The Genome of Hawaii Virus and its Relationship with other Members of the caliciviridae

Hawaii virus (Hu/NLV/GII/Hawaii virus/1971/US), a member of the genus Norwalk-like viruses (NLVs) in the family Caliciviridae, has served as one of the reference strains for the fastidious caliciviruses associated with epidemic gastroenteritis in humans. The consensus sequence of the RNA genome of Hawaii virus was determined in order to establish its relatedness with other members of the family. The RNA genome is 7,513 nucleotides (nts) in length, excluding the 3-end poly (A) tract, and is organized into three major open reading frames (ORF1, nts 5–5,104; ORF2, nts 5,085–6,692; and ORF3, nts 6,692–7,471). Phylogenetic analysis showed the closest relatedness of Hawaii virus throughout its genome to Lordsdale virus, a Genogroup II NLV. Analysis of the predicted secondary structure of the RNA from the 5-end of the genome and the putative beginning of the subgenomic RNA showed the presence of two hairpin structures at both ends that are similar to each other and to those of other NLVs.     

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.     

Cloning and Sequencing of Complete cDNA of Japanese Encephalitis Virus YL Strain in Taiwan

We determined the complete nucleotide sequence of the YL strain of Japanese encephalitis virus and its amino acid sequence was deduced. Our results displayed that the genome of YL strain contained a single open reading frame of 10,296 nucleotides (nts) which was flanked by untranslated region (UTR) containing 95 bases at the 5-end and 586 bases at the 3-end, respectively. Comparison of sequences showed that the overall amino acid sequence and 3 UTR of YL were similar to those of the virulent strain JaGAr01. However, some significant amino acid differences of viral envelope (E) protein were observed between YL and JaGAr01; the amino acid sequence of E protein in YL strain possessed RGG_(387–389) tripeptide instead of RGD_(387–389) in JaGAr01 and in other strains; and another amino acid is K₍₁₃₈₎ in YL, not E₍₁₃₈₎ found in others. These differences suggested that the YL strain impairs in viral attachment to the cell surface and loses neuroinvasiveness, and therefore this strain was used as a live attenuated vaccine.     

Nucleotide Sequences of Double-Stranded RNA Segments from a Hypovirulent Strain of the White Root Rot Fungus Rosellinia necatrix: Possibility of the First Member of the Reoviridae from Fungus

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×10⁶ to 2.95×10⁶. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5-end (5-ACAAUUU-3) and at the 3-end (5-UGCAGAC-3) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.     

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.     

Nucleotide Sequence and Phylogenetic Analysis of Cucurbit Yellow Stunting Disorder Virus RNA 2

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5 to 3 direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.     

Characterisation of the first complete genome sequence of the roe deer (Capreolus capreolus) papillomavirus

The complete genomic DNA of a novel roe deer (Capreolus capreolus) papillomavirus (CcPV1) was amplified and sequenced from fibropapillomatous skin lesions of a Hungarian roe deer. Viral DNA was detected by a pair of degenerate primers and the remaining genomic sequence was amplified by a long-template high-fidelity PCR and sequenced. The CcPV1 genome was 8032 bp long and contained open reading frames (ORFs) typical for Delta-papillomaviruses (E6, E7, E1, E2, E4, E5, E9, L2, and L1) and a 799 bp long untranslated regulatory region (URR). Phylogenetic analysis based on the 3861 bp long concatenated sequence of the E1-E2-L2-L1 ORFs and on separate alignments of all major ORFs using both neighbour-joining and maximum parsimony methods placed CcPV1 on a distinct branch between Ovine papillomavirus 1 and the other deer papillomaviruses within the Delta-papillomavirus genus, although pairwise nucleotide alignments of L1 ORF sequences determined highest identities with European Elk Papillomavirus (71.2%) and Reindeer Papillomavirus (70.3%).     

Completion of the Porcine Epidemic Diarrhoea Coronavirus (PEDV) Genome Sequence

The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5 end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.     

Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus

Summary. The complete nucleotide sequence of Strawberry pallidosis associated virus (SPaV), a newly identified member of the genus Crinivirus, family Closteroviridae has been determined. RNA 1 is 8067 nucleotides long and encodes at least three open reading frames (ORFs). The first ORF (ORF 1a) specifies a multifunctional protein that has papain-like proteinase, methyltransferase and RNA helicase domains. The RNA-dependent-RNA polymerase is encoded in ORF 1b and is probably expressed by a +1 ribosomal frameshift. The 3 ORF of RNA 1 encodes a small protein with two potential transmembrane helices. RNA 2 is 7979 nucleotides long and encodes 8 ORFs, similar in amino acid sequence and arrangement with those of other criniviruses. SPaV encodes the largest structural protein of closteroviruses sequenced to date as the minor coat protein of the virus has molecular mass of approximately 80kDa. The 3 non-translated regions share nucleotide sequence identities of about 56% and the predicted folding of the non-translated regions is similar. Phylogenetic analyses reveal that SPaV is related most closely to Abutilon yellows virus and Beet pseudo-yellows virus, another virus that has been identified recently to cause identical symptoms on strawberry indicator plants as SPaV.     

