during the kinetics of viral multiplication were obtained from A-15 in both lines, although in AP-61 they tend to be equal from the 4th day on. The strain A-15 showed a particular behaviour of these biological properties on comparing them with the other strains under study, which may be related to changes found in its neucleotide sequence.

[Determination of verotoxins in strains of Escherichia coli O157:H7]. / Determinación de verotoxinas en cepas de Escherichia coli O157:H7.

We performed an study to find out the main virulence factor in verotoxigenic Escherichia coli: the production of verotoxins in 50 non sorbitol-fermenting Escherichia coli strains (possible enterohemorrhage) which were isolated from children with acute diarrheas in the City of Havana and referred to the National Reference Laboratory for acute diarrheal diseases in "Pedro Kourí" Institute. By using the agglutination technique with latex particles of E. coli O157:H7, we determined whether the verotoxins belonged to this serotype, we also researched the production of verotoxins in Vero cell culture. Ninety-six percent of the total number of strains were positive in the qualitative determination of this factor which was more frequently observed after 24 hours.

[Study of various biological properties of 3 strains of dengue 2 with differences in their nucleotide sequences]. / Estudio de algunas propiedades biológicas de 3 cepas de dengue 2 con diferencias en sus secuencias nucleotídicas.

Some biological properties of Dengue-2 strains such as A-15 (isolated in Cuba in 1981); Jamaica (isolated in Jamaica in 1981) and New Guinea "C" (NG"C") standard strain differing in their nucleotide sequences were studied. The results showed that the cytopathic effect in C6/36 HT cell line occurred earlier in A-15 strain and that fluorescence was first detected in Jamaica and A-15 strains. This seems to indicate that rapid detection of strains does not have any relation to neither their history of passage nor the original isolation system. A-15 and NG"C" strains exhibited an heterogeneous pattern formed by big and small plaques but average size of plaques in NG"C" was lower whereas Jamaica showed only small plaques. The most neurovirulent strain in mice was NG"G" followed by A-15 whereas Jamaica was not neurovirulent at all. These results indicate that A-15 has a different biological behaviour which is probably due to intrinsic differences. It should be taken into account that 7 amino acid changes were found in the envelope protein which may have affected the expression of some biological properties.

Meningoencefalitis virales por Enterovirus en Cuba en el período 1990-1995 / Viral meningoencephalities due to enteroviruses in Cuba during 1990-1995

Se describen los resultados del estudio de enterovirus como agentes causanes de meningoencefalitis viral (MEV) en Cuba, desde 1990 hasta 1995. En este período fueron estudiados 586 muestras de heces, 108 líquidos cefalorraquídeos y 1095 sueros pareados para un total de 1789 muestras, procedentes de 1458 pacientes diagnosticados clínicamente con esta patología. Las muestras para el aislamiento viral se inocularon en dos sistemas celulares diferentes, encontrándose 225 muestras positivas a enterovirus que representan el 32,42 o/o del total; el mayor número de aislamientos (217) fue a partir de heces, en células diploides de fibroblastos de pulmón humano (PHuE-1). Las determinaciones de anticuerpos se realizaron por prueba de neutralizacion en micrométodo, enfrentándolos con 10 antígenos de enterovirus (Echovirus 4, 6, 9, 11 y Coxsackievirus B1-6) y, en períodos epidémicos, además con el virus aislado. En los años estudiados se produjeron tres brotes epidémicos por Coxsackievirus A9 (1990-91), Echovirus 30 (1994) y Coxsackievirus B5 (1995). Es de señalar que desde 1970 los Coxsackievirus A9 y Echovirus 30 se vincularon en nuestro país, por primera vez, con epidemias de MEV. En los sueros pareados se obtuvo 66,84 o/o de positivos, siendo la mayor positividad a los Echovirus 6 y 11. Al considerar en conjunto la positividad por aislamiento y serología, más del 80 o/o de los casos estudiados pudieran tener ua explicación por algún enterovirus, lo uqe demuestra la importancia de estos agentes como causantes de MEV en Cuba

Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

[The standardization of an ultramicro ELISA assay for the detection of IgG antibodies to the respiratory syncytial virus]. / Normalización de un ensayo de ultramicroELISA para la detección de anticuerpos IgG al virus sincitial respiratorio.

