Sequence of rice hoja blanca tenuivirus RNA-2

The sequence of rice hoja blanca tenuivirus RNA-2 is analysed and compared to its counterpart in rice stripe tenuivirus. The RNA encodes two proteins, in an ambisense arrangement. The 94 kD pc2, located in the complementary sense RNA, has several features typical of viral membrane (glyco)proteins, and also has regions of local homology to the glycoproteins of the Phleboviruses (Bunyaviridae). The 23 kD pv2 lies in the viral sense RNA and has two small conserved domains that are almost exclusively found in retro-viral membrane glycoproteins. Its genome location is analogous to the NSm protein of several of the Bunyaviridae species, which is thought to have a membrane-related function. The two open reading frames are separated by a large intergenic region which, in common with the other tenuivirus ambisense RNA segments, has a short region that is highly conserved between RStV and RHBV. The significance of these results with respect to the virus structure and gene expression is discussed.The GenBank accession number of the sequence reported in this paper is L54073.     

Nucleotide sequence and genome organization of the medium RNA of Iris yellow spot virus from the United States

Iris yellow spot tospovirus (IYSV) of the family Bunyaviridae causes a serious disease in onion in the USA and other parts of the world. Inspite of its economic importance, the complete genomic sequence of IYSV from the USA is not available. The genome structure and organization of the medium (M) RNA of a Washington (WA) isolate of IYSV were determined and compared to the corresponding region of two isolates previously described from Brazil and The Netherlands. Sequence analysis showed that the M-RNA was 4,817 nucleotides long and potentially coded for the movement protein (NSm) in the viral sense and the glycoprotein precursor (Gn and Gc) in the viral complementary sense. The predicted sizes of NSm and Gn/Gc precursor were 34.7 and 128.84 kDa, respectively. The two open reading frames are separated by a 380 nucleotide intergenic region. Phylogenetic analysis of the NSm and Gn/Gc genes from the WA isolate showed grouping that reflected their respective serogroups. The WA isolate formed a close cluster with the two previously reported IYSV isolates and the IYSV cluster was distinguishable from other tospovirus species. This is the first report of complete genomic sequence of the M-RNA of IYSV from the US. Sequences reported here is available in NCBI GenBank under the following accession no.: FJ361359.     

Sequence Analysis of the Glycoproteins of <Emphasis Type="Italic">Tomato Chlorotic Spot Virus</Emphasis> and <Emphasis Type="Italic">Groundnut Ringspot virus</Emphasis> and Comparison with other Tospoviruses

The tospoviruses Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV) cause high economic losses in several vegetable crops in Brazil. The glycoprotein precursor coding sequence was still not available for these two viruses. In this study, the 3' 4kb M RNA of TCSV and GRSV genome was cloned and sequenced. The sequences were compiled with the available 5' region sequence (NS_M gene and 5' UTR) of the same isolates. The M RNA of TCSV was deduced as formed by 4,882 nucleotides, while of GRSV by 4,855 nucleotides. Both M RNA comprised two ORFs in an ambisense arrangement. The vcRNA ORF coded for viral glycoprotein (G1/G2) precursor of TCSV (128.46kDa) and for glycoprotein precursor of GRSV (128.16kDa). Comparison of the TCSV and GRSV glycoprotein precursor proteins with those of other tospoviruses showed the highest identity with Tomato spotted wilt virus (81 and 79%, respectively). The amino acid sequence comparison of glycoprotein precursor between TCSV and GRSV revealed a high identity of 92%. However, the nucleotide sequence of the M RNA intergenie region showed only 78%. Phylogenetic analysis was done based on glycoprotein precursor and on M RNA intergenic region of tospoviruses and parameters on tospovirus taxonomic classification were discussed.     

Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence,genome organization and homology to other tospoviruses

Summary The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22–34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82–86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis virus (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.     

