Detection and characterization of waterborne gastroenteritis viruses in urban sewage and sewage-polluted river waters in Caracas, Venezuela.

The detection and molecular characterization of pathogenic human viruses in urban sewage have been used extensively to derive information on circulating viruses in given populations throughout the world. In this study, a similar approach was applied to provide an overview of the epidemiology of waterborne gastroenteritis viruses circulating in urban areas of Caracas, the capital city of Venezuela in South America. Dry season sampling was conducted in sewers and in a major river severely polluted with urban sewage discharges. Nested PCR was used for detection of human adenoviruses (HAds), while reverse transcription plus nested or seminested PCR was used for detection of enteroviruses (HuEVs), rotaviruses (HRVs), noroviruses (HuNoVs), and astroviruses (HAstVs). HRVs were fully characterized with genotype-specific primers for VP4 (genotype P), VP7 (genotype G), and the rotavirus nonstructural protein 4 (NSP4). HuNoVs and HAstVs were characterized by sequencing and phylogenetic analysis. The detection rates of all viruses were >or=50%, and all sampling events were positive for at least one of the pathogenic viruses studied. The predominant HRV types found were G1, P[8], P[4], and NSP4A and -B. Genogroup II of HuNoVs and HAstV type 8 were frequently detected in sewage and sewage-polluted river waters. This study reveals relevant epidemiological data on the distribution and persistence of human pathogenic viruses in sewage-polluted waters and addresses the potential health risks associated with transmission of these viruses through water-related environmental routes.

Construction and biological properties of yellow fever 17D/dengue type 1 recombinant virus.

Dengue virus, a mosquito-borne flavivirus, is one of the most formidable public health threats in tropical and subtropical regions. As yet, there is no licensed vaccine to protect against the disease. A chimeric yellow fever (YF) 17D/dengue (DEN) type 1 virus was constructed by replacing the pre-membrane and envelope genes of YF 17D virus with those from DEN 1 VeMir95 virus, a Venezuelan isolate. The chimeric YF 17D/DEN 1 VeMir95 virus was regenerated from full-length infectious clones stably propagated in Escherichia coli by transfection of Vero cells with in vitro transcribed RNA. The chimeric virus proliferated efficiently in Vero cells ( approximately 6.6 log(10) plaque-forming units/ml). The chimeric virus was not neurovirulent to 3-week-old Swiss Webster mice inoculated by the intracerebral route, in contrast to the YF 17DD vaccine strain that was lethal for 90% of the mice. The YF 17D/DEN 1 virus at Passage 6 was more attenuated for rhesus monkeys than the YF 17DD commercial vaccine after intracerebral inoculation according to the standard neurovirulence test. This virus is a potential candidate to be included in a tetravalent DEN vaccine formulation. The availability of the cloned cDNA allows further structure/function studies on the viral envelope.

Evaluation of a commercial enzyme immunoassay for the detection of norovirus antigen in fecal samples from children with sporadic acute gastroenteritis.

The Ridascreen Norwalk-like virus enzyme immunoassay was compared with (RT)-PCR on 92 stool samples collected from children with sporadic acute gastroenteritis. Homogenization and pre-dilution of the whole stool sample resulted in high specificity (97.5%) and moderate sensitivity (60%). This assay may be useful to screen outbreaks for norovirus, but limited to detect the virus in sporadic cases of diarrhea.

Calicivirus infection in human immunodeficiency virus seropositive children and adults.

BACKGROUND: The importance of enteric viral infections in HIV-related diarrhea is uncertain. Human caliciviruses have emerged as a leading cause of acute diarrhea worldwide. OBJECTIVES: To evaluate the importance of calicivirus infections in HIV-related diarrhea. Study design 151 fecal samples collected from children and adults infected with HIV, with and without diarrhea, were examined. In addition, 89 fecal samples from non HIV-infected children and adults were also tested. Samples were analyzed by RT-PCR using primer sets specific to Norovirus genogroup I or genogroup II as well as primers designed to react with both Noroviruses and Sapovirus genus. RESULTS: Viruses were detected with equal frequencies in stools from HIV infected and non-infected adults (12%). However, specimens from HIV infected children were more likely than those of HIV-negative children to have caliciviruses (51% versus 24%, P<0.05). Viral infections were not significantly associated with diarrhea neither in children nor in adults, regardless of HIV status. Viruses genetically related to the common Lordsdale virus (Norovirus genogroup II) and London/92 virus (Sapovirus) clusters were detected circulating among children. CONCLUSIONS: These results suggest that caliciviruses may be an important opportunistic pathogen in children infected with HIV.

