Principles of vaccination and possible development strategies for rational design.

Historically, apart from hygiene, vaccination can be considered as one of the most successful accomplishments of public health in the 20th century. It has lead to some of the greater public health triumphs ever, including the eradication of naturally occurring smallpox and in the control of diseases such as polio. In addition there has been a significant reduction in disease burden imposed by measles, mumps, hepatitis, influenza, diphtheria, haemophilus influenza B and many other infections.

Avoiding biohazards in medical, veterinary and research laboratories.

Personnel in medical, veterinary or research laboratories may be exposed to a wide variety of pathogens that range from deadly to debilitating. For some of these pathogens, no treatment is available, and in other cases the treatment does not fully control the disease. It is important that personnel in laboratories that process human or microbiological specimens follow universal precautions when handling tissues, cells, or microbiological specimens owing to the increasing numbers of individuals infected with hepatitis C and HIV in the US and the possibility that an individual may be asymptomatic when a specimen is obtained. Similar precautions must be followed in laboratories that use animal tissues owing to the possibility of exposure to agents that are pathogenic in humans. Personnel with conditions associated with immunosuppression should evaluate carefully whether or not specific laboratory environments put them at increased risk of disease. We offer here some general approaches to identifying biohazards and to minimizing the potential risk of exposure. The issues discussed can be used to develop a general safety program as required by regulatory or accrediting agencies, including the Occupational Safety and Health Administration.

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.

Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes.

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract. We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30- to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% +/- 12.5% of the total virus yield compared with 0.23% +/- 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms.

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.

Electron microscopy of buffalo green monkey kidney cells persistently infected with hepatitis A virus and immunolocalization of HAV antigens.

Studies were carried out to analyse the ultrastructural changes and the distribution of hepatitis A virus (HAV)/antigens at subcellular level in buffalo green monkey kidney (BGMK) cells persistently infected with HM-175 strain of HAV. HAV infected BGMK cells showed distinct abnormalities in the endoplasmic reticulum and cytoplasmic membrane as compared to uninfected cells. The abnormalities were characterized by wavy arrays, structures like myelin, annulate lamellae, cytoplasmic inclusion bodies and vesicles. The wavy arrays within the cytoplasm of the host cells appeared to represent degenerating membranes. A complex myelin like body was found in close association with a group of virus like particles. Annulate lamellae like structures involving single paired membrane were detected infrequently whereas the cytoplasmic vesicles were numerous in these cells. An indirect immunogold technique was utilized to localize the HAV antigenin infected cells. A high density immunogold label for HIV like particles was predominantly detected in cytoplasmic vesicles. These results suggest a strong association of membrane substructure in vesicle forms with the compartmentalized replication of HAV within persistently infected host cells.

A cytopathic and a cell culture adapted hepatitis A virus strain differ in cell killing but not in intracellular membrane rearrangements.

Aside from a common gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype, the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Nevertheless, several cp HAV strains have been isolated during the past decade. In FRhK-4 cells infected with HM175/24a, a fast-growing cp strain, increasing amounts of viral RNA, detected by fluorescence in situ hybridization, indicated viral RNA replication. An ultrastructural analysis of the infected cells revealed a tubular-vesicular network in close proximity to the rough endoplasmic reticulum. Infection of the same cell type with a cell culture adapted (cc) strain, HM175/P35, divulged membrane alterations indistinguishable from the network induced by the cp strain. The overall appearance of the tubular-vesicular network resembles membrane alterations induced by other picornaviruses. However, the shape of the vesicle-like structures is rather oblong and tubular and, thus, seems to be specific for HAV. By electron microscopic immunocytochemistry (IEM), proteins 2B and 2C were found exclusively on the membranes of the network. Proteins expressed from the open reading frame of the cc HAV variant or 2B proteins originating from HM175 cp, cc, or the wt strain expressed in the absence of other HAV proteins induced membrane alterations resembling those seen in HAV-infected cells. The induction of similar structures suggests that protein 2B is involved in the rearrangement of cellular membranes. In all cases, IEM demonstrated that the 2B protein was closely associated with altered membranes. The extent of membrane changes did not seem to increase for both the cp strain and the cc strain during the infectious cycle. Late in the infection and shortly before the culture died off, a large number of cells infected with HM175/24a showed typical signs of apoptosis, whereas the cc strain did not induce cell killing in the same type of cells. Therefore, we conclude that cell death in HM175/24a-infected cells is induced by apoptosis rather than by cytopathology.