The glycoprotein gene of Chrysanthemum stem necrosis virus and <Emphasis Type="Italic">Zucchini lethal chlorosis virus</Emphasis> and molecular relationship with other tospoviruses

Two tospoviruses, Chrysanthemum stem necrosis virus (CSNV) and Zucchini lethal chlorosis virus (ZLCV), cause economical losses in several ornamental and vegetable crops in Brazil. The nucleocapsid gene and movement protein sequences had already been reported for both viruses, though the glycoprotein precursor gene sequence was not available. In this study, cDNA fragments (ca. 4 kb) of the M RNA 3' portion of CSNV (isolate Chry-1) and ZLCV (isolate 1038), including the complete glycoprotein precursor gene, partial NSm gene, and the entire intergenic and 3' untranslated regions, were cloned and sequenced. The sequences were assembled with the corresponding 5' region sequence (NSm gene and 5'UTR) of the same isolates to build up the complete sequence of the M RNA segment of both species. The M RNA of CSNV was 4,828 nucleotide-long, while of ZLCV 4,836 nucleotides. Both M RNA molecules comprised two ORFs in an ambisense arrangement. The vcRNA coded for the viral glycoprotein (Gn/Gc) precursor gene of CSNV and ZLCV (both with 127.5 kDa). Comparison of deduced amino acids of the CSNV and ZLCV glycoprotein precursor genes with those of other tospoviruses showed the highest identity with that of Tomato spotted wilt virus (86%) and with that of CSNV (82%), respectively. However, the nucleotide sequence of the intergenic and 3' untranslated regions of CSNV and ZLCV shared lower identities with other tospoviruses. The glycoprotein precursor gene is thought to be a good candidate as additional classification parameter for Tospovirus taxonomy. The presence of the RGD motif in both Gc proteins indicated that they are typical American tospoviruses, which was confirmed by phylogenetic analysis. The membrane topology of both glycoproteins is discussed.     

The NSm proteins of Rift Valley fever virus are dispensable for maturation, replication and infection

Rift Valley fever (RVF) virus belongs to the Bunyaviridae family of segmented negative-strand RNA viruses and causes mosquito-borne disease in sub-Saharan Africa. We report the development of a T7 RNA polymerase-driven plasmid-based genetic system for the virulent Egyptian isolate, ZH501. We have used this system to rescue a virus that has a 387 nucleotide deletion on the genomic M segment that eliminates the coding region for two non-structural proteins known as NSm. This virus, DeltaNSm rZH501, is indistinguishable from the parental ZH501 strain with respect to expression of structural proteins and growth in cultured mammalian cells.     

The S RNA segment of Sandfly Fever Sicilian virus: evidence for an ambisense genome

The complete nucleotide sequence of the S RNA segment of Sandfly Fever Sicilian (SFS) virus (Phlebovirus, Bunyaviridae) was determined from overlapping cDNA clones and by primer extension. The RNA is 1746 nucleotides in length and has two large open reading frames (ORF), one of which (24.8 kDa) is viral-complementary in sense, and the other (30.4 kDa) is in the viral sense. This ambisense genome arrangement has been seen in another member of the Phlebovirus genus, Punta Toro (PT) virus (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306), but not in representatives of either the Bunyavirus or Hantavirus genera of the Bunyaviridae. Comparison of the predicted amino acid sequences for SFS virus with the recognized products of PT S RNA (T. Ihara, Y. Matsuura, and D. H. L. Bishop, 1985, Virology 147, 317-325; H. A. Overton, T. Ihara, and D. H. L. Bishop, 1987, Virology 157, 338-350) indicated that the 24.8-kDa ORF encodes the nucleoprotein (N) of SFS virus, and the 30.4-kDa ORF codes for a nonstructural protein (NSs). Subgenomic messenger RNAs, from which these two proteins are presumably translated, were detected in virus-infected cells.     

Genetic organisation of <Emphasis Type="Italic">Iris yellow spot virus</Emphasis> M RNA: indications for functional homology between the G<Subscript>(C)</Subscript> glycoproteins of tospoviruses and animal-infecting bunyaviruses

Summary.  The complete nucleotide sequence (4838 nucleotides) of Iris yellow spot virus (IYSV) M RNA indicates, typical for tospoviruses, the presence of two genes in ambisense arrangement. The vRNA ORF codes for the potential cell-to-cell movement (NSm) protein (34.8 kDa) and the vcRNA ORF for the viral glycoprotein (G1/G2) precursor (128.6 kDa). Multiple sequence alignment of the NSm and G1/G2 precursor proteins of IYSV with those of other tospoviruses, showed highest homologies to Peanut bud necrosis virus (PBNV) and Watermelon silver mottle virus (WSMV). The potential cell-to-cell movement protein of tospoviruses is highly conserved (40–70% identity), with the exception of the first 60 N terminal amino acids, a domain that clearly diverged. For the G1 and G2 viral glycoproteins, blast searches revealed a significant homology between the C-terminally located tospoviral G1 (G_(C)) protein with the counterpart of the animal-infecting bunyaviruses, suggesting a functional homology for these proteins. Received January 15, 2002; accepted July 10, 2002     

