Relationship between apoptosis and the BH2 domain sequence of the VP5 peptide of infectious pancreatic necrosis virus

Objective. To determine whether the level of apoptosis induced by infectious pancreatic necrosis virus (IPNV) is related to the amino acid sequence of the BH2 domain of the VP5 protein and the level of infectivity. Materials and methods. Three IPNV strains were used, the VP2 protein gene was amplified for genotyping and the VP5 sequence was also obtained. The infectivity of the strains was calculated using the viral titer obtained at 12, 24, 36 and 45 hpi in CHSE-214 cells. The percentage of apoptosis in infected cells was visualized by TUNEL assay and immunohistochemistry (caspase 3 detection). Results. The V70/06 and V33/98 strains corresponded to genotype Sp, while V112/06 to VR-299; the amino acid analysis of the V70/06 strain allows its classification as middle virulent strain and V33/98 and V112/06 strains as low virulent ones; infection with the V112/06 strain produced a lower viral titer (p<0.05). The VP5 gene of the 3 strains showed four homologous domains to Bcl-2, however, the BH2 domain was truncated in V70/06 and V33/98 (12 kDa), being complete (15kDa) in V112/06, which also showed the Trp155 residue, equivalent to Trp188 considered as a critical factor for the function of Bcl-2. The average apoptosis was below 12%, showing no differences between strains (p>0.05). Conclusions. The results showed that the differences in the BH2 sequence of the VP5 protein, infectivity and the VP2 sequence are not associated with the modulation of apoptosis

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNVcauses great economic loss and is one of themost frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV

Infectious pancreatic necrosis virus (IPNV) enumeration through epifluorescence microscopy: technical aspects

A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody

Chilean IPNV isolates: Robustness analysis of PCR detection

Background: The genomes of several infectious pancreatic necrosis viruses (IPNVs) isolated in Chile were sequenced with a single amplification approach for both segments A and B. The resulting sequences were then used to determine the conservation of the primer-binding regions used in polymerase chain reaction (PCR)-based diagnostic methods proposed in the literature. Thus, the robustness of each technique was studied, particularly the eventual effect of further mutations within the primer-binding sites. Results: On analysis, most methods currently used to detect Chilean IPNV varieties were deemed adequate. However, the primers were designed to be genogroup specific, implying that most detection methods pose some risk of detecting all strains prevalent in the country, due to the coexistence of genogroups 1 and 5. Conclusions: Negative resultsmust be interpreted carefully given the high genomic variability of IPNVs. Detection techniques (quantitative reverse transcription (qRT)-PCR) based on degenerate primers can be used to minimize the possibilities of false-negative detections

Virus de la peste porcina africana proteínas estructurales y enzimas asociadas al viron

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Virología y Genética Molecular, Fecha de lectura:10-07-197

Examen rápido de microorganismos en aguas de lastre

Se examinaron las comunidades microbianas del agua de lastre mediante microscopía de epifluorescencia. Se realizaron análisis cualitativos y cuantitativos con el fin de probar si los perfiles microbianos encontrados en diferentes barcos estaban relacionados con sus itinerarios de viaje y/o el manejo de sus tanques de agua de lastre. Las concentraciones de VLPs (¿virus-like particles¿) y bacterias presentaron diferencias evidentes en barcos que arribaron a un mismo puerto. Estas diferencias son coherentes con los respectivos itinerarios de viaje. Además, la extensión del tiempo de confinamiento del agua de lastre en un tanque determina que algunas formas bacterianas predominen progresivamente sobre otras. Lo anterior permitiría, en principio, determinar si el agua de lastre ha sido renovada en mar abierto antes de arribar a puerto. Los resultados presentados se restringen al examen del agua de lastre de unos pocos barcos. Sin embargo, pensamos que una mayor investigación en esta área determinará que la microscopía de epifluorescencia será cada vez más útil para caracterizar microbiológicamente las aguas de lastre, contribuyendo así a develar su historia reciente

Examen rápido de microorganismos en aguas de lastre

Se examinaron las comunidades microbianas del agua de lastre mediante microscopía de epifluorescencia. Se realizaron análisis cualitativos y cuantitativos con el fin de probar si los perfiles microbianos encontrados en diferentes barcos estaban relacionados con sus itinerarios de viaje y/o el manejo de sus tanques de agua de lastre. Las concentraciones de VLPs (¿virus-like particles¿) y bacterias presentaron diferencias evidentes en barcos que arribaron a un mismo puerto. Estas diferencias son coherentes con los respectivos itinerarios de viaje. Además, la extensión del tiempo de confinamiento del agua de lastre en un tanque determina que algunas formas bacterianas predominen progresivamente sobre otras. Lo anterior permitiría, en principio, determinar si el agua de lastre ha sido renovada en mar abierto antes de arribar a puerto. Los resultados presentados se restringen al examen del agua de lastre de unos pocos barcos. Sin embargo, pensamos que una mayor investigación en esta área determinará que la microscopía de epifluorescencia será cada vez más útil para caracterizar microbiológicamente las aguas de lastre, contribuyendo así a develar su historia reciente

Polyadenylation, methylation, and capping of the RNA synthesized in vitro by African swine fever virus

African swine fever (ASF) virus synthesizes in vitro four classes of capped and polyadenylated RNAs with sedimentation coefficients of about 16,12, 10, and 7 S, free poly(A) with a sedimentation coefficient of about 4 S and a material, smaller than 4 S, labeled with S-adenosyl[methyl-3H]methionine and partially polyadenylated. The average chain length of free and RNA-bound poly(A) is about 14 and 33 nucleotides, respectively. The following CAP structures were present in the RNA: m7GpppAm (79%), m7GpppGm (13%), GpppAm (5.8%), and GpppGm (2.3%). Neither CAP 2 structure nor internal methylations were detected in the RNA.This work was supported by grants from Comité Conjunto Hispano-Norteamericano para la Cooperacidn Cientifica y T&mica and Comision Administradora de1 Dezcuento Complementario