The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3-nontranslated region (3-NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic-and antigenomic-sense transcripts. The results show that the 3-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondarystructure elements located at the 3 end of the HAV RNA. p38 also interacted with a 5-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3D pol -coding sequence and the 3-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis.
The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aerhiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.
Summary. Hepatitis E virus (RNA and antigen) was detected in serial passagesof FRhK-4 cells after they had been co-cultivated with primary kidney cells derived from cynomolgus monkeys experimentally infected with this virus.Hepatitis E virus (HEV) is the etiological agent of enterically transmitted human hepatitis E, acute jaundice disease widely spread in many tropical countries [2,4]. Under laboratory conditions the infection has been reproduced in several species of non-human primates after inoculation with HEV-containing clinical specimens [1,5,8,11]. Numerous attempts to grow this virus in cultivated cells using conventional cell culture inoculation techniques have so far proved unsuccessful. In this paper we report the establishment of cell lines which harbour the HEV.The experiments were performed as follows: (1) Cynomolgus monkeys (Macaca faseicularis) were inoculated with the HEV (2598 Osh strain originated from Soviet Central Asia) as described earlier [1,3]. (2) As soon as the levels of serum alanine aminotransferase became pathologically elevated the animals were sacrified; the kidneys were removed aseptically and used for preparation of primary cell monolayers following the usual procedure [6]. 'The cells were cultivated for 14 days at 37 °C in 500ml flat glass flasks in 0.5% lactalbumin hydrolysate medium supplemented with 15% of fetal calf serum (Flow). (3) The primary cells were sub-passaged 1-2 times in the Eagle's MEM with 10% of fetal calf serum at 7-8 day intervals. (4) These cells were allowed to proliferate until sub-confluent monolayers had been formed, then the medium was decanted and a suspension of fresh fetal rhesus monkey kidney (FRhK-4) cells [13]
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