Virus isolate from carp: genetic characterization reveals a novel picornavirus with two aphthovirus 2A-like sequences.

Picornaviruses have been isolated from a variety of hosts, mainly mammals and birds. Here, we describe the sequence analysis of carp picornavirus 1 (CPV-1) F37/06 that was isolated from an organ pool (heart, brain, liver) of a common carp (Cyprinus carpio). This carp perished after an accidental discharge of liquid manure into a fish pond and presented without obvious clinical symptoms. Experimental intraperitoneal infection of young carp with CPV-1 revealed no clinical signs, but the virus was re-isolated from various organs. Sequence analysis of almost the complete genome (7632 nt excluding the poly-A tract) revealed a novel picornavirus clade. In phylogenetic trees, the polymerase sequence clusters with parechoviruses, duck hepatitis A virus, eel picornavirus and aquamavirus A. The ORF includes 6807 nt and encodes a polyprotein of 2269 amino acids. CPV-1 has a genome layout like that of picornaviruses except for the presence of two aphthovirus 2A-like NPGP sequence motifs: VPg+5'UTR[1AB-1C-1D-2A1(npgp)/2A2(npgp)-2B-2C(ATPase)/3A-3B(VPg)-3C(pro)-3D(pol)]3'UTR-poly-A. 2A1(npgp) and 2A2(npgp) are separated by 133 amino acids. The proteins 2A2(npgp), 2B, 3A and 3B(VPg) have no significant similarity to the corresponding proteins of other picornaviruses. Amino acid identities of the orthologous proteins P1, 2C, 3C(pro) and 3D(pol) range from 16.4 to 40.8 % in the eel picornavirus/CPV-1 comparison. 3D(pol) shows the closest similarity to eel picornavirus, with an amino acid identity of 40.8 %, followed by human parechovirus (36.5 %), duck hepatitis A virus (32.7 %) and swine pasivirus (29.3 %). Both the unique genome organization and low sequence similarity support the assignment of CPV-1 to a novel picornavirus species within a novel genus.

Genomic analysis of a Canadian equine rhinitis A virus reveals low diversity among field isolates.

Equine rhinitis A virus (ERAV) is an ubiquitous virus, routinely identified in equine respiratory infections; however, its role in disease and genetic features are not well defined due to a lack of genomic characterization of the recovered isolates. Therefore, we sequenced the full-length genome of a Canadian ERAV (ERAV/ON/05) and compared it with other ERAV sequences currently available in GenBank. The ERAV/ON/05 genome is 7,839 nucleotides (nts) in length with a variable 5'UTR and a more conserved 3'UTR. When ERAV/ON/05 was compared to other reported ERAV isolates, an insertion of 13 nt in the 5'UTR was identified. Further phylogenetic analysis demonstrated that ERAV/ON/05 is closely related to the ERAV/PERV isolate, which was isolated in 1962 in the United