Nucleotide sequence of the S RNA of Lassa virus (Nigerian strain) and comparative analysis of arenavirus gene products

The nucleotide sequence of the small (S) genomic RNA of Lassa virus (strain GA391, of Nigerian origin) has been determined. The RNA has features which conform to those seen in most other arenavirus S RNAs which have been characterised, including conserved terminal sequences, an ambisense arrangement of the coding regions for the precursor glycoprotein (GPC) and nucleocapsid (N) proteins and an intergenic region capable of forming a base-paired "hairpin" structure. Comparison of the nucleotide sequence with that of the Josiah strain of Lassa virus (from Sierra Leone) reveals considerable nucleotide divergence in the third base of codons in the reading frames of all three proteins, although the resulting protein sequences are highly conserved, with 92, 94 and 91% identical residues for the mature glycoproteins G1 and G2 and the N protein, respectively. Sequence alignments of the available arenavirus structural proteins and dendrograms summarising the relationships between the viral proteins are presented.     

Analysis of the Intracellular Transport Properties of Recombinant La Crosse Virus Glycoproteins

The G1 and G2 glycoproteins of La Crosse virus, a member of theBunyavirusgenus of the Bunyaviridae, are encoded as a single open reading frame (ORF) in the viral middle-sized RNA segment. The primary product from this ORF is processed, either cotranslationally or shortly after translation, into the two glycoproteins and a nonstructural protein, NSm, of unknown function. We have expressed La Crosse glycoproteins using vaccinia vectors and studied their processing and localization. When expressed in the native G2-NSm-G1 configuration, both G1 and G2 targeted to the Golgi apparatus as shown by their colocalization with wheat germ agglutinin and acquired resistance to endoglycosidase H. When expressed independently, G2 was targeted to the Golgi apparatus but G1 was retained in the endoplasmic reticulum, indicating that a G1–G2 association is required for Golgi targeting of G1. In contrast to results with other members of the Bunyaviridae, we found that expression of G1 and G2 from separate vectors did not lead to the transport of the G1–G2 complex to the Golgi. However, disruption of the NSm region with a foreign sequence did not interfere with transport of the complex. When a portion of the β-galactosidase gene was inserted in frame into NSm, the glycoproteins derived from this construct were processed and targeted properly and were capable of mediating cell-to-cell fusion.     

The complete nucleotide sequence of a capsicum chlorosis virus isolate from Lycopersicum esculentum in Thailand

Summary. The complete nucleotide sequence of a tospovirus isolated from Lycopersicum esculentum in Thailand was determined. The L RNA comprises of 8912 nt and codes for the RNA-dependent RNA-polymerase (RdRp) (2877 aa). Two ORFs are located on the M RNA (4823 nt) encoding the non-structural (NSm) protein (308 aa) and the viral glycoprotein precursors (Gn/Gc) (1121 aa) separated by an intergenic region of 433 nt. ORFs coding for the non-structural (NSs) and nucleocapsid (N) protein, 439 aa and 275 aa, respectively, were identified on the S RNA (3477 nt) separated by an intergenic region of 1202 nt. The N protein of the Thailand isolate was most closely related to that of capsicum chlorosis virus (CaCV), sharing an amino acid sequence identity of 92.7%. Additionally, multiple sequence analyses revealed significant similarities to tospoviruses of the species Watermelon silver mottle virus and to several putative tospovirus entries in GenBank. Based on these alignments it is proposed to refer to all these different viruses as isolates of CaCV.     

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.     

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.     

