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[Application of the nucleotidic sequencing of VP1 to the identification of human Enterovirus].

The introduction of a mollecular method to identify the Entoviruses based on the amplification sequecing and phylogenetic analysis of protein VPI was described. It was proved that this method reduces significantly the time required for the identification of the isolated Entoviruses and that it is very useful in the characterization of isolates which are difficult to typify by the routine immunoloigical reagents. As it is a very fast technqiue, its use is very important during epidemics to determine the causal agent rapidly.

Enterovirus↗

PubMed1986

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus.

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Animals↗

PubMed1997

Evidence that virion-associated VP1 of avibirnaviruses contains viral RNA sequences.

The VP1 encoded by genomic segment B of birnaviruses is generally known to exist as a genome-linked protein (VPg) and as a "free" polypeptide of 90 kDa in virus particles. The guanylylation activity associated with infectious bursal disease virus (IBDV) was demonstrated by incubating purified virus in presence of [alpha 32P] GTP; optimum activity in the 90 kDa form of VP1 was seen in low salt concentration in the presence of 4 mM magnesium ions over a wide range of incubation temperatures. The IBDV VP1 was shown to lack guanyl transferase activity. Northwestern (RNA-protein) blot analysis of purified virus using a radiolabelled cDNA probe consisting of 3' and 5' ends of genomic segment B indicated that both forms of virion-associated VP1 contained viral RNA sequences of which those linked to VPg corresponded to the two genome segments and those linked to the 90 kDa VP1 were probably a short oligonucleotide of the terminal viral RNA sequences.

Animals↗

PubMed1993

BK virus antigenic variants: sequence analysis within the capsid VP1 epitope.

DNA sequences for the VP1 gene which codes for the major capsid protein of BK virus (BKV) and may be responsible for antigenic variability were determined for seven BKV isolates. The observed sequence differences and those previously reported correlate with the typing of isolates into four groups by haemagglutination inhibition. Amino acid coding alterations were found to be clustered within residues 61 to 83. Each antigenic group was found to have a characteristic amino acid sequence between residues 61 and 83. Several clones originating from a single isolate, although differing slightly in restriction enzyme digestion patterns, were found to be identical in this region. The VP1 sequences of three of the four groups were analysed by hydropathy plots and two hydrophilic areas of high antigenicity were identified. One of these corresponds to residues 61 to 83 and it is postulated that this region is the epitope responsible for serotypic differences between BK isolates.

Amino Acid Sequence↗

PubMed2004

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002.

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.

Animals↗

PubMed2007

Emergence of recent echovirus 30 lineages is marked by serial genetic recombination events.

In an earlier report, different variants of echovirus 30 (E-30), an enterovirus serotype, were identified during two outbreaks in 1997 and 2000. Here, the diversity of E-30 was investigated over a longer period (1991-2005) and the variations in four genomic segments were determined in 52 isolates involved in meningitis cases, to characterize the evolutionary processes underlying the emergence of lineages. Phylogenetic analysis of the VP1 sequences showed that five phylogenetic variants succeeded one another. When a partial 3CD segment was examined, the five variants split further into 10 lineages. Phylogenetic groupings observed with both the VP1 and 3CD sequences were clearly related to the calendar time of virus isolation. The rapid turnover of lineages during the study period was not associated with variations in amino acid residues in either the VP1 or the 3CD sequences, indicating major evolutionary contraints in E-30. The variation patterns were examined further along a subgenomic segment of 4878 nt in 13 virus isolates, representative of the 10 lineages. Breakpoints detected in the similarity profiles were investigated by bootscanning and maximum-likelihood phylogenetic analysis of virus genes. Evidence of several past recombination events was observed in the middle of the genome and predicted recombination crossover sites were mapped with precision. The contribution of recombination to the evolution of E-30 is substantial. It is associated tightly with the emergence of new genetic lineages and certain recombinants have undergone epidemic spread.

Enterovirus B, Human↗

PubMed2025

Global Evolution and Transmission Dynamics of Enterovirus D68.

