Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates.

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.

tRNA genes were found in Piscirickettsia salmonis 16S-23S rDNA spacer region (ITS).

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.

In vivo effect of EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide) on experimental infected rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) fry with infectious pancreatic necrosis virus.

The in vivo antiviral effect of 5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide (EICAR) was evaluated in coho salmon and rainbow trout fry, experimentally infected with infectious pancreatic necrosis virus (IPNV). Treatment consisted of a daily bath of 2 h in 0.4 microg ml(-1) or 0.8 microg ml(-1) of EICAR, for approximately 20 days. The behavior of the fish was studied for 45 days post-infection. The survival of the infected treated groups was compared with the survival of non-infected and infected untreated control groups. The results showed that the survival of coho salmon and rainbow trout fry in the infected group treated with both doses of EICAR was similar to the survival observed in the healthy control group (approximately 94%). While, the survival of the infected and untreated control fish was 56% for salmon and 28% for trout, there were no significant difference in the weight of coho salmon fry between those treated with EICAR and non-infected and infected untreated control groups. However, in rainbow trout there was a statistically significant weight decrease in infected untreated group. Finally, the analysis of tissue samples of the fish by reverse transcription associated with the polymerase chain reaction (RT-PCR) suggest that EICAR have decreased the viral load in infected treated fry. Consequently, the results indicate that EICAR is an effective inhibitor of IPNV replication in vivo and could be a promissory antiviral compound for the treatment of IPNV disease.

Inhibitory effects of EICAR on infectious pancreatic necrosis virus replication.

Recently, the antiviral 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) was shown to inhibit the replication of the infectious pancreatic necrosis virus (IPNV). In order to obtain more information about the mechanism of the antiviral action of EICAR we studied its effect on viral macromolecules synthesis. EICAR was found to inhibit IPNV messenger and genomic RNA synthesis. To inhibit viral RNA synthesis, EICAR must be added at least 3 h before the start of RNA synthesis. This suggests that EICAR does not directly affect the viral RNA polymerization process. Moreover, the antiviral action of EICAR was reversed by the exogenous addition of guanosine (5-50 microg/ml), but not adenosine or cytidine (10-100 microg/ml). Our findings suggest that the antiviral action of EICAR is mediated by a reduction of the intracellular guanosine 5'-triphosphate (GTP) pool level, as has been observed with ribavirin and EICAR in other biological systems.

Detection of the infectious hematopoietic necrosis virus directly from infected fish tissues by dot blot hybridization with a non-radioactive probe.

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.

Inhibitors of infectious pancreatic necrosis virus (IPNV) replication.

In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-beta-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC50) were 0.01 micrograms/ml and 0.5 micrograms/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC50) were 50 micrograms/ml and 100 micrograms/ml, respectively, and the concentrations that reduced [methyl-3H] thymidine incorporation by 50% (IC50) were 0.5-1 and 50 micrograms/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50-100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.

[Evaluation of an immunoenzyme technique (ELISA) to diagnose typhoid fever]. / Evaluación de un ensayo inmunoenzimático (ELISA) para el diagnóstico de la fiebre tifoídea.

The efficiency of an ELISA method, designed to detect polyvalent IgG and IgM antibodies to Salmonella typhi polysaccharide was evaluated in patients admitted or convalescing from typhoid fever and in control subjects. Polyvalent antibodies to S typhi were demonstrated in 28/30 (93%) typhoid patients, 0/11 bacteremic patients (E coli or S paratyphi A) and 0/15 asymptomatic individuals. Widal test showed significant anti-0 agglutinin values (> = 1: 160) in only 12/30 (40%), 1/11 (9%) and 0/15 subjects from each group respectively. Typhoid patients tested on admission or at discharge showed similar high reactivity rates to ELISA. On the contrary, the Widal test detected only 2/15 (13%) patients on admission (p < 0.02) and 10/15 (67%) at discharge. These results and additional immunoblotting tests suggest that ELISA developed to detect polyvalent anti-LPS antibodies could represent a highly specific diagnostic tool to confirm typhoid fever in endemic areas.

