Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.Equine arteritis virus (EAV) is the type member of the family Arteriviridae (38), which was recently grouped together with the coronaviruses and the toroviruses in the newly established order of the Nidovirales (4, 13). Other members of the Arteriviridae are Porcine reproductive and respiratory syndrome virus, Lactate dehydrogenase-elevating virus, and Simian hemorrhagic fever virus.EAV is a spherical, enveloped virus with a diameter of 50 to 60 nm (18, 30) and a positive-stranded RNA genome of about 12,700 nucleotides (nt) (8). The virion envelope is derived from intracellular host cell membranes and contains two major and three or four minor structural proteins (12,38,39). The envelope surrounds an isometric nucleocapsid of about 35 nm (18), which is composed of the genomic RNA and multiple copies of the nucleocapsid protein (N).The EAV replicase is produced in the form of two large polyproteins: the open reading frame 1a (ORF1a) protein and the ORF1ab protein, the C-terminal part of which is expressed by ORF1a/1b ribosomal frameshifting (8). An apparently complete proteolytic processing scheme for the ORF1a and ORF1ab nonstructural polyproteins, which results in the generation of 12 end products (nsp1 to nsp12) and a large number of processing intermediates, was recently obtained (41,46,47,50).The EAV structural proteins are translated from a 3Ј-coterminal nested set of subgenomic (sg) mRNAs, which also carry a common 5Ј leader sequence derived from the 5Ј end of the genome (11). The mechanism of sg mRNA transcription, which resembles that of the coronaviruses in many aspects, involves a discontinuous step of which many details remain to be elucidated (4,24,38,48).Little is known about the genome replication of arteriviruses at the molecular level. The genomic 3Ј end is polyadenyla...

Chimeric yellow fever virus (YF) RNAs were constructed in which the YF structural genes were replaced by the hepatitis C virus (HCV) structural genes or fusions between the YF and HCV structural genes. Interestingly, RNA replication required nucleotide complementarity between the 3-located conserved sequence 1 and an RNA sequence located in the 5 end of the YF capsid sequence. The (chimeric-)HCV structural proteins were efficiently expressed and processed, and the native E1/E2 heterodimer was formed. However, no indication for the production of HCV-like particles was obtained. Yellow fever virus (YF) is the type member of the genusFlavivirus in the family Flaviviridae, a group of viruses that also contains the genera Pestivirus and Hepacivirus (for recent reviews, see references 5 and 23). The YF genome is a positivestranded RNA molecule of approximately 11 kb that contains a 5Ј end cap structure. The 3Ј end is nonpolyadenylated but instead contains an extensive secondary RNA structure (17,27,28). The genome encodes for a single polyprotein of over 350 kDa that is co-and posttranslationally processed by host and viral proteases (23). The capsid (C) protein is released from the N-terminal part of this polyprotein by the viral protease NS2B-3. The envelope proteins prM and E are processed by signal peptidase cleavages, while the YF nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) are processed from the remainder of the polyprotein by host proteases and the viral NS2B-3 protease.Hepatitis C virus (HCV) has been classified as a separate genus (Hepacivirus) in the family Flaviviridae. HCV encodes a polyprotein of approximately 3,000 amino acids. Co-and posttranslational cleavage of the polyprotein generates at least 10 polypeptides: the putative structural proteins C, E1, and E2 and several nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). In addition, a small hydrophobic polypeptide (p7) is encoded between the E2 and NS2 genes. The function of this polypeptide is unknown, but studies on the closely related bovine viral diarrhea virus p7 protein indicate that it has a function in virion formation (18). The C protein is the putative nucleocapsid protein, and E1 and E2 are the HCV envelope glycoproteins (15, 25) that form heterodimers stabilized by noncovalent interactions (11). These heterodimers are believed to constitute the native prebudding complex (11) and do not leave the endoplasmic reticulum (ER) due to retention signals present in the C termini of E1 and E2 (8, 9, 14).Studies on HCV have been hampered by the lack of an efficient cell culture system. Recently, a system that allowed the replication of selectable full-length HCV genomes in a hepatoma cell line was established (19,26). Despite the expression of HCV structural proteins, no evidence for the assembly of virions could be obtained.As a tool to study the expression of the HCV structural proteins and possibly HCV assembly, chimeric YF/HCV genomes were constructed in which the structural genes of YF-17D were replaced by the structu...

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates,. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

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