Hepatitis A en niños / Hepatitis A in children

La Hepatitis A (HVA), también llamada hepatitis infecciosa, transmitida por alimentos, epidémica,ictericia catarral o epidémica, entre otros, es una enfermedad producida por un agente viral que se trasmite por vía fecal oral y generalmente su curso es autolimitado, aunque, puede progresar ahepatitis fulminante ocasionando la muerte a una proporción pequeña de los infectados. Pertenece al géner o Hepatovir us de la Familia Picornaviridae. La HVA, tiene una distribución universal, aunque con grandes diferencias geográficas en cuanto a su prevalencia, ocurre en forma esporádica y epidémica en todo el mundo, con una tendencia a presentarse en ciclos. La HVA, tiene un periodo de incubación prolongado, entre 15 a 50 días, con un promedio de 29 días, lo que hace difícil relacionar los síntomas con algún alimento o bebida ingerida. El diagnostico de la HVA, se basa en la detección de anticuerpos contra el VHA tipo IgM e IgG. El tratamiento básicamente es de soporte, sintomático y en casos de falla hepática, el trasplante es la única opción. La inmunoglobulina confiere inmunidad pasiva a corto plazo mientras la vacuna provee una protección activa a largo plazo.

Hepatitis A (HVA), also called infectious hepatitis, foodborne, epidemic, or epidemic or catarrhaljaundice, among others, is a disease caused by a viral agent that spreads through fecal-oral routeand usually self-limited course, although fulminant hepatitis can progress to causing death to a small proportion of those infected. Is a Hepatovirus genus of the Picornaviridae Family. The HVA, has a worldwide distribution, but with large geographical differences in its prevalence, occurs in sporadic and epidemic worldwide, with a tendency to occur in cycles. The HVA, has a long incubation period between 15 to 50 days, with an average of 29 days, making it difficult to correlate symptoms with food or drink intake. The diagnosis of HVA was based on the detection of antibodies against HAV IgM and IgG.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus.

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TCID50] ml(-1) to <1 log TCID50 ml(-1) within 24 h and from 3.82 log TCID50 ml(-1) to <1 log TCID50 ml(-1) within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus.

Immunochemiluminescent focus assays for the quantitation of hepatitis A virus and rotavirus in cell cultures.

Two new immunological methods, the luminescent immunofocus assay (LIFA) and the luminescent immunofocus inhibition assay (LIF-IA), are described for the quantitation of cytopathic and non-cytopathic viruses propagated on cell culture monolayers. These methods use enhanced chemiluminescent detection to identify foci (luminescent immunofoci, LIF) of virus-infected cells. Viruses are propagated in susceptible cells under an agarose overlay, inactivated with ultraviolet irradiation, lifted onto nitrocellulose membranes, and probed with virus-specific monoclonal or polyclonal antibody followed by a second antibody conjugated to horseradish peroxidase. Membranes are then treated with a luminol-based detection reagent and exposed to light sensitive film for up to 10 min. The film is developed and foci appear as dark, discrete spots which are proportional to the dose of each virus. The LIFA detected both cytopathic and non-cytopathic hepatitis A viruses (HAV) and simian rotavirus. For the cytopathic HAV, the LIFA and plaque counts were comparable. The LIF-IA was developed for HAV using virus-specific antiserum which effectively attenuated LIF formation. The LIFA and LIF-IA may be completed 5 days faster than conventional radioimmunofocus assays for HAV and rotavirus and do not require the use of radiolabeled antibodies, offering safety advantages and making these techniques more adaptable for general use. Luminescent immunofocus assays should be useful for the detection and quantitation of virtually any cytopathic or non-cytopathic virus that can be propagated in monolayer cultures when virus-specific antiserum is available.

Adaptation of primate cell-adapted hepatitis A virus strain HM175 to growth in guinea pig cells is independent of mutations in the 5' nontranslated region.

