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.     

The cloning and sequencing of the virion protein genes from a British isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes.

Previous analysis of porcine respiratory coronavirus (PRCV) mRNA species showed that mRNAs 2 and 3 were smaller than the corresponding transmissible gastroenteritis virus (TGEV) mRNA species (Page et al. (1991) J. Gen. Virol. 72, 579-587). Sequence analysis showed that mRNA 3 was smaller due to the presence of a new putative RNA-leader binding site upstream of the PRCV ORF-3 gene. However, this observation did not explain the deletion observed in PRCV mRNA 2. Polymerase chain reaction (PCR) was used to generate cDNA from the 3' coding region of the putative polymerase gene to the poly (A) tail of PRCV for comparison to the equivalent region from TGEV. The PRCV S protein was found to consist of 1225 amino acids, which had 98% similarity to the TGEV S protein. However, the PRCV S gene contained a 672 nucleotide deletion, corresponding to 224 amino acids (residues 21 to 245 in TGEV S protein), 59 nucleotides downstream of the S gene initiation codon. The PRCV genome from the ORF-3 gene to the poly (A) tail was sequenced for comparison to TGEV in order to identify other potential differences between the two viruses. Four ORFs were identified that showed 98% similarity to the TGEV ORF-4, M, N and ORF-7 genes. No other deletions or any PRCV specific sequences were identified.     

Complete genome sequence of arracacha mottle virus

Arracacha mottle virus (AMoV) is the only potyvirus reported to infect arracacha (Arracacia xanthorrhiza) in Brazil. Here, the complete genome sequence of an isolate of AMoV was determined to be 9,630 nucleotides in length, excluding the 3′ poly-A tail, and encoding a polyprotein of 3,135 amino acids and a putative P3N-PIPO protein. Its genomic organization is typical of a member of the genus Potyvirus, containing all conserved motifs. Its full genome sequence shared 56.2 % nucleotide identity with sunflower chlorotic mottle virus and verbena virus Y, the most closely related viruses.     

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.     

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     

Nucleotide sequence analysis of Plum pox virus isolate W3174: evidence of a new strain

The nucleotide sequence of Plum pox virus (PPV) isolate W3174 was determined. The virus genome consists of 9788 nucleotides (nt), excluding the poly(A) tail at the 3′-terminus, with 5′- and 3′-untranslated regions (UTRs) of 146 and 219 nt, respectively. The deduced polyprotein consists of 3141 amino acid (aa) residues, with the coat protein region consisting of 993 nt (331 aa). In comparisons with PPV-D, -M, -EA and -C isolates, nucleotide identity levels ranged from 79 to 80% for the entire genome and from 78 to 79%, 78 to 81%, and 92 to 95% for the NIb, CP, and 3′-UTR regions, respectively. The majority of nucleotide substitutions in the NIb region of W3174 are silent, while substitutions in the CP region are not silent, giving aa identities of 89–91% and 79–81%, respectively. The HC-Pro region contains the KITC and PTK motifs, and the DAG motif is located at positions 12–14 of the deduced CP aa sequence, all associated with aphid transmission. Phylogenetic analysis based on the complete genome sequence, the NIb, CP, and 3′-UTR region were performed. PPV-W3174 consistently formed a distinct clade or group, when compared to members of all four recognized strains of PPV, indicating that it is genetically distinct. These results are consistent with serological and nucleic acid-based strain typing data and justify recognition of this isolate as representative of a new strain identified as PPV-W.     

Complete nucleotide sequence and genomic organization of a primate calicivirus, Pan-1

Summary.  The primate calicivirus, Pan-1, was originally isolated from several primate species. It displayed typical calicivirus morphology by electron micro-scopy. We determined the genomic sequence of Pan-1 by cDNA cloning and direct RNA sequencing. Pan-1 shares a similar genomic organization and a high degree of sequence identity with feline caliciviruses. The Pan-1 genome contains 8 304 nucleotides, plus a poly-A tail, and is longer than any other calicivirus strains with a completely known sequence. The extra sequences of Pan-1 include a unique 424-nucleotide sequence at the 5′ end of ORF1, additional amino acids at the N-terminus of the capsid, and a longer 3′ UTR. Received March 20, 1998 Accepted July 22     

Absence of a coding region for the helper component-proteinase in the genome of cucumber vein yellowing virus,a whitefly-transmitted member of the <Emphasis Type="Italic">Potyviridae</Emphasis>

Summary. The complete nucleotide sequence of isolates of Cucumber vein yellowing virus (CVYV) has been determined. The viral genome comprises 9734 nucleotides, excluding a 3′-terminal poly(A) sequence. The genome of CVYV has a 5′-non coding and a 3′ non coding region of respectively 67 and 240 nucleotides. The RNA of CVYV encodes a single polyprotein of 3148 amino acid residues and has a deduced genome organization and motifs typical for a member of the family Potyviridae. However, CVYV is atypical because it lacks a coding sequence region for the putative helper-component as well as conserved helper-component-proteinase motifs which may account for its vector relations. All the present coding regions were compared to those from several members of the Potyviridae family. CVYV is most closely related to Sweetpotato mild mottle virus confirming its assignation to the genus Ipomovirus, despite similarities with tritimoviruses.     