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni.

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Characterization of the antibody reactivity to synthetic peptides from different parts of the hepatitis C virus genome.

Infection by hepatitis C virus (HCV)*, the aetiologic agent responsible for the majority of non-A-non-B posttransfusion hepatitis, is detected by assaying for antibodies against structural and nonstructural recombinant proteins or synthetic peptides. The aim of this study was to characterize the antibody reactivity of selected sera against antigenic peptides spanning immunodominant regions of the core, NS4 and NS5 HCV proteins. Reactivity to synthetic peptides was determined by enzyme immunoassay (EIA) for 11 selected sera from blood donors (good responders), for 27 selected sera from hemodialysis patients (poor responders), all positive for HCV antibodies (tested by different second and third-generation assays), and for 7 negative sera. Some peptides from the core and the NS4 region were widely recognized by the tested sera. Sera not reactive with core, NS4, or NS5 region by some immunoblot assays exhibited reactivity against peptides from these proteins. Autoimmune reactivity associated with HCV infection was evaluated by using a synthetic peptide derived from the GOR peptide; 8/11 HCV-positive sera were found reactive against this peptide. No correlation was found between reactivity to any of the peptides tested and the presence of HCV RNA in the serum or with HCV genotype. The EIA reactivity of peptides from the core region suggested a multideterminant antigenic structure, where reactivity of each epitope may be differentially affected by neighboring amino acids depending on individual sera. This situation was particularly evidenced in selected sera from poor responder specimens where a more restricted antibody response to core peptides was observed. Reactivity of sera from HCV-infected patients with synthetic peptides from the core, NS4, and NS5 regions indicated the presence of multiple linear epitopes (particularly in the core region) that may be used in a mixture for immunodiagnosis; however, the length and exact position of the synthetic peptides must be chosen carefully.

Preliminary characterization of Escherichia coli isolated from pigs with diarrhoea in Venezuela.

The aetiology of neonatal porcine diarrhoea was studied in 15 different herds located in the north-western region of Venezuela. Of 56 strains of Escherichia coli analyzed, 16 (28.6%) were shown to produce heat-stable (STa) enterotoxin, as detected by infant mouse assay. Only four of these STa+ isolates also possessed the K88 pilus antigen, two were 987P+ and none possessed the K99 antigen, leaving 10 STa+ samples in which no pilus antigen was identified. Among the 40 STa negative samples were six K88+ specimens, one K99+, four 987P+, one which reacted as K88+ + K99+ and one K88+ + 987P+. Considering as pathogenic any strain showing at least one of the characters studied, pathogenic E. coli were detected with an overall frequency of 42.9%, being more prevalent during the second week of life. An electrophoretic analysis of the plasmid content of the field isolates of E. coli, revealed the presence of numerous species of extrachromosomal DNA, although no direction association could be made between a particular plasmid and any of the pathogenic characteristics identified. Results of Southern blot analysis indicate that the STa enterotoxin was preferentially encoded within an endemic plasmid of 4.9 Md. Other plasmids present in the E. coli isolates could be related to antibiotic resistance. With the exception of one strain, all E. coli isolates were resistant to more than one of the nine drugs tested; multiresistant E. coli were frequently isolated, including four strains which were resistant to seven antibiotics.

Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela.

The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983-February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4-6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.

Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.

A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions. Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal. Staining with EB and fluorescein diacetate was mutually exclusive. The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations. Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B. The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.

Identification of picobirnavirus, viruses with bisegmented double stranded RNA, in rabbit faeces.

Picobirnaviruses are a novel group of viruses recently found in the faeces of several species of vertebrates. Examination by polyacrylamide gel electrophoresis of rabbit faecal samples collected in one animal facility revealed the viruses in 23 (11 per cent) of 211 samples. Further analysis by electron microscopy and caesium chloride isopycnic centrifugation confirmed the presence of picobirnaviruses in the samples. The oral inoculation of three newly weaned rabbits with purified viruses resulted in the excretion of a virus with an electropherotype similar to the inoculum, by two of the three inoculated animals. Maximal viral shedding was detected 13 days after inoculation. No sign of diarrhoea was observed either in the inoculated animals or in the virus excreting animals surveyed. No antibody activity could be detected in the paired serum samples taken from the inoculated animals.