Infección por virus hepatitis A y embarazo: hallazgos sonográficos / Hepatitis A virus infection and pregnancy: ultrasonographic findings

Presentamos un caso de infección materna por virus hepatitis A en una mujer de 20 años que cursaba un embarazo de 17 semanas. El seguimiento sonográfico mostró compromiso fetal caracterizado por ascitis, heteregeneidad hepática, intestino hiperecogénico y oligohidroamnios. Similares hallazgos han sido publicados en 2 casos reportados de infección intrauterina por este virus

Hepatitis virales / Viral hepatitis

En esta revisión se describen los virus hepatotropos actualmente conocidos, su epidemiología con referencia especial a los datos nacionales relativos a los virus A, B, C y E; su historia natural y sus aspectos clínicos más relevantes. Se enfatizan, además los diferentes marcadores virales serológicos, para el diagnóstico de infección aguda o crónica. Se incluyen también los diferentes tratamientos y las medidas de prevención (pasivas o activas) recomendadas actualmente

Enterically transmitted hepatitis. Hepatitis A and E viruses.

Enterically-transmitted hepatitis is caused by hepatitis A virus and hepatitis E virus. The most important agent is hepatitis A virus, which is distributed worldwide and infects all age groups. Most infections in children are minimally symptomatic and immunity is long-lasting, so severe disease tends to occur in nonimmune adults. Hepatitis E virus is found in the developing world and has a greater propensity for symptomatic infection of children. Both agents are transmitted via contaminated water, often through food vehicles.

Excretion of hepatitis A virus (HAV) in adults: comparison of immunologic and molecular detection methods and relationship between HAV positivity and infectivity in tamarins.

Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools.

Recovery and detection of enterovirus, hepatitis A virus and Norwalk virus in hardshell clams (Mercenaria mercenaria) by RT-PCR methods.

A method for recovery of enteric viruses from hardshell clams (Mercenaria mercenaria) has been developed and evaluated. Seeded 50-g samples of clam tissue homogenates were processed by adsorption elution precipitation, two fluorocarbon extractions and PEG precipitation. Clam concentrates were assayed by infectivity and by RT-PCR after guanidinium isothiocyanate (GIT) extraction and/or an indirect immunomagnetic capture (IC) of the virus using paramagnetic beads. GIT extraction removed PCR inhibitors and allowed a reliable RT-PCR detection of viral RNA. The detection sensitivity of GIT extraction-RT-PCR was < 1 PFU of poliovirus 1, < 10 PFU of HAV and 1-11 PCRU of Norwalk virus. IC was very effective for additional concentration and purification of enteric viruses from clam concentrates removing most RT-PCR inhibitors. The sensitivity of this method was comparable to the GIT extraction and the sample volume tolerance for PCR was increased about 10-fold. Both methods gave similar efficiency for virus detection in samples seeded with low virus levels. The procedure developed in this study is effective for enteric viruses detection in hardshell clams by RT-PCR.

Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.

Persistence of infectious hepatitis A virus and its genome in artificial seawater.

The stability of the hepatitis A virus (HAV) genome detectable by RT-PCR in artificial sterile seawater seeded with HAV has been compared to that of HAV detectable in cell culture. The HAV genome was detectable by RT-PCR for 232 days while virus particles were detectable in cell culture for only 35 days. This difference in stability indicates that detection of the HAV genome by RT-PCR is not a reliable indicator of the survival of HAV detectable in cell cultures. However, before these results can be extrapolated to stability in natural seawater, the effect of additional elements in the natural environment, such as bacteria, fungi and suspended matter, on the stability of the HAV genome and cell culture infectious HAV particles, will have to be examined.

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.

Evidence that childhood acute lymphoblastic leukemia is associated with an infectious agent linked to hygiene conditions.

OBJECTIVES: The incidence of acute lymphoblastic leukemia (ALL) in children has shown temporal and geographic variation during the 20th century, with higher rates in developed nations appearing in the first half of the century, but with persisting low rates in developing nations. We sought to assess the relation of childhood ALL with hygiene conditions, an aspect of socioeconomic development affecting rates of exposure to infectious agents. METHODS: Infection patterns for hepatitis A virus (HAV), an agent with a fecal-oral route of transmission, were used to indicate hygiene conditions in different populations, with emphasis on instructive United States and Japanese data. A catalytic model was fit to these data, estimating the HAV force of infection and age-specific seroprevalence rates over time. These analyses were used to assess the temporal relationship of changes in HAV infection rates to changes in childhood leukemia mortality and incidence rates. RESULTS: We observed an inverse relationship between HAV infection prevalence and rates of childhood leukemia. Further, decreases in the HAV force of infection in the United States and Japan appear to have preceded increases in childhood leukemia rates. We describe a model based on a putative leukemia-inducing agent with a change in infection rate over time correlated with that of HAV that describes well the temporal trends in childhood leukemia rates for White children in the US and for Japanese children. CONCLUSION: The data suggest that improved public hygiene conditions, as measured by decreased prevalence of HAV infection, are associated with higher childhood ALL incidence rates. The model that we present supports the plausibility of the hypothesis that decreased childhood exposure to a leukemia-inducing agent associated with hygiene conditions leads to higher rates of ALL in children by increasing the frequency of in utero transmission caused by primary infection during pregnancy (or by increasing the number of individuals infected in early infancy because of lack of protective maternal antibodies).

