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PubMed1990

cDNA-cloning and expression of VP1-specific sequences of foot-and-mouth disease virus types A5 and O1.

The RNA genome of foot- and mouth disease virus strains A5 Westerwald and O1 Lausanne has been reverse-transcribed and cloned in lambdaphages or plasmids. Identification of cDNA-clones containing VP1-specific sequences was achieved by hybridization, restriction mapping, and sequence analysis. VP1-coding cDNA-fragments were subcloned into the expression vector pEX which led to synthesis of fusion proteins with beta-galactosidase. These fusion proteins reacted with anti-VP1 antibodies on a Western blot, but were not capable of inducing neutralizing antibodies to mice. This seemed to suggest a tertiary structure of the VP1-epitopes unlike those of native VP1. Other attempts are discussed to construct VP1-fusion proteins folding similarly to the native viral protein structure.

Amino Acid Sequence↗

PubMed2003

Detection of MEF-1 laboratory reference strain of poliovirus type 2 in children with poliomyelitis in India in 2002 & 2003.

BACKGROUND & OBJECTIVES: Significant progress has been made towards eradication of poliomyelitis in India. Surveillance for acute flaccid paralysis (AFP) has reached high standards. Among the 3 types of polioviruses, type 2 had been eliminated in India and eradicated globally as of October 1999. However, we isolated wild poliovirus type 2 from a small number of polio cases in northern India in 2000 and again during December 2002 to February 2003. Using molecular tools the origin, of the wild type 2 poliovirus was investigated. METHODS: Polioviruses isolated from stool samples collected from patients with AFP were differentiated as wild virus or Sabin vaccine-like by ELISA and probe hybridization assays. Complete VP1 gene nucleotide sequences of the wild type 2 poliovirus isolates were determined by reverse transcriptase polymerase chain reaction (RT-PCR), followed by cycle sequencing. VP1 nucleotide sequences were compared with those of wild type 2 polioviruses that were indigenous in India in the past as well as prototype/laboratory strains and the GenBank database. RESULTS: Wild poliovirus type 2 was detected in stool samples from 6 patients with AFP in western Uttar Pradesh and 1 in Gujarat. In addition, the virus was isolated from one healthy contact child and from environmental sewage sample in Moradabad where three of these patients were reported. These isolates were identified as genetically closely related to laboratory reference strain MEF-1. Molecular characterization of the isolates confirmed that there was no evidence of extensive person-to-person transmission of the virus in the community. INTERPRETATION & CONCLUSION: Laboratory reference strain (MEF-1) of poliovirus type 2 caused paralytic poliomyelitis in 10 patients in September 2000 and November 2002 to February 2003. The origin of the virus was some laboratory as yet not identified. This episode highlights the urgent need for stringent containment of wild poliovirus containing materials in the laboratories across the country in order to prevent recurrence of such incidents.

Capsid Proteins↗

PubMed1998

VP1-coding sequences of recent isolates of foot-and-mouth disease virus types A, O and Asia1.

A large part of the capsid protein VP1-coding sequence of foot-and-mouth disease virus, isolated between 1993 and 1996 in Europe, was amplified by the reverse transcription-dependent polymerase chain reaction (RT-PCR). The same was done with some non-European virus isolates, especially those against which vaccines were currently produced. The products were sequenced, and the sequences aligned. The alignment comprises sequences of the types A, O and Asia 1. Although the provenance of virus introduced to Europe remains unknown, genetic relation to some other isolates was indicated. Several genotypes of the virus were found to circulate in the field since years.

Aphthovirus↗

PubMed2004

Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses.

Noroviruses (NoV), previously called "Norwalk-like viruses", have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid protein (VP1) is the reference genomic region for establishing genotypes. In this study, we analyzed complete NoV VP1 sequences (n=100) and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup specific, broadly reactive, degenerate primer sets (GI and GII). The region D primers were evaluated in a single-tube one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NoV strains from both outbreaks and sporadic cases. In total, 95% of the samples tested positive using the new region D primer sets. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) including three new genetically distinct clusters, two of which were unresolved using region A sequences. Phylogenetic analysis of the complete VP1 sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In summary, we successfully developed and evaluated a broadly reactive RT-PCR assay for reliable genotyping of GI and GII noroviruses.

Base Sequence↗

PubMed2006

Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine.

