Complete nucleotide sequence and genome organization of a Chinese isolate of tobacco bushy top virus<Superscript>*</Superscript>

Summary.  The complete nucleotide sequence of a Chinese isolate of tobacco bushy top virus (TBTV), designated TBTV-Ch, was determined from cDNA generated from double-stranded RNA extracted from diseased tobacco. The genome is 4152 nucleotides (nt) in size, contains four putative open reading frames (ORFs) and untranslated regions of 10 nt and 645 nt at the 5′ and 3′ ends, respectively. In genome organization and in the amino acid sequence of its potential products, the RNA of TBTV-Ch is similar to other umbraviruses sequenced to date. The results suggested that TBTV should be regarded as a definitive species of the genus Umbravirus. Received May 15, 2002; accepted September 6, 2002     

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3′ untranslated region of RNA

Summary.  The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3′ untranslated region (UTR). To investigate the effect of the 3′UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA▵, that lacked the RNA region from nt 1286 to 8785. A series of MA▵ RNAs that differ in the primary structure of their 3′UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3′X resulted in enhancement of the translation efficiency. Translation of MA▵ RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA▵(1041) RNA composed of nt 1 to 1041 but MA▵(8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3′UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA▵ RNA. Received May 10, 2000 Accepted October 11, 2000     

Molecular characterization of a satellite RNA associated with blackcurrant reversion nepovirus

Summary.  A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M_r of 44 220. The coding region was bordered by a 5′ leader sequence of 25 nt and a 3′-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5′-terminal consensus sequence and a 40 nt motif (located at positions 264–303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs. Received March 22, 1999/Accepted August 30, 1999     

2a型丙型肝炎病毒5’非编码区的RACE护增及二级结构的分析

目的：获得真全长中国大陆2a型丙型肝炎病毒（HCV）5’非编码区（5’UTR）cDNA，并分析其一级结构和二级结构，为进一步研究其在HCR复制、翻译中的调控机制、开发新的抗HCV药物奠定基础。方法：利用逆转录套式聚合酶链反应（RT－PCR）联合限制性内切酶长度多态性分析（RFLP）初步筛选出1例2a型HCV感染者，采用cDNA末端快速扩增技术（RACE）扩增出一条约800bp的cDNA片段，A－T克隆，用RFLP与PCR鉴定重组子，全自动序列分析仪测定插入子序列，RNAdraw预测二级结构。结果：RACE法获得真全长2a型HCV 5’端序列。5个克隆中，3个克隆含全长5’UTR序列，与HCV－1，HC－C2，HC－J6，HC－J8相比，同源性分别为93．6％－94．4％，92．1％－93．0％，98．8％－99．7％，96．2％－96．5％，与2a型标准株HC－J6相比，21，170，222，247，339位不同，分别为G，A，C，C，T,但这些突变不影响其二级结构。另外2个克隆为5’端缺失突变株，分别缺失54bp和144bp。结论：RACE技术快速、有效、实用，可用效获得病毒基因组的末端序列；依此获得中国大陆2a型HCV的5’UTR cDNA；在感染者血液中存在5’端部分缺失的HCV基因片段。     

Characterization of the large (L) RNA of peanut bud necrosis tospovirus

Summary.  The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing ‘core polymerase region’ were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 5′ and 3′ termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 nt complementary sequence with one mismatch. Comparisons of the predicted amino acid sequence of the L protein of PBNV with other members of Bunyaviridae suggest that the L protein of PBNV is a viral polymerase. The L protein had highest identity in the ‘core-polymerase domain’ with the corresponding regions of other tospoviruses, tomato spotted wilt virus and impatiens necrotic spot virus. Received October 17, 1997 Accepted June 16, 1998     

Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera

Summary. A potexvirus was isolated from creeping phlox (Phlox stolonifera) plants from a commercial nursery in Pennsylvania. The virus was serologically related to clover yellow mosaic virus, plantain virus X, potato virus X, and potato aucuba mosaic virus, and was most closely related to papaya mosaic virus (PapMV). The sequence of a PCR fragment obtained with potexvirus group-specific primers was distinct from that of PapMV; the coat protein (CP) gene and 3′ untranslated region (UTR) were closely related to Alternanthera mosaic virus (AltMV), previously reported only from Australia. The host range was similar to that of the Australian isolate (AltMV-Au), and the phlox isolate reacted strongly with antiserum to AltMV-Au. The full sequence of the phlox isolate was more closely related to PapMV throughout the genome than to any potexvirus other than AltMV-Au, for which only the CP and 3′UTR sequences are available. The phlox isolate was therefore named AltMV-PA (for Pennsylvania), and the full 6607 nt sequence is presented¹. Additional AltMV isolates from creeping phlox (AltMV-BR and AltMV-SP) and trailing portulaca (Portulaca grandiflora; AltMV-Po) were also isolated, suggesting that AltMV may be widespread, and may have been mis-diagnosed in the past as PapMV. AltMV has the potential to spread to other ornamental crops. ¹The full sequence of AltMV-PA has been deposited in GenBank as Accession Number AY863024; the 3′-terminal sequences of AltMV-PA, AltMV-BR, AltMV-SP, and AltMV-Po have been deposited under Accession numbers AY850929, AY850928, AY850931, and AY850930, respectively.     

Genome analysis of hepatitis C virus strain 274933RU isolated in Russian Federation

Five overlapping cDNA fragments of hepatitis C virus (HCV) isolate 274933RU, obtained by RT-PCR, were amplified and cloned. Complete nucleotide RNA sequence has been determined. The genomic organization of 274933RU was, from 5' to 3' terminals, 5' UTR (341 nt), polyprotein ORF (9033 nt), 3' UTR (40 nt except for the poly(U-UC) and polypyrimidine stretch), and X-tail (98 nt). Phylogenetic analysis of the core and NS5 genes showed that the isolated strain belonged to HCV 1b subtype.     

Genomic sequence of the RA27/3 vaccine strainof rubella virus

Summary.  The sequence of the genome of the RA27/3 vaccine strain of rubella virus (RUB) was determined. In the process, several discrepancies between the previously reported genomic sequences of two wild RUB strains (Therien and M33) were resolved. The genomes of all three strains contain 9 762 nucleotides (nts), exclusive of the poly A tract. In all three strains, the genome contains (), a 40 nt untranslated region (UTR), an open reading frame (ORF) of 6 348 nts that encodes nonstructural proteins, a 123 nt UTR between the two genomic ORFs, a 3 189 nt ORF that encodes the structural proteins, and a 62 nt UTR. The end of the subgenomic RNA was found to correspond to a uridine residue at nt 6 436 of the genomic RNA. At the nucleotide level, the sequence of the three strains varied by 1.0 to 2.8%, while at the amino acid level, the sequence varied by 1.1 to 2.4% over both ORFs. The RA27/3 sequence will be of use in identification of the determinants of its attenuation, in vaccine production control and in development of second generation RUB vaccines based on recombinant DNA technology. Received June 24, 1996 Accepted January 8, 1997     

The Complete Nucleotide Sequence of a Pakistani Isolate of Watermelon Mosaic Virus Provides Further Insights into the Taxonomic Status in the Bean Common Mosaic Virus Subgroup

Watermelon mosaic virus (WMV) is a potyvirus with a worldwide distribution, but is mostly found in temperate and Mediterranean regions. The complete nucleotide (nt) sequence of a Pakistani isolate of WMV (WMV-Pk) was determined and compared with French isolate (WMV-Fr) and other closely related potyviruses. WMV-Pk showed overall identities of 94.4% (nt) and 96% (amino acid; aa) with the WMV-Fr. However, variability was observed in the 5′ UTR and P1 region. Although sequence identities over most of the genome were well above 90% at both the nt and aa levels, reaching 99.6% (aa) in the CP and 100% (aa) in the 6K1 and 6K2, thereby suggesting that WMV-Pk and WMV-Fr are identical strains, but the sequence identities in the P1 region were only 80.6% (aa) and 82.8% (nt), while that in the 5′ UTR was 82%. These differences may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in two different agro-ecological zones. The sequence of WMV-Pk is very close to that of Soybean mosaic virus (SMV) over most of the genome, except for the N-terminal region, which is subject to recombination between SMV and Peanut stripe virus (PSV)/Bean common mosaic virus (BCMV), as revealed by Simplot and phylogenetic analyses of N- and C-terminal P1, HC-Pro, and 5′ UTR regions of the genome.     

Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate

Summary.  Presently, one of the most economically important pathogens affecting swine is the porcine reproductive and respiratory syndrome virus (PRRSV). This virus is prevalent in herds throughout the world and continues to pose a significant threat as newer and more virulent disease phenotypes emerge. In this report we describe the full-length nucleotide sequence of a Canadian PRRSV isolate, designated PA8. A consecutive sequence of 15,411 nucleotides was obtained from a set of overlapping cDNA clones. In order to determine the extent of genetic variation among isolates recovered from swine in Canada and the US, as well as to understand the molecular mechanisms governing the evolution of PRRSV, the full-length sequence of PA8 was compared with that of two US isolates, VR2332 and 16244B. The genomic sequence of PA8 shared 98.2% and 99.2% identity with 16244B and VR2332, respectively. The untranslated regions (UTR) at the 5′ and 3′ ends of the genome were very well conserved. Notable exceptions include an eight nucleotide difference at the 5′ end of the 5′ UTR of VR2332 relative to PA8 and 16244B and a two nucleotide difference in the 3′ UTR of PA8 relative to VR2332 and 16244B. In contrast to PA8 and VR2332, 16244B possessed two nucleotide differences within the RNA pseudoknot structure of the ribosomal frameshift region between open reading frame (ORF)1a and ORF1b. Amino acid differences were distributed throughout the genome, however they appeared to be most extensive in Nsp1β and ORF5 of the nonstructural and structural coding regions, respectively, suggesting that the evolutionary pressure to conserve these viral genes is somewhat lower. Received January 10, 2000/Accepted May 22, 2000     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

Molecular characterization of a distinct potyvirus from whitegrass in China

Summary.  A potyvirus isolated from perennial whitegrass (Pennisetum centrasiaticum Tzvel.) in North China was characterized at the molecular level. The 3′ terminal nucleotide (nt) sequence of 1669 nt of the viral RNA genome has been determined, which covered the coding region of the C-terminal part of the large nuclear inclusion protein (NIb, RNA polymerase), capsid protein (CP) gene and the 3′ nontranslated region (NTR). The CP gene consisted of 909 nt (including the stop codon) encoding 302 amino acid residues, and the 3′ NTR was 241 nt in length excluding the polyadenylated tract. Sequence comparison of the amino acids of CPs showed that this virus was most closely related to Sorghum mosaic virus and Maize dwarf mosaic virus with percent identities of 77% to 78% while that of the 3′ NTRs suggested that it was most closely related to Zea mosaic virus with identity of 72%. This virus isolate was to some extent closely related to other members of the Sugarcane mosaic virus subgroup of potyviruses for the CP amino acid sequences. Phylogenetic analyses of the sequences indicated that this virus isolate represented a distinct potyvirus, and the name Pennisetum mosaic virus (PenMV) is proposed. Received November 22, 2002; accepted January 8, 2003 Published online March 21, 2003     

Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus

Summary.  The complete nucleotide (nt) and deduced amino acid (aa) sequences of isolates Ali, U, Her (from potato, Solanum tuberosum) and TamMV (from tamarillo, Solanum betacea) of Potato virus A (PVA, genus Potyvirus) were determined and compared with the previously reported sequence of PVA isolate B11. Most parts (proteins) of the polyprotein showed over 95% aa sequence similarity. The cylindrical inclusion (CI) protein and the 6K 1 protein were the most conserved proteins among the five isolates. TamMV was the most different isolate. Sequence similarity between TamMV and the other isolates was the lowest in regions close to the 5′-end [5′-non-translated region (NTR) and P1 region] and 3′-end (N-terminus of coat protein) of the genome. However, the termini of the genome (the first 60 nt of the 5′-NTR and the entire 3′-NTR) were highly similar in all five isolates. A frameshift region in the replicase (NIb) was identified the PVA isolates Ali, B11, Her and U, as compared to TamMV and other potyviruses. Received May 25, 1999/Accepted July 23, 1999     

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida

Summary.  With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750 – 800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a M_r of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3′ end of the viral genome encompassing the 3′ non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3′ non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested. Accepted November 5, 1997 Received July 25, 1997     

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3′ untranslated region (10 nucleotides in length) and 2 insertions in the 3′ untranslated region involving a total of 5 nucleotides. Interestingly, from the 5′ UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3′ UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus. Jin-Sheng Wen and Wen-Zhong Zhao equally contributed to this work. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number: EF011023.     

Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates belonging to the European genotype 1 have recently emerged in South Korea, suggesting potential problems for disease control. In the present study, we attempted to determine the complete nucleotide sequence of the first Korean type 1 PRRSV isolate, designated KNU-07. The full-length genome of KNU-07 was found to be 15,038 nucleotides in length, which was 60 nucleotides shorter than the type 1 prototype strain Lelystad due to a notable 60-bp deletion within the nonstructural protein 2 (NSP2). The KNU-07 genome was shown to consist of a 221-nucleotide (nt) 5′ untranslated region (UTR), a 14,703-nt protein-coding region, and a 114-nt 3′ UTR, followed by a 42-73-bp poly(A) tail. A nucleotide sequence comparison of the KNU-07 genome with 20 complete PRRSV genomes revealed a 10.5–13.3% and 39.5–40.3% divergence from type 1 and type 2 strains, respectively, at the genome level, indicating a high similarity to the virus strains commonly identified as the European genotype. In order to investigate genetic variation and to understand the molecular evolution of the type 1 isolate in Korea, extensive phylogenetic analyses were performed using the ORF5 and ORF7 nucleotide sequences of published type 1 PRRSV isolates. The data further indicated that the newly emerging type 1 isolate KNU-07 belongs to the recently proposed pan-European subtype 1. Taken together, the results of this study describe the genomic characterization of the type 1 PRRSV isolated in South Korea, suggesting a recent introduction of the virus typical for this genotype that has commonly appeared worldwide.     

Full-genome Nucleotide Sequence of a Hepatitis C Virus Variant (Isolate Name VAT96) Representing a New Subtype within the Genotype 2 (Arbitrarily 2k)

Hepatitis C virus (HCV), a single-stranded RNA virus of the family Flaviviridae, has a wide range of genetic heterogeneity: 6–11 genotypes (or 6 clades) have been known and each genotype comprises multiple subtypes. Here we report the entire nucleotide sequence of an HCV isolate from a patient in Moldova with chronic hepatitis (isolate name VAT96). The genetic organization of VAT96 was, from 5 to 3 ends, 5UTR (341 nt), polyprotein ORF (9099 nt), 3UTR (38 nt except for the poly-U and poly-pyrimidine stretch), and X-tail (98 nt). Comparison of the polyprotein amino acid sequence of VAT96 with those of known full-genome isolates assigned VAT96 to the genotype 2 (or clade 2), and further phylogenetic analysis based on a 447-nt sequence that covers part of the C and E1 regions suggested that VAT96 represents a new subtype within the genotype 2, arbitrarily designated 2k VAT96 was unique in that it possessed a U residue prior to GCC at the 5 end of its genome while all the other full-genome HCV sequences start with GCC or ACC. In addition, the polyprotein ORF of HCV-VAT96, like HCV-BEBE1 of 2c, encoded several additional amino acids in excess, compared to 2a and 2b sequences. Despite these characteristics that may be unique to VAT96, the 98-nt sequence of the X-tail of VAT96 was highly homologous to those of other isolates with different genotypes so far reported.     

<Emphasis Type="Italic">Melandrium yellow fleck bromovirus</Emphasis> infects <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and has genomic RNA sequence characteristics that are unique among bromoviruses

Melandrium yellow fleck bromovirus (MYFV) systemically infected Arabidopsis thaliana, although the susceptibility of several A. thaliana accessions to MYFV differed from their susceptibility to the other two bromoviruses infecting A. thaliana. We constructed full-length cDNA clones of MYFV genomic RNAs 1, 2, and 3 and determined their complete nucleotide sequences. Similar to Broad bean mottle bromovirus, (1) the 5′-terminal nucleotide of the MYFV genomic RNAs was adenine, and (2) the “D-arm” was absent from the tRNA-like structure in the 3′ untranslated regions (UTRs) of MYFV RNAs. As unique characteristics, MYFV RNA3 lacked the poly(A) tract in the intercistronic region and contained a directly repeated sequence of about 200 nucleotides and polypyrimidine tracts of heterogeneous lengths in the 5′ UTR. Co-infection experiments using RNA3 clones with or without the duplicated sequence demonstrated that the duplication contributed to the competitive fitness of the virus in Nicotiana benthamiana.