Previous studies of hepatitis A virus (HAV) genotypes after adaptation of wild-type virus to growth in cell cultures of primate origin identified determinants for growth in cell culture in the viral 2B and 2C protein-coding regions of the genome and demonstrated that an increased growth efficiency in a particular cell line was achieved by subsequent mutations in the 5' nontranslated region (5'NTR). The results reported in this study demonstrate that the passage of HAV adapted to primate BS-C-1 cells in guinea pig cells resulted in increased growth efficiency in the rodent cells and decreased growth efficiency in BS-C-1 cells. This adaptation occurred without mutation in the 5'NTR, but the viral 2B and 2C proteins seem to play a role during adaptation to the new environment, as one mutation occurred in each protein. Although the data presented here do not clearly identify which region of the viral genome underwent mutations to improve the interaction of the viruses with guinea pig proteins, they do confirm that the 5'NTR is not the only region responsible for providing host cell-specific information.

Rapid concentration and detection of hepatitis A virus from lettuce and strawberries.

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.

Hepatitis A vaccine administration: comparison between jet-injector and needle injection.

INTRODUCTION: Type A hepatitis virus (HAV) is a serious health problem throughout the world and can be spread via fecal-oral contact. Both immune globulin and an HAV vaccine provide protection, but the vaccine gives complete protection. Efficacy of methods of vaccination in relation to the formation of anti-HAV antibodies is unclear; thus, this study seeks to determine if significant differences exist between the syringe as compared to the jet injection technique. The purpose of this study was to compare in a randomized trial Biojet jet-injection system to a needle-syringe method. To determine if a significant difference between these two methods in seroconversion rates or geometric mean titers of anti HAV antibody occurs at day 15, 30, and 210 days after vaccination. METHOD: Anti-HAV IgG(-) adult hospital employees were randomized to receive 1440 EL.U of hepatitis a vaccine (HAVRIX(R)) in 2 doses by either needle or jet-injector (Biojector(R)) system at month 0 and 6. HAV seroconversion titer results were measured by the Boehringer-Mannheim method. RESULTS/DISCUSSION: A higher proportion of persons who received HAV vaccine via the Biojector(R) seroconverted with anti-HAV level >/=20 mIU at day 15, 30, and month 7 when compared with a needle injection.Side-effect profiles reported by participants in both methods were below those identified in current published and insert information, but the Biojector(R) had greater local reactivity in all categories when compared to the needle method.

Uncoating kinetics of hepatitis A virus virions and provirions.

When the growth kinetics of immature hepatitis A virus provirions and mature virions were monitored, distinct eclipse phases were noted for both types of particles. Strikingly, uncoating of virions occurred around 4 h postinfection, while uncoating of provirions occurred predominantly between 8 and 10 h postinfection. It is proposed that the heterogeneous mixture of infectious hepatitis A virus particles (virions and provirions) typically present in inocula is responsible for the normally asynchronous nature of hepatitis A virus uncoating kinetics.

Conformational changes in the hepatitis A virus capsid in response to acidic conditions.

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.

Inactivation of hepatitis A virus in heat-treated mussels.

Hepatitis A is a widespread infectious disease world-wide. In Italy, shellfish consumption was shown to be a major risk factor for hepatitis A infection, especially when these products are eaten raw or slightly cooked. The aim of the present study was to evaluate Hepatitis A virus (HAV) resistance in experimentally contaminated mussels treated at different temperatures (60, 80 and 100 degrees C) for various times. The presence of HAV was evaluated by cell culture infection and reverse transcriptase-polymerase chain reaction confirmation. The experiments, carried out on HAV suspension and contaminated mussel homogenate both containing about 10(5) 50% tissue culture infectious dose ml-1, showed that, under our experimental conditions, the treatments at 60 degrees C for 30 min, 80 degrees C for 10 min and an immersion at 100 degrees C for 1 min were not sufficient to inactivate all the viruses; it was necessary to prolong the treatment at 100 degrees C for 2 min to completely inactivate the virus. Thus it is advisable to eat only cooked shellfish, paying particular attention to the times and temperatures used in the cooking process, since evidence suggests that the shellfish body may protect the virus from the heat effect.

Validation of the heat treatment step used in the production of diaspirin crosslinked hemoglobin (DCLHb) for viral inactivation.

