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PubMed2025

Molecular characterization of JC virus in progressive multifocal leukoencephalopathy cases from India.

Progressive Multifocal Leukoencephalopathy (PML) is a rare, often fatal demyelinating disease of the central nervous system caused by reactivation of the John Cunningham virus (JCV) in immunocompromised individuals. Despite an estimated 2.4 million people living with HIV in India, the reported incidence of PML remains lower than in Western countries, likely due to underdiagnosis, underreporting, and distinct host genetic and viral factors. The rising number of individuals on immunosuppressive therapies, including organ transplant recipients and those with autoimmune disorders, further emphasizes the need to study JC virus diversity in the Indian context. This study aimed to characterize the genetic diversity of JCV in India by sequencing the VP1 and non-coding control region (NCCR) from cerebrospinal fluid (n=30) of confirmed PML cases using Sanger sequencing. VP1 sequencing (n=23) revealed a predominance of genotypes 2 (subtypes 2D, 2A, 2B) and 3A. NCCR analysis (n=17) showed extensive rearrangements relative to the archetype form, with most sequences classified as Type II-R. Structural variations, including deletions, duplications, and insertions were common, particularly in blocks D, C, and F. Transcription factor binding sites (TFBS) were identified for TATA box, Tst-1, SP-1, p53, CEBPB, AP-1, NF-1, EGR-1, GF-1, CRE-TAR and NFkB. Additional TFBS were created due to rearrangements, often spanning two blocks. These findings underscore the genomic diversity of JCV in India and highlight the need for continued molecular surveillance to better understand its implications for high-risk populations.

Leukoencephalopathy, Progressive Multifocal↗

PubMed2002

Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences.

Chicken anaemia virus (CAV) was detected in the bursa of Fabricius of a 4-week-old chicken obtained from an outbreak of acute infectious bursal disease in Bangladesh. Repeated attempts to grow this virus in MDCC-MSB1 cells were not successful. A full-length PCR amplicon of the genome of this strain, designated as BD-3 CAV, was cloned and sequenced. The complete nucleotide sequence and the deduced amino acid sequence were compared with those of 12 other CAV strains. The genetic analysis of the amino acid sequences of VP1 indicated the possible existence of genetic groups among CAV strains, as BD-3 CAV along with four other strains (CIA-1, L-028, Isolate 704 and TR-20) formed a distinct lineage. These strains have four signatory amino acids in VP1, such as 75I/T, 97L, 139Q and 144Q, out of which the latter two are located in a small hydrophilic peak.

Amino Acid Sequence↗

PubMed2003

Multiple viral determinants mediate myopathogenicity in coxsackievirus B1-induced chronic inflammatory myopathy.

Mice infected with myopathic coxsackievirus B1 Tucson (CVB1(T)) develop chronic inflammatory myopathy (CIM) consisting of hind limb weakness and inflammation. Amyopathic virus variants are infectious but attenuated for CIM. In this report, viral clones, chimeras, and sequencing were used to identify viral determinants of CIM. Chimeras identified several regions involved in CIM and localized a weakness determinant to nucleotides 2493 to 3200 of VP1. Sequencing of multiple clones and viruses identified five candidate determinants that were strictly conserved in myopathic viruses with one located in the 5' untranslated region (UTR), three in the VP1 capsid, and one in the 3C protease. Taken together, these studies implicate Tyr-87 and/or Val-136 as candidate determinants of weakness. They also indicate that there are at least two determinants of inflammation and one additional determinant of weakness encoded by myopathic CVB1(T).

Animals↗

PubMed2006

Molecular typing of enteroviruses associated with viral meningitis in Cyprus, 2000-2002.

Human enteroviruses are responsible for a wide spectrum of clinical diseases affecting many different organ systems. Although infection is usually asymptomatic, infections of the central nervous system manifested as meningitis or encephalitis can pose a serious public health problem, especially during outbreaks. In this study, samples from 218 patients diagnosed with enteroviral meningitis between January 2000 and December 2002 were analysed in order to assess the epidemiology of human enteroviruses as a cause of viral meningitis in Cyprus. A new typing strategy, based on partial sequencing of the 5' non-coding region (5'NCR), prediction of type, and selection of type-specific primers for sensitive VP1 PCR amplification, was developed. As clustering in the 5'NCR was concordant with clustering in the VP1 region, quick and reliable typing by VP1 sequencing was achieved without virus isolation in cell culture. The most frequent enterovirus serotypes identified were Human echovirus 30 (55.5%), Human echovirus 13 (15.1%), Human echovirus 6 (13.8%) and Human echovirus 9 (8.3%). Human coxsackieviruses B2, B1 and B5, Human echovirus 4, Human enterovirus 71 and Human coxsackievirus A6 represented rather rare serotypes. This is the first molecular epidemiological study of enterovirus meningitis in Cyprus. Serotype distribution corresponded basically with observations in other European countries, suggesting the spread of enteroviruses by tourism.

