Eleven single nucleotide polymorphisms and one triple nucleotide insertion of the human TGF-β III receptor gene

We found 11 single nucleotide polymorphisms and one triple nucleotide insertion in the cDNA of the human transforming growth factor β (TGF-β) III receptor gene (TGFBR3) located on 1p33–p32, encoding betaglycan, a component of the TGF-β receptor system. Inside the 5′ untranslated region (UTR), a G→A polymorphism was identified at position 311. In the open reading frame (ORF), a non-conservative T→C polymorphism was identified at position 392, and three conservative polymorphisms were found at positions 563 (G→A), 1548 (G→A), and 2370 (C→T). A triple nucleotide insertion (GCA) was identified at position 1419. Inside the 3′ UTR, six polymorphisms were identified: four G→A, at positions 2918, 3055, 3098, and 3355; one T→A, at position 3183; and one G→C, at position 3966. In addition to these changes, some divergences from the published sequence were observed in all 12 chromosomes tested. These included, in the ORF, an additional C after position 555, two additional G after position 563, and an additional T after position 1388. No T was found at position 1394. The alterations translate to a changed amino acid sequence. Inside the 3′ UTR, additional discrepancies were identified. The discovered changes and polymorphisms may be useful for further genetic studies of TGFBR3 receptor deficiencies. Received: December 22, 1999 / Accepted: February 25, 2000     

Sequence analysis of the <Emphasis Type="Italic">Meq</Emphasis> gene in the predominant Marek’s disease virus strains isolated in China during 2006–2008

The main aim of the present study were to investigate sequence diversity in the Meq gene of Marek’s disease viruses (MDV) isolated in China and to determine the most prevalent MDV strains. The 19 MDV strains were isolated from dead or diseased chickens from different chicken farms in China during 2006–2008, and the Meq gene was sequenced from each of these strains. Sequence analysis showed that all of the isolates contained an open reading frame of 1020 nucleotides, which encoded a 339 amino acid peptide. Compared with reference MDV strains, 12 of the 19 MDV isolates possessed two amino acid substitutions, (T → A) at position 139 and (P → R) at position 176, one isolate shared sequence similarity with the attenuated strain CVI988, and five of the other six isolates exhibited one amino acid change (P → T) at position 177 or 176. The 19 MDV isolates shared between 99.0 and 100% nucleotide sequence homology, and between 97.7 and 100% amino acid sequence homology. The nucleotide and amino acid sequence identity between the 19 MDV isolates and the 25 reference MDV strains varied from 97.6 to 100% and 94.4 to 100%, respectively. Based on the phylogenetic relationships between Meq gene sequences, Chinese MDV isolates constituted a separate clade to MDV reference strains, demonstrating that a different genotype of MDV was prevalent in China between 2006 and 2008.     

A transforming plasmid from HSV-2 transformed cells contains rat DNA homologous to the HSV-1 and HSV-2 genomes

Morphologically transformed rat (3Y1) cell lines were established following transfection with HSV-2 mtrII DNA sequences (0.585 to 0.601 map units). The mtrII sequences were cloned in plasmids containing the neor gene. Cells resistant to the antibiotic G-418 were passed into soft agarose, and clonal lines were established from individual colonies. The DNAs from two cell lines examined by Southern blot hybridization were shown to contain the original transfected viral DNA sequences in a fashion consistent with a multiple and complex pattern of integration. From one cell line, an approximately 20-kbp plasmid was isolated after transformation of bacteria with Hirt supernatant DNA. This plasmid was capable of rapidly transforming rat cells at a greater than 1000-fold higher frequency than the mtrII DNA. This plasmid consists mainly of unique sequence rat DNA with two copies of the HSV-2 mtrII region DNA (HSV-2 genomic map unit location of ca. 0.595) present at sites distant from each other. The rat DNA in the rescued plasmid is homologous to the putative focus-forming sequences present in the HSV-2 mtrIII (0.53 to 0.58 map units) and the colinear HSV-1 DNA. The genomic copy of these rat sequences in four HSV-2 mtrII transformed cell lines appears to have undergone rearrangement. These data provide evidence that the HSV-2 mtrII sequences are involved in transformation, and that the HSV-2 mtrII region may affect transformation by rearranging the cellular sequences that are homologous to mtrIII.     

