Hepatitis E virus infection in Latin America: a review.

Data reported during recent years reveal the complex picture of the epidemiology of hepatitis E virus (HEV) infection in Latin America. Whereas in countries like Argentina and Brazil is almost identical to the characteristic of most countries from North America and Europe, HEV in the Caribbean and Mexico involves the water-borne, non-zoonotic viral genotypes responsible for epidemics in Asia and Africa. Nevertheless, Latin America has been considered a highly endemic region for hepatitis E in the scientific literature, a generalization that ignores the above complexity. In addition, reports from isolated Amerindian communities, which display well known, important and very specific epidemiological features for hepatitis B and D virus infections are neither taken into account when considering the epidemiology of hepatitis E in the region. This review updates compilation of the available information for the HEV infection, both among humans and other mammals, in Latin America, discusses the strengths and the weaknesses of our current knowledge, and identifies future areas of research.

Molecular characterization of sewage-borne pathogens and detection of sewage markers in an urban stream in Caracas, Venezuela.

Molecular characterization of two sewage-borne pathogens identified hepatitis A virus (HAV) subgenotype IA and Giardia duodenalis assemblages A and B as predominant genotypes circulating in an urban area of Venezuela. This study reveals epidemiological features of human pathogens of worldwide distribution and the efficacy of molecular methods for accurate assessment of sewage pollution.

Hepatitis A virus genetic diversity in Venezuela: exclusive circulation of subgenotype IA and evidence of quasispecies distribution in the isolates.

Hepatitis A virus (HAV) infection is highly prevalent in Latin America, including Venezuela. Subgenotype IA seems to circulate in an almost exclusive fashion, except in Brazil. The aim of this study was the molecular characterization of the HAV infection in Venezuela, in order to characterize the circulating strains and to analyze the presence of quasispecies in sporadic cases and an epidemic outbreak. A total of 125 (113 sera and 12 feces) samples positive for anti-HAV IgM from sporadic cases and epidemic outbreak, were submitted to hemi-nested RT-PCR for amplification of the VP1 N terminus or complete region of the HAV genome. Sequences obtained from 96 Venezuelan isolates were used for phylogenetic analysis. The quasispecies distribution was evaluated by cloning of HAV amplicons. Phylogenetic analysis of HAV sequences from Venezuela showed the exclusive circulation of subgenotype IA, but with co-circulation of two lineages, not found in other countries. The genetic variability found among Venezuelan strains was also analyzed by single-strand conformation polymorphism (SSCP). This technique allowed the detection of intra-strain variability, which was indeed related to the presence of quasispecies populations in the isolates. The quasispecies heterogeneity was higher in some isolates derived from sporadic cases compared to the one observed in the outbreak. The molecular characterization of HAV isolates from Venezuela showed the circulation of a unique subgenotype IA, but with the presence of diverse strains and quasispecies inside the viral populations.

SARS-CoV-2 host tropism: An in silico analysis of the main cellular factors.

Recent reports have shown that small and big felines could be infected by SARS-CoV-2, while other animals, like swines and mice, are apparently not susceptible to this infection. These findings raise the question of the role of cell factors associated with early stages of the viral infection in host selectivity. The cellular receptor for SARS-CoV-2 is the Angiotensin Converting Enzyme (ACE2). Transmembrane protease serine 2 (TMPRSS2) has been shown to prime the viral spike for its interaction with its receptor. GRP78 has also been proposed as a possible co-receptor. In this study, we used several bioinformatics approaches to bring clues in the interaction of ACE2, TMPRSS2, and GRP78 with SARS-CoV-2. We selected several mammalian hosts that could play a key role in viral spread by acting as secondary hosts (cats, dogs, pigs, mice, and ferrets) and evaluated their predicted permissiveness by in silico analysis. Results showed that ionic pairs (salt bridges, N-O pair, and long-range interactions) produced between ACE2 and the viral spike has an essential function in the host interaction. On the other hand, TMPRSS2 and GRP78 are proteins with high homology in all the evaluated hosts. Thus, these proteins do not seem to play a role in host selectivity, suggesting that other factors may play a role in the non-permissivity in some of these hosts. These proteins represent however interesting cell targets that could be explored in order to control the virus replication in humans and in the intermediary hosts.

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.

A simplified methodology for purification of peanut (Arachis hypogaea) agglutinin.

The agglutinin from peanut (Arachis hypogaea) was readily isolated by affinity chromatography on acid-treated Sepharose 6B. The recovered lectin (50 mg/100 g seeds) appeared as a single band of Mr 32,000 on gel electrophoresis and its specific haemagglutination titre on desialylated human A red blood cells was very high (2(15)).

[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.

[High prevalence of hepatitis B and C markers in an indigent community in Caracas, Venezuela]. / Alta prevalencia de marcadores de hepatitis B y C en una comunidad de indigentes de Caracas, Venezuela.