Enterovirus D68 (EV-D68), a serotype of the enterovirus species D, has garnered significant attention due to outbreaks reported in 2014, 2016, and 2018. In this study, 36 Chinese EV-D68 strains were isolated, sequenced, and combined with all EV-D68 VP1 sequences from GenBank to form a data set of 1679 sequences. This data set served as the basis for phylogenetic, evolutionary dynamics, phylogeographic, and key amino acid site mutation analyses of EV-D68. Based on the VP1 region, EV-D68 is classified into four genotypes (A-D), and seven subgenotypes (B1-B3, D1-D4), with B3 and D3 being the predominant subgenotypes. Bayesian skyline plots indicated that genotypes B and D experienced multiple population expansions, aligning with reported EV-D68 outbreaks. Phylogeographic analyses of the B3 subgenotypes revealed sequences from Europe and North America clustering into a single evolutionary branch, suggesting significant transmission between these regions. Additionally, mutation analysis identified VP1-98 as a high-frequency mutation site, differing significantly between the previously prevalent A and C genotypes and the currently prevalent B and D genotypes. However, the functional implications of this mutation require further investigation. This study provides a solid theoretical basis for epidemiological research, disease surveillance, and prevention efforts related to EV-D68.

Enterovirus Infections↗

PubMed2001

Molecular characterization of foot-and-mouth disease virus isolated from ruminants in Taiwan in 1999-2000.

In 1999, 10 sporadic outbreaks of cattle foot-and-mouth disease (FMD) occurred in Taiwan. By the time, infection was limited to the Chinese yellow cattle (a native species of beef cattle in Mainland China), which did not develop vesicular lesions under field conditions. Five viruses isolates obtained from individual farms were confirmed to be the serotype O FMD virus (O/Taiwan/1999). During January-February 2000, however, this virus has spread to dairy cattle and goat herds, causing severe mortality in goat kids and vesicular lesions in dairy cattle. Partial nucleotide sequence of the capsid coding gene 1D (VP1) was determined for the virus isolates obtained in this study. Phylogenetic analysis of the VP1 sequences indicated that the O/Taiwan/1999 viruses shared 95-97% similarities to the virus strains isolated from the Middle East and India. The species susceptibility of the O/Taiwan/1999 virus was experimentally studied in several species of susceptible animals, showing that the virus did cause generalized lesions in dairy cattle and pigs, however, it would not cause vesicular lesions on the Chinese yellow cattle and the adult goats. These studies suggested that the O/Taiwan/1999 virus was a novel FMD virus of Taiwan and it presented various levels of susceptibility in cattle species.

Amino Acid Sequence↗

PubMed2005

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Para, Brazil.

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Para showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Acute Disease↗

PubMed2001

Evidence for positive selection in foot-and-mouth disease virus capsid genes from field isolates.

The nature of selection on capsid genes of foot-and-mouth disease virus (FMDV) was characterized by examining the ratio of nonsynonymous to synonymous substitutions in 11 data sets of sequences obtained from six different serotypes of FMDV. Using a method of analysis that assigns each codon position to one of a number of estimated values of nonsynonymous to synonymous ratio, significant evidence of positive selection was identified in 5 data sets, operating at 1-7% of codon positions. Evidence of positive selection was identified in complete capsid sequences of serotypes A and C and in VP1 sequences of serotypes SAT 1 and 2. Sequences of serotype SAT-2 recovered from a persistently infected African buffalo also revealed evidence for positive selection. Locations of codons under positive selection coincide closely with those of antigenic sites previously identified with the use of monoclonal antibody escape mutants. The vast majority of codons are under mild to strong purifying selection. However, these results suggest that arising antigenic variants benefit from a selective advantage in their interaction with the immune system, either during the course of an infection or in transmission to individuals with previous exposure to antigen. Analysis of amino acid usage at sites under positive selection indicates that this selective advantage can be conferred by amino acid substitutions that share physicochemically similar properties.

Amino Acid Sequence↗

PubMed2006

Genetic and antigenic diversity among noroviruses.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Amino Acid Sequence↗

PubMed1997

Genotype-specific RNA probes for direct identification of wild polioviruses by blot hybridization.

We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genotypes. Hybrids were detected by a sensitive chemiluminescence assay, capable under normal diagnostic conditions of detecting specific wild poliovirus sequences in samples containing up to a 100-fold excess of Sabin vaccine strain-related sequences of the same serotype.

Base Sequence↗

PubMed2026

Evaluation of amplicon-based nanopore sequencing for foot-and-mouth disease viruses in clinical and environmental samples.