5' Untranslated Regions↗

PubMed1990

Heterotypic protection induced by synthetic peptides corresponding to three serotypes of foot-and-mouth disease virus.

Synthetic peptide vaccines of the general sequence Cys-Cys(200-213)-Pro-Pro-Ser-(141-158)-Pro-Cys-Gly, where the numbered residues refer to VP1 sequences of three different strains of foot-and-mouth disease virus, have been evaluated in cattle and guinea pigs. High levels of serotype-specific (homotypic) antiviral and antipeptide antibody were produced with each peptide. The A- and O-serotype peptides provided complete protection of guinea pigs against their respective virus challenges. The C-serotype peptide appeared to be less effective than the other peptides. In cross-protection studies (heterotypic) in guinea pigs, it was possible to protect A-serotype peptide-vaccinated animals against O-virus challenge and vice versa. Some heterotypic protection was also achieved with the C-serotype peptide. The heterotypic protection observed related more to the presence of cross-reactive antipeptide antibody than to neutralizing antibody.

Amino Acid Sequence↗

PubMed1996

A protective anti-peptide antibody against the immunodominant site of the A24 Cruzeiro strain of foot-and-mouth disease virus and its reactivity with other subtype viruses containing the same minimum binding sequence.

A synthetic peptide vaccine of the general sequence Cys-Cys-(200-213)-Pro-Pro-Ser-(l41-158)-Pro-Cys-Gly(peptide A40), where the numbered residues refer to the VP1 sequence of foot-and-mouth disease virus (FMDV) strain A24 Cruzeiro, has previously been shown to elicit neutralizing and protective antibodies in guinea-pigs and cattle. To examine this immunogenic tract in more detail monoclonal antibodies (MAbs) were raised to this peptide. One such MAb C1.1, which recognized the homologous peptide, bound to native virus, neutralized infectivity in vitro and passively protected mice from challenge. Using overlapping dodecameric peptides the minimum binding 'footprint' of this MAb incorporated residues 149-154 which were respectively Gly-Ser-Leu-Ala-Ala-Arg. Since this 'footprint' occurs in several other A subtype strains of FMDV, the extent to which MAb C1.1 could cross-react was also examined. Using a liquid-phase competition ELISA, only viruses with a sequence that encompassed the same minimum binding 'footprint', namely A27 Cundinamarca Colombia/76, A Argentina/79, and A Venceslau Brazil/76 reacted with similar affinity against MAb C1.1. However, further serological examination of C1.1 with these viruses by indirect ELISA, in vitro neutralization and passive protection showed clear functional disparity. In contrast to the liquid-phase ELISA, the ability of C1.1 to react with electrostatically bound virus varied significantly depending on the subtype examined. Moreover, the capacity of this MAb to neutralize these subtypes showed wide divergence which was mirrored by the protection data.

Amino Acid Sequence↗

PubMed1995

Characterization of an acid-resistant mutant of foot-and-mouth disease virus.

A foot-and-mouth disease virus mutant which is stable at pH 6.4 has been isolated from a virus of serotype A. In contrast to the parent (P) virus, which gave a mixture of large and small plaques in BHK21 cells and in a bovine kidney cell line, the acid-resistant (AR) virus gave small plaques which did not increase markedly in size after 24 hr. The infectivity titer of the acid-resistant virus was about 100-fold lower in suckling mice than in BHK21 cells, whether the inoculation was made intraperitoneally or intracerebrally, whereas the parent virus gave similar titers in both systems. Furthermore, in mice the AR virus reached its end point two to three times more slowly. The diameter of the AR virus was almost 20% less than that of the P virus and it had a more distinct topography, but the two viruses cosedimented in sucrose gradients. However, the buoyant density in CsCl of the AR virus was slightly lower (1.42 compared with 1.43 g/cc) in coruns. The RNAs and capsid proteins of the two viruses gave similar profiles in sucrose gradients and by SDS-PAGE, respectively. However, isoelectric focusing of the capsid proteins revealed considerable differences between the two viruses. Whereas the P virus gave four protein bands, corresponding to VP1-VP4, the AR virus gave one band for VP4, two for VP3, two for VP2, and four for VP1. Sequence analysis of the genes coding for the capsid protein regions of the two viruses showed four changes (one silent), resulting in an Ala-3-->Ser substitution in VP1 and Glu-131-->Lys and Asp-133-->Ser substitutions in VP2.