[The IgG antibody response in patients colonized by Helicobacter pylori]. / Respuesta de anticuerpos IgG en pacientes colonizados por Helicobacter pylori.

The IgG antibody response specific to Helicobacter pylori was evaluated through ELISA in a group of 92 gastric patients colonized by this bacteria. 74 had gastritis and 19 gastroduodenal ulcer. Three control groups were studied in a similar way: normal adult volunteers (n = 17), adults with E coli or S typhi bacteremia (n = 30) and normal infants (n = 30). IgG antibody response to H pylori was demonstrated in 98% of colonized patients and 0% of infants. Asymptomatic individuals and those with bacteremia had high rates of antibody response (76 and 90% respectively), although this rate and also the titers of antibody response were significantly lower than that of colonized patients (p < 0.05). ELISA reactive sera from colonized patients and asymptomatic individuals evidenced a similar antibody pattern when tested by blotting. This profile was absent in non reactive sera, including those with high antibody titers to C jejuni. The presence of specific IgG antibodies to H pylori in the majority of colonized gastric patients and asymptomatic adults suggests that this infection is very common in our population.

Respuesta de anticuerpos IgG en pacientes colonizados por helicobacter pylori / Antibodies to helicobacter pylori in colonized patients with gastric symptoms and in asymptomatic groups

The IgG antibody response specific to Helicobacter pylori was evaluated through ELISA in a group of 92 gastric patients colonized by this bacteria. 74 had gastritis and 19 gastroduodenal ulcer. Three control groups were studied in a similary way: normal adult volunteers (n=17), adults with E coli or S typhi bacteremia (n=30) and normal infants (n = 30). IgG antibody response to H pylori was demonstrated in 98% of colonized patients and 0% of infants. Asymptomatic individuals and those with bacteremia had high rates of antibody response (76 and 90% respectively), although this rate and also the titers of antibody response were significantly lower than that of colonized patients (p < 0.05). ELISA reactive sera from colonized patients and asymptomatic individuals evidenced a similar antibody pattern when tested by blotting. This profile was absent in non reactive sera, including those with high antibody titers to C jejuni. The presence of specific IgG antibodies to H pylory in the majority of colonized gastric patients and asymptomatic adults suggest that this infection is very common in our population

Role of the inner protein capsid on in vitro human rotavirus transcription.

The inner protein shell of human rotavirus consists of a single polypeptide called VP6 which was removed from the single-shelled virus by treatment with CaCl2, leaving the viral core. The core thus obtained was unable to transcribe. However, the addition of a supernatant containing VP6 in the absence of Ca2+ restored the transcriptional activity. VP6 obtained from different electropherotypes and serotypes was able to restore transcriptional activity to homologous and heterologous cores. Viral cores obtained after incubation with purified VP6 had electron microscopic characteristics, polypeptide compositions, and transcription products similar to those of the single-shelled virus. The results suggested the successful in vitro reconstitution of the single-shelled virus.

In vitro transcription of human pararotavirus.

Purified human pararotavirus obtained from stool samples from a 6-month-old infant was characterized. Electron microscopy of the viral particles subjected to different treatments suggested that the protein shells differed from those described for rotavirus. Treatment with both EDTA or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the presence or absence of Mg2+ seemed to convert the virions into core particles by removal of both the outer and inner shells, and no particles equivalent to single-shelled rotavirus were observed. Different procedures were used to activate the human pararotavirus-associated RNA-dependent RNA polymerase. The enzyme was not activated by chelating agents or by thermal shock as in rotavirus. Activation by thermal shock occurred only in the presence of the four ribonucleoside triphosphates and Mg2+. However, the polymerase of pararotavirus was found to be similar to those described for rotaviruses. When in vitro transcripts were analyzed, 11 RNA species having a migration pattern similar to that of the original genomic RNA were detected.