In the present study, a genomic analysis of full VP1 sequence region of 15 clinical re-isolates (14 healthy vaccinees and one bone marrow tumor patient) was conducted, aiming to the identification of mutations and to the assessment of their impact on virus fitness, providing also insights relevant with the natural evolution of Sabin strains. Clinical re-isolates were analyzed by RT-PCR, sequencing and computational analysis. Some re-isolates were characterized by an unusual mutational pattern in which non-synonymous mutations outnumbered the synonymous ones. Furthermore, the majority of amino-acid substitutions were located in the capsid exterior, specifically in N-Ags, near N-Ags and in the north rim of the canyon. Also mutations, which are well-known determinants of attenuation, were identified. The results of this study propose that some re-isolates are characterized by an evolutionary pattern in which non-synonymous mutations with a direct phenotypic impact on viral fitness are fixed in viral genomes, in spite of synonymous ones with no phenotypic impact on viral fitness. Results of the present retrospective characterization of Sabin clinical re-isolates, based on the full VP1 sequence, suggest that vaccine-derived viruses may make their way through narrow breaches and may evolve into transmissible pathogens even in adequately immunized populations. For this reason increased poliovirus laboratory surveillance should be permanent and full VP1 sequence analysis should be conducted even in isolates originating from healthy vaccinees.

Amino Acid Substitution↗

PubMed2004

Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome.

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.

Capsid Proteins↗

PubMed1986

Relationship of Theiler's murine encephalomyelitis viruses to the cardiovirus genus of picornaviruses.

Sequence analysis of VP1 in the DA strain of Theiler's murine encephalomyelitis viruses (TMEV) showed that 13 of the first 23 N-terminal amino acids were identical to those in the corresponding protein of encephalomyocarditis virus. There was little similarity to the corresponding VP1 sequences of poliovirus types 1, 2 and 3, coxsackievirus B3, human rhinoviruses 2 and 14, human hepatitis A virus or foot-and-mouth disease virus. These results, as well as serological relationships detected by immunoblotting, suggest that the TMEV are more closely related to the cardioviruses than to the enteroviruses with which they are presently classified. This newly recognized relationship suggests potential for recombinant infectious cDNA studies between TMEV and cardioviruses.

Amino Acid Sequence↗

PubMed2001

[Nucelotide sequence of an ECHO virus isolated from a meningiti].

BACKGROUND: To determine the nucleotide sequences of W strain (Zhaoyuan/W/87) isolated from a meningitis patient, and compare them with the sequences of relational enteroviruses. METHODS: Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA was amplified by PCR, the products of PCR were purified. Then RNA sequence analysis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. RESULTS: The partial nucleotide sequences of 5 UTR, region 2C and 3D of W strain (Zhaoyuan/W/87) were respectively 244, 275 and 353 nucleotides long. The complete nucleotide sequences of region VP1 were 876 nucleotides long coding 292 amino acids. In comparison with the VP1 sequence of two ECHO29 strains in Genbank through internet, the homogeneity of nucleotide sequence were 82.1% and 81.7%, that of amino sequence were 95.1% and 94.4%. CONCLUSIONS: W strain Zhaoyuna/W/87) was a mutation strain of ECHO29.

Amino Acid Sequence↗

PubMed1995

A 10-amino-acid linear sequence of VP1 of foot and mouth disease virus containing B- and T-cell epitopes induces protection in mice.

The area of foot and mouth disease virus (FMDV) comprising residues 140 and 160 of capsid protein VP1 has been used extensively as an immunogen in natural and experimental hosts. A detailed epitope mapping of this region, however, has not been reported. For this purpose a synthetic peptide containing the residues 135 to 160 (p135-160) of VP1 of FMDV O1 Campos was analyzed for its T- and B-cell epitopes. The p135-160 is highly immunogenic, either by itself or coupled to a carrier protein (BSA), elicits a long-lasting neutralizing antibody response in mice, and provides solid protection against virulent challenge. By using a set of synthetic 10mer overlapping peptides, which cover the entire sequence 135-160 of VP1, we have shown that at least four discrete B epitopes are regularly distributed along the peptide. Although immunization with each of the 10mers coupled with BSA as a carrier protein induced peptide-specific antibody responses, individually none of the 10mers was able to induce neutralizing antibodies. However, anti-135-160 antibodies sorted by immunoaffinity chromatography using each of the 10mers revealed the existence of at least four discrete neutralizing sites: one spanning residues 135-144, at least two more between residues 140 and 154, and another in the region 150-160. Moreover, T-cell epitopes were identified, both by antigen-dependent proliferation assays and by adoptive cell transfer. By both methods, a T-cell epitope was located in the area comprising residues 135-144; the cell transfer experiment, which seems to be more sensitive, also identified a second T-cell epitope between residues 150 and 160. Interestingly, when the region 135-144, which contains both B- and T-cell epitopes, was in a tandem repeat configuration it induced a strong neutralizing antibody response in mice and solid protection against the challenge.

Amino Acid Sequence↗

PubMed2026

Molecular Epidemiology of Non-Polio Enterovirus: Insights From L20B Cell Line Adaptation From Children With Acute Flaccid Paralysis in Pakistan.