[Development of an enzyme assay for detection of hepatitis B virus core IGM antibodies]. / Desarrollo de un ensayo enzimático para la determinación de anticuerpos IgM anti-cápside del virus de la Hepatitis B.

Detection of IgM anti-core (anti-HBcAg) antibodies of Hepatitis B Virus (HBV) is an useful marker for hepatitis B virus (HBV) acute infection. The aim of this study was to perform an immunodiagnostic assay for the detection of IgM anti-HBcAg antibodies. Hepatitis B core antigen (HBcAg) was produced by a recombinant clone of Escherichia coli and used for the development of the immunoassay. An IgM capture enzyme immunoassay (EIA) was selected for the detection of IgM anti-HBcAg antibodies. A total of 110 human plasma or sera were tested by the capture EIA and a commercial assay. The capture EIA yielded 99% of sensitivity and 93% specificity, when compared with the commercial test. The capture EIA developed here is of interest for epidemiological studies, particularly for endemic regions in South America.

Infection with transfusion-transmitted virus (TTV) in humans and other primates in Venezuela.

Tranfusion-transmitted virus (TTV), a single-stranded circular DNA virus that chronically infects humans and other animals, displays a high degree of genetic diversity and was originally thought to be associated with hepatitis. The prevalences of TTV infection among different populations of humans and non-human primates from Venezuela have now been evaluated, using serum samples and three different detection tests. All three tests were PCR-based, one involving a hemi-nested PCR and primers based on the N22 open-reading-frame-1 region (N22-PCR), another employing 55 cycles with primers from the more conserved untranslated region (UTR-PCR), and the other using a hemi-nested PCR with primers from the same region (HUTR-PCR). The overall prevalences of human infection appeared much higher with the HUTR-PCR (52%) than with the N22-PCR (13%) or the UTR-PCR (5%). When the products amplified by N22-PCR from 28 human isolates of TTV were sequenced, only two genotypes of the virus were detected. The non-human sera tested came from primates kept in a zoo in north-western Venezuela. TTV DNA was detected, by HUTR-PCR, in both of the chimpanzee sera tested but not in any of the sera from the 11 New-World primates or the other 12 Old-World primates that were investigated. The results, particularly those of the HUTR-PCR, indicate that TTV infection is common in Venezuela, especially in populations, such as many Amerindian groups, who live under poor sanitary conditions. Although TTV infection may be relatively rare among non-human primates from the New World, this will have to be investigated further, using many more samples collected throughout the Americas.

Isolation of plaque variants differing in virulence from the 17D strain of yellow fever virus.

Two preparations of yellow fever vaccine (17D) were studied by clonal analysis. From one (17D-England) three types of clones were isolated, each differentiated by plaque size in Vero cells and virulence for intracerebrally inoculated mice: small plaque (SP) and large plaque (LP) clones were lethal, whereas medium plaque (MP) clones failed to kill at doses up to 10(6) p.f.u. Analysis of 24 randomly selected clones showed the original vaccine to be a mixture of predominantly MP and SP variants; 58% and 42% respectively. A single passage in suckling mice modified this composition to 90% SP and 10% LP variants. The response of six inbred strains of mice to intracerebral inoculation of the MP variant varied from complete resistance to complete susceptibility. From another 17D substrain (17D-South Africa) two types of variants were isolated (a SP avirulent type and a LP type of intermediate virulence), both different from the previously described variant.

Serological and genomic characterization of two porcine rotaviruses with serotype G1 specificity.

Two porcine rotavirus strains, C60 and C95, which had been previously shown to be reactive in an enzyme-linked immunosorbent assay with serotype G1-specific monoclonal antibodies, were classified as G1 by cross-neutralization tests and on the basis of the homology of the sequenced VP7 gene. This report confirms that porcine rotavirus strains with a G1 serotype occur in nature.

Replication of virulent and attenuated strains of yellow fever virus in human monocytes and macrophage-like cells (U937).

