Hepatitis E virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention.

Hepatitis E virus (HEV), the sole member of the genus Hepevirus in the family of Hepeviridae, is the major cause of several outbreaks of waterborne hepatitis in tropical and subtropical countries and of sporadic cases of viral hepatitis in endemic and industrialized countries. Transmission of HEV occurs predominantly by the fecal-oral route although parenteral and perinatal routes have been implicated. The overall death rate among young adults and pregnant women is 0.5-3% and 15-20%, respectively. HEV is a small non-enveloped particle that consists of a polyadenylated single-strand RNA molecule containing three discontinuous and partially overlapping open reading frames. There are four major genotypes of HEV and a single serotype. At present, there are approximately 1,600 sequences of HEV that are already available at INSDC of both human and animal isolates. Diagnostic and molecular assays have been described for the accurate differentiation of ongoing from remote infection of HEV. Identification and characterization of swine HEV in the United States, Japan, and many other countries and their close relationship to locally characterized human HEV found in the same geographic areas prove that HEV is indeed a zoonotic virus and that domestic swine, wild deer, and boars are reservoirs of HEV in nature. A cell culture system for the propagation of the virus has been described, and a very successful phase 2 vaccine trial has been completed. This review summarizes the current knowledge on the molecular biology, clinical features, transmission, diagnosis, epidemiology, and prevention of HEV.

Verses, viruses, and the vulnerability of the blood supply in industrialized countries.

In the last 30 years, tremendous progress in identifying transfusion-transmitted viruses such as HBV, HCV, and HIV in industrialized countries has been achieved. Currently, the residual risk of transmitting these viruses through transfusion is very low especially after the introduction of "minipool" nucleic acid-amplification tests. Despite these major technical advances, there remains a legitimate concern as to the transmission of other blood-borne infectious agents through blood transfusion. Among these agents are HBV mutants, occult HBV, and HCV infections, malaria, Chagas, West Nile, dengue, and vesiviruses, bacterial infections such as Yersinia enterocolitica, and tick borne diseases such as human monocytic ehrlichiosis, human granulocytic ehrlichiosis, Rocky Mountain spotted fever, and Lyme and prion diseases such as Creutzfeldt and variant Creutzfeldt. Most of these agents are very rarely transmitted by transfusion in industrialized countries. However, an awareness of their possible transmission is essential for the control of spread of these diseases among the public by human-to-human transmission via blood transfusion. This review summarizes the current status of prevalence and diagnosis of these emerging diseases and also updates our knowledge on recently discovered non-pathogenic blood-borne viruses such as GB virus C and Torque Tenoviruses.

Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes.

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq-, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1-D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man.

Quantitation of hepatitis B surface antigen by an automated chemiluminescent microparticle immunoassay.

A fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) was used for the detection and quantitation of hepatitis B surface antigen (HBsAg). The assay is capable of processing up to 800 HBsAg tests per hour. The concentration of HBsAg is determined by utilizing a previously generated Architect HBsAg calibration curve. Architect HBsAg QT sensitivity was found to be around 0.2ng/ml which is equivalent or superior to other known and commercially available enzyme immunoassays and/or chemiluminescent immunoassays. We performed a quantitative study of HBsAg, HBeAg, HBV-DNA and HBV-DNA polymerase in over 733 sera obtained from 43 chronic hepatitis B carriers. Serum HBsAg levels detected by Architect HBsAg QT were found to be higher in HBeAg-positive than in anti-HBe-positive HBV chronic carriers and correlated with the level of serum HBV-DNA and HBV-DNA polymerase.

Selectively primed adaptive driver RDA (SPAD-RDA): an improved method for subtractive hybridization.

A modification of the Representational Difference Analysis (RDA) method for subtractive hybridization, termed Selectively Primed Adaptive Driver (SPAD) RDA, is described. It differs from conventional RDA primarily in the manner by which initial driver (D) and tester (T) amplicon complexities are determined, and by optimizing the composition of D with respect to T for each round of subtraction. Total nucleic acid is extracted from serum or plasma and converted to double-stranded DNA/cDNA. A polymerase chain reaction (PCR) primer containing a selective nucleotide(s) at its 3'-end is used to generate amplicons of reduced complexity. Parallel subtractions are carried out, D vs. T (DT) for enrichment of tester-unique sequences and D vs. D (Driver Control or DC) to generate an optimized driver for use in the subsequent round. Following each round, agarose gel electrophoresis is used to visually identify any DT-unique bands through a side-by-side comparison of DT and DC subtraction products. In comparison to conventional RDA, SPAD-RDA achieved greater enrichment of viral sequences from an HCV infected chimpanzee, resulting in isolation of 13.7% of the viral genome, and an overall enrichment for HCV sequences of 239-fold. Virus fragments were also obtained from an HCV-infected human sample subtracted against non-paired human driver sequences. J. Med. Virol. 71:150-159, 2003.

DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice.

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.

Detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral L1 genome segment.

A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.

Detection of mammalian reovirus RNA by using reverse transcription-PCR: sequence diversity within the lambda3-encoding L1 gene.

Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 +/- 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.

Identification of a novel variant of hepatitis E virus in Austria: sequence, phylogenetic and serological analysis.

We isolated a novel hepatitis E virus (HEV-Au1) variant from a patient in Austria suffering from acute viral hepatitis, who had no known risk factors for acquiring hepatitis E. The clinical presentation and initial serological findings have been reported previously. In this paper we report the results of sequence and phylogenetic analysis of HEV products from viral RNA isolated from acute phase serum. The results show that HEV-Au1 is significantly divergent from other HEV isolates. The nucleotide identity of analysed fragments from the novel isolate ranges from 76.6 to 78.4% when compared to isolates from endemic regions and 84.6 to 87.9% when compared to isolates from non-endemic regions. Divergent results were obtained when serum samples taken from the convalescent phase of disease were tested with three different immunoassays (EIAs). An EIA based on United States isolate-specific peptides showed enhanced reactivity whereas EIAs based on recombinant proteins derived from prototype HEV strains from Burma and Mexico were unable to detect antibodies to HEV (anti-HEV) in late phase serum. The findings verify the presence of an additional HEV variant in an industrialized country and provide information about possible problems in detecting anti-HEV.

Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals.

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34.1% in volunteer blood donors, 39.6% in commercial blood donors, 59.6% in non-A-GB hepatitis cases, 81.7% in injectable drug users and 95.9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A-GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.