BACKGROUND: Non-polio enteroviruses (NPEVs) are increasingly implicated in acute flaccid paralysis (AFP), often resembling poliomyelitis and complicating eradication efforts. In Pakistan, limited molecular surveillance has hindered comprehensive characterization. The L20B cell line, designed for poliovirus detection, occasionally supports NPEV replication, challenging AFP case interpretation. METHODS: Between January 2021 and December 2022, 4615 stool samples from AFP cases in children ≤15 years were analyzed. Of these, 435 were identified as NPEVs via L20B cytopathic effects and intertypic differentiation reverse transcription-polymerase chain reaction. VP1 sequencing was performed on 218 representative isolates, yielding 153 high-quality sequences (70.2%). The 224/222 primer set showed superior amplification. Phylogenetic analysis used MUSCLE alignment and the Neighbor-Joining method in MEGA X, with statistical evaluation of epidemiological data. RESULTS: NPEVs were frequently found in L20B-positive AFP cases, highlighting the cell line's limited specificity. Most cases involved children under 5, with a slight male bias. Enterovirus B was predominant (98.0%), especially Echovirus 7 (20.3%) and Echovirus 11 (10.5%), followed by Coxsackievirus B1 and Echovirus 33 (5.9% each). Geographic clustering was noted in Punjab (45.1%), Khyber Pakhtunkhwa (30.7%) and Sindh (20.3%), with seasonal peaks in late summer and early autumn. Phylogenetic data revealed localized Enterovirus B circulation with minimal genetic variation. CONCLUSIONS: The detection of diverse NPEVs in L20B-positive AFP cases emphasizes their relevance in post-polio surveillance. Incorporating routine VP1 sequencing, optimized primer use, and targeted seasonal and regional monitoring is vital to reduce diagnostic uncertainty and inform public health strategies.

Humans↗

PubMed2005

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1.

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.

Animals↗

PubMed2000

Resolution of the pathways of poliovirus type 1 transmission during an outbreak.

An outbreak of poliomyelitis with 20 cases occurred in Israel, Gaza, and the West Bank from October 1987 to October 1988. The wild type 1 poliovirus associated with the outbreak was most closely related to viruses found in the Nile Delta. The epidemiologic links among patients involved in the outbreak and patients with community-acquired infections during the outbreak were inferred from the evolutionary relationships among isolates of the outbreak virus. Complete VP1 sequences (906 nucleotides) were determined for 12 clinical and 4 sewage isolates. A total of 58 nucleotide differences were found among the 16 isolates; 74% of all substitutions were synonymous third-position transitions. An evolutionary tree, representing both the pathways of VP1 sequence evolution and the inferred chains of virus transmission during the outbreak, was constructed under the assumption that each substitution had occurred only once. The combined epidemiologic and molecular data suggest that a single founder strain was introduced into Israel from the vicinity of Gaza in the fall of 1987. Poliovirus circulation was apparently localized to southern communities during the winter and spread north by the following summer into the Hadera subdistrict of Israel, where it radiated via multiple chains of transmission into other communities in northern Israel and the West Bank. The close sequence matches (>99%) between clinical and sewage isolates from the same communities confirm the utility of environmental sampling as a tool for monitoring wild poliovirus circulation.

Base Sequence↗

PubMed1983

Comparison of the major antigenic determinants of different serotypes of foot-and-mouth disease virus.

Complete nucleotide sequences which code for the capsid protein VP1 of two foot-and-mouth disease virus serotypes, O1Campos/Brazil/58 and C3Indaial/Brazil/71, have been determined. Ten available VP1 sequences (three serotype O, three serotype C, and four serotype A) were aligned and compared. Our evidence suggests that O1BFS/Britain/68 and O1K/Germany/66 are closely related to O1Campos/Brazil/58. Significant variations were observed between the nucleotide sequences of C3Indaial determined by two different laboratories. These differences are probably the result of virus adaptation and propagation in different laboratories. In one of the isolates (C3Biogen), a 13-base-pair stem and 13-base-pair loop structure is located in the 134-158 amino acid variable region. Isolates of different serotypes differ at two highly variable regions, amino acid positions 42-60 and 134-158, but isolates of the same serotype show major differences only in the variable region between amino acids 134 and 158. Since the remaining amino acid sequence of VP1 is highly conserved, we conclude that the 134-158 amino acid variable region is involved in subtype specificity, whereas both variable regions contribute to serotype differences.

Amino Acid Sequence↗

PubMed2025

Molecular Epidemiology of Coxsackievirus A10 Associated With Hand, Foot and Mouth Disease From 2021 to 2024 in Shenzhen, China.