An ultramicro ELISA assay of double antibody for the detection of IgG antibodies to the respiratory syncytial virus (RSV) was standardized. It was used a RVS antiprotein F monoclonal antibody produced by the Genetic Engineering and Biotechnology Center (GEBC) in Havana. The use of this antibody allowed to include crude antigenic preparations instead of purified fractions, which caused a significant reduction of the reactivity obtained with the antigen control. The assay conditions were determined by crossed titration. It was obtained a sensitivity of 97.2%, a coincidence of 91%, and a specificity of 83.3% of the UMELISA as regards the complement fixation. The results may be qualitatively expressed or by antibody titres using only one serum dilution (1:40) and a pattern curve.

[A comparison of the NCI-H292 line with other continuous lines for the multiplication of respiratory viruses]. / Comparación de la línea NCI-H292 con otras líneas continuas para la multiplicación de virus respiratorios.

The NCI-H292 continual line of mucoepidermoid cells of the human lungs has been reported to be useful for the propagation of many viruses, mainly Adenovirus and Paramyxovirus. It is stated the possible substitution of primary cultures of monkey kidney for NCI-H292 in order to isolate such agents. In the present paper it is evaluated the utility of this line for multiplying the respiratory syncytial viruses Adenovirus 3 and 7, and the parainfluenza viruses 1, 2, and 3, in comparison with the continual cellular lines traditionally used for the propagation of these viruses, whose strains were inoculated this time in the Vero, HEp-2, and HeLa lines, according to their know sensitivities as well as in NCI-H292 simultaneously. The viral multiplication was detected by the appearance of the cytopathic effect or by hemadsorption. As a result, it was demonstrated the multiplication capacity of the NCI-H292 line for Adenoviruses 3 and 7 and parainfluenza 3, being more useful for their multiplication than the traditionally used lines.

[The physicochemical characterization of the agents isolated during the outbreak of epidemic neuropathy in Cuba. I]. / Caracterización físico-química de los agentes aislados durante el brote de neuropatía epidémica en Cuba. Parte I.

This paper reports on the physical and chemical characteristics of agents isolated from serum samples of patients presenting with epidemic neuropathy. The behaviour of isolated enteroviruses was as described for such viruses. Mild cytopathogenic effects-producing agents behaved in a variable form regarding sensitivity to chloroform; on the other hand they were neither sensitive to phosphonoacetic acid (PAA) nor to guanidine hydrochloride (GHC1) and grew in cells previously treated with bromodeoxyuridine (BDUR). These results suggest the presence of agents resembling enteroviruses and enveloped viruses. Further studies for the characterization of such agents need to be performed.

[The physicochemical characterization of the strains isolated during the outbreak of epidemic neuropathy in Cuba. II]. / Caracterización físico-química de los agentes aislados durante el brote de neuropatía epidémica en Cuba. Parte II.

The presence of 2 agents such as a Cox A9 strain and another mild cytopathogenic effect-producing strain, both isolated from patients presenting with epidemic neuropathy is reported in this paper. A mild cutopathogenic effect which was propagated in successive dilutions was developed in the dilution 10(-4) by means of the neutralization test of a Coxsackie A9 virus with its homologous hyperimmune serum. A gradient in saccharose was performed in a mild cytopathogenic effect-producing strain and a typical cytopathogenic effect of an Enterovirus developed from one of the fractions passed in tissular cultures in the presence phosphonoacetic acid (PAA). The possible pathogenic role of these viruses are discussed.

[The factors affecting the growth of the agent, producing a mild cytopathic effect, isolated in patients with epidemic neuropathy]. / Factores que influyen en el crecimiento del agente productor del efecto citopático ligero aislado en pacientes con neuropatía epidémica.

Certain factors influencing upon the growth of the agent producer of mild cytopathogenic effect and isolated from the cerebrospinal fluid of patients presenting with epidemic neuropathy are described in this paper. It was demonstrated that a concentration of NaHCO3 was essential for the occurrence of the cytopathogenic effect. Determined concentrations of MnCl2 allowed for the visualization of the cytopathogenic effect and increased viral yields.

[Standardization of the plaque method for viruses isolated in patients with epidemic neuropathy]. / Normalización del método de placas para los virus aislados en pacientes con neuropatía epidémica.

This paper reports on the necessary conditions for plaque development in mild cutopathogenic effect-producing agents which were isolated from samples of the cerebrospinal fluid of patients presenting with epidemic neuropathy.