Hepatitis B and C serological markers were evaluated in indigent patients living in a workhouse in Caracas, Venezuela. Three out of 146 specimens were reactive for HBsAg (2.1%) and 41 for anti-HBc (28%). Eight sera were reactive for antibodies against HCV, and 7 were confirmed by immunoblot assays (4.8%). Four out of these 7 HCV-positive sera were also HCV-RNA positive. The prevalence for viral hepatitis markers was significantly higher than that found in blood donor population in Caracas. The probable risk factors associated with this high prevalence of blood-borne viral hepatitis were: promiscuity, transfusion, drug addiction, alcoholism and history of reclusion. This high prevalence of hepatitis B and C compels the precaution for medical and paramedical staff attending this kind of patients.

Inhibition of hepatitis B virus and human immunodeficiency virus (HIV-1) replication by Warscewiczia coccinea (Vahl) Kl. (Rubiaceae) ethanol extract.

The primary objective of this study was to search for natural products capable of inhibiting hepatitis B virus (HBV) replication. The research design, methods and procedures included testing hydro-alcoholic extracts (n = 66) of 31 species from the Venezuelan Amazonian rain forest on the cell line HepG2 2.2.15, which constitutively produces HBV. The main outcomes and results were as follows: the species Euterpe precatoria, Jacaranda copaia, Jacaranda obtusifolia, Senna silvestris, Warscewiczia coccinea and Vochysia glaberrima exerted some degree of inhibition on HBV replication. The leaves of W. coccinea showed a significant antiviral activity: 80% inhibition with 100 µg mL⁻¹ of extract. This extract also exerted inhibition on covalently closed circular deoxyribonucleic acid (cccDNA) production and on HIV-1 replication in MT4 cells (more than 90% inhibition with 50 µg mL⁻¹ of extract). Initial fractionation using organic solvents of increasing polarity and water showed that the ethanol fraction was responsible for most of the antiviral inhibitory activities of both the viruses. It was concluded that Warscewiczia coccinea extract showed inhibition of HBV and HIV-1 replication. Bioassay-guided purification of this fraction may allow the isolation of an antiviral compound with inhibitory activity against both viruses.

Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia.

The objective of this study was the evaluation of the genetic diversity found in HBV circulating among Venezuelan Amerindians and the general population in Colombia. Phylogenetic analysis of the S region in 194 isolates showed that genotype F is highly predominant in Colombia and Venezuela. This might be related to the genetic background of the population. F3 is the main subgenotype which circulates in both countries. Phylogenetic analysis of 61 complete genome sequences of HBV American genotypes confirms the presence of two genotypes F and H, and 4 F subgenotypes. In Venezuela, subgenotypes F1, F2, and F3 circulate in East and West Amerindians, while only F3 was found among South Amerindians. Japreira community derived from Yucpa Amerindians around 150 years ago. However, several Japreira HBV sequences were forming a clade that can be classified as subgenotype 2b, differing from Yucpa sequences that belong mainly to subgenotype F3. The apparent absence of correlation between the phylogenetic groupings of HBV isolates with the ethnical origin in aboriginal populations might be suggesting a recent origin of HBV American subgenotypes, or a genetic drift effect.

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.

Antigenicity of adult Schistosoma mansoni alkaline phosphatase.

Antibodies to the alkaline phosphatase (AP) of Schistosoma mansoni in infected human and mice sera were evaluated by a direct solid-phase AP immunoadsorption assay (APIA) and by Western blot and immunostaining. APIA consisted of (a) solid-phase capture of immunoglobulins from infected human or mice, (b) immunoadsorption of the enzyme antigen by the antibodies, and (c) detection of the enzymatic activity. By this procedure the appearance of the anti-AP response in mice was detected around 50 days post-infection; this response was not specific for an AP of a given schistosome strain and it was not induced by an autoimmunity phenomenon. Fourteen out of 15 sera from infected people tested by APIA showed a clear antibody response against this enzyme. Immunoblots in non-reducing conditions supported APIA results indicating that the parasite AP was specifically recognized by the antibodies present in infected human and mice sera. These results suggest the possible usefulness of the schistosome AP as a marker for S. mansoni infection.

Schistosoma mansoni: surface membrane isolation with lectin-coated beads.

Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are located within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.

[Production and characterization of monoclonal antibodies directed against the hepatitis B surface antigen]. / Producción y caracterización de anticuerpos monoclonales dirigidos contra el antígeno de superficie del virus de la hepatitis B.

Hepatitis B virus surface antigen (HBsAg) found in a commercial vaccine was use as immunogen and antigen for the production and selection of murine monoclonal antibodies against this viral antigen. Antibody relative avidity was determined based on their capture capacity. Competition studies, and the differential recognition pattern of vaccine preparations showed that high avidity IgG1 antibodies were directed against two distinct antigenic regions. Among them, 6F4 was most suitable for the detection of HBsAg in a sandwich ELISA system, using an IgM antibody, 5E8, as capture. However, the combined use of 6F4 with another which does not recognize the same epitope did not improve the sensitivity of the assay. On the other hand, the combination of 6F4 with a lower avidity IgG1 antibody, 6G10, increased the universe of antigenic diversities recognized by this system. An enzyme immunoassay for detection of HBsAg is proposed, using 3 monoclonal antibodies: one IgM as capture and two IgG1 for detection.