Nucleotide sequence of the genome of a citrus isolate of olive latent virus 1

Summary The 3699 nt genome of olive latent virus 1 (OLV-1), described years ago from Southern Italy as a putative sobemovirus, was completely sequenced. OLV-1 genomic RNA was not polyadenylated and had a structure virtually identical to that of species of theNecrovirus rather than theSobemovirus genus. Five open reading frames (ORFs) were identified, of which the 5-proximal encoded a 23 K protein and ended with an amber codon whose readthrough could yield a putative 82 K product. This polypeptide had extensive sequence similarity with polymerases of serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D) and species of the familyTombusviridae and related genera (Dianthovirus andMachlomovirus). Two small ORFs followed, which encoded polypeptides of 8 K and 6 K, respectively. The 6 K product had extensive homology with the comparable 6 K protein of TNV-A and was also related to the 11 K protein of shallot latent carlavirus, one of the triple block polypeptides involved in cell-to-cell virus movement. The 3-proximal ORF was in the same position as the coat protein (CP) cistron of necroviruses and encoded a 30 K product related to CP of both TNV-A and -D. Computer-assisted comparative analysis of structural and non-structural proteins of OLV-1, TNV-A and TNV-D disclosed an overall distant relationship between OLV-1 and TNV-D. OLV-1 genome appeared homologous to that of TNV-A, but differences from TNV-A were the absence of the small ORF downstream of the CP cistron and in the low degree of sequence identity in CP (39% aa identity). OLV-1 is serologically distantly related to TNV-A and even more distantly related to TNV-D. We propose that OLV-1 is a necrovirus species in its own right.The sequence data reported in this paper have been deposited in the EMBL database and given the accession number X85989.     

Sequence of the 3′ half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5

We have determined the nucleotide sequence of the 3-terminal half of the RNA genome of Murray Valley encephalitis virus (MVE) using seven overlapping cDNA clones; an estimated 80–90 nucleotides at the extreme 3-end remain to be sequenced. In conjunction with previous sequence data for the 5 half (16), we can conclude that the MVE genome contains a long open reading frame of 10,302 nucleotides that encodes a polyprotein of 3434 residues. Comparison of the MVE deduced amino acid sequence with that of other flaviviruses shows that MVE is most closely related to Japanese encephalitis virus, consistent with serological studies. Using N-terminal amino acid sequence analysis, three nonstructural proteins (NS1, NS3, and NS5) have been identified and mapped on the MVE genome. MVE NS3 contains sequence motifs suggesting that its amino terminus may function as a serine protease. The central region of NS3 (in the linear amino acid sequence) has motifs that are found in NTP-binding proteins and helicases. MVE NS5 contains a conserved Gly-Asp-Asp sequence that is thought to be essential for RNA-dependent RNA polymerases.     

Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5 terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3 terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.     

Nucleotide sequence of the 3′-terminal region of citrus tatter leaf virus RNA

The 3-terminal sequence of citrus tatter leaf virus lily isolate (CTLV-L) was determined from cloned cDNA. The sequence contains two open reading frames (ORFs). ORF1 encodes a protein that contains consensus sequences associated with the RNA-dependent RNA polymerase. ORF2, which is in a different reading frame within ORF1, can encode a 36 kD protein, putatively identified as a movement protein. CTLV-L coat protein (CP) was found to be located in the C-terminal region of the polyprotein encoded by ORF1. Evolutionary relationships and classification of capilloviruses is discussed.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession number D14455.     

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.     

