Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.     

Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus

The sequence of the genome of tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) coding for structural proteins and nonstructural protein NS1 has been previously reported (A. G. Pletnev, V. F. Yamshchikov, and V. M. Blinov, 1986, FEBS Lett. 200, 317-321; Yamshchikov and Pletnev, 1988, Nucleic Acids Res. 16, 7750. Now we have cloned and sequenced the genomic RNA that encodes all nonstructural proteins. Together with our earlier sequence analyses, these data show that the TBE genome is 10,477 bases in length with a single open reading frame extending from nucleotides 127 to 10,363, encoding 3412 amino acids. The 5'- and 3'-noncoding regions have stem-loop structures. The polyprotein precursor is proteolytically cleaved, apparently by a mechanism resembling that proposed for the expression of polyproteins of the other flaviviruses, such as yellow fever and Kunjin viruses. The deduced TBE gene order is 5'-C-pre(M)M-E-NS1-NS2A-NS2B-NS3-ns4a-NS4B -NS5-3'. The genome structure and the polyprotein of TBE virus is similar to mosquito-borne flaviviruses, although TBE virus is transmitted by ticks. Comparison of the sequence homology of polyproteins of flaviviruses suggests that TBE virus is more closely related to yellow fever virus than to other serological subgroups of flaviviruses. The hydrophobicity profile of the TBE polyprotein is similar to those of other flaviviruses. Nonstructural proteins NS2A, NS2B, ns4a, and NS4B are extremely hydrophobic, suggesting that these proteins are likely associated with cellular membranes. Proteins E, NS1, NS3, and NS5 are the most conserved and these proteins may be involved in the general activities related to viral reproduction.     

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome

The sequence of the 5'-end of the genome of dengue 2 (Jamaica genotype) virus has been previously reported (V. Deubel, R. M. Kinney, and D. W. Trent, 1986, Virology 155, 365-377). We have now cloned and sequenced the remaining 75% of the genomic RNA that encodes the nonstructural proteins. The complete genome is 10,723 bases in length with a single open reading frame extending from nucleotides 97 to 10,269 encoding 3391 amino acids. The 3'-noncoding extremity presents a stem- and loop-structure and contains a repeated oligonucleotide sequence. Comparisons of the nucleotide sequences of the genomes of dengue 2 viruses of different topotypes reveal 90-95% similarity, with 64-66% similarity evident between dengue viruses of different serotypes. The amino acid sequence of the polyprotein of dengue 2 Jamaica virus shows 97, 68, 50, and 44% similarity with those of other dengue 2, dengue 1, or dengue 4, West Nile, and yellow fever viruses, respectively. Despite amino acid sequence divergence, the hydrophobic profile of the flavivirus proteins is highly conserved. Proteins NS1, NS3, and NS5 are the most conserved. Conserved amino acid stretches present in all flavivirus proteins may be involved in common essential biological functions.     

Genetic characterization of Yokose virus, a flavivirus isolated from the bat in Japan

Yokose virus (strain Oita-36) was isolated from the bat in Japan in 1971. In the present study, we determined complete nucleotide sequences of Yokose virus using RT-PCR and RACE techniques. Yokose virus genome consists of 10,857 nucleotides in length (accession no. AB114858), containing a single open reading frame (3425 amino acids) encoding 11 viral proteins. We deduced the boundaries of each protein in the polyprotein sequence according to the protein cleavage sites of other flaviviruses. The nucleotide sequences of the 5' and 3' nontranslated region (NTR) and amino acid sequences of individual proteins of the virus were compared with those of six other flaviviruses including Japanese encephalitis virus, dengue-2 virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, and Rio Bravo virus or Modoc virus. Yokose virus demonstrated the highest similarity to yellow fever virus. Yokose virus also has CS1 motif, which are well-conserved specifically in mosquito-born flaviviruses, in its 3' NTR. When a part of the NS5 amino acid sequence (345 amino acids) was compared with those of other four flaviviruses, Entebbe bat virus, Sokuluk virus, Sepik virus, and yellow fever virus, the three former viruses are more closely related to Yokose virus than yellow fever virus. Human sera from dengue-virus-infected case and yellow fever vaccine reacted with the viral proteins. Moreover, human serum from a yellow fever vaccine weakly neutralized Yokose virus. Our results suggest that there are cross-reactive antigenicities among Yokose virus and other flaviviruses.     