Organization of the middle RNA segment of snowshoe hare Bunyavirus

The genetic organization of the M RNA segment of snowshoe hare (SSH) virus, a member of the Bunyavirus genus of the family Bunyaviridae, has been determined. The middle (M) RNA segment has a single open reading frame (ORF) of 1441 amino acids. We have used amino- and carboxy-terminus sequencing and synthetic peptides to map proteins within the ORF. The order of the proteins translated from the single large open reading frame is G2, NSm, G1. The G2 protein extends from amino acids 14 to 299. The molecule is 286 residues long, with a computed nonglycosylated molecular weight of 31,973 Da. It is preceded by a cleaved 13 amino acid signal sequence. G2 includes a long highly hydrophobic sequence and contains three potential N-linked glycosylation sites. The G1 protein occupies the C-terminal end of the open reading frame from amino acids 474 to 1441 (968 amino acid residues) and has a computed nonglycosylated, molecular weight of 108,981 kDa. It has two potential N-linked glycosylation sites, and a potential transmembrane region followed by a potential cytoplasmic domain at the C-terminal end. If membrane associated it has an orientation of N-terminus outer, C-terminus inner. Limited trypsin digestion removes a 33-kDa fragment from the N-terminal end, leaving a virion-associated truncated G1 molecule (amino acids 762 to 1441) with a single N-linked glycosylation site. Between the G2 and G1 molecules there are 174 amino acids, sufficient to code for 19 kDa of protein. Some antibodies raised against peptides within this region react with proteins of 11 kDa (NSm) and 10 kDa present in infected cell lysates, but the exact relationship of these proteins to the open reading frame remains to be determined.     

The genome structure of turnip crinkle virus

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.     

The complete genome sequence of polygonum ringspot virus

The complete genome sequence of polygonum ringspot virus (PolRSV), genus Tospovirus, family Bunyaviridae, was determined. This is the first report of the complete genome sequence for a European tospovirus isolate. The large RNA of PolRSV was 8893 nucleotides (nt) in size and contained a single open reading frame of 8628 nucleotides in the viral-complementary sense, coding for a predicted RNA-dependent RNA polymerase of 330.9 kDa. Two untranslated regions of 230 and 32 nucleotides were present at the 5′ and 3′ termini, respectively, which showed conserved terminal sequences, as commonly observed for tospovirus genomic RNAs. The medium and small (S) RNAs were 4710 and 2485 nucleotides in size, respectively, and showed 99 % homology to the corresponding genomic segment of a previously partially characterized PolRSV isolate, Plg3. Protein sequences for G_N/G_C, N and NSs were identical in length in the two PolRSV isolates, while an amino acid insertion was observed for the NSm protein of the newly characterized isolate. The noncoding intergenic region of the S RNA was very short (183 nt) and was not predicted to form a hairpin structure, confirming that this unique characteristic within tospoviruses, previously observed for Plg3, is not isolate specific.     

Analysis of the genomic sequence of a human metapneumovirus

We recently described the isolation of a novel paramyxovirus from children with respiratory tract disease in The Netherlands. Based on biological properties and limited sequence information the virus was provisionally classified as the first nonavian member of the Metapneumovirus genus and named human metapneumovirus (hMPV). This report describes the analysis of the sequences of all hMPV open reading frames (ORFs) and intergenic sequences as well as partial sequences of the genomic termini. The overall percentage of amino acid sequence identity between APV and hMPV N, P, M, F, M2-1, M2-2, and L ORFs was 56 to 88%. Some nucleotide sequence identity was also found between the noncoding regions of the APV and hMPV genomes. Although no discernible amino acid sequence identity was found between two of the ORFs of hMPV and ORFs of other paramyxoviruses, the amino acid content, hydrophilicity profiles, and location of these ORFs in the viral genome suggest that they represent SH and G proteins. The high percentage of sequence identity between APV and hMPV, their similar genomic organization (3'-N-P-M-F-M2-SH-G-L-5'), and phylogenetic analyses provide evidence for the proposed classification of hMPV as the first mammalian metapneumovirus.     

Dugbe nairovirus M RNA: Nucleotide sequence and coding strategy

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.     

Sequence analysis of the medium RNA segment of three Simbu serogroup viruses,Akabane, Aino,and Peaton viruses

The sequence analysis was carried out for the medium (M) RNA segment of the Akabane virus (AKAV), Aino virus (AINV), and Peaton virus (PEAV) of the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. The complementary sequences of the M RNA segments of AKAV, AINV, and PEAV contain a single large open reading frame (ORF), like other orthobunyaviruses. The ORFs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. The identity of the M segment among these viruses is remarkably low, although previous researchers reported that the small RNA segments are highly conserved. Because the M segment codes for the viral surface glycoproteins G1 and G2, the variability of the M segment may affect the antigenicity of these viruses. Phylogenetic studies based on the M and S segment sequences suggested that genetic reassortment has been occurring among ancestral viruses of the three Simbu serogroup viruses throughout their evolution.