Foot-and-mouth disease (FMD) causes severe global economic loss, necessitating rapid viral characterization. Nanopore sequencing provides a simple, real-time workflow suitable for on-site outbreak response, addressing the limitations of conventional methods. In this study, we optimized a previously published amplicon-based protocol and used this method to characterize a diverse range of samples (vesicular fluid, epithelium, serum, nasal/oral swabs, and environmental samples) collected during FMD outbreaks in 2025 in the Republic of Korea. Of the 129 samples collected, we successfully recovered complete genomes from 37 samples and VP1 sequences from 85 samples. Amplifying the S-fragment in isolation and separately barcoding each pool of PCR amplicons markedly improved sequence recovery. Furthermore, sequencing success depended on viral load and sample type. Based on comparisons with real-time RT-PCR results, whole-genome sequence (WGS) recovery exceeded 77.3% at cycle threshold (Ct) values ≤25 across all clinical samples. In the Ct > 30 category, serum samples yielded the highest WGS recovery rates (44.4%). This rate was markedly higher than the success rates observed for epithelium (20.0%) and nasal swabs (9.1%), whereas oral swabs and environmental samples failed to yield any sequences (0%). However, VP1 recovery from environmental samples reached 80% at Ct ≤ 30 (8/10), providing an approach to enable non-invasive monitoring. These findings demonstrate that amplicon-based nanopore sequencing is a practical method for the rapid generation of genomic data during FMD outbreaks.IMPORTANCEAlthough rapid detection and genomic data analysis are crucial for effective foot-and-mouth disease (FMD) control, the collection of these data can be challenging for certain sample types and impacted by reduced viral loads that result from nationwide FMD vaccination. This study provides a practical solution through large-scale evaluation of an optimized amplicon-based nanopore sequencing protocol to enhance the sequencing success rates for both clinical and environmental samples. Using a modified protocol to enhance genome recovery, we demonstrated that sequence data could be retrieved from diverse sample types (even with high real-time RT-PCR cycle threshold values). We identified serum as the most suitable sample, with environmental sample sequencing allowing for non-invasive monitoring during outbreaks. These results support the use of nanopore sequencing for rapid genomic analysis, particularly in outbreak responses, such as rapid surveillance, emergency vaccine selection, and epidemiological monitoring.

Foot-and-Mouth Disease↗

PubMed1995

Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors.

Simian virus 40 (SV40) sequences for large tumor antigen (T-ag) were recently detected in a significant fraction of certain human brain tumors of early childhood (Bergsagel et al., N. Engl. J. Med. 326, 988-993, 1992). In the current study, we sought to determine whether authentic SV40 was present in the choroid plexus and ependymoma tumors previously examined. Polymerase chain reaction and DNA sequence analysis revealed authentic SV40 regulatory region and major capsid (VP1) sequences in 14 of 17 tumors tested. Only one 72-basepair element was detected in the SV40 enhancer region of positive tumor samples, an arrangement designated as "archetypal." The C terminus of the T-ag gene was detected in the same 14 tumors and was sequenced from 5 tumors; some nucleotide changes were found that would result in amino acid changes in T-ag. Infectious SV40 was isolated from one sample after lipofection of tumor DNA into monkey kidney cells. Sequence analysis of the rescued virus SVCPC revealed (i) an archetypal regulatory region, (ii) nucleotide changes in the C terminus of the T-ag gene that distinguished it from SV40 laboratory strains 776 and SV40-B2 and from human isolate SVPML-1, and (iii) identity with previous human brain tumor isolate SVMEN in the three genomic regions sequenced. No human-isolate-specific distinguishing features were detected among the viral sequences analyzed. Thus, authentic SV40 is present in humans and associated with two tumor types known to be induced experimentally by the virus.

Amino Acid Sequence↗

PubMed1994

Animal-derived antigenic variants of foot-and-mouth disease virus type A12 have low affinity for cells in culture.

We recently have shown that binding of foot-and-mouth disease virus (FMDV) to cells in culture requires an arginine-glycine-aspartic acid (RGD) sequence in the G-H loop of the capsid protein VP1 (P. W. Mason, E. Rieder, and B. Baxt, Proc. Natl. Acad. Sci. USA 91:1932-1936, 1994). In this report, we show that FMDV type A12 viruses found in infected bovine tongue tissue (BTT) differ from their tissue culture-grown derivatives at amino acid residues near the RGD. Viruses genetically engineered to contain VP1 sequences found in animal tissue (BTT viruses) were antigenically different from their tissue culture derivatives and bound to BHK cells more poorly than did the tissue culture-adapted viruses. Passage of the genetically engineered BTT viruses in BHK cells resulted in the rapid selection of variants with cell-binding properties, antigenic characteristics, and sequences typical of tissue culture-adapted viruses. These data indicate that residues near the RGD are critical for cell binding and that interpretations of antigenic variation of FMDV can be affected by virus cultivation in vitro.