The study aimed to investigate epidemiological profile and molecular characteristics of coxsackievirus A10 (CVA10) associated with hand, foot and mouth disease (HFMD) in Shenzhen, China and comparatively analyze genomes of CVA10 strains related to differential clinical phenotypes. A total of 3170 clinical specimens collected between 2021 and 2024 were examined for CVA10 using real-time RT-PCR. Complete VP1 sequences and near-complete genome sequences of CVA10 were determined by RT-PCR methods and sequencing. Sequences were analyzed using a series of bioinformatics programs. Two (33.33%) out of 6 severe cases were infected with CVA10. The detection rate of CVA10 associated with mild HFMD ranged from 1.21% to 6.11% in 2021-2024, with an overall detection rate of 3.73%. There was no significant difference in the infection rate of CVA10 between males and females or different age groups. The CVA10 infections mainly occurred in Spring (March to May) and Summer (June to August) in Shenzhen. Of the 74 VP1 sequences determined, 71 (95.95%) of them were detected in the sub-genotype C2, 3 (4.05%) were assigned to the genotype D. Genomic sequence analysis indicated that the genotype D of CVA10 of this study derived from genetic recombination between CVA10 and CVA16 in 3A-3D coding region (nucleotide position: 5075-6896). Different variable sites were observed in the two CVA10 strains associated with different severe complications when compared to CVA10 strains associated with mild diseases. In conclusion, CVA10 associated with HFMD circulated at a low level in Shenzhen in 2021-2024, with C2 as the predominant genotype. Recombinant genotype D of CVA10 was introduced first to Shenzhen in 2024. The study emphasizes the importance of continuous molecular surveillance of CVA10.

Humans↗

PubMed1998

Prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient.

VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by approximately 10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of approximately 3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started approximately 9.3 years earlier. This estimate is about the time (6. 9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.

Adult↗

PubMed2002

Molecular phylogeny and proposed classification of the simian picornaviruses.

The simian picornaviruses were isolated from various primate tissues during the development of general tissue culture methods in the 1950s to 1970s or from specimens derived from primates used in biomedical research. Twenty simian picornavirus serotypes are recognized, and all are presently classified within the Enterovirus genus. To determine the phylogenetic relationships among all of the simian picornaviruses and to evaluate their classification, we have determined complete VP1 sequences for 19 of the 20 serotypes. Phylogenetic analysis showed that A13, SV19, SV26, SV35, SV43, and SV46 are members of human enterovirus species A, a group that contains enterovirus 71 and 11 of the coxsackie A viruses. SA5 is a member of human enterovirus species B, which contains the echoviruses, coxsackie B viruses, coxsackievirus A9, and enterovirus 69. SV6, N125, and N203 are related to one another and, more distantly, to species A human enteroviruses, but could not be definitely assigned to a species. SV4 and SV28 are closely related to one another and to A-2 plaque virus, but distinct from other enteroviruses, suggesting that these simian viruses are members of a new enterovirus species. SV2, SV16, SV18, SV42, SV44, SV45, and SV49 are related to one another but distinct from viruses in all other picornavirus genera, suggesting that they may comprise a previously unknown genus in Picornaviridae. Several simian virus VP1 sequences (N125 and N203; SV4 and SV28; SV19, SV26, and SV35; SV18 and SV44; SV16, SV42, and SV45) are greater than 75% identical to one another (and/or greater than 85% amino acid identity), suggesting that the true number of distinct serotypes among the viruses surveyed is less than 20.

Animals↗

PubMed1999

Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages.

The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13). Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus. In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1. Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage. One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9. Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool. It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity.

Amino Acid Sequence↗

PubMed2000

Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1.

Three peptides corresponding to the 2-100 amino acids of VP1 unique sequence (VP1-F1), to the 99-227 amino acids of VP1 unique sequence (VP1-F2) and to the 237-781 amino acids of VP1 protein common to VP2 (VP1-F3 = VP2) were produced by prokaryotic expression. The three peptides, which span the entire VP1 structural protein of parvovirus B19 and also the entire VP2 protein, were used to evaluate the immunoreactivity against linear epitopes of these fragments in a large number of serum samples taken in different clinical situations with regards to B19 infection and in some commercial preparations of aspecific immunoglobulins. The data demonstrated that the specific VP1-F1 fragment, corresponding to the amino-terminal half of the VP1 unique region, is immunodominant and can elicit a long lasting immune response in comparison with VP1-F2 and VP1-F3 = VP2. Data regarding the presence of specific IgG to the three fragments in commercial preparations of immunoglobulins demonstrated that the dominant immune response was also against VP1-F1 linear epitopes while IgG against VP1-F2 and IgG against VP1-F3 = VP2 could be found only in high concentrations of Ig preparations. The reported data can be useful as a basis for the development of a B19 recombinant vaccine.

Adolescent↗