Short report: phylogenetically distinct hepatitis E viruses in Pakistan.

Hepatitis E, which is enterically transmitted, is the most common cause of acute hepatitis in much of Asia. Phylogenetic analysis of several isolates of hepatitis E virus (HEV) from Asia suggests that transmission of this virus is geographically restricted. In Sarghoda, Pakistan, HEV Sar-55 was isolated from a 1987 outbreak. It belongs to the Central-Asian cluster of the Asian sub-genotype. We now report the complete sequence of a second Pakistan HEV from a 1988 outbreak in Abbottabad. The Abbottabad nucleotide sequence was compared with 15 other complete HEV sequences using statistical methods of phylogenetic analysis. The analysis showed that Abbottabad HEV belongs to the South Asia cluster of the Asian sub-genotype. The sequence differences of the 2 Pakistan isolates recovered only one year apart suggest that HEV of 2 distinct origins circulate in Pakistan.

Identification of a novel hepatitis E virus in Nigeria.

Sporadic cases of acute hepatitis E among ten native Nigerian adults were reported in Port-Harcourt (Nigeria). Hepatitis E virus (HEV) was detected in serum and/or faecal samples of seven patients by RT-PCR of the open reading frame (ORF)-1 polymerase region and the 3'-end of ORF2. Restriction analysis widely used to distinguish genotypes I and III showed that all Nigerian strains have a pattern similar to the Mexican strain (NotI, nt 286; SmaI, nt 397; no KpnI restriction site) but displayed a BsmI restriction site at nt 213 as do most African HEV strains sequenced so far. Sequence analysis performed from internal ORF1 and ORF2 PCR products displayed strong homogeneity between the HEV isolates, determining a regional cluster. Phylogenetic analysis of nucleotide sequences revealed that these strains were more related to the Mexican prototype genotype III (87% homology in ORF1, 80% homology in ORF2) than to either the African strain genotype I (74% homology in ORF1, 77% homology in ORF2) or the USA strain genotype II (75% homology in ORF1, 77% homology in ORF2). Genetic divergence up to 15% in ORF2 with the Mexican genotype clearly defined a new subgenotype within genotype III. At the amino acid level, Nigerian strains showed more homology with genotype III (96%) than with genotype I (92%). This study clearly determined the co-existence of genotypes I and III in Africa. These Nigerian HEV strains belonging to genotype III, but sharing some properties with genotype I, could be one of the missing links between African and Latin American HEV and could help us to determine the phylogenetic evolution of HEV from the ancestral virus.

[Geographical distribution of hepatitis E virus genotypes]. / Répartition géographique des génotypes du virus de l'hépatite E.

Hepatitis E virus (HEV) is the major agent of acute hepatitis in developing countries where the infection occurs sporadically or in large waterborne epidemics. HEV, classified in the Caliciviridae, is not culturable. The detection of HEV RNA by RT-PCR in serum and stool samples is reliable during the 7 to 15 days following the onset of the disease. Restriction endonuclease analysis, cloning and sequencing of PCR products allow a phylogenetic analysis of HEV isolates. Although they belong to a single serotype, strains recovered from different geographical regions display a significant genetic heterogeneity. Sequencing data from ORF1 and ORF2 regions has led to the characterization of 3 distinct genotypes: genotype I gathering the Asian and African subgenotypes; genotype II gathering swine and human US strains; genotype III limited to the Mexico prototype. Novel variants are currently described from Africa (Nigeria), China and Europe (Greece and Italy). Each genotype appears to be related to a well defined geographical area. Nevertheless, a genetic variability is observed within endemic regions such as Asia or Africa. Nigerian endemic isolates especially could represent an intermediate stage in the evolutionary process towards genetic diversity. The animal reservoir, proved by the detection of HEV sequences by PCR among pigs in Nepal and in the USA, could help to resolve unanswered questions about the origin of HEV genotypes, their spread and evolution.

Phylogenetic analysis of hepatitis E virus isolates from Egypt.