[Characteristics of experimental models of hepatitis A in Papio hamadryas]. / Kharakteristika éksperimental'nykh modelei gepatita A na pavianakh gamadrilakh.

Hepatitis A (HA) was induced in 14 Papio hamadryas by strain VHA-PH isolated from this species of monkeys with spontaneous infection, strain VHA-MM isolated from Macaca mulatta, and a unique strain VHA-H3 isolated from a patient; this latter strain is pathogenic for Macaca mulatta in experiment. All infected seronegative animals developed a disease with virological, serological, biochemical, and morphological signs characteristic of human HA, but the duration of these signs manifestation varied. Virus in the feces and an increased level of SGPT were detected periodically starting from days 3-26 to 24-135, and in 4 monkeys even later (up to days 163-238). Morphologic changes in the liver, typical of acute hepatitis, were observed from days 10-46 to days 16-130. Strain VHA-H3 is less pathogenic for papios. HA models on Papio hamadryas infected with strains VHA-PH and VHA-MM can help solve many research and practical problems.

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.

Membrane permeability induced by hepatitis A virus proteins 2B and 2BC and proteolytic processing of HAV 2BC.

The ability to rearrange membranes is a unique feature of nonstructural proteins 2B, 2C, and 2BC of some picornaviruses. To analyze in detail membrane binding of the respective proteins of hepatitis A virus (HAV), they were transiently expressed in the vaccinia/T7 system, and their effect on membrane permeability was studied using beta-galactosidase as reporter. Although 2C had no effect, the significantly increased reporter activity observed in the extracellular space of 2B- and 2BC-expressing cells points to a specific effect of HAV proteins 2B and 2BC on membrane permeability. In biochemical fractionation studies, HAV 2C and 2BC showed properties of intregral membrane proteins, whereas 2B was associated with membranes as a peripheral protein. Proteinase 3C-mediated cleavage of precursor 2BC in vivo was most efficient when the enzyme was coexpressed in its precursor forms P3 or 3ABC, which both include the membrane-anchoring domain 3A. 3ABC showed the same solubility pattern as 2BC, suggesting that colocalization of 2BC and 3ABC might be required for the efficient liberation of 2B and 2C and occurs on membranes that have been proposed as the site of viral RNA replication.

[Viral hepatitis of enteric origin]. / Les hépatites virales d'origine entérique.

Hepatitis viruses of oral-fecal origin are responsible for a high morbidity and mortality throughout the world, even if they never result in chronic hepatitis. Two viruses, the virus of hepatitis A (VHA) and of hepatitis E (VHE) are at present the cause of severe viral hepatitis of enteric origin. Water is the principle vector in the spread of these viruses. However, the epidemiological aspects vary according to the pathogenic agent. VHA is excreted in a highly concentrated form in the feces for a relatively short period of time. Since it resists in an exterior environment, the virus remains infectious for a long time. VHE is excreted for a short period of time and in low concentrations. The viral particles are fragile in vitro and their variability in the environment is little known. The possible reservoir role of certain animals has been envisaged. Epidemics arise especially in countries suffering from poor hygiene and massive water pollution. Hepatitis A should no longer be considered a benign disease of childhood. The progress made in hygiene and economic development in industrialized countries have made contacts with this virus scarce, rendering the populations more receptive to it and epidemics more widespread. When the sickness occurs later in life, infection is more often symptomatic and can be serious, resulting sometimes long-term indisposition. Hepatitis E has a vast distribution throughout the world and manifests itself either in epidemic or endemic-sporadic form in many poor countries. In developed countries, it comes about mostly as a result of imported pathology, even if there exists a "substratum" of infection in these areas. The main clinical aspects, such as we were able to study them in 39 cases of military men from Tchad, Guyana and Somalia, are comparable to those of hepatitis A. The reasons for the particular gravity of symptoms in pregnant women are unknown. These affections have no specific treatment. In the field of prevention, vaccination is at present the best means for hepatitis A prophylaxis. Until a vaccine against hepatitis E is found, prevention depends on hygiene, sanitation measures et distribution of drinking water.