Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR

The genomic cloning and sequence of hepatitis E virus (HEV) from an epidemic in North India is reported. We describe here a simple method wherein the viral RNA was reverse transcribed and then amplified in a single step using an extra long polymerase chain reaction procedure. The full genome nucleotide sequence of this HEV isolate (called Yam-67) was made up of 7191 nucleotides, excepting the poly(A) tail and had three open reading frames: ORF1 coding for 1693 amino acids (aa), ORF2 coding for 659 aa and ORF3 coding for 122 aa. This North Indian isolate of HEV showed close sequence homology to other HEV isolates from India and Asia, but was distant from the Chinese genotype 4, Japanese, Mexican and US isolates. There is no indication from sequence analysis that this may be an atypical strain of HEV, as reported earlier.     

Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae

The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3' and 5' ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51-53 to a UAG stop codon at bases 6765-6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5' end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.     

Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome

Three serologically distinct groups of hantaviruses have been associated with severe, moderate, and mild forms of hemorrhagic fever with renal syndrome (HFRS). To gain a better understanding of the genetic variation among these viruses, we cloned and sequenced the M and the S genome segments of Sapporo rat virus, an etiologic agent of moderate HFRS, and compared the predicted gene products to those of Hantaan virus, and the H?lln?s strain of Puumala virus, which are etiologic agents of severe and mild HFRS, respectively. The SR-11 S segment consisted of 1769 nucleotides and had an open reading frame (ORF) in the virus-complementary sense RNA with a coding capacity of 429 amino acids. Deduced amino acids from the SR-11 S segment ORF displayed 83% homology with those of Hantaan nucleocapsid (N) protein. Comparison of the S segment ORFs of all three viruses revealed 58% homology. No evidence for additional nonstructural protein(s) encoded by the SR-11 S segment was obtained. The SR-11 M segment consisted of 3651 nucleotides and had an ORF in the virus-complementary sense RNA with a coding capacity of 1134 amino acids. Amino acid sequences predicted from the SR-11 M segment ORF were 75% homologous with those encoding Hantaan G1 and G2 envelope glycoproteins. Comparison of the deduced amino acid sequences of the M segment ORFs of SR-11, Hantaan, and H?lln?s viruses revealed a 43% homology for amino acids constituting the G1 proteins and a 55% homology for amino acids constituting the G2 proteins of the three viruses. The envelope proteins of SR-11 virus were localized within the M segment ORF by amino-terminal sequence analysis of purified G1 and G2. G1 initiated at amino acid 17 and G2 at amino acid 647 within the ORF. Five potential asparagine-linked glycosylation sites were identified in the SR-11 G1 coding sequences, four of which were conserved between Hantaan and SR-11 viruses and three of which were conserved among all three viruses. One potential glycosylation site was identified in the SR-11 G2 coding sequences and was conserved among Hantaan, SR-11 and H?lln?s viruses. Cysteine residues were highly conserved within the M segment ORFs of all three viruses, suggesting a similar structure and function of the G1 and G2 proteins.     

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain H?lln?s B1 coding for the viral RNA polymerase

The L RNA segment of the nephropathia epidemica virus (NEV) strain H?lln?s B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.     

Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan

We identified hepatitis E virus (HEV) RNA in serum from a Japanese patient with acute hepatitis, who had never been abroad. The full-genome nucleotide sequence of the HEV isolate (JRA1) from this patient was composed of 7227 nucleotides excepting the poly(A) tail and had ORF1 coding for 1703 amino acids (aa), ORF2 coding for 660 aa, and ORF3 coding for 122 aa. This Japanese strain showed approximately 87% nucleotide similarity to human and swine strains reported from the United States, while it had only 73-76% similarity to Asian and Mexican strains. Here we report the characteristics of the HEV-JRA1 isolate, which might be the first example of an indigenous strain(s) of HEV in Japan.     

Complete sequence of the Pepino mosaic virus RNA genome

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.     

RNA segment 5 of broadhaven virus, a tick-borne orbivirus, shows sequence homology with segment 5 of bluetongue virus

The sequence of Broadhaven (BRD) virus segment 5, the major genetic determinant of serotype, is 1658 nucleotides in length and contains a single open reading frame (ORF) having the coding capacity for a protein of Mr 52.5K. Comparison of the ORF of segment 5 of BRD virus with published sequences of bluetongue virus (BTV) revealed 30% nucleotide homology and 31% amino acid homology with the protein encoded by segment 5 of BTV serotype 10. Significant homology was not shown with segment 2 of BTV, the major genetic determinant of the BTV serotype. The sequences at the 3' and 5' ends determined for BRD segment 5 were similar to the respective 3' and 5' regions of BTV. The sequence data provide evidence of an evolutionary relationship between two ecologically distinct groups of orbiviruses and demonstrate changes that have occurred in the functions of genetically related genomic segments.     