Amino Acid Sequence↗

PubMed2026

Molecular Epidemiology and Pathogenicity Evaluation of Porcine Teschovirus in Tibetan Pigs on the Qinghai-Tibet Plateau of China.

Porcine teschovirus (PTV) is the causative agent of porcine diarrhea and multisystem disorder and poses a global threat to the health of domestic pigs. However, its epidemic and pathogenic characteristics in Tibetan pigs, which are a unique indigenous breed in the Qinghai-Tibet Plateau of China, remain largely unexplored. Here, we conducted a comprehensive investigation during 2024-2025, in which 303 diarrheic fecal samples were collected from 21 farms across eight counties in Ganzi Tibetan Autonomous Prefecture, with an average altitude of 3433 m. RT-PCR testing identified 141 PTV-positive samples, yielding a high positivity rate of 46.5% (141/303). From these positives, 27 VP1 sequences were cloned and sequenced; phylogenetic analysis revealed that 16 strains belonged to Teschovirus A, three strains were divided into Teschovirus B, and three strains formed the interspecies recombinant genotypes (PTV-15/16). Interestingly, five novel strains were classified as undefined genotypes, indicating an extensive genetic diversity among PTV strains circulating in Tibetan pigs. Furthermore, a PTV strain, designated PTV-SCgz-01, was successfully isolated in PK-15 cells, with a near-complete genomic sequence of 7081 nucleotides. Phylogenetic analysis based on the polyprotein and the VP1 genes indicated that it belonged to genotype PTV-4, whereas recombination analysis revealed that PTV-SCgz-01 is a natural recombinant with parental strains derived from HNMY (PTV-4) and China/SWU-ZG2/2018 (PTV-6). Experimental infection of 17-day-old Tibetan piglets demonstrated that this isolate induces severe watery diarrhea. Notably, the virus also caused severe pulmonary hemorrhage and mild cerebral hyperemia with neuronal degeneration, and it had a high mortality rate (40%), suggesting that strain PTV-SCgz-01 has strong pathogenic potential for 17-day-old Tibetan piglets. Our findings provide a more comprehensive molecular epidemiology of PTVs in Tibetan pigs and underscore the need for viral surveillance and control in this unique pig population.

Animals↗

PubMed1987

Nucleic acid sequence of the VP1 region of attenuated MS-1 hepatitis A virus.

The nucleotide sequence of the VP1 region of marmoset-attenuated hepatitis A virus (HAV), MS-1, was determined by incorporative dideoxynucleotide sequencing of the RNA obtained from purified, liver-derived virus. Comparison of this nucleotide sequence to those of four previously published isolates revealed that one of the isolates, HM-175, which was obtained from Australia and passed three times in marmosets, had a 8.5-11% nucleotide variability compared to the remaining four isolates which were isolated from North American sources. This nucleotide variability does not result in amino acid differences with the exception of two of the North American isolates, which were derived from tissue culture passage. These isolates have been shown to contain regions of variability generated by nucleotide insertions and/or deletions, while the remaining three isolates, including the Australian isolate, demonstrate limited amino acid differences within the VP1 molecule.

Amino Acid Sequence↗

PubMed1991

Two mechanisms of antigenic diversification of foot-and-mouth disease virus.

The amino acid replacements that underlay the diversification of the main antigenic site A (VP1 residues 138 to 150) of foot-and-mouth disease virus (FMDV) of serotype C have been identified. Sixteen new VP1 sequences of isolates from 1926 until 1989 belonging to subtypes C1, C2, C3, C4, C5, and unclassified are reported. The reactivities in enzyme-linked immunoelectrotransfer blot assays of capsid protein VP1 with a panel of neutralizing monoclonal antibodies that recognize sites A or C (the VP1 carboxy-terminus) have been correlated with the amino acid sequence at the relevant epitopes. The analyses involving the immunodominant site A reveal two mechanisms of antigenic change. One is a gradual increase in antigenic distance brought about by accumulation of amino acid replacements at two hypervariable segments within site A. A second mechanism consists of an abrupt antigenic change manifested by loss of many epitopes, caused by one replacement at a critical position (particularly Ala (145)----Val or His (146)----Gln). The identification of the amino acid substitutions responsible for such large antigenic changes provides new information for the design of synthetic anti-FMD vaccines. However, the screening of isolates from six decades suggests that the virus, even within the confines of a single serotype, has exploited a minimum of its potential for antigenic variation.

Amino Acid Sequence↗