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PubMed1985

Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A.

The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.

Aphthovirus↗

PubMed1991

Splice site skipping in polyomavirus late pre-mRNA processing.

Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site.

3T3 Cells↗

PubMed2002

Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: diagnostic implications in penaeid shrimp.

Anecdotal industry reports of Taura syndrome (TS) epizootics in a Taura syndrome virus (TSV) tolerant strain of Penaeus stylirostris and collected evidence of field TS epizootics in P. stylirostris suggested that a distinct new TSV strain might have emerged since 1994. The Ecuadorian 1992 TSV genome published in GenBank is virtually identical to the Hawaiian 1994 TSV isolate (HI94TSV) used as reference throughout this investigation. Three other geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris). Selected TSV diagnostic methods set forth by the Office International des Epizooties were utilized as the basis for isolate analysis. By Southern blot, TSV probes P15 and Q1 reacted specifically with all the diagnostic reverse transcription polymerase chain reaction (RT-PCR) fragments. Additionally, labeled RT-PCR amplicons from the TSV isolates amplified by routine diagnostic RT-PCR primers gave positive in situ hybridizations with TSV, indicating that all 4 isolates shared homology. By Western blot, immuno-dot blot, and immunohistochemistry, all TSV-purified isolates reacted with TSV polyclonal antibody (PAb). However, with TSV monoclonal antibody (MAb) 1A1 all isolates, except SIN98TSV, reacted, indicating that the difference in isolate SIN98TSV is within VP1, the target for MAb 1A1. The amino acid (AA) sequence of SIN98TSV VP1, MX99TSV VP1 and SON2KTSV VP1 has a 98% homology with the reference HI94TSV VP1. A span of 12 AAs are identified in SIN98TSV VP1 containing significant AA substitutions which may account for a conformational change of the antigenic epitope sufficient to prevent MAb 1A1 from binding. The implications of these results with respect to the antibody-based diagnosis of TSV are discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Viral protein 1 sequence analysis of three infectious bursal disease virus strains: a very virulent virus, its attenuated form, and an attenuated vaccine.

Comparisons between sequences of very virulent, virulent, and attenuated strains of the infectious bursal disease virus (IBDV) may indicate sites on the genome co-inciding with virulence. In an attempt to detect if such sites exist on the coding region of segment B, viral protein 1 (VP1) (encoded for by segment B) of a very virulent Israeli virus, IL3; its attenuated strain, IL4; and the attenuated Winterfield vaccine 2512 were cloned and sequenced. A comparison was made among them and with six other published sequences of segment B. Six nucleic acids distinguished between IL3 and IL4, three of which were predicted to be expressed as amino acids. A striking similarity between the VP1 sequences of 2512 and P2 (an attenuated German strain) was discovered. Although conclusions could not be drawn concerning attenuation sites on VP1, the analysis performed on the VP1 sequences of the two Israeli strains and the Winterfield 2512 strain sheds light on the phylogeny of IBDV and contributes to the accumulating information that may lead to the identification of virulence-related sites of this virus.

Amino Acid Sequence↗

PubMed1988

VP1 of serotype C foot-and-mouth disease viruses: long-term conservation of sequences.

The nucleotide sequences of the VP1-coding regions of several isolates of serotype C3 foot-and-mouth disease virus (FMDV) were determined. The deduced amino acid sequences were compared with those of serotype C1 FMDV. The results provide evidence for two different lineages of FMDV C3 and document the potential for both long-term conservation and rapid evolution of FMDV.

Amino Acid Sequence↗

PubMed1995

Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.

Amino Acid Sequence↗

PubMed1984

Biotechnological approach to a new foot-and-mouth disease virus vaccine.

