Molecular cloning and complete nucleotide sequence of genomic RNA of the AIK-C strain of attenuated measles virus

Twelve cDNA clones covering the entire genome of the AIK-C strain of a seed for live measles vaccine were obtained, and the nucleotide sequences were determined. The full viral genomic RNA consists of 15,894 nucleotides. Comparisons of the nucleotide sequence and the deduced amino acid sequence between the AIK-C and other Edmonston strains revealed the following changes: 56 nucleotide differences and one C residue insertion, 31 amino acid changes, and 19 silent mutations.     

Partial nucleotide sequence of the Japanese encephalitis virus genome

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.     

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.     

Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty

As part of a study of the molecular basis of pathogenicity of echovirus 9, the complete nucleotide sequence of the mouse-virulent echovirus 9 strain Barty was determined. Excluding the poly(A) tail, the complete RNA genome is composed of 7451 bases. The postulated open reading frame extends from nucleotide (nt) 741 to 7349 and predicts a polyprotein of 2203 amino acids (aa). As compared with the sequence of the echovirus 9 prototype strain Hill, which is apathogenic for newborn mice, 1492 nt are exchanged, leading to 9% divergence of the deduced amino acid sequence. The foremost difference between both strains is located at the C-terminus of the capsid protein VP1. In the case of strain Barty, an additional 10 aa fragment, including an RGD motif, is inserted.     

Molecular cloning and nucleotide sequence of the genome of hog cholera virus

A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV). This clone served as a probe for screening different size-selected cDNA libraries. After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame. Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs. With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.     

Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process

The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA₁₄-14-2 and its parental virulent strain SA₁₄, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5 and 3 untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5- and 3-terminal untranslated regions contain one- and two-point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.     

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.     

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.     

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA

Summary.  The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3 803 nucleotides and has five open reading frames (ORFs). The 5′ ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria. Accepted August 25, 1997 Received June 18, 1997     

Comparative sequence analysis of four complete primary structures of plum pox virus strains

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5 terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3 terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5 and 3 ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5 end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%). The sequence analysis of the coat protein region of the four complete and one partial sequence indicates that the Hungarian plum pox virus strain diverges at the larger extent, similar to the El Amar strain, from which only less than half of the sequence is available.     

The complete nucleotide sequence and genome organization of barley mild mosaic virus (Na1 strain)

Summary The 5-terminal half of RNA 1 and whole of RNA 2 of barley mild mosaic bymovirus Na1 strain (BaMMV-Na1) were sequenced to give together with the published data its complete genomic sequence. BaMMV-Na1 RNA 1 and RNA 2 consist of 7 263 and 3 516 nucleotides, excluding the 3 poly A tails, respectively. RNA 1 encodes a single large polyprotein of 2 258 amino acids (M_r 256 K), containing eight putative functional proteins, and RNA 2 also encodes one polyprotein of 891 amino acids (M_r 98 K), containing two functional proteins. These functional proteins are arranged in the same manner as in RNA 1 and RNA 2 of barley yellow mosaic bymovirus (BaYMV), and significant amino acid sequence homology (25–58%) exists between the proteins of the two viruses. The BaMMV-Na1 proteins show less amino acid sequence homology (18–32%) with the corresponding proteins of potyviruses or rymoviruses than with those of Ba YMV. Comparisons of the BaMMV-Na1 proteins with the corresponding proteins of other partially sequenced BaMMV isolates show 87–98% amino acid sequence identity. There is 91–94% nucleotide sequence identity between the 3 non-coding regions (NCRs) in RNA 1 or RNA 2 of BaMMV-Na1 and other BaMMV isolates, but only 68–72% identity between the 5 NCRs in RNA 2 of BaMMV-Na1 and other BaMMV isolates.The nucleotide sequence data described in this paper will appear in the DDBJ, EMBL and GenBank nucleotide databases under the accession numbers D83408 (RNA 1) and D83409 (RNA 2).     