Hepatitis E virus (HEV) genome was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in fecal samples of two sporadic cases of hepatitis E in Cairo Egypt. Sequence of the complete putative structural region [open reading frame (ORF)-2] and complete region of unknown function (ORF-3) was determined for the two HEV isolates. Phylogenetic analysis of the nucleotide sequences was performed using neighbor joining or maximum parsimony methods of tree reconstruction. Direct correspondence between the HEV evolutionary trees and geographic origin of the HEV isolates was observed. Three genotypes of HEV were identified: genotype I (Asia-Africa), genotype II (US), and genotype III (Mexico). Genotype I was further divided into two subgenotypes (Asia and Africa). In the Asian subgenotype, three smaller genetic clusters were observed (China-like sequences, Burma-like sequences, and sequence from a fulminant case of HEV). The segregation of all these genetic clusters was supported by the high level of bootstrap probabilities. Four regions of the HEV genome were used for phylogenetic analysis. In all four regions, Egyptian HEV isolates were grouped in a separate African clade.

Experimental African HEV infection in cynomolgus macaques (Macaca fascicularis).

Experimental infection with hepatitis E virus (HEV) from Africa has not been investigated. Our purpose was to study hepatitis E produced by HEV from Chad (North Africa) and to analyze the genetic sequence of the HEV obtained after animal passage. An HEV-containing fecal sample from Chad was intravenously inoculated in four cynomolgus macaques. When serum Alanine Amino Transferase (ALT) levels rose, open liver biopsy and bile aspiration were performed. In all the monkeys, an ALT rise occurred 25 to 32 days after inoculation and new anti-HEV was detected by Enzyme Immuno Assay (EIA). Hepatic histopathology was consistent with acute viral hepatitis. HEV was detected by polymerase chain reaction (PCR) in bile (3/4 animals) and feces (2/4 animals) and by imunoelectron microscopy (IEM) in the inoculum and one bile specimen. A genetic variant HEV was identified in one monkey. The Chad HEV produced hepatitis E with pathophysiologic and histopathologic findings similar to those observed with HEV from other geographic origins. A genomic variant HEV population was produced after one passage in a macaque.

Role of hepatitis E virus in sporadic cases of acute and fulminant hepatitis in an endemic area (Chad).

Forty-one patients with acute or fulminant hepatitis and 86 control patients were entered into a study of sporadic, acute, and fulminant hepatitis in the N'Djamena area of Chad in 1993. Acute hepatitis B was diagnosed in nine (22%) patients and acute hepatitis E in 27 (66%) patients. No acute hepatitis A was observed and 10% of the patients had serologic markers of hepatitis C virus (HCV) infection. Dual acute hepatitis B and E were observed in four patients (10%) and acute HEV infection was associated with chronic hepatitis B surface antigen carriage in 16 (39%). Epidemiologic findings concerning HBV from Chad suggest that these patients had undiagnosed chronic liver disease due to HBV, with acute deterioration caused by superimposed HEV replication. Moreover, it is obvious that in developing countries only the most severe cases of hepatitis are seen in hospital settings and a large proportion of them are related to superinfection with HBV and HEV. Antibody to HEV was observed in 22% of the control patients. This observation and the fact that epidemic and sporadic cases of HEV are observed in Chad indicates that HEV is highly endemic in this country.

Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence.

The purpose of this study was to analyze partial nucleotide sequences and derived peptide sequences of hepatitis E virus (HEV) from two outbreaks of hepatitis E in Africa (Chad 1983-1984; Algeria 1978-1980). A portion of ORF3 and the major portion of ORF2 were amplified by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The PCR products were sequenced directly or after cloning into the pCRII vector. Sequences were then compared to the corresponding regions of reported full length HEV sequences. In the ORF2 and ORF3 regions, the homology between the Algerian and the Chad isolates at the nucleic acid level was 92 and 95%, respectively. At the peptide level the homology was 98% in both regions. In these regions, both strains are more related to Asian strains at the nucleic acid level (89 to 95%) and at the amino acid level (95 to 100%) than to the Mexico strain. At the peptide level the differences are less apparent. Both African isolates have amino acid changes in common with some reference strains although the Chad isolate has three unique changes. These African strains of HEV, based on the ORF2 and ORF3 phylogenetic trees, appear to be a distinct phylogenetic group, separate from the Mexican and Asian strains.

Short report: polymerase chain reaction detection of hepatitis E virus in north African fecal samples.