Animals↗

PubMed2003

Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design.

Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11-25) and VP3(102-121), in rabbits immunised with either Freund's adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund's protocol.

Amino Acid Sequence↗

PubMed1983

Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing.

A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VP0 were isolated from sucrose gradient purified virus by chromatofocusing in a pH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4 did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.

Animals↗

PubMed2003

Evidence of recombination in natural populations of hepatitis A virus.

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. At least seven HAV genotypes have been identified all over the world, including four human genotypes (I, II, III, and VII) and three simian strains (IV, V, and VI). Phylogenetic analysis using full-length VP1 sequences revealed that human strain 9F94 has a close genetic relation with strain SLF-88 (sub-genotype VII). Nevertheless, the same analysis using full-length VP2 or VP3 sequences revealed that strain 9F94 has a close genetic relation with strain MBB (sub-genotype IB). To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossing over had taken place in the VP1 capsid protein. These findings indicate that capsid-recombination can play a significant role in shaping the genetic diversity of HAV and, as such, can have important implications for its evolution, biology, and control.

Capsid Proteins↗

PubMed1993

Detection of foot-and-mouth disease viral sequences in clinical specimens and ethyleneimine-inactivated preparations by the polymerase chain reaction.

The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.

Amino Acid Sequence↗

PubMed2026

Balancing under constraint: Structural insights into norovirus evolution and antigenic innovation.

Norovirus is the leading cause of acute viral gastroenteritis worldwide. While genomic studies have revealed its diversity and evolutionary patterns, the structural mechanisms driving viral adaptation remain poorly understood. Here, we establish a comprehensive structural database of norovirus VP1 P-domains across nine genogroups (GI-GIX) through large-scale AlphaFold2 predictions. By integrating phylogenetic analysis of VP1 sequences and structures, we demonstrate that sequence and structural evolution show overall concordance under purifying selection, yet significant local discrepancies reveal distinct patterns of convergent evolution shaped by structural constraints and functional divergence. Focusing on the predominant GII.4 genotype, we found that compared to near-full-genome and nucleotide trees, only the VP1 amino acid tree reliably clustered GII.4 variants in chronological order as monophyletic groups. We further identify a hierarchical evolutionary strategy: positive selection may drive structural hypervariability in major antigenic epitopes D and C for immune escape, with epitope D exhibiting pronounced structural flexibility that complicates its structural characterization, whereas coevolutionary analysis uncovers a broad network of compensatory interactions spanning multiple epitopes, with striking enrichment in epitope A. These epitopes exhibited a pattern of "sequence plasticity with structural conservation", maintained by coevolutionary constraints that preserve conformational integrity. Together, these findings suggest that norovirus vaccine strategies targeting the structurally conserved conformations of epitopes A and G could overcome the limitations of traditional strain-specific approaches, offering a pathway toward broad protection against evolving viral diversity.

Norovirus↗

PubMed2006

Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies.

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type 1 diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction.

Autoantibodies↗

PubMed1995

A method for determining the position and size of optimal sequence regions for phylogenetic analysis.

The availability of fast and accurate sequencing procedures along with the use of PCR has led to a proliferation of studies of variability at the molecular level in populations. Nevertheless, it is often impractical to examine long genomic stretches and a large number of individuals at the same time. In order to optimize this kind of study, we suggest a heuristic procedure for detection of the shortest region whose informational content can be considered sufficient for significant phylogenetic reconstruction. The method is based on the comparison of the pairwise genetic distances obtained from a set of sequences of reference to those obtained for different windows of variable size and position by means of a simple index. We also present an approach for testing whether the informative content in the stretches selected in this way is significantly different from the corresponding content shown by the larger genomic regions used as reference. Application of this test to the analysis of the VP1 protein gene of foot-and-mouth-disease type C virus allowed us to define optimal stretches whose informative content is not significantly different from that displayed by the complete VP1 sequence. We showed that the predictions made for type C sequences are valid for type O sequences, indicating that the results of the procedure are consistent.

Animals↗

PubMed2000

Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel.