Trichovirus,a new genus of plant viruses

Summary The genusTrichovirus embraces five viral species (two definitive and three tentative) with similar biological, morphological, physicochemical, and ultrastructural properties. Viral replication is likely to occur in the cytoplasm, where virions accumulate in more or less loose bundles or paracrystalline aggregates. The genome is a 3 polyadenylated, positive-sense, single stranded RNA of 7.5–8.7 kb in size. In definitive species (apple chlorotic leaf spot and potato T viruses), the genome is constructed of three slightly overlapping open reading frames coding for replication-related proteins (ORF 1), a putative movement protein (ORF 2), and the coat protein (ORF 3), respectively. ORFs 2 and 3 are probably expressed through subgenomic RNAs. Grapevine virus A (GVA) and grapevine virus B (GVB), two tentative species, may express an extra small open reading frame at the 3 terminus, encoding, in the case of GVB, a polypeptide with homologies with the small RNA-binding protein of carlaviruses. The taxonomic relevance of this difference in genome organization remains to be ascertained.     

Fine structure of the vaccinia virus gene encoding the precursor of the major core protein 4 a

Summary A 6008 base pair fragment of the vaccinia virus DNA containing the gene for the precursor of the major core protein 4 a, which has been designated P4 a, was sequenced. A long open reading frame (ORF) encoding a protein of molecular weight 102,157 started close to the position where the P4 a mRNA had been mapped. Analysis of the mRNA by S1 nuclease mapping and primer extension indicated that the 5 end defined by the former method is not the true 5 end. This suggests that the P4 a coding region is preceded by leader sequences that are not derived from the immediate vicinity of the gene, similar to what has been reported for another late vaccinia virus mRNA. The sequenced DNA contained several further ORFs on the same, or opposite DNA strand, providing further evidence for the close spacing of protein-coding sequences in the viral genome.     

A highly conserved nucleotide string shared by all genomes of human papillomaviruses

The nucleotide string TAAAACGAAAGT is the longest perfect homology shared by all sequenced human papillomavirus genomes. This nucleotide string, which was also found to be highly specific for human papillomavirus genomes, shares the same genomic position in all viral types (5 end of the E1 open reading frame) and putatively codes in every case for the same amino acids. One possible evolutionary model was used to estimate the probability of random occurrence of the nucleotide string in 10 human papillomavirus genomes. It assumed that the universal string had been subjected to the same mutation rate as the entire E1 open reading frame. The estimated probability was found to be very low, suggesting that the conservation of the string could not have resulted from random divergence and that its conservation among human papillomaviruses is likely to reflect the occurrence of biological constraints. It is speculated that this nucleotide string may be required to code for amino acids indispensable for the nuclear localization of E1-coded peptides or to bind cellular factors affecting viral replicative functions. Definitive evidence is expected to come from oligonucleotide-protein binding experiments and from site-directed mutagenesis of cloned HPV genomes. This motif, universal among human papillomaviruses, is being successfully used in the design of consensus primers from the early region.     

Predicted stem-loop structures and variation in nucleotide sequence of 3′ noncoding regions among animal calicivirus genomes

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The RNA Fold computer program was used to analyze 3-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3-terminal sequences are 40–46 nucleotides in length and 72–91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of –2.1 to –18.2 kcal/mole. The RHDV genomic 3-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of –35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3 noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.Disclaimer: No endorsements are herein implied. Brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standards of the products, and the use of the names by the USDA implies no approval of the products to the exclusion of others that may also be suitable.     

Nucleotide sequence of the 3′ terminal region of the RNA of two filamentous grapevine viruses

Summary The 3 terminal region of grapevine virus A (GVA) and grapevine virus B (GVB), encompassing 1883 and 2136 nucleotides, respectively, was sequenced by the deoxynucleotide chain termination method. Three putative open reading frames (ORF) were identified in both genomic viral RNAs, denoted 1 to 3 in the 5 to 3 direction. ORF 1 encoded a polypeptide with estimated M_r of 31 kDa (GVA) and 36.5 kDa (GVB), possessing the G/D motif of the 30 K superfamily movement proteins, and showing good alignments with putative movement proteins of trichoviruses and capilloviruses. ORF 2 was identified as the coat protein (CP) cistron, coding for polypeptides with an estimated M_r of 21.5 kDa (GVA) and 21.6 kDa (GVB). These CPs showed substantial sequence homology with one another and with CPs of tricho- and capilloviruses, but not of closteroviruses. ORF 3 potentially coded for two small polypeptides with estimated M_r of 10 kDa (GVA) and 14 kDa (GVB). The ORF 3 product of GVB (14 K), but not that of GVA, shared some homology with the 3 terminal polypeptides of different plant viruses that exhibit the zinc finger domain of proteins with nucleic acid-binding properties. GVA and GVB have many properties in common with trichoviruses but possess an extra open reading frame (ORF 3). Whether this finding may have a bearing on the classification of these viruses is unclear. However, until the taxonomic significance of this difference in genome structure is established, it seems plausible to include GVA and GVB as tentative species in theTrichovirus genus.