Nucleotide sequence of capsid protein gene of rice tungro bacilliform virus

Summary The sequence of 5,028 nucleotides, including one open reading frame (ORF), of rice tungro bacilliform virus (RTBV) dsDNA was determined. The predicted translational product comprises 1,675 amino acids and has M_r of 194, 134 (p194). The amino acid sequences of three tryptic fragments from the 32k capsid protein of RTBV (p32) were found in the predicted translational product indicating that the ORF codes for the RTBV capsid protein.     

Comparison of the RNA polymerase genes of marine birnavirus strains and other birnaviruses

Summary.  The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3–99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4–20.8% divergences in the coding region, which gave 10.1–11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1–85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1–95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III. Received August 19, 2002; accepted October 30, 2002     

Comparison of the sequence of the secretory glycoprotein A (gA) gene in Md5 and BC-1 strains of Marek's disease virus type 1

DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV 1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV 1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV 1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted.     

Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli

We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease.     

Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus

The nucleotide sequence of the 60.1- to 65.5-m.u. region of Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined. Seven large open reading frames were identified. Two open reading frames potentially encoding gene products of 143 and 38 kDa were found in the counterclockwise direction upstream of the p6.9 gene. Four additional open reading frames were found in the opposite direction. Analysis of the predicted amino acid sequence of the 143-kDa gene revealed a potential leucine zipper motif, a putative nuclear localization signal, and seven amino acid motifs previously identified in a number of proteins involved in NTP binding and DNA/RNA unwinding. The mutation in a DNA replication defective temperature-sensitive mutant was fine mapped to the carboxy terminus of the ORF1(p143) gene. Sequence analysis of the mutation site identified a single base change of a guanine to an adenine, resulting in the substitution of a methionine for valine. This mutation resides seven amino acids downstream of the putative NTP-binding motif of the ORF1(p143) gene product and results in a DNA negative mutant. Together these data strongly suggest that the ORF1(p143) gene product is a baculovirus helicase.     

Identification and characterization of the guinea-pig cytomegalovirus glycoprotein H gene

Summary Subunit vaccines which target viral envelope glycoproteins offer promise for the prevention of congenital cytomegalovirus (CMV) infection. The guinea pig model of CMV infection is uniquely well suited to testing vaccines for prevention of congenital infection, since, in contrast to other animal cytomegaloviruses, the guinea pig CMV (GPCMV) crosses the placenta, producing intrauterine infection. Antibody to the CMV glycoproteins B (gB) and H (gH) appears to be important in conferring protective immunity. Unfortunately, little is known about specific GPCMV envelope glycoproteins. Sequencing of GPCMV genome fragments was therefore undertaken to test whether GPCMV encodes a gH homologue. Partial sequencing of theHind III A fragment of the GPCMV genome revealed an open reading frame of 2 169 nucleotides capable of encoding a protein of 723 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gH coding sequences of other herpesviruses. The GPCMV gH ORF encodes 12 highly conserved cysteine residues, contains 9 potential N-linked glycosylation sites, and has a predicted M_r of 81.6 kDa. Northern blot hybridizations with gH-specific probes identified an abundant 5.1 kb mRNA with expression kinetics of an early gene. A polyclonal antiserum raised against a synthetic peptide derived from the deduced amino acid sequence of the gH ORF identified a virion-associated protein with an approximate M_r of 85-kDa, the putative GPCMV gH, in immunoblot assays.The nucleotide sequences reported in this paper have been submitted to the GenBank database and assigned the accession number U49361.     

Molecular variation of hop mosaic virus isolates

Hop mosaic virus (HpMV), a member of the genus Carlavirus, is importance to hop production worldwide. We identified variation in nucleic and amino acid sequences among 23 HpMV isolates from Australia, the USA, the Czech Republic, South Africa and Japan using a 1,455-bp fragment covering the 3′ end of the virus genome including ORFs 4, 5 and 6. Three clusters of two or more isolates were identified in phylogenies of the total nucleotide sequence and the coat protein (ORF5) amino acid sequence. Two of these clusters combined in analyses of ORF4 and ORF6 amino acid sequences. Isolates from within and outside of Australia were found in each cluster, indicating that sequence variation was not associated with geographic source. Monitoring of HpMV variants in the field and evaluation of the impact of variants on vector association, rate of spread, and hop yield and quality can now be undertaken.     

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.     

Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an Mr 62,000 polypeptide

Marker transfer procedures were used to locate the site of mutation in the genome of a previously characterized (B. Moss, E. N. Rosenblum, and P. Grimley, 1971), Virology 45, 135-148) rifampicin-resistant (RifR) vaccinia virus isolate. Starting with a cosmid library prepared from the mutant genome, recombination with successively smaller DNA fragments was shown to transfer drug resistance to wild-type vaccinia virus. In this manner, the mutation was mapped within a 485-bp DNA segment in the central region of the genome at the extreme right end of the HindIII D fragment. Nucleotide sequencing indicated that this DNA segment differed from the homologous region of wild-type DNA by a single C/G----A/T substitution. Sequencing of the flanking 2195 bp revealed two tandem nonoverlapping open reading frames (ORFs) encoding putative polypeptides of Mr 16,908 and 61,840. The RifR mutation resulted in a predicted glutamine----lysine change only 27 amino acids from the NH2 terminus of the longer ORF. A predicted asparagine to aspartic acid substitution, found in another RifR vaccinia virus mutant by J. Tartaglia and E. Paoletti (Virology 147, 394-404, 1985), mapped near the carboxyl terminus of the same ORF. These data suggest a model in which head-to-tail interaction between Mr 61,840 polypeptides occurs and in which rifampicin blocks virus assembly by preventing this association.     

Nucleotide sequence and phylogenetic analyses of the DNA polymerase gene of Anticarsia gemmatalis nucleopolyhedrovirus

The DNA polymerase from Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was identified and sequenced, and its amino acid sequence was compared with other viral DNA polymerases to identify conserved regions and to reconstruct a phylogenetic tree. The sequence analysis of the AgMNPV DNA polymerase gene revealed the presence of a 2976 nucleotides open reading frame (ORF) encoding a polypeptide of 991 amino acid residues with a predicted molecular mass of 114.7 kDa. Among the baculovirus DNA polymerase genes identified to date, the AgMNPV DNA polymerase gene shared maximum amino acid sequence identity with the DNA polymerase gene of Choristoneura fumiferana nucleopolyhedrovirus defective strain (CfDEFNPV) (94%). The alignment of 140 virus sequences, 23 of them from baculovirus, showed that, of the 10 conserved regions identified, 5 are exclusive to baculoviruses (R1, R5, R9, R6 and R10), only 2 of them (R6 and R10) previously described as such in the literature. Our analysis, based on their positions in the AgMNPV DNA polymerase model, suggests that R9 and R10 could interact with DNA. Phylogenetic analysis of DNA polymerase sequences places the enzyme from AgMNPV within the cluster containing the polymerases of Group I Nucleopolyhedrovirus and suggests that the AgMNPV DNA polymerase is more closely related to that of CfDEFNPV than to those of other baculoviruses.     

Polymorphism of extracellular superoxide dismutase (EC-SOD) gene: relation to the mutation responsible for high EC-SOD level in serum

Summary Extracellular superoxide dismutase (EC-SOD) with amino acid substitution R213G generated by the nucleotide substitution 760C→G in the heparin binding domain is responsible for the high EC-SOD level in serum. We identified the two DNA polymorphic sites in the coding region of EC-SOD gene related to the 760C→G and determined the allele frequencies. The polymorphisms were A and G at nucleotide position (nt.) 241 and C and T at nt. 280 near the N-terminal. The haplotype frequencies in Japanese were 241A280C: 0.45, 241G280T: 0.37, and 241G280C: 0.18. The haplotype of 241A280T did not exist. The mutation 760C→G must occur on the allele having the haplotype of 241G280T.     

Characterization of the gene encoding glycoprotein C of duck enteritis virus

A total of 2,718 bp of DNA fragment was amplified from the C-KCE strain of duck enteritis virus (DEV) genome using thermal asymmetric interlaced PCR. This newly identified viral DNA fragment contained two non-overlapping open reading frames (ORFs) oriented from the 5′ to 3′ direction. The first ORF was comprised of 43.5% G + C and contained the full-length genomic sequence of the UL44 gene (1,296 bp) encoding 431 amino acid residues of DEV glycoprotein C (gC). The second ORF encoded a partial peptide of the UL43 gene. The sequences of DNA and deduced amino acids of the DEV gC gene shared high homology with other members of known herpesviruses, supporting the classification of DEV. Phylogenetic analysis of the DEV gC gene revealed that the gC gene had a close evolutionary relationship with the subfamily of Alphaherpesvirinae.