Complete genomic sequence of Pepper vein banding virus (PVBV): a distinct member of the genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Summary. The complete genomic sequence of Pepper vein banding virus (PVBV), a potyvirus infecting chilli and other solanaceous plants in south India, was determined and compared with those of other potyviruses. The viral genome contained 9711 nucleotides, excluding the poly-A tail. The length of the 5- and 3-untranslated regions (UTR) were 163 and 281 nucleotides respectively. As for other potyviruses, the PVBV genome has a single open reading frame (ORF) starting at nucleotide 164 and ending at nt 9430, which encodes a polyprotein of 3088 amino acid residues. There are nine putative conserved cleavage sites within the polyprotein, which can result in ten functionally distinct protein products. Phylogenetic analysis of the potyviral polyprotein sequences showed that PVBV is a distinct species of this genus.     

The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain

Summary The complete nucleotide sequence of the RNA genome of turnip mosaic virus Japanese strain (TuMV-J) has been determined from five overlapping cDNA clones and by direct sequencing of viral RNA. The RNA sequence was 9833 nucleotides in length, excluding a 3 terminal poly(A) tail. An AUG triplet at position 130–132 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3164 amino acid residues. Interestingly, a different amino acid sequence (continuous twenty amino acids) within the cytoplasmic inclusion protein between TuMV-J and Canadian strain of TuMV was observed, caused by an insertion and a deletion of nucleotides.DDBJ/EMBL/GenBank accession number D83184.     

Nucleotide sequence of barley stripe mosaic virus RNAα: RNAα encodes a single polypeptide with homology to corresponding proteins from other viruses

The complete nucleotide sequence of RNAα from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5′-terminal sequence of 91 nucleotides and a 3′-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (αa) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the as polypeptide also has limited homology with the 58K (βb) protein encoded by BSMV RNAβ and includes a consensus sequence found in mononucleotide-binding polypeptides.     

Sequence and gene structure of the hepatitis E virus isolated from Myanmar

Hepatitis E virus (HEV) is a causative agent of enterically transmitted non-A, non-B hepatitis. Hepatitis E occurs not only in sporadic forms but also in epidemic outbreaks in the developing world. We have revealed the nucleotide and predicted amino acid sequences of full cDNA of HEV isolated from sporadic hepatitis E of Myanmar. The genome is 7194 nucleotides long, followed by a poly(A) tail, and has three open reading frames. The nonstructural gene is located in the 5 terminus, while the structural gene is situated in the 3 terminus. Our HEV strain has 98.5% nucleic acid identity with the HEV strain cloned by workers at Genelabs Incorporated from Myanmar. The difference is point nucleotide substitutions. There is a high degree of nucleotide relatedness among HEVs isolated from the same geographical location.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the following accession number: D10330.     

Structural Organization of the Genome of SARS-Associated Coronavirus (Strain SoD) Isolated on the Territory of the Russian Federation

Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.     

Hepatitis E virus genotype 4 isolated from a patient with liver failure: full-length sequence analysis showing potential determinants of virus pathogenesis

The full-length genome sequence of a genotype 4 strain of Hepatitis E virus (HEV) (CHN-NJ-H2011) from a patient (in Nanjing, China) with liver failure has been determined. Phylogenetic analysis showed that CHN-NJ-H2011 belongs to genotype 4, subtype 4h. Comparative sequence analysis carried out on a 301-bp fragment of ORF2 showed that CHN-NJ-H2011 shares high nucleotide sequence identity (94.3–94.7 %) with porcine viruses (ch-shsw1 and Ch-estw2) isolated in the same geographical region, pointing to the strong possibility of zoonotic transmission of HEV genotype 4. A broader comparison with other genotype 4 isolates revealed 12 unique amino acid substitutions in ORF1 and three in ORF2 that might serve as signatures of disease severity for genotype 4 HEV infection.     

Genomic characterization of an enterovirus 97 strain isolated in Shandong,China

The genomic characterization of human enterovirus 97 (EV97) strain isolated from an acute flaccid paralysis case in Shandong province, China in 1999, is described. The strain, designated as 99188/SD/CHN/1999/EV97 (abbreviated as 99188), had a genome of 7394 nucleotides. Compared with other EV97 strains, it had 81.3–83.3% nucleotide similarity and 94.0–95.4% amino acid similarity in VP1 coding region, and it had 81.4% complete genomic similarity with prototype strain BAN99-10355. The most striking feature was the deletion of 18 nucleotides in the 3′ end of VP1 coding region, combined with two deletions and one insertion in 5′ and 3′ untranslated regions. All these findings demonstrated the strain 99188 had a distant genetic relationship with other EV97 strains. In the phylogenetic trees generated from VP1 and 3D sequences of human enterovirus species B (HEV-B), the lineages of strain 99188 were not congruent, suggesting the event of recombination. Similarity plot analysis further provided the evidence of recombination with other strains of HEV-B in P2 and P3 coding region. This is the first finding of EV97 in China and the third genomic sequence of EV97 reported.     