汉滩病毒Z10株G1糖蛋白编码区克隆及序列分析

目的 汉滩病毒浙１０（Ｚ１０）株Ｇ１糖蛋白编码区的克隆、核苷酸序列分析，提供我国应用最为广泛的肾综合征出血热（ＨＦＲＳ）疫苗株序列资料。方法 反转录ＰＣＲ法扩增Ｇ１基因片段，克隆入ｐＧＥＭ－Ｔ载体，双脱氧链终止法测定核苷酸序列。结果 测定１４４９个核苷酸，可编码４８３个氨基酸。与Ｉ型７６／１１８、Ｌｅｅ、Ｈｏｊｏ株比较核苷酸同源性分别为８７％、８６％、８６％，与Ⅱ型Ｒ２２株同源性为６７％。比较氨基     

Complete genome sequence of attenuated low-temperature Thiverval strain of classical swine fever virus

The Thiverval vaccine strain of classical swine fever virus (CSFV) was derived from virulent Alfort strain through the serial passages in cells at 29–30°C. In this study, we determined the complete genome sequence of this strain and found that its genome contains one open reading frame (ORF) that encodes a polyprotein with 3,898 amino acids. The 5′-UTR of Thiverval is 373 nt long with only one mutation at position 220. In contrast, the length of 3′-UTR is highly heterogeneous ranging from 233 to 259 bp. The heterogeneity of length of the 3′-UTR was due to an insertion of a variable length of T-rich sequence ranging from 6 to 32 nt. The insertion may change the structure and free energy of the 3′-UTR, resulting in a destabilization of the 3′-UTR. Sequence alignment of Thiverval and other CSFV strains showed 85.2–99.6% identities at the nucleotide level and 92.5–99.5% at the amino acid level. The phylogenetic tree analysis of the complete ORF, partial region of E2, and NS5B suggests that the CSFV Thiverval strain belongs to genetic group 1 and subgroup 1.1. The results from this study provide insight into the molecular mechanism of the attenuation of Thiverval vaccine strain.     

The genome of choristoneura fumiferana nuclear polyhedrosis virus: Molecular cloning and mapping of the EcoRI, BamHI, SmaI, XbaI and Bg1II restriction sites

The DNA genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) was extracted from purified virus preparations and analyzed with restriction endonucleases EcoRI, BamHI, HindIII, SmaI, XbaI and Bg1II. The sizes of the fragments were calculated with respect to mobilities of standard adenovirus-2 DNA fragments. The total size of the genome was calculated to be about 125 kb. Most of the CfMNPV genome was then cloned as EcoRI fragments in the plasmid vector pBR322. The clones were authenticated by electrophoretic analysis in the presence of EcoRI-digested CfMNPV DNA and by hybridization of ³²P nick-translated clones to Southern blots of different enzyme digests of the genome. Data from such hybridization experiments complemented with other mapping procedures were used to construct restriction maps for BamHI, EcoRI, XbaI, Bg1II and SmaI enzymes.     

Nucleotide sequence of the nucleoprotein gene of the RC·HL strain of rabies virus,a seed strain used for animal vaccine production in Japan

By using a phage vector (lambda ZAP II) and the mRNA extracted from IMR-32 cells infected with the RC·HL strain of rabies virus, we constructed a cDNA library from which four nucleoprotein (N)-specific cDNA clones were obtained by Southern blot hybridization. These clones contained a cDNA insert of about 1.4 kb, in which the longest open reading frame was the same length as that reported for the N cDNA of three fixed strains, CVS, PV, and SAD B19. When the nucleotide and deduced amino acid sequences were compared between the RC·HL and the three strains, homology was within the range of 91.5–91.8% and 95.1–96.0%, respectively. Of 183 nucleotides of the RC·HL N-cDNA that were not identical to that of the corresponding site of at least one of the three strains, 41 were shared with the CVS strain, whereas only three were shared with either of the other two strains. In the amino acid sequence, we found 29 residues that were not shared in common with all of the four strains, 11 of which were the substitutions with radically different amino acids that might cause conformational changes of the protein, and, in addition, five of which were located in the region close to the C terminus. The number of such amino acid substitutions between the RC·HL and CVS strains was smaller than that of the other three strains. These results are not inconsistent with the presumption that the RC·HL and CVS strains originated from the same laboratory strain of the Pasteur viruses.     

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

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

Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus

The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus (BVDV) has been molecularly cloned and the nucleotide sequence determined. The cloned sequence was 12,573 nucleotides in length, corresponding to a molecular weight of 4.3 X 10(6), having a base composition of 32.2% A, 25.7% G, 22.1% U, and 20.0% C. However, the sequences at the 5' and 3' termini of the RNA have not been unequivocally established. A single major open reading frame extending the length of the molecule was found in the viral-sense (positive polarity) sequence. This open reading frame was capable of encoding 3988 amino acids, representing 449 kDa of protein.