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40 degrees C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A(481) to G in the 5' untranslated region and the VP1 amino acid I(143) to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for approximately 6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.

5' Untranslated Regions↗

PubMed2025

Opportunities for machine learning to predict cross-neutralization in FMDV serotype O.

Accurately estimating cross-neutralization between serotype O foot-and-mouth disease viruses (FMDVs) is critical for guiding vaccine selection and disease management. In this study, we developed a machine learning approach to estimate r1 values-an established measure of antigenic similarity-using VP1 sequence data and published virus neutralization titer (VNT) results. Our dataset comprised 108 serum-virus pairs representing 73 distinct FMDV strains. We applied Boruta feature selection and random forest classifiers, optimizing model performance through tenfold cross-validation and sub-sampling to address class imbalance. Predictors included pairwise amino acid distances, site-specific polymorphisms, and differences in potential N-glycosylation sites. Using a 0.3 r1 threshold to define cross-neutralization, the final model achieved high accuracy (0.96), sensitivity (0.93), and specificity (0.96) in training, and performed robustly on independent test sets - accuracy was 0.75 (95% CI 0.60 and 0.90), F1 score 0.86% and PPV 0.77. Importantly, key VP1 residues-positions 48, 100, 135, 150, and 151-emerged as strong predictors of antigenic relationships. Our results demonstrate the utility of integrating routinely generated genomic data with machine learning to inform vaccine candidate selection and anticipate immune interactions among circulating FMDV strains. This approach offers a practical tool for accelerating vaccine decision-making and can be adapted to other FMDV serotypes. The latest version of the r1 predictive model is available for access via a Shiny dashboard (https://dmakau.shinyapps.io/PredImmune-FMD/).

Foot-and-Mouth Disease Virus↗

PubMed2004

Characterization of the complete genomic sequence of genotype II hepatitis A virus (CF53/Berne isolate).

The complete genomic sequence of hepatitis A virus (HAV) CF53/Berne strain was determined. Pairwise comparison with other complete HAV genomic sequences demonstrated that the CF53/Berne isolate is most closely related to the single genotype VII strain, SLF88. This close relationship was confirmed by phylogenetic analyses of different genomic regions, and was most pronounced within the capsid region. These data indicated that CF53/Berne and SLF88 isolates are related more closely to each other than are subtypes IA and IB. A histogram of the genetic differences between HAV strains revealed four separate peaks. The distance values for CF53/Berne and SLF88 isolates fell within the peak that contained strains of the same subtype, showing that they should be subtypes within a single genotype. The complete genomic data indicated that genotypes II and VII should be considered a single genotype, based upon the complete VP1 sequence, and it is proposed that the CF53/Berne isolate be classified as genotype IIA and strain SLF88 as genotype IIB. The CF53/Berne isolate is cell-adapted, and therefore its sequence was compared to that of two other strains adapted to cell culture, HM-175/7 grown in MK-5 and GBM grown in FRhK-4 cells. Mutations found at nucleotides 3889, 4087 and 4222 that were associated with HAV attenuation and cell adaptation in HM175/7 and GMB strains were not present in the CF53/Berne strain. Deletions found in the 5'UTR and P3A regions of the CF53/Berne isolate that are common to cell-adapted HAV isolates were identified, however.

Base Sequence↗

PubMed2004

Cross-reactivity among sapovirus recombinant capsid proteins.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences. Human SaV strains are noncultivable, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV. In this study, rVP1 constructs of SaV GI, GII, and GV strains were expressed in a baculovirus expression system. The structures of the GI, GII, and GV VLPs, with diameters of 41-48 nm, were morphologically similar to those of native SaV. However a fraction of GV VLPs were smaller, with diameters of 26-31 nm and spikes on the outline. This is the first report of GII and GV VLP formation and the first identification of small VLPs. To examine the cross-reactivities among GI, GII, and GV rVP1, hyperimmune rabbit antisera were raised against Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1. Western blotting showed the GI antisera cross-reacted with GV rVP1 but not GII rVP1; GII antisera cross-reacted weakly with GI rVP1 but did not cross-react with GV rVP1; and GV antisera reacted only with GV rVP1. Also, hyperimmune rabbit and guinea pig antisera raised against purified GI VLPs were used to examine the cross-reactivities among GI, GII, and GV VLPs by an antigen enzyme-linked immunosorbent assay (ELISA). The ELISA showed that the GI VLPs were antigenically distinct from GII and GV VLPs.

Animals↗