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PubMed1989

Sequences of capsid protein VP1 of two type A foot-and-mouth disease viruses.

We have sequenced the nucleotides of the regions that encode the capsid protein VP1 of the foot-and-mouth disease viruses (FMDV) A5Bernbeuren/1984 and A Iran/1987. Amino acid sequences and secondary protein structures are provided. Both proteins consist of 212 amino acids. The sequences and secondary structures are compared to those of FMDV A22/CCCP/64, a strain previously endemic in the Near East. Nucleotide divergency among the three sequences is highest for FMDV A5Bernbeuren/1984 (18% compared to 13% for each other case). Thirty amino acid divergencies are observed between A22/CCCP/64 and A5 Bernbeuren/1984 or A Iran/1987, whereas the latter two differ by 27 residues. The secondary structures of all three proteins are different. A Iran/1987 is considered to belong to a thus far unknown subtype.

Amino Acid Sequence↗

PubMed1995

Sequence and phylogenetic analysis of the VP1 gene in two cell culture-adapted HAV strains from a unique pathogenic isolate.

The nucleotide sequences of the VP1 coding region of two newly characterized, cell culture-adapted hepatitis A virus (HAV) strains (RG-SB11 and RG-SB16) were analyzed and compared with homologous regions of previously characterized HAV strains of human or monkey origin, and at different levels of tissue-culture adaptation. In particular, HM175wt and its derivative strains and MBB, LCDC1, PA21, and AGM27 isolates were considered. RG-SB11 and RG-SB16 HAV strains were derived from a pathogenic isolate from an acutely infected patient, purified from stool, and subjected to different strategies of adaptation. Several nucleotide differences were observed, but high conservation was found in the predicted VP1 protein sequences, which confirms structural constraints for this region. Furthermore, comparative amino-acid sequence analysis of VP1 from all HAV isolates studied has shown, particularly for those from naturally infected monkeys, that differences are limited to the amino and carboxy-terminal part of the molecule. The results of phylogenetic analysis have confirmed the common origin of the RG-SB11 and RG-SB16 strains. The complete nucleotide sequences of the VP1 coding region of the RG-SB11/16, HM175 derivative strains and of other HAV strains has shown that branch-length evolution can give a measure of the evolution of HAV during adaptation processes.

Amino Acid Sequence↗

PubMed1990

Comparison of vaccine strains and the virus causing the 1986 foot-and-mouth disease outbreak in Spain: epizootiological analysis.

RNAs of the most recent foot-and-mouth disease virus isolated in Spain (A5Sp86) during the 1986 outbreak, and of the three vaccine strains in use at that time in that country, have been compared. Although these viruses are serologically indistinguishable, differences have been found among them by T1 fingerprinting. This genetic heterogeneity affects the immunogenic VP1 gene, with amino acid changes located at the carboxyterminal end of the molecule. VP1-coding sequences obtained have been compared with those previously reported for European A5 FMDVs and it has been possible to trace their phylogenetic origin. The most parsimonious evolutionary tree obtained shows that the viruses analyzed are closely related to those previously isolated in 1983 in Spain, Portugal and Morocco. In spite of the VP1 sequence homology shown by this group of viruses, the genetic distances among field isolates and vaccine strains are significantly shorter than the distances found among field isolates. Thus, a significant relationship among virus recovered from recent outbreaks and the vaccine strains in use at that time in Spain, has been obtained.

Amino Acid Sequence↗

PubMed2000

Polyomavirus persistence in lymphocytes: prevalence in lymphocytes from blood donors and healthy personnel of a blood transfusion centre.

BK and JC polyomaviruses (BKV and JCV) are widespread in humans and are thought to persist and reactivate under immune alterations. In addition to the kidney, lymphoid cells have been proposed as a site of latency. However, while this was shown to occur in immunocompromised patients, discordant data were published for healthy humans. To help to solve this issue, an extensive study (231 healthy subjects) was carried out on peripheral blood mononuclear cells (PBMC) from blood donors of two towns and from operators of a blood transfusion centre. To discriminate between past and recent infection, nested PCRs for BKV and JCV non-coding control region (NCCR) and VP1 DNA sequences were carried out. Twenty-two per cent of subjects had BKV NCCR, but only 7% also had BKV VP1, as detected by PCR assays of similar sensitivities; the latter positivity was found to decrease with age. In both towns, the BKV WW archetypal DDP strain, subtype I, was found. Only 0.9% of subjects contained JCV DNA, for both NCCR and VP1. Blood operators presented a statistically significant increased prevalence of BKV NCCR (3. 0-fold) and BKV VP1 (9.4-fold) sequences with respect to blood donors of comparable ages, suggesting the possibility of occupational risk of BKV (re)infection or reactivation. Since the possibility of amplifying BKV VP1 sequences from PBMC of healthy humans is lost with age, this suggests that PBMC are not a site of polyomavirus persistence in healthy individuals and that detection of BKV VP1 DNA in PBMC is probably indicative of recent infection or reactivation.