Major contributions towards the development of an absolutely safe FMDV vaccine are evident. With the identification of VP1 as the immunogenic protein, it is possible to manufacture a subunit vaccine via biotechnology. DNA sequences encoding the VP1 protein can be introduced into a bacterium with ease; under the appropriate conditions, large amounts of VP1 can be produced in a short time. The accumulation of amino acid sequences generated by recombinant DNA techniques allows identification of antigenic domains, which are the basis of variability among serotype and subtype viruses. As a result, vaccine production by chemical synthesis of short peptides corresponding to the antigenic determinants is greatly facilitated. At present, results from experimental vaccines employing genetically engineered or chemically synthesized VP1 antigens against homologous virus infection are encouraging. The current approach of preparing vaccine is to utilize the antigenic specificity of the virus. Since FMDV undergoes antigenic drift, variants not neutralized by type-specific serum will arise. An alternative approach is to prepare vaccines based on antigenic sites shared among all serotype and subtype viruses.

Adjuvants, Immunologic↗

PubMed1993

Identification of group-common linear epitopes in structural and nonstructural proteins of enteroviruses by using synthetic peptides.

Synthetic peptides were employed in enzyme-linked immunosorbent assays to identify group-common linear epitopes in the structural and nonstructural proteins of enteroviruses. Nine linear epitopes were recognized by using sera from patients with heterotypic immunoglobulin G antibody responses to enterovirus infections. The most-reactive peptides were derived from conserved regions of the amino-terminal part of VP1, whereas peptides representing sequences from other conserved regions of VP1, as well as VP2, VP3, and VP4, and from a nonstructural region showed no or poor reactivity. These findings may be useful in the development of serological tests for the diagnosis of infections caused by a broad range of enteroviruses.

Amino Acid Sequence↗

PubMed1994

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.

Base Sequence↗

PubMed2005

[Functional analysis of promoters of Vibrio cholerea typing phage VP1 with reporter system].

Phage VP1 infects and lyses Vibrio cholerae. The VP1 genome is a circular double-strand DNA and its size is 32176 base pairs. Analysis of the sequence of the VP1 genome revealed the presence of 15 putative promoter sequence. The activities of these putative promoters in V. cholerae were assayed by transformation of reporter gene plasmid and phage infection together. Promoter regions were ligated into pRS1274/BamH I/EcoR I. Then transformed into E. coli JM109 and all of clone display blue. The recombinant plasmids were transformed into V. cholerae 7743 deltaZ by electroporation, then bacteriophage VP1 infect transformant. The time-course expressing lacZ gene and detecting change of beta-galactosidase enzyme activity in V. cholerae transformants at latent period, indicated P17 probably is a early promoter; P2 and P3 and P9 etc are medium-term promoters; P18 is a late promoter.

Bacteriophage Typing↗

PubMed1998

PCR detection of JC virus DNA in the brain tissue of a 9-year-old child with pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a rare cerebral tumor of young adults with a slow growth and a good prognosis. Due to its peculiar histopathological findings, the tumor resemble to the lytic phase of progressive multifocal leukoencephalopathy (PML), a JC Virus (JCV) induced disease. For these reasons, the presence of JCV genoma and viral particles were searched for by means of nested polymerase chain reaction (nPCR) and electron microscopy (EM) in a 9-year-old child with PXA. Although EM did not reveal any viral particles, nPCR did reveal genomic sequences of the LT, R, and VP1 regions of JCV. Sequence analysis showed that the R region was mutated with respect to the archetypal form thus yielding the Mad 4 variant of JCV previously reported as being oncogenic in animals. We suggest that JCV may have played a role in the development of this tumor.

Astrocytoma↗

PubMed2005

Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis.

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.

Abscisic Acid↗

PubMed2004

Molecular characterization of coxsackievirus B5 isolates.