Cloning and sequence analysis of the genes encoding the nonstructural proteins of Langat virus and comparative analysis with other flaviviruses.

Langat virus, a member of the family Flaviviridae is antigenically very similar to highly pathogenic tick-borne encephalitis viruses. We cloned and sequenced the complete nonstructural gene-coding region of Langat virus (strain TP21) and compared the deduced amino acid sequences of each nonstructural protein to those of other flaviviruses. By alignment with the reported amino acid sequences of the nonstructural proteins of several flaviviruses, we were able to predict proteolytic cleavage sites and identify sequence motifs, which are highly conserved among flaviviruses. Sequence similarity calculations revealed that the NS3 and NS5 proteins are the most highly conserved of the flavivirus nonstructural proteins. The NS3 and NS5 proteins of Langat virus contained specific peptide sequences that have been demonstrated to be associated with helicase or polymerase activities, respectively. The NS1 protein of Langat virus displayed complete homology of potential N-linked glycosylation sites and cysteine residues with the NS1 proteins of other tick-borne flaviviruses, suggesting a highly conserved NS1 protein structure. The data presented in this report serve to complete the entire sequence of the Langat virus-coding region and provide the basis for comparison of this naturally attenuated virus to the other highly virulent tick-borne flaviviruses.     

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.     

Localization and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins

To locate epitopes on internal antigens of rabies virus, purified N and NS proteins of the nucleocapsid were cleaved at methionine, tryptophan or glutamic acid residues, transferred to nitrocellulose and immunostained using monoclonal antibodies (MAbs) specific for N and NS proteins, respectively. Five MAb-positive fragments of N protein and one fragment of NS protein were located after NH2-terminal amino acid sequence analysis within the deduced amino acid sequences of N and NS proteins. Antigenic analysis of synthetic overlapping peptides corresponding to the amino acid sequences of these fragments localized two major antigenic sites of N protein and one antigenic site of NS protein. Like the N- and NS-specific MAbs, anti-peptide antisera produced against the different synthetic antigens either reacted in a type-common fashion with all rabies virus strains, or in a type-specific manner with a restricted number of strains. The synthetic peptides corresponding to the three antigenic regions of the N and NS proteins also stimulated proliferation of human T lymphocytes derived from vaccinees who received inactivated rabies virus vaccine. This suggested that the antigenic regions of N and NS proteins are recognized by both B and T cells.     

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.     

Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein

The cDNAs of rabies virus (the CVS strain) encoding the structural proteins (G, N, NS, and M) were cloned. Of these clones, the nucleotide sequence of the cDNA encoding the nucleoprotein was determined to compare with those of other strains of rabies virus. The comparison confirmed that the nucleotide sequences and deduced amino acid sequences are highly conserved among strains including an avirulent strain.     

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.     

Complete nucleotide sequence of the Japanese encephalitis virus genome RNA

The complete nucleotide sequence of the Japanese encephalitis virus (JEV) genome RNA was determined. The JEV genome contains 10,976 nucleotides and encodes a single long open reading frame (ORF) of 10,296 nucleotides corresponding to 3432 amino acid residues. This long polypeptide is thought to be cleaved into three structural proteins and several nonstructural proteins of the virus. The genetic location of the three structural proteins was determined by comparing the deduced amino acid sequence from the nucleotide sequence with the N-terminal amino acid sequences that were determined from the three purified structural proteins. The C-terminal region of the ORF may encode a RNA-dependent RNA polymerase which has significant sequence homology with those of other RNA viruses.     