Epidemics of enterically-transmitted non-A, non-B hepatitis were described in 1983-1984 involving French soldiers in Chad and in 1979-1980 in residents of Algeria. Hepatitis E virus (HEV) was subsequently implicated by serology. In this study, the presence of HEV in patient stool specimens from both outbreaks and from sporadic cases in residents of Chad (1994) was documented. This virus was detected in fecal suspensions by antibody capture of the virus and reverse transcriptase-polymerase chain reaction amplification of the viral RNA in the 3' end of open reading frame 2. Two of five epidemic cases from Chad (1983-1984) were positive, as well as one of five sporadic cases from Chad (1994), and two of three epidemic cases from Algeria (1979-1980). Of these 13 patients, 12 had detectable anti-HEV IgG in their serum. These results confirmed that HEV was the cause of hepatitis in at least five of these 13 patients.

Use of digoxigenin-labeled RNA probe to test hepatitis A virus antiviral drugs.

A nucleic acid hybridization assay was used to evaluate inhibitory activity of antiviral compounds against hepatitis A virus (HAV) in cell culture and compared to radioimmunoassay by analysis of variance procedure. The 5' genomic end of the HM-175 strain was used as digoxigenin-labeled RNA probe. Dot-blot examination showed a reduction of detectable HAV RNA in infected cells when treated with amphotericin B. An antiviral dose-effect was shown by statistical analysis of densitometric measures of hybridization signals. Comparison between molecular hybridization assay and radioimmunoassay by analysis of variance procedure showed the equivalence of both methods. Data previously obtained on selected drugs by antigen and infectious titres determinations were confirmed by hybridization assay and make possible digoxigenin-labeled RNA probe use to measure an antiviral dose-effect for screening of hepatitis A antiviral compounds.

Development of an RNA/RNA hybridization assay for the detection of the HAV CF53 strain.

A quick and sensitive dot-blot assay using non-radioactive labelled RNA probes was developed for the detection of the CF53 strain of hepatitis A virus (HAV) in cell culture. The cDNA of the 5' end of the HM175 strain was inserted in a transcription vector pSPT18 and was used to synthesize 32P- or digoxigenin-labelled RNA probes. These RNA probes specifically detected the RNA of the CF53 strain and can be used to detect HAV in PLC/PRF/5 cells. The sensitivity of non-radioactive tests was comparable to that of radiolabelled probes.

Studies on mechanism of action of glycyrrhizin against hepatitis A virus replication in vitro.

Glycyrrhizin (GL) achieved a concentration-dependent inhibition of the replication of hepatitis A virus (HAV) in PLC/PRF/5 cells. GL has been shown to inhibit an early stage of the HAV replication. GL was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labelled virions to cells. GL inhibited HAV penetration of the plasma membrane as measured by the amount of infective virus no longer neutralizable by specific antibody over time.

[Detection of hepatitis A virus by riboprobe labeled with digoxigenin: comparison of methods]. / Détection du virus de l'hépatite A par ribosonde marquée à la digoxigénine: comparaison de méthodes.

A riboprobe (RNA probe), corresponding to the 5' end of the HM175 hepatitis A virus (HAV) genome, was synthetized in vitro and was digoxigenin-labeled. Then the riboprobe was used to detect the CF53 HAV strain. Conditions of virus denaturation (with or without SDS and proteinase K, timing of assay) to release viral RNA were tested by dot-blot hybridization on a ten fold dilution of HAV suspension. Densitometric measures of dot-blot spots allowed to appreciate optimization of the method. Sensitivity of hybridization was compared with sensitivity of radioimmunoassay (RIA) and cell culture methods. Hybridization signals and scale of HAV suspension were consistent when 0.05% SDS, 0.17 micrograms/ml Proteinase K, 37 degrees C, 30 mn or 3 hours are used. 8.10(2) TCID50 HAV was detected by hybridization with digoxigenin-labeled RNA probes. Detection threshold was the same as radioimmunoassay and lower comparatively to cell culture.

[Water and viral hepatitis]. / L'eau et les hépatites virales.

The main agents responsible for acute viral hepatitis throughout the world are the hepatitis A virus (HAV) and the hepatitis E virus (HEV). Both are transmitted by fecal-oral route and can provoke large epidemics, HAV in developed countries and HEV in developing countries. Water is a major vehicle of spread. However, two different epidemiological patterns have to be distinguished: level of HAV excretion is short but high. Because of its resistance to physical and chemical agents, HAV remains infectious for a long time under environmental conditions. Progress in hygiene have nearly stopped the circulation of HAV in industrialized countries, making populations more susceptible to the infection and increasing the epidemic risk. Conventional measures sometimes fail to prevent HAV infections. Vaccine is currently the best way for hepatitis A prophylaxis; HEV is excreted briefly and at low concentrations. Viral particles are fragile in vitro and their viability in environment is not yet understood. Epidemics mainly occur in countries with poor sanitary conditions, resulting from heavy water pollutions. High case-fatality rates are observed, especially among pregnant women. The control of enterically transmitted viral hepatitis remains a major public health challenge. Virological surveillance of waste water could improve strategies based on hygiene, sanitation and supply of drinking water.