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.     

Sequence and analysis of the capsid protein of Nudaurelia capensis ω virus, an insect virus with T = 4 icosahedral symmetry

We have determined the nucleotide sequence of the RNA2 segment of the Nudaurelia capensis ω virus genome. It was found to consist of 2448 nucleotides and contained one long open reading frame (ORF) encoding the 644 residue capsid protein. The deduced amino acid sequence of this protein reveals a positively charged amino terminus, a characteristic exhibited by several other viral capsid proteins, that is thought to be important for interactions between the capsid and the genomic RNA. There are 366 and 150 bases of untranslated sequence on the 5′ and 3′ ends, respectively. The ORF encoding the capsid protein initiates at the second AUG from the Fend. The 5′ proximal AUG specifies a short ORF (30 codons) which terminates 1 base before the initiation codon for the coat protein. Our analysis also revealed the presence of a second, previously unidentified polypeptide associated with Nudaurelia capensis ω virus particles. The amino terminal sequence of this protein corresponds to a portion of the long ORF beginning at codon 571. The lack of an initiation codon near this sequence indicates that the small polypeptide is most likely produced as a carboxy terminal cleavage product from a 70-kDa capsid protein precursor, yielding the previously identified 62-kDa protein and the 8-kDa protein that we have observed. The putative cleavage site would be at an Asn/Phe pair, somewhat resembling known cleavage sites (Asn/Ala) in the T = 3 Nodaviridae. In addition, we have found that there is also a second polypeptide similar in size to that from Nudaurelia capensis ω virus associated with particles of Nudaurelia capensis β virus, the type member of Tetraviridae.     

Sequence analysis of the entire RNA genome of a sweet potato chlorotic fleck virus isolate reveals that it belongs to a distinct carlavirus species

Since the paucity of information on sweet potato chlorotic fleck virus (SPCFV) had precluded its classification, we have determined the complete nucleotide sequence of the single-stranded RNA genome of a Ugandan isolate of SPCFV. The genome is 9104 nucleotides long (excluding the poly(A) tail) and potentially includes six open reading frames (ORFs). Based on genomic organisation and sequence similarity, SPCFV appears to be a member of the genus Carlavirus (family Flexiviridae). However, SPCFV is distantly related to typical carlaviruses, as most of its putative gene products share amino acid sequence identities of <40% with those of typical carlaviruses. Its closest relative is melon yellowing-associated virus, a proposed carlavirus from Brazil, with which it shares ORF5 and ORF6 amino acid sequence identities of 61 and 46%, respectively.     

Genetic characterization and sequence heterogeneity of a canadian isolate of Swine hepatitis E virus

Swine hepatitis E virus (HEV) is a newly identified potentially zoonotic agent that is possibly transmitted to humans from pigs. Swine HEV is prevalent in pig populations and does not cause abnormal clinical symptoms in infected pigs, further implicating a likelihood of a risk of transmission to humans by normal contact. To date in North America, only one strain of swine HEV (strain US swine) has been fully sequenced. In the present study, we identified a swine HEV isolate from pigs in Canada, designated the Arkell strain, and determined the full length of the genomic sequence. The genome of Canadian strain Arkell consisted of 7,242 nucleotides, excluding the poly(A) tail of at least 15 A residues. The genome contained three open reading frames (ORFs), ORF1, ORF2, and ORF3, which had coding capacities for proteins of 1,708, 660, and 122 amino acids, respectively. Comparative analysis of the full-length genomic sequence indicated that the sequence of strain Arkell was distinct from those of all other known HEV isolates by 13 to 27% and shared the highest degrees of identity with human HEV isolates US-1 and US-2, HEV isolate US swine, and the human and swine HEV isolates recently isolated in Japan. On the basis of sequence similarities and phylogenetic analyses, HEV strain Arkell was grouped into genotype 3. The sequence of the Arkell swine HEV isolate differed from those of HEV isolate US swine and HEV isolate Japan swine by 13 and 14%, respectively. To date, two isolates of swine HEV (isolates Arkell and SK3 [D. Yoo et al., Clin. Diagn. Lab. Immunol. 8:1213-1219, 2001]) have been identified in Canadian pigs, and their sequences also differ from each other by 11.8%. Our studies indicate that, as with human HEV strains, swine HEV isolates exhibit extensive genetic heterogeneity.