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.

Antiserum generated by DNA vaccine binds to hepatitis E virus (HEV) as determined by PCR and immune electron microscopy (IEM): application for HEV detection by affinity-capture RT-PCR.

Previously, we have described that injection of an expression vector containing hepatitis E virus (HEV) open reading frame 2 (HEV-ORF-2) generated a strong antibody response in mice. To characterize the reaction of this antiserum with native HEV and to evaluate its potential diagnostic application, we tested the antiserum's ability to bind HEV using immune electron microscope (IEM) and affinity-capture reverse transcription polymerase chain reaction (RT-PCR) amplification. Antiserum to ORF-2 aggregated HEV virions as seen by electron microscopy, providing direct evidence that ORF-2 encodes a structural protein. Antiserum also captured HEV for RT-PCR amplification. This antiserum bound HEV from diverse origins (Asia, Africa, Mexico) at virus concentrations found in patient fecal specimens and bile from inoculated non-human primates. The specificity of the affinity binding was demonstrated when pre-immune sera or sera collected from mice injected with control DNA vector (lacking the HEV ORF-2 gene) failed to bind HEV for RT-PCR amplification and IEM. Specific RT-PCR amplification was confirmed by restriction enzyme digestion of PCR products. The sensitivity of the binding was evaluated by RT-PCR amplification of serially diluted bile containing a genetically divergent HEV, Mexico'86. HEV was detected in a 10(-8) dilution of this bile. This is the first report that antibodies elicited by a DNA vaccine recognize native HEV. Our results indicate that ORF-2 encodes a structural protein and that antiserum to this protein enables simple, sensitive, and specific HEV detection by affinity-capture RT-PCR.

The socioeconomic impact of hepatitis E in Nepal.

Hepatitis E disease is responsible for substantial morbidity in Nepal. A socioeconomic analysis was performed to describe the costs and the effects of hepatitis E disease (HE) on health status in a Nepalese population living in the Kathmandu Valley. A modified health status index was used to quantify healthy days lost associated with HE. One hundred thirty-four individuals recently recovered from HE were interviewed in June 1998. The median age was 22 years and 60% were female. Study participants were sick and bedridden for a median of 22 and 10 days, respectively. The median healthy days lost per individual was 35 (768,000 total per region). The median cost of illness per individual, including direct and indirect, was $37 ($1,238,676 total per region). The percentage of yearly income lost for wage earners totaled 19.4%. Hepatitis E disease is associated with significant costs and loss of healthy days in Nepal. Further research is warranted to understand and limit this common disease.

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.

Hepatitis E virus: complete genome sequence and phylogenetic analysis of a Nepali isolate.

The complete nucleotide sequence of the Nepali strain TK15/92 of hepatitis E (HEV) was determined. It showed the highest sequence homology with the Burmese B1 strain, but closer evolutionary relatedness to the Indian strains. Difficulties in reverse-transcribing and amplifying the hypervariable region in ORF1 suggested that strong secondary structures might be intrinsically responsible for the high mutational rate observed in this region of the HEV genome.

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.

Rates of hepatitis E virus infection and disease among adolescents and adults in Kathmandu, Nepal.

To determine hepatitis E virus (HEV) infection and disease rates in the Kathmandu Valley of Nepal, serum was collected from 757 healthy Nepalese (ages 12-48 years) during March and September 1992 and September 1993. At each visit, reports of interval illness were obtained. Sera were examined for IgG to HEV, using a commercially available kit. Seroconversion was used as a marker for HEV infection, and an episode of hepatitis E was defined as a history of jaundice with seroconversion. Seroprevalence ranged from 16% to 31% and increased with age, whereas both infection and disease rates decreased with age. Infection and disease rates were as high as 99/1000 and 45/1000 person-years, respectively. These results highlight the importance of sporadic hepatitis E as a public health problem among adolescents and young adults in this region.

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.

Malaria among United States troops in Somalia.

PURPOSE: United States military personnel deployed to Somalia were at risk for malaria, including chloroquine-resistant Plasmodium falciparum malaria. This report details laboratory, clinical, preventive, and therapeutic aspects of malaria in this cohort. PATIENTS AND METHODS: The study took place in US military field hospitals in Somalia, with US troops deployed to Somalia between December 1992 and May 1993. Centralized clinical care and country-wide disease surveillance facilitated standardized laboratory diagnosis, clinical records, epidemiologic studies, and assessment of chemoprophylactic efficacy. RESULTS: Forty-eight cases of malaria occurred among US troops while in Somalia; 41 of these cases were P falciparum. Risk factors associated with malaria included: noncompliance with recommended chemoprophylaxis (odds ratio [OR] 2.4); failure to use bed nets (OR 2.6); and failure to keep sleeves rolled down (OR 2.2). Some patients developed malaria in spite of mefloquine (n = 8) or doxycycline (n = 5) levels of compatible with chemoprophylactic compliance. Five mefloquine failures had both serum levels > or = 650 ng/mL and metabolite:mefloquine ratios over 2, indicating chemoprophylactic failure. All cases were successfully treated, including 1 patient who developed cerebral malaria. CONCLUSIONS: P falciparum malaria attack rates were substantial in the first several weeks of Operation Restore Hope. While most cases occurred because of noncompliance with personal protective measures or chemoprophylaxis, both mefloquine and doxycycline chemoprophylactic failures occurred. Military or civilian travelers to East Africa must be scrupulous in their attention to both chemoprophylaxis and personal protection measures.

Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis).

The pathogenesis of experimental hepatitis E has not been thoroughly investigated. The purpose of this study was to more accurately document the events in this disease. Cynomolgus macaques were inoculated intravenously with bile or feces containing hepatitis E virus (HEV). Serum, bile, and liver specimens were evaluated with light microscopy, immune electron microscopy, immunofluorescence microscopy, EIA, and polymerase chain reaction. In the third week, there were histopathologic changes and HEV antigen (HEVAg) in liver, HEV in bile, and alanine aminotransferase (ALT) elevations. Widespread pathologic changes were detected during the fourth week and antibody to HEV (anti-HEV) and peak ALT values in the fifth or sixth week. By the sixth week, HEVAg had disappeared but pathologic changes persisted. This study supports the concept that experimental hepatitis E has an initial phase in which hepatic HEV replication is accompanied by the onset of hepatitis and a later phase in which the appearance of anti-HEV is accompanied by progression of the hepatitis.