Adolescent↗

PubMed2005

The minor receptor group of human rhinovirus (HRV) includes HRV23 and HRV25, but the presence of a lysine in the VP1 HI loop is not sufficient for receptor binding.

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.

Amino Acid Sequence↗

PubMed2006

Molecular genetics of the BK virus.

The BK Virus (BKV) genome is a double-stranded, circular DNA molecule with genetic organization similar to other polyomaviruses, and high homology to JC Virus (JCV) and SV40. The archetypal form of BKV noncoding regulatory region (NCRR) is the infectious form of BKV that replicates in the urothelium and is excreted in the urine. Rearranged forms of the NCRR are found in kidney and other tissues often in association with disease. BKV strains can be assigned to genotype/serotype groups based on sequence variation in the VP1 gene. Sequencing of the complete genomes from patient samples will enhance BKV phylogenetic studies and identify genotypic differences and naturally occurring mutations in BKV that may correlate with incidence and/or severity of a disease. This chapter is a review of the molecular genetics of the BK virus in respect to BKV disease.

BK Virus↗

PubMed1998

Expression of the major core structural proteins VP3 and VP7 of African horse sickness virus, and production of core-like particles.

The genome segments encoding the seven structural proteins of African horse sickness virus (AHSV), including the largest coding for VP1, were cloned and sequenced. Analysis of the VP1 sequence supports the putative identity of this protein as an RNA polymerase. The genes encoding the two major core proteins, VP3 and VP7, were also cloned and expressed by both in vitro translation and by means of recombinant baculoviruses. Co-infection of insect cells with VP3 and VP7 recombinant baculoviruses resulted in the intracellular formation of multimeric particles with a diameter of 72 nm, which structurally resembled authentic AHSV cores (core like particles: CLP). The complete genome of AHSV has now been cloned and sequenced.

African Horse Sickness Virus↗

PubMed2002

The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection.

The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75 degrees C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.

Amino Acid Sequence↗

PubMed1983

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment.

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C1-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O1K, A(10)61, A12 and C3 Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919-1931; Kleid et al., Science 214 (1981) 1125-1129; Boothroyd et al., Gene 17 (1982) 153-161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285-8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.

Amino Acid Sequence↗

PubMed2006

Analysis of the genetic and the corresponding antigenic variability of the VP1 3' end of ECHO virus type 11 and ECHO virus type 30.

The enteroviruses (EV), RNA viruses belonging to the Picornaviridae family, have a high genetic variability due to the absence of the efficient proofreading and post replicative repair activities associated with the RNA polymerase. In the present work, we studied the genetic and the antigenic variability of ECHO virus types 11 (E11) and 30 (E30), which are the most isolated echoviruses serotypes in clinical and environmental samples. We established on the 3' end of the VP1 gene, consensus sequences of E11 and E30 by alignment of 67 E11 and 247 E30 published sequences in GenBank. Our results of sequences comparison showed that the majority of the mutational sites are situated on the third nucleotide of the codon. These mutations were without consequence on the antigenic sequences of the VP1 protein. Thus, E11 and E30 have a high genetic variability (1/3 of the nucleotides are variable), but a relative antigenic conservation. The analysis of the intertypic antigenic variability between E11 and E30 was obtained by the alignment of the corresponding amino acids sequences relative to the N-terminal part of the VP1 protein. Two discriminating parts were highlighted, probably representing antigenic sites for neutralisation antibodies.

Amino Acid Sequence↗

PubMed1993

Peptide antisera targeted to a conserved sequence in poliovirus capsid VP1 cross-react widely with members of the genus Enterovirus.

