Quantitative method of viral pollution determination for large volume of water using ferric hydroxide gel impregnated on the surface of glassfibre cartridge

Quantitative method of viral pollution determination for large volume of water using ferric hydroxide gel impregnated on the surface of glassfibre cartridge filter. The use of ferric hydroxide gel, impregnated on the surface of glassfibre cartridge filter enable us to recover 62.5% of virus (Poliomylitis type I, Lsc strain) exsogeneously added to 400 liters of tap-water. The virus concentrator system consists of four cartridge filters, in which the three first one are clarifiers, where the contaminants are removed physically, without significant virus loss at this stage. The last cartridge filter is impregnated with ferric hydroxide gel, where the virus is adsorbed. After the required volume of water has been processed, the last filter is removed from the system and the viruses are recovered from the gel, using 1 liter of glycine/NaOH buffer, at pH 11. Immediately the eluate is clarified through series of cellulose acetate membranes mounted in a 142mm Millipore filter. For the second step of virus concentration, HC1 1N is added slowly to the eluate to achieve pH 3.5-4. MgC1, is added to give a final concentration of 0.05M and the viruses are readsorbed on a 0.45 , porosity (HA) cellulose acetate membrane, mounted in a 90 mm Millipore filter. The viruses are recovered using the same eluent plus 10% of fetal calf serum, to a final volume of 3 ml. In this way, it was possible to concentrate virus from 400 liters of tap-water, into 1 liter in the first stage of virus concentration and just to 3 ml of final volume in a second step. The efficiency, simplicity and low operational cost, provded by the method, make it feasible to study viral pollution of recreational and tap-water sources.

The viral nature of Toddia França, 1912

Toddia França, 1912 under the light microscope occurs as inclusion corpuscles in the cytoplasm of erythrocytes of cold-blooded vertebrates sometimes accompanied by crystalloid bodies. Its position among the protozoans or the viruses has been discussed by some authors, but remained unclear. To elucidate this problem we studied Toddia from a Brazilian frog (Leptodactylus ocellatus) by electron microscopy. In the cytoplasm of the infected cells we found no protozoan, but rather virus-like particles often hexagonal in outline, averaging 195 nm excluding their two involving membranes, and presenting a central area of variable electron density. Particles at different stages of development were generally found around or on area lighter density than the cytoplasm. which resembled a virus synthesis site. At high magnification, the nuclear or cytoplasmic crystals allied to Toddia resembled the crystalline lattice of the inclusion bodies associated with the polyhedrosis viruses and poxviruses from insects, of the capsules of granulosis viruses and of other protein crystals in ultrathin sections. Cytochemical tests in Toddia corpuscles displayed exclusively the presence of deoxyribonucleic acid. These findings indicate that Toddia is not a protozoan and demonstrate that it is in all probability a viral inclusion corpuscle. Taking into account the nucleic acid type found in its structure (DNA) and the hexagonal shape usually shown in ultrathin sections by its component particles, which have a cytoplasmic site of synthesis and assembly, we tentatively relate Toddia with the so-called "Icosahedral Cytoplasmic Deoxyriboviruses". We believe that the present paper gives the first report of virus-like particles in L. ocellatus.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.

Extrachromosomal nucleic acids in bovine babesia

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.

An outbreak of diarrhoea associated with rotavirus serotype 1 in a day care nursery in Rio de Janeiro, Brazil

Faeces from 17 children less than 1.6 years old 15 adultsmore than 22 years old were collected during an outbreak of gastroenteritis in aday care nursery and screened for the presence of adenovirus and rotavirus by enzyme immunoassay (EIARA) and other viruses by electron microscopy (EM) and polycrylamide gel electrophoresis (PAGE). Ten samples (58.8 per cent) from childrenand one (6.7 per cent) from adults were positive for rotavirus and all samples were negative for bacteria and parasites. No other viruses were observed in EM. An enzyme immunoassay test using monoclonal antibodies (MAb-EIA) to determine the subgroup(s) and the serotype(s) of rotavirus was performed and the results showedthat all positive samples belong to serotype 1, subgroup II of group A rotaviruses. In PAGE test all samples had the same profile and the 10 and 11 dsRNA segments corresponed to the "long" profile of group A of rotaviruses. These results corroborated the MAbEIA results and indicate a sole source of infection. The majorsymptoms observed were: vomiting (60 per cent), fever (70 per cent) and diarrhoea (100 per cent). In previous years (1989 to 1991) we observed only rotavirus serotype 2 in this same day care nursery, but no outbreak was reported.

Development of a vaccine strategy against human and bovine schistosomiasis: background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output. The use of appropriate monoclonal antibody probes has allowed the demonstration that the inhibition of parasite fecundity following immunization was related to the inhibition of enzymatic activity of the molecule. Epitope mapping of Sm28 GST has indicated the prominent role of the N and C terminal domains. Immunization with the corresponding synthetic peptides was followed by a decrease of 70% of parasite fecundity and egg viability. As a preliminary step towards phase I human trials, vaccination experiments have been performed in cattle, a natural model for Schistosoma bovis. Vaccination of calves with the S. bovis GST has led to a reduction of ever 80% of egg output and tissue egg count. Significant levels of protection were also observed in goats after immunization with the recombinant S. bovis GST. Increasing evidence of the participation of IgA antibodies in protective immunity has prompted us toward the development of mucosal immunization. Preliminary results indicate that significant levels of protection can be achieved following oral immunization with live attenuated vectors or liposomes. These studies seem to represent a promising approach towards the future development of a vaccine strategy against one of major human parasitic diseases.