Virulent and attenuated strains of yellow fever virus were compared for their ability to grow in cultures of unstimulated leucocytes and monocytes derived from human peripheral blood, and of a macrophage-like cell line of human origin, U937. The extent of virus growth in leucocyte cultures varied depending on the strain of virus, multiplicity of infection, presence of diluted antibody in the culture medium but independently of the flavivirus immune status of the donor. The same pattern of differential growth was observed in the three types of cultures used. Although strain related variation in growth occurred within both virulent and attenuated strains, most of the attenuated strains produced higher virus yields than the virulent ones, suggesting that replication in this cell system is not related to the expression of virulence for the host. Replication in human monocytes as an in vitro marker of immunogenity for substrains of 17 D vaccine virus is discussed.

Kinetics of heat inactivation of Venezuelan equine encephalomyelitis virus.

Thermal inactivation of Venezuelan Equine Encephalomyelitis Virus (VEEV) was studied at temperatures from 26 degrees to 55 degrees C. Inactivation of infectivity took place by two thermodynamically different reactions, one of which predominated at temperatures below 44 degrees C and the other at higher temperatures. The presence of 1 or 2 M NaCl stabilized the VEE virus at low temperatures but enhanced the inactivation at high temperatures. This latter effect at temperatures higher than 50 degrees C, is associated with the occurrence of two-component survival curves. The different effects of hypertonic NaCl concentrations at the two ranges of temperature, are related to different mechanisms of inactivation operating at each range (protein denaturation and nucleic acid-RNA breakdown). Different kinetics of thermal inactivation at 55 degrees C were observed between virus strains with different virulence. However, no significant correlations was found between the virulence of the eleven VEE virus strains studied and their thermostability at 37 degres and 55 degrees C.

Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.

Replication of alphaviruses in cultures of donkey monocytes.

Representative strains of Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) were compared for their ability to grow in cultures of unstimulated leucocytes and monocytes derived from donkey peripheral blood. Replication of epizootic and vaccine strains of VEEV, but not of enzootic strains was observed in this system. Only a minority of monocytes supported virus replication as detected by immunofluorescence, electron microscopy and infectious center assays. EEEV did not appear to replicate in this cell system although virus attached to and was internalized by monocytes.

Identification of viruses with bi- and trisegmented double-stranded RNA genome in faeces of children with gastroenteritis.

Polyacrylamide gel electrophoresis of nucleic acid extracted from faecal samples of diarrhoeic children revealed the presence of group A rotavirus in 50 (23.4%) samples and group C rotavirus in 1 (0.5%) sample out of 214 tested. One other sample showed the presence of three bands (with apparent length of 2.92, 2.37 and 1.32 kbp) which by enzymatic digestion analysis, were shown to consist of double-stranded RNA (dsRNA). The sample was shown by electron microscopy to contain virus particles with a diameter of 32-34 nm. On the basis of morphology and genomic characteristics, this virus closely resembles a virus hitherto described only in chickens by Leite et al. in 1990 and tentatively named "picotrirnavirus". From the same group of 214, one sample containing a "picobirnavirus" was also identified. Thus, small icosahedral viruses with either a bior trisegmented dsRNA genome appear to infect humans. However, their pathogenic potential remains to be established.

Cross-reactive, serotype- and monotype-specific neutralization epitopes on VP7 of serotype G3 and G5 porcine rotavirus strains.

VP7 specific monoclonal antibodies raised against serotype G5 porcine rotavirus strains isolated in Venezuela showed either a serotype G5- or monotype-specific pattern of reactivity by neutralization against a panel of 53 group A rotavirus isolates representative of all established G serotypes. Monoclonal antibodies raised against two G3 porcine strains were either specific for a subset of porcine G3 strains or reactive with another subset of porcine G3 strains and with most G5 strains. Neither were reactive with G3 strains from other species. Analysis of neutralization resistant mutants selected with these monoclonal antibodies indicated that epitopes defined by cross-reactive, serotype- and monotype-specific monoclonal antibodies overlap functionally and that binding and neutralization by these antibodies depended on specific amino acid residues in the region A or C of VP7. Results indicate that a high degree of monotypic variation occurs among G5 and G3 porcine rotavirus strains and the existence of at least one common epitope shared by G5 and G3 porcine strains, in the major neutralization domain of these VP7s.