Rabbits were immunized with synthetic peptides derived from an immunodominant region of the VP1 capsid protein of enteroviruses. This region shows a high degree of homology among all sequenced members of the genus. Peptide-induced antisera were used for immunoperoxidase staining of cell cultures infected with 41 different serotypes of enterovirus. Specific cytoplasmic staining was readily seen in all but two cases. Echovirus type 22 was previously known to differ genetically from the rest of the enteroviruses, and hence, a negative result was expected. Surprisingly, one of the tested serum samples reacted with echovirus 22-infected cells. Coxsackievirus A7-infected cells could be reliably stained with only one of the tested serum samples. For the remaining 39 serotypes, scattered infected cells resulting from 1 to 2 days of incubation with diluted inocula were easily scored as positive before the cytopathic effect became visible. The same antibodies were also used in a sandwich-type enzyme immunoassay to demonstrate poliovirus antigens in cell extracts as early as 3 h after a high-multiplicity infection. These antibodies are candidates for enterovirus group reagents, being potentially useful in both the laboratory diagnosis of enterovirus infections and research on enterovirus-host interactions.

Amino Acid Sequence↗

PubMed1979

Polyoma viruses with mutations at endonuclease HindII site 1: alterations at the COOH terminus of VP1.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.

Capsid↗

PubMed2003

Sequencing of 'untypable' enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types.

The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 'untypable' isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19.4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15.7-18.2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24.2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.

Adult↗

PubMed1987

Neutralization of foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites.

Seven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformation-dependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP1. The sequence of most or all of the VP1 coding region of 10 escape mutants of that site plus three parental isolates was determined by primer extension sequencing. At least five amino acids were found to be involved but in only one case (residue 148 of VP1) did a change at that residue produce complete resistance to neutralization. Partial resistance was produced by changes at residues 144, 154 or 208 of VP1 or another residue(s), as yet undefined, that is probably in one of the other capsid polypeptides. Thus the site defined by these mutants was made up of at least three regions, the region involving residues 144 to 154 of VP1, the region encompassing residue 208 from the COOH terminus of VP1, plus a region, probably of VP2 or VP3, encompassing the undefined residue(s).

Amino Acid Sequence↗

PubMed2003

Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997-2000.

Human enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae) has been responsible for sporadic cases and outbreaks of hand-foot-and-mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like disease in Europe, the U.S.A., Australia and Asia. Recently, there has been an increase in EV71 activity in the Asia-Pacific region, with many outbreaks of HFMD associated with brainstem encephalitis manifesting as neurogenic pulmonary oedema with a high case fatality rate. In 1997, and again in 2000, EV71 outbreaks occurred in peninsular Malaysia. Variations in VP1 gene sequences have been shown to divide all known EV71 field isolates into three distinct genogroups (A, B and C). Consequently we examined the VP1 gene sequences of 43 EV71 strains isolated in peninsular Malaysia between 1997 and 2000 in order to determine the genogroup prevalence over the period. In this study we show that four subgenogroups (B3, B4, C1 and C2) of EV71 circulated in peninsular Malaysia between 1997 and 2000. Subgenogroups B3, B4 and C1 have been identified as the primary cause of the outbreaks of EV71 in peninsular Malaysia. Subgenogroup C1 also displayed endemic circulation from 1997 to 2000 and subgenogroup C2 was present at a low level during the 1997 outbreak.

Amino Acid Sequence↗

PubMed1996

Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.

Amino Acid Sequence↗

PubMed1995

Identification and site-directed mutagenesis of the primary (2A/2B) cleavage site of the hepatitis A virus polyprotein: functional impact on the infectivity of HAV RNA transcripts.

The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site. Gln836-->Asn, and Gln836-->Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.

3C Viral Proteases↗

PubMed1996

Serological and molecular analysis of serotype O foot-and-mouth disease virus isolated from disease outbreaks in India during 1987-91.

Foot-and-mouth disease virus (FMDV) type O outbreaks have been reported frequently in vaccinated cattle in India. Twenty-five field isolates, recovered from outbreaks in vaccinated and unvaccinated cattle between 1987 and 1991, were analyzed in relation to the vaccine strain (R2/75) by complement fixation, serum neutralization and partial nucleotide sequencing of the VP1 gene. These sequences were compared with the viral sequences in GenEMBL database. Although the Indian type O viruses were close to the European type O1 viruses, they constituted a separate group of type O FMDVs. One of the field viruses, isolated from an outbreak in vaccinated cattle and designated as BAK/90, showed significant serological and nucleotide sequence variations from the vaccine strain. Phylogenetic analysis showed that the BAK/90 and R2/75 viruses belong to separate subgroups. The other isolates were found to be serologically related to both the BAK/90 and the vaccine strain. The BAK/90 strain gave broader antigenic coverage, showed better immunogenicity, and yielded larger amounts of 146S particles in suspension cultures as compared with R2/75. Taken together, these results favour inclusion of the BAK/90 strain in the vaccine to provide adequate protection against the field variants of type O FMDV currently circulating in India.

Amino Acid Sequence↗