Rotaviruses as a cause of nosocomial, infantile diarrhoea in Northern Brazil: pilot study

Faecal samples were obtained from 190 children, aged 0 to 5 years, admitted to a public hospital in Belém, Pará, Brazil. These patients were placed in a pediatric ward with 40 beds distributed in six rooms. Case were classified into three groups: (a) nosocomial: children who developed gastroenteritis 72 hr or later after admission; (b) community-acquired: patients admitted either with diarrhoea or who had diarrhoea within 72 hr following admission; (c) non-diarrhoeic: those children who had no diarrhoea three days before and three days after collection of formed faecal sample. Specimens were routinely processed for the presence of rotaviruses, bacteria and parasites. Rotaviruses were detected through enzyme-linked immunosorbent assay (ELISA) and subsequently serotyped/electrophoretyped. Rotaviruses were the most prevalent enteropathogens among nosocomial cases, accounting for 39 % (9/23) of diarrhoeal episodes; on the other hand, rotaviruses ocurred in 8.3 % (11/133) and 9 % (3/34) of community-acquired and non-diarrhoeic categories, respectively. Mixed infections involving rotavirus and Giardia intestinalis and rotavirus plus G. intestinalis and Entamoeba histolytica were detected in frequencies of 8.6 and 4.3 %, respectively, in the nosocomial group. The absence of bacterial pathogens in this category, and the unusual low prevalence of these agents in the other two groups may reflect the early and routine administration of antibiotics following admission to this hospital. Rotavirus serotype 2 prevailed over the other types, accounting for 77.8 % of isolates from nosocomial diarrhoeal episodes. In addition, at least five different genomic profiles could be observed, of which one displayed an unusual five-segment first RNA cluster. Dehydration was recorded in all cases of hospital-acquired, rotavirus-associated diarrhoea, whereas in only 57 % of nosocomial cases of other aetiology. It was also noted that nosocomial, rotavirus-associated diarrhoeal episodes occur earlier (7 days), following admission, if compared with those hospital-acquired cases of other aetiology (14 days).

Quantitation of HIV-1 RNA Viral Load Using NASBA Methodology and Comparison with other Surrogate Markers for Disease Progression

In this study, HIV-1 viral load quantitation determined by Nucleic Acid Sequence Based Amplification (NASBA) was compared with other surrogate disease progression markers (antigen p24, CD4/CD8 cell counts and b-2 microglobulin) in 540 patients followed up at São Paulo, SP, Brazil. HIV-1 RNA detection was statistically associated with the presence of antigen p24, but the viral RNA was also detected in 68% of the antigen p24 negative samples, confirming that NASBA is much more sensitive than the determination of antigen p24. Regarding other surrogate markers, no statistically significant association with the detection of viral RNA was found. The reproducibility of this viral load assay was assessed by 14 runs of the same sample, using different reagents batches. Viral load values in this sample ranged from 5.83 to 6.27 log (CV = 36 %), less than the range (0.5 log) established to the determination of significant viral load changes.

Adenosine Deaminase and Guanosine Deaminase Activities in Sera of Patients with Viral Hepatitis

In order to investigate purin and primidin metabolism pathways in hepatitis, adenosine deaminase (ADA) and guanosine deaminase (GDA) activities in sera of patients with different types and manifestations of viral hepatitis disease (A, B, C, D, E, chronic, acute) were investigated and compared with the control group of healthy individuals. Hepatitis cases were classified with respect to their serological findings and clinics. When compared all the hepatitis cases with the controls, levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase enzymes, as well as ADA and GDA, were significantly higher than the control group (p<0.01). Levels of ADA and GDA in hepatitis cases were determined as 26.07±11.98 IU/l and 2.37±1.91 IU/l, respectively. When compared their ADA and GDA levels amongst the classified hepatitis groups, there was no difference in ADA levels amongst cases (p>0.05). However, GDA levels in hepatitis A group were closed to the controls. Increase in serum ADA activities in hepatitis forms may be dependent on and reflect the increase in phagocytic activity of macrophages and maturation of T-lymphocytes, and may be valuable in monitoring in viral hepatitis cases.

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Rabies in tamarins (Callithrix jacchus) in the State of Ceará, Brazil, a distinct viral variant?

Presently, the State of Ceará reports the largest percentage of human rabies cases originated from wild animals in Brazil, transmitted by the principal simian species, the tamarin (Callithrix jacchus), found in various locations throughout the State, but concentrated along the coast. Epidemiological studies indicated that possibly the same virus caused the deaths in humans and non-human primates. This rabies virus seem to be different from all other identified so far.