Sequences of Chandipura virus N and NS genes: evidence for high mutability of the NS gene within vesiculoviruses

The nucleotide sequence of the 3' end of the genome of Chandipura (CHP) virus, including the complete sequences of the nucleocapsid (N) and phosphoprotein (NS) genes was determined, principally from cloned cDNAs of the N and NS mRNAs. The NS mRNA of CHP virus is 908 bases in length and encodes a protein of 293 amino acids. Comparison of the CHP virus NS protein sequence with those of vesicular stomatitis virus of the New Jersey serotype (VSV (NJ)) and of the Indiana serotype (VSV (IND] revealed homologies of only 23 and 21%, respectively, with no consecutive stretches of more than four amino acids identical among the three sequences. As with the two VSV serotypes, the highest homology between the NS proteins of CHP and VSV was in a 20-amino acid region near the carboxy termini of the proteins. Of the potential phosphorylation sites, there are eight conserved serine or threonine residues among the three sequences. Despite the dissimilarity among primary sequences of the NS proteins, their overall structure, as assessed by amino acid composition and by the relative hydropathicities of the sequences, has been conserved throughout evolution. The N mRNA of CHP virus is 1291 bases long and encodes a protein of 422 amino acids. In contrast to the NS protein, the CHP N protein is at least 50% homologous to the N proteins of each of the VSV serotypes. We have identified a region near the center of these N protein sequences which is conserved among members of both the rhabdovirus and paramyxovirus families. This extent of conservation of the N protein sequences underscores the high rate of mutability of the NS protein sequences among the vesiculoviruses.     

The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83

Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5'- and 3'-noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser(TRD) to Thr(TC-83) substitution in nonstructural protein (nsP) three at amino acid position 260. The two silent mutations (one each in E1 and E2), one amino acid substitution in the E1 glycoprotein, and five substitutions in the E2 envelope glycoprotein of TC-83 virus were reported previously (B.J.B. Johnson, R.M. Kinney, C.L. Kost, and D.W. Trent, 1986, J. Gen. Virol. 67, 1951-1960). The genome of TRD virus was 11,444 nucleotides long with a 5'-noncoding region of 44 nucleotides. The carboxyl terminal portion of VEE nsP3 contained two peptide segments (7 and 34 amino acids long) that were repeated with high fidelity. The open reading frame of the nonstructural polyprotein was interrupted by an in-frame opal termination codon between nsP3 and nsP4, as has been reported for Sindbis, Ross River, and Middelburg viruses. The deduced amino acid sequences of the VEE TRD nsP1, nsP2, nsP3, and nsP4 polypeptides showed 60-66%, 57-58%, 35-44%, and 73-71% identity with the aligned sequences of the cognate polypeptides of Sindbis and Semliki Forest viruses, respectively. The lack of homology in the nsP3 of the viruses is due to sequence variation in the carboxyl terminal half of this polypeptide.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.     

Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus.

Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins.     

Cloning and Sequence Analysis of a Non-Structural Gene of an Aquareovirus

The nucleotide and deduced amino acid sequence of genome segment 11 encoding a nonstructural protein of an aquareovirus strain SBR have been determined. Nucleotide sequence analysis showed that the genome segment 11 of SBR virus is 780 nucleotides long and contains a major open reading frame that codes for a polypeptide of 236 amino acids with a predicted molecular weight of 25,504 Da. The second reading frame of genome segment 11 was 480 nucleotides long and codes for a polypeptide of 145 with a predicted molecular weight of 15,715 Da. The genome segment 11 contains 24 nontranslated nucleotides at the 5′-end and 48 nontranslated nucleotides at the 3′-end. This gene codes for two nonstructural polypeptides NS29 and NS15. Comparison of the deduced amino acid sequence of this gene with the published sequences of other members of the family Reoviridae indicated no sequence relatedness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe

Summary The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96–100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57–59% and 78–81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes.     

中国大陆庚型肝炎病毒NS区部分基因序列分析及变异 …

目的 研究中国庚型肝炎患者分离的病毒基因的特异性及变异规律。方法 应用逆转录－巢式聚合酶链反应（ＰＣＲ）技术从１６例庚型肝炎病毒（ＨＧＶ）感染住院病人血清中扩增了ＨＧＶＮＳ５区部分基因片段。经纯化后采用双脱氧链末端终止法进行序列分析。