A series of experiments was performed to assess the ability of the heat treatment step used in the manufacture of diaspirin crosslinked hemoglobin (DCLHb) to inactivate viruses. In-process solutions (reaction mixtures after the crosslinking process) from six different manufacturing lots were used as test media in a 1:680 scaled down system in which the key process parameters used in the large scale production were duplicated. The inactivation of five different viruses (Bovine Viral Diarrhea Virus, Pseudorabies Virus, Human Immunodeficiency Virus 1, Porcine Parvovirus and Hepatitis A Virus) was evaluated. Each validation experiment consisted of spiking the solution at 37 degrees C with virus, heating to 74 +/- 1 degrees C over a period of 30 minutes, holding at 74 +/- 1 degrees C for 90 minutes and cooling from 74 +/- 1 degrees C to less than 10 degrees C over a period of 30 minutes. Duplicate experiments were performed with each of the viruses with the exception of Human Immunodeficiency Virus 1, for which three experiments were performed. In each experiment samples were removed before, during, and after heating for the purpose of determining virus titer and evaluating key process parameters. The results obtained from these experiments confirmed that the key process parameters in these experiments using the scaled down test system reproduced those of the large scale manufacturing process. The results of the virus assays showed at least a 7 log reduction was accomplished by the heat treatment for each of the viruses tested.

Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation.

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

Increased infectivity of hepatitis A virus cDNA clones with engineered 5'-terminal extra-cistronic sequences.

The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepatovirus group, is a single-stranded molecule, ca. 7.5 kb in length of positive polarity. Translation of this uncapped RNA starts at the 10th (or 11th) AUG triplet (position 734-36), by a mechanism of internal initiation of translation. The long sequences extending between the uncapped 5'-end and the translation initiation site contain two (instead of just one) pyrimidine-rich tracts (PRTs) spanning nucleotides 94-140 and 711-724, respectively. The latter lies only 11 nucleotides upstream from the initiation site of translation, and the question arose as to whether the notoriously poor replication ability of HAV was a consequence of a down regulation of translation due to the too short "spacer" sequence intervening between the 3'-PRT and the initiation of the main open reading frame. To address this issue, a series of full-length HAV cDNA clones were constructed in which the "spacer" sequence (normally 11 nts) was brought to 45 nts. Following transfection of COS-1 cells with these constructs, the amount of HAV (+)-strand RNA was determined by dot hybridization using a strand-specific RNA probe. HAV cDNA clones carrying a 45-nt "spacer" increased two-fold the rate of (+)-strand viral RNA synthesis, suggesting that the poor translation ability of HAV RNA may be one of the mechanisms responsible for the lengthy replication cycle of HAV.

Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses.

A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of Bacteroides fragilis HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of B. fragilis HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable.

Utilization of chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus to study the molecular basis of virulence.

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5' or 3' half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3' half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.

A cytopathogenic, apoptosis-inducing variant of hepatitis A virus.

A cytopathogenic variant of hepatitis A virus (HAV(cyt/HB1.1)) was isolated from persistently infected BS-C-1 cells by serial passages in FRhK-4 cells. This virus shows a rapid replication pattern and high final titers are obtained, which are main characteristics of cytopathogenic HAVs. Sequencing of the nontranslated regions and the coding regions for 2ABC and 3AB revealed that mutations are distributed all over these regions and that certain mutated sites correspond to those in other cytopathogenic HAV variants. Investigating the mechanisms causing the cytopathic effect in FRhK-4 cells infected with this variant, we found that an apoptotic reaction takes place.

Influence of the 5' noncoding region of hepatitis A virus strain GBM on its growth in different cell lines.

Previous sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK-4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5' noncoding region (5'NCR). Multiple nucleotide changes occurred in the 5'NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5'NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5'NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5'NCRs from different GBM variants by replacing the 5'NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5'NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5'NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5'NCR.

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.

Growth of hepatitis A virus in murine cells.

In order to investigate the growth of hepatitis A virus (HAV) in murine cells, L929 cells of the established mouse cell line were transfected with the virion RNA or infected with the virions and examined for the formation of negative-strand RNA and the rise of the viral infectivity titer. In both the transfected and infected cells, the formation of negative-strand HAV RNA was assayed by the reverse transcription-polymerase chain reaction (RT-PCR). In the transfected cells, infectious HAV of an average titer of 10(1.8) TCID50/dish was obtained. The experiment with the virion infection was further extended by using other mouse cell lines, namely Balb/3T3 clone A31, NIH/3T3, and Swiss/3T3. Here, only NIH/3T3 cells were found capable to support the formation of negative-strand HAV RNA. Thus some murine cell lines are considered to have a complete cellular machinery for supporting the growth of HAV, though the efficiency of virus growth therein was considerably lower as compared to that in the susceptible primate cells.