Recombination and phylogeographical analysis of Lily symptomless virus

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.     

粉尘螨变应原第5组分基因克隆及分子特征分析

目的 获得粉尘螨变应原第5组分的编码基因(Der f5)并了解其分子特征.方法 用RNAiso试剂盒提取粉尘螨总RNA,根据GenBank已公布的Der f5核酸序列设计引物,用RT-PCR扩增获得其编码基因,插入pMD19-T Simple载体进行序列测定和分析.结果 获得的Der f5基因与参考序列(GenBank AY283283)同源性达97.8%,含1个完整的开放阅读框架(ORF),由132个氨基酸组成,信号肽位于1～19AA、跨膜区域位于1～19AA,为细胞外疏水性蛋白.二级结构由延伸主链(1.52%)、无规则卷曲(7.58%)和α螺旋(90.91%)组成.具有酪蛋白激酶Ⅱ磷酸化位点2个.其氨基酸序列与屋尘螨变应原第5组分相似率为78%.结论 成功克隆了 Der f5,并初步预测得其分子特征.     

The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1

The complete nucleotide sequence of red clover necrotic mosaic virus (RCNMV) RNA-1 has been determined. RNA-1 is 3889 nucleotides in length with a 5' terminal m7GpppA cap. The RNA contains three large open reading frames (ORFs): the 5' proximal ORF, encoding a 27-kDa polypeptide; the internal ORF, coding for a 57-kDa polypeptide; and the 3' terminal ORF, encoding the 37-kDa capsid protein. The sequence results confirm in vitro translation of 27-, 50-, and 37-kDa products but do not account for the observed 90-kDa product. A translational frameshift event from the 27- to the 57-kDa ORFs is proposed to explain the synthesis of the observed 90-kDa in vitro product. The putative translational frameshift region is structurally similar to several retrovirus frameshift regions and the putative barley yellow dwarf virus (BYDV) frameshift regions. Extensive amino acid homology was observed in the 57-kDa downstream ORF with the downstream domains of the carnation mottle virus (CarMV), turnip crinkle virus (TCV), maize chlorotic mottle virus (MCMV) readthrough, and BYDV fusion proteins. The 57-kDa ORF contained the conserved "GDD" motif. A significant alignment between the capsid proteins of RCNMV, CarMV, and TCV was also observed. Given the extensive amino acid sequence similarity of RCNMV, CarMV, and TCV polymerase and capsid proteins, we speculate that they are closely related, evolutionarily.     

Sequence and genomic organization of a rabbit hemorrhagic disease virus isolated from a wild rabbit

Rabbit hemorrhagic disease virus (RHDV) is a member of the caliciviridae family. The nucleotidic sequence of a full-length cDNA of one RHDV isolate (RHDV-SD) is reported. The genome is 7437 bases long and includes two ORFs, ORF1 (7034 b) and ORF2 (353 b), coding for the polyprotein and the Vp12, respectively. The coding sequence for the second structural protein (the capsid protein, Vp60) is located at the 3 end of ORF1. Comparison of RHDV-SD with the German RHDV isolate revealed 470 nucleotide substitutions (96% homology). The deduced amino acid sequences of the two isolates are closely related (98% identity), and no hypervariable region could be identified either in the structural or nonstructural proteins.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z29514.     

Complete sequence of the S RNA of lymphocytic choriomeningitis virus (WE strain) compared to that of Pichinde arenavirus

Previous studies have reported that the 3' half of the small, S, RNA species of the WE strain of lymphocytic choriomeningitis (LCM) virus codes for the viral nucleoprotein in a subgenomic, viral-complementary, mRNA species (Romanowski, V. and Bishop, D.H.L. (1985) Virus Res. 2, 35-51). The complete sequence of the LCM-WE S RNA has now been obtained, indicating that the 5' half of the RNA codes for the viral glycoprotein precursor in a viral-sense sequence that does not overlap the N gene. It is concluded that, like Pichinde virus (Auperin, D. et al. (1984) J. Virol. 52, 897-904), LCM has an ambisense S RNA coding strategy. The LCM-WE S RNA is 3375 nucleotides in length, has a size of 1.14 X 10(6) Da and base composition of 26.1% A, 23.2% C, 21.5% G, 29.2% U. The 3' and 5' end sequences of the S RNA are complementary for some 30 nucleotides, depending on the arrangement. The non-coding regions at the two ends are 77 (5') and 60 (3') nucleotides long. The glycoprotein precursor has a primary amino acid size of 56293 Da and is rich in potential glycosylation sites as well as histidine and cysteine residues. It has both amino and carboxy proximal hydrophobic regions. The LCM-WE S RNA and predicted protein sequence data have been compared to those of Pichinde arena-virus. Extensive RNA and protein sequence homology exists for the two S RNA species, although the homology for the glycoprotein sequences of the two viruses (39%) is less than the 50% observed for the two viral nucleoproteins.     