Antiviral activity of carrageenan on hepatitis A virus replication in cell culture.

Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5. No cytotoxic effects were detected with concentrations of carrageenans up to 200 micrograms/ml. The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50% (CD50) to the effective dose that inhibited 50% of viral antigen expression (ED50), were greater than 400 with iota-carrageenan, greater than 222 with lambda-carrageenan and greater than 10 with kappa-carrageenan. The selectivity index of ribavirin (reference substance) was only 5. The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity. lota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.

Inhibition of hepatitis A virus replication in vitro by antiviral compounds.

Forty antiviral compounds were screened for inhibitory effect on hepatitis A virus (HAV) antigen expression in the human hepatoma cell line PLC/PRF/5. Ribavirin, amantadine, glycyrrhizin, and pyrazofurin were selected in this screening test and were studied further. The selectivity indices of these four compounds, calculated as the ratio of 50% cytotoxic dose (determined by the trypan blue exclusion and by inhibition of [3H] leucine incorporation) to the 50% effective dose (determined by the viral antigen expression), were 4.6 and 3.0 with ribavirin, 5.3 and 5.9 with amantadine, 15.2 and 16.9 with glycyrrhizin, and 45.4 and 74.6 with pyrazofurin. All four compounds resulted in concentration-dependent reductions of HAV antigen expression and HAV infectivity. Ribavirin, amantadine, pyrazofurin, and glycyrrhizin emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.

[Comparative study of plasmid resistance to mercury of 2 bacterial strains of animal origin]. / Etude comparative de la résistance plasmidique au mercure de deux souches bactériennes d'origine animale.

Bacterial resistance to mercury has been studied in two different strains from animal origin, Salmonella typhimurium 9205 and Escherichia coli 467. These two strains are resistant to mercuric chloride but sensitive to phenylmercury, and thus belong to the group of bacteria that possess a "narrow" spectrum resistance. The presence of plasmids within the cells has been demonstrated through conjugation experiments and direct detection of extrachromosomal DNA in transconjugants. These plasmids, termed p9205-1 and p467-30, differ from each other by both their size (100 and 50 Kbp, respectively) and structure, as shown by the restriction patterns arising from digestion by nucleases BamHI, HindIII, PstI and EcoRI. Each plasmid has been treated with enzyme SalI to yield DNA fragments that have been cloned into pBR322. Two recombinant plasmids, p9205-1/Sal and p467-30/Sal, have thus been constructed, each of them harboring a 2 Kbp fragment that appears to contain the merA gene coding for mercuric reductase. From the analysis of the restriction maps of these recombinant plasmids as well as the functional behaviour of the bacteria that they are able to transform, it can be concluded that they are identical.

[Plasmid resistance to antiseptics. Demonstration of methods]. / Etude de la résistance plasmidique aux antiseptiques. Mise au point de méthodes.

Susceptibility to chlorhexidine, benzalkonium chloride and mercury chloride was studied for 70 Gram negative strains (56 Enterobacteriaceae and 14 Pseudomonas aeruginosa) recovered from humans or animals. Minimal inhibitory concentration (MIC) was determined on solid agar (replica plating) and in a liquid medium (laser nephelometry). For resistant strains, plasmidic mediation was looked for using conjugation. Isolation of lysates by the miniprep analytic method and physicochemical characterization of plasmids by agar electrophoresis were carried out using conjugative strains. MICs obtained using the two methods differed by one dilution at the most. Both methods yielded reproducible results. Regardless of the origin of strains, a resistant population was identified only for mercury chloride. This resistance was plasmid-mediated and transferred singly or associated with resistance to other antibiotics.

[Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium]. / Adaptation à la lignée cellulaire PLC/PRF/5 du virus de l'hépatite A libéré dans le surnageant de culture.

The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th). The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5.