Coxsackie B viruses of serotype 5 are associated frequently with sporadic cases of neurological diseases, epidemics of meningitis, and chronic diseases such as cardiomyopathy and diabetes. In this article, 15 strains of Coxsackievirus B5 isolated from patients with neurological disorders and healthy people were investigated by partial sequencing in the 5' half of the VP1 region and compared to other published sequences of Coxsackievirus B5, in the same genomic region. All Coxsackievirus B5 sequences showed less than 25% nucleotide difference between each other and a minimum of 27.8% of divergence with prototype sequences from other Coxsackievirus B serotypes. Within the Coxsackievirus B5 group of sequences, four clusters were individualized and may correspond to four genotypes: one genotype with large geographical distribution, containing most recent strains that have circulated from 1984 to 2000, another genotype represented by the prototype Faulkner strain, isolated in the early 1950s, and two intermediate genotypes, comprising strains isolated from 1970 to 1999 and closely related to swine vesicular disease virus. This study confirms the ability of partial sequencing in VP1 to determine serotype and to genetically characterize Coxsackievirus B5 field isolates. It gives a first approach on the molecular epidemiology of these viruses, which have a particular importance in human health.

Algeria↗

PubMed1997

The quiescent/colorless alleles of viviparous1 show that the conserved B3 domain of VP1 is not essential for ABA-regulated gene expression in the seed.

A series of vp1 alleles distinguish at least two classes of maturation-related genes that are regulated by the VP1 factor and abscisic acid (ABA). The intermediate vp1-c821708 and vp1-McW alleles have quiescent (non-viviparous) anthocyanin-deficient phenotypes while maintaining significant levels of maturation-specific gene expression in the developing embryo. However, expression of the C1 regulatory gene of the anthocyanin pathway is not detected in these mutants. Reduced steady-state levels of structurally altered VP1 proteins are detected in quiescent mutant embryos. The VP1-McW protein sequence lacks the highly conserved region encoded by exons 3-5 of the Vp1 gene. A sensitive RT-PCR assay was used to rule out significant amounts of intact transcripts in the vp1-McW mutant that could account for the quiescent phenotype. In transient expression assays, the VP1-McW protein and other mutants with a truncated B3 domain of VP1 retained a strong capacity to synergistically enhance ABA-regulation of the Em-GUS reporter gene; whereas transactivation of both Em-GUS and C1-sh-GUS genes in the absence of hormone was strongly inhibited. These results indicate that the largest conserved region in VP1 homologs (B3) is critical for gene activation at low or insignificant ABA dosages; whereas the N-terminal domain provides a key interface with ABA signaling pathways in the developing seed.

Abscisic Acid↗

PubMed2003

Characterization of tissue tropism determinants of adeno-associated virus type 1.

Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.

Amino Acid Sequence↗

PubMed2003

Sequential changes in hepatitis A virus genotype distribution in Estonia during 1994 to 2001.

Hepatitis A virus (HAV) isolates from a large outbreak and from non-outbreak cases in Estonia were characterized by sequencing the aminoterminal VP1 region. From January 1998 to December 1999, a total of 1084 cases of hepatitis A were reported to the Harjumaa-Tallinn and Ida-Virumaa Health Protection Services in Estonia. The attack rate was highest among males aged 15-29. Initial cases were noted to be associated with injecting drug use. IgM anti-HAV positive sera were available from 107 hospitalized outbreak cases and from 68 patients sampled during 1994 to 2001. HAV RNA was detected in 42% of sera from 1994-1996 and in 88% of sera from 1998-2001. It was possible to obtain HAV sequences from 83 outbreak and 29 background cases. The outbreak strain was represented by five different sequences, all belonging to subtype IIIA. During the outbreak, this IIIA strain also spread into the general population. All available non-outbreak isolates from 1994 to 2001 but one belonged to genotype IA and formed distinct clusters as compared to isolates from other parts of the world. One subtype IIIA isolate from 1995 was unrelated to the outbreak strain. Subtype IA had been dominating in Estonia during 1994-2001, but the outbreak strain from 1998 to 1999 was IIIA. This subtype was encountered previously in addicts in Sweden during the 1980s and in Norway at the end of the 1990s. This study supports the use of limited sequencing within the aminoterminal VP1 region for studying the molecular epidemiology of hepatitis A.

Adolescent↗

PubMed2006

Full-length sequence analysis of four IBDV strains with different pathogenicities.

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.

3' Untranslated Regions↗

PubMed1986

A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus.

In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.

Antigens, Polyomavirus Transforming↗