[Low impact of silent hepatitis B virus infection on the incidence of post-transfusion hepatitis in Venezuela]. / Bajo impacto de la infección silente por el virus de la hepatitis B en la incidencia de hepatitis postransfusional en Venezuela.

OBJECTIVE: Silent infection by hepatitis B virus (HBV) occurs in the absence of serological markers for the virus. This type of occult infection is generally chronic, asymptomatic, and associated with low levels of viral replication. This study determined the presence of HBV DNA in the sera of blood donors who were negative for serological markers that were tested during screening, with the goal of evaluating the impact of silent HBV infection in posttransfusion hepatitis B in Venezuela. METHODS: A total of 2,075 sera were tested in 53 serum pools of 25-50 donations (0.5-1.0 mL from each sample). The pools were subjected to ultracentrifugation prior to DNA extraction by the proteinase K, phenol/chloroform method. RESULTS: No HBV DNA was found in any of the pools by nested polymerase chain reaction, using primers for highly conserved regions of the genes that code for the surface antigen and for the viral capsid. Aminotransferase levels were normal in 98% of 200 sera that were tested. CONCLUSIONS: These results suggest that there is a low risk of acquiring posttransfusion hepatitis B in Venezuela.

[Usefulness of the PCR technique (polymerase chain reaction) in the follow-up of patients infected with hepatitis C virus. Preliminary communication]. / Utilidad de la Técnica de PCR (reacción en cadena de la polimerasa) en el seguimiento de pacientes infectados con el virus de la hepatitis C. Comunicación preliminar.

UNLABELLED: In the study we show the usefulness of PCR (polymerase chain reaction) to follow patients with chronic hepatitis, infected with hepatitis C virus (HCV) of Centro Médico de Caracas. The study included 14 patients: 12 anti-HCV positive, 1 with chronic autoimmune hepatitis and 1 classified as non B-non C hepatitis. The patients were divided in 3 groups: Group 1 (5 pretreatment patients, anti-HCV+), 4 with increase in ALT and PCR positive, 1 with normal ALT and PCR negative. Group 2 (7 treated with recombinant interferon alpha 2b), 4 without normalization of ALT and PCR positive, 3 with normalization of ALT and PCR negative. Group 3 (control) 2 patients anti-HCV negative and PCR negative. Two posttreatment patients could be genotyped: one patient was infected with 1a and showed an early relapse with treatment and the other was infected with genotype 1b, which is reported to be more refractory to antiviral treatment. CONCLUSIONS: the results show a 100% correlation between biochemical markers of HCV infected patients and the presence of viral RNA detected by PCR. the usefulness of determination of genotype to assess any prognostic value of this parameter in Venezuela is discussed.

Immunodiagnosis of Schistosomiasis mansoni with APIA (alkaline phosphatase immunoassay).

The previously shown antigenicity of Schistosoma mansoni (JL venezuelan strain) alkaline phosphatase (Mg2+, pH 9.5) allowed its use in an immunodiagnosis assay, that consisted in the immunoadsorption of the enzymatic activity. Protein-A coated polyvinyl plates were used as solid phase to capture IgG from sera of infected human patients. After buffered saline washings, the plates were incubated with an enzyme-rich fraction (a n-butanol aqueous extract of adult worm obtained from pairs). Immunoadsorbed alkaline phosphatase (AP) was revealed by adding rho-nitrophenyl phosphate. Anti-AP antibodies were detected in 93% of coproparasitologically proven S. mansoni-infected venezuelan patients but not in parasite-free control sera and sera from patients infected with parasitosis other than schistosomiasis. The APIA did not correlate with cure but the anti-AP antibody response was progressively reduced after treatment. The use of an AP substrate amplifying system allowed an improvement of the assay sensitivity without loss of specificity. The data suggest that the APIA could be used as a marker of infection by S. mansoni.

A double sandwich monoclonal enzyme immunoassay for detection of hepatitis B surface antigen.

An enzyme immunoassay for detection of hepatitis B surface antigen based on the use of 3 monoclonal antibodies (mAbs) was developed: an IgM as capture and 2 IgG1 for detection. The system biotin-streptavidin was compared with direct conjugation of mAbs to peroxidase and was preferred because of its higher signal to noise ratio. The possibility of simultaneous addition of human serum and biotin-mAb was discarded because of an evident prozone effect with some sera containing high HBsAg levels. The conjugation of biotin to IgG1 mAbs through a spacer arm (amidocaproyl) and the use of a highly sensitive substrate (tetramethylbenzIdine) improved the assay detection limit by about 10 times.