Molecular analysis of the gene encoding the immunodominant phenotypically varying P270 protein ofTrichomonas vaginalis

Trichomonas vaginalisis a flagellated protozoan responsible for the most common non-viral sexually transmitted disease. The immunogen P270 was previously found to be up-regulated in expression and to undergo phenotypic variation between surface versus cytoplasmic localization in trichomonads harbouring a dsRNA virus. In this report, we characterize the entirep270open reading frame (ORF) and the unknown flanking 5- and 3-unique, non-repeat coding sequences of the gene in addition to untranslated regions. Consistent with an earlier report (Dailey & Alderete, 1991,Infect. Immun.59: 2083–88), a significant portion of the gene consists of a tandemly repeated 333 bp element that contains the sequence coding for the epitope DREGRD detected by murine monoclonal antibody and antibody from the sera of patients. The non-repeat coding regions for the 5- and 3-ends were 69 nucleotides (23 amino acids) and 1183 nucleotides (395 amino acids), respectively. Sequencing of repeat elements showed them to be identical, affirming the highly-conserved nature of this element throughout the gene. The start codon was immediately preceded by the 12 nucleotide consensus sequence (TCATTTTTAATA) found in other trichomonad protein-coding genes. A very AT-rich, non-coding region was identified upstream of thep270ORF. P270 appears to contain a leader sequence at the amino-terminus and transmembrane domain at the carboxy-terminus. No significant homology was found with any reported proteins at either the nucleotide or amino acid level.     

Molecular characterization of the RNA S segment of nephropathia epidemica virus strain H?lln?s B1

The S segment RNA of nephropathia epidemica virus (NEV) strain H?lln?s B1 was isolated by molecular cloning of the corresponding cDNA. The RNA is 1785 nucleotides long with the 3' and 5' termini being complementary for 23 bases. The viral messenger-sense RNA contains one major open reading frame (ORF) with a coding capacity of 433 amino acids encoding a 49-kDa polypeptide. Compared to the Hantaan S segment cDNA sequence there is a nucleotide homology of 60 and 61% at the amino acid level. Many of the amino acid differences are conservative exchanges. The C-termini of the NEV and Hantaan nucleocapsid proteins are nearly identical and the hydrophilicity profiles are very similar. In contrast, the following differences are significant: The calculated isoelectric points of the NEV and Hantaan nucleocapsid proteins are 5.6 and 6.7, respectively. The most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the C-terminus of NEV and close to the N-terminus of Hantaan virus nucleocapsid polypeptides.     

Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.     

Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara elegans (Thielaviopsis basicola)

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35-45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.     

Characterization of the early region 3 and fiber genes of Ad7

The nucleotide sequence and the predicted amino acid sequences for open reading frames (ORFs) encoded in the Bam-Hl D fragment of Ad7 (Gomen) DNA show an organization and conservation of potential polypeptides between Ad3 and Ad7. Five ORFs encoded within early region 3 (E3) and shared with the corresponding region of Ad3 can be identified; four of these potential coding regions also share homology to ORFs found in E3 of Ad2 and Ad5. The fiber gene of late region 5 (L5) is also apparent within this region; S1 mapping experiments show that the 5' and 3' boundaries of the main exon in fiber mRNA lie at each end of the proposed fiber ORF. The predicted amino acid sequence for Ad7 fiber shares 60% amino acid homology to Ad3 fiber, but only 20% to Ad2 fiber. Surprisingly, there are three regions of partial amino acid homology near the N- and C-termini of the predicted fiber gene sequences from Ad2, Ad3, Ad5, and Ad7; these conserved regions may be important for interaction with penton base, for proper folding of the shaft of the molecule, or for recognition of the cellular receptor to which adenovirus attaches during infection.     

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene

Summary The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.GenBank Accession No. U24432.