Long,nearly identical untranslated sequences at the 3′ terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain)

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3 UTR) immediately 5 of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3 UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned the accession number Z34265.     

Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).     

The 3′-nucleotide sequence of an ordinary strain of potato virus S

The sequence of 1346 nucleotides of the 3-terminal region of an ordinary strain of potato virus S was determined from cloned cDNA. This portion of the viral RNA contains two major open reading frames (ORFs), encoding the coat protein of M_r33K and a protein of M_r11K 3 to the coat protein, both of which showed high homology, 93% and 79%, respectively, with the equivalent proteins of the Andean strain. A major block of amino acids that differ between the ordinary and Andean strains was present in both the coat protein and the 11K. These blocks may be responsible for the substantial differences between the ordinary and Andean strains at the symptom and aphid transmissibility levels.     

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5′ trailer region

Summary. Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5′ trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.     

Determination of the 5′- and 3′-Terminal Sequences Completes the Sequences of the Two Double-stranded RNAs of Penicillium stoloniferum Virus S

The two genomic segments of Penicillium Stoloniferum virus S (PsV-S), a member of the Partitiviridae, were recently sequenced and published. We independantly sequenced PsV-S and showed that the original sequence was missing nucleotides at both the 5′ and 3′ termini of both segments. We determined the correct sequence in three independent experiments and found the segments to be 1753 bp (encoding the RNA-dependant RNA polymerase) and 1581 bp (encoding the Capsid Protein). Homology was shown between the 5′ and 3′ ends of PsV-S and other members of the Partitiviridae. The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers AM040148 and AM040149.     

On the variability of the 3′ terminal sequence of the turnip mosaic virus genome

Summary The sequence of the 3-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212 nts upstream of the 3 poly(A)-tract. Determination of the N-terminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the 3-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212 nts) than the 3-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3-NTR of the TuMV-Chi isolate which also measures 212 nts. The 3-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3-NTR of TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp 12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3-NTR of approximately 212 nucleotides in length.     

Nucleotide sequence of the 3′ terminal region of the genome of four Lettuce mosaic virus isolates from Greece and Yemen

Summary.  Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1 ¹ and mo1 ² and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR. Received July 1, 1998 Accepted March 25, 1999     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3′-UTR of the genomic RNA 3

Summary.  In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3′ termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3′ untranslated terminal region (3′-UTR) of the genomic RNA 3. Received March 15, 2000 Accepted September 13, 2000     

Determination of the nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus genome

Summary.  The nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3′ and 5′ non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5′ NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5′ very end, and the extra 9 nucleotides in the US-2 strain. In the 3′ NCR, swine HEV shared 90–91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58–65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV. Received April 20, 2001 Accepted July 10, 2001     

Rice tungro spherical virus: Nucleotide sequence of the 3′ genomic half and studies on the two small 3′ open reading frames

Rice tungro spherical virus (RTSV) consists of a single-stranded RNA genome of about 12 kilobases that contains one large open reading frame, ORF 1 and two small ORFs 2 and 3 at its 3 end (Shen et al., 1993, Virology 193:621–630); it was suggested that ORF 2 was expressed via a frameshift. To study the genomic information of RTSV and the variation between different RTSV isolates, the 3 half of a Philippine isolate and parts of a Thai and an Indian isolate were cloned and sequenced. Significant sequence differences were found in ORF 2 and in the 3 non-translated region. Additional stop codons have been revealed in the previously described ORF 2 in several independent clones from the three different virus isolates, the most conserved stop codon in the middle of ORF 2 being confirmed by direct RNA sequencing. These results suggest that ORF 2 could only express a peptide of about 5 kDa instead of 12 kDa as proposed earlier. Polyclonal antisera were raised against ORF 2 and 3 proteins as fusions with glutathione-S-transferase. Using these antisera we failed to detect any virus-specific peptides in extracts from infected rice plants and in virus preparations. The nucleotide sequence of the 3 end of our RTSV isolates contains several small ORFs and does not contain a repeat of 256 nucleotides found in the published sequence. These results indicate that RTSV could contain an unusually long 3 non-coding region of 1240 nucleotides in length.     

Polymorphism of the 5′ terminal region of Citrus tristeza virus (CTV) RNA: Incidence of three sequence types in isolates of different origin and pathogenicity

Sequences of the 5' terminal region of the genomic RNA from eight isolates of Citrus tristeza virus (CTV) were previously classified into three types (I, II and III), with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. Sequencing of an additional 58 cDNA clones from 15 virus isolates showed that all sequences could be unequivocally assigned to one of the three types previously established. The relative frequency of each sequence type was assessed in 57 CTV isolates of different geographic origin and pathogenic characteristics by RT-PCR with sets of type-specific primers using CTV dsRNA as template. None of the isolates yielded amplification of the type I or II sequences alone, but in 19 of them type III sequences were the only amplification product detected. Within isolates containing more than one sequence type, eight had type II and III sequences, 11 had type I and III sequences, and 19 had sequences of the three types. Isolates containing only type III sequences caused only mild to moderate symptoms in Mexican lime, an indicator species for most CTV isolates, whereas isolates causing stem pitting in sweet orange an/or grapefruit, generally contained sequences type II. None of the sequence types could be traced to a precise geographic area, as all types were detected in isolates from at least nine of the 12 countries from which samples were taken.     

Sequence analysis of the 5′ ends of tomato spotted wilt virus N mRNAs

Summary Messenger RNAs transcribed from the tomato spotted wilt virus (TSWV) RNA genome have characteristic extra non-templated heterogeneous sequences at their 5 ends which may be the result of a cap-snatching event involving cellular mRNAs. In order to investigate the genetic origin of these extra sequences and to gain more insight in the process of cap-snatching as performed by TSWV, nucleocapsid protein (N) mRNAs derived from the TSWV S RNA were cloned and sequenced. Twenty clones were obtained which contained 5-proximal sequences of non-viral origin, ranging in length from 12 to 21 nucleotides. None of the sequences analyzed were identical and no base preference at the endonucleolytic site was observed.     

Sequence determination of the extreme 5′ end of equine arteritis virus leader region

The extreme 5 end of the leader sequence of four equine arteritis virus (EAV) strains was obtained by using rapid amplification of cDNA end method (5 RACE), and sequenced. Seventeen more nucleotides were added upstream of the 5 end of the EAV published genomic sequence. A common feature among the analyzed EAV isolates was the presence of an AUG start codon within the added sequence and the appearance of an intraleader open reading frame (ORF) of 111 nucleotides which was predicted to encode a peptide of 37 amino acids. The role of this putative intraleader ORF has yet to be determined.     

Sequence variation in 5′ termini of rubella virus genomes: changes affecting structure of the 5′ proximal stem-loop

Summary Variation within a 523 nucleotide region proximal to the 5 terminus of seven rubella virus strains has been anlysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5 untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.     

Analysis of the genetic and the corresponding antigenic variability of the VP1 3′ end of ECHO virus type 11 and ECHO virus type 30

The enteroviruses (EV), RNA viruses belonging to the Picornaviridae family, have a high genetic variability due to the absence of the efficient proofreading and post replicative repair activities associated with the RNA polymerase. In the present work, we studied the genetic and the antigenic variability of ECHO virus types 11 (E11) and 30 (E30), which are the most isolated echoviruses serotypes in clinical and environmental samples. We established on the 3′ end of the VP1 gene, consensus sequences of E11 and E30 by alignment of 67 E11 and 247 E30 published sequences in GenBank. Our results of sequences comparison showed that the majority of the mutational sites are situated on the third nucleotide of the codon. These mutations were without consequence on the antigenic sequences of the VP1 protein. Thus, E11 and E30 have a high genetic variability (1/3 of the nucleotides are variable), but a relative antigenic conservation. The analysis of the intertypic antigenic variability between E11 and E30 was obtained by the alignment of the corresponding amino acids sequences relative to the N-terminal part of the VP1 protein. Two discriminating parts were highlighted, probably representing antigenic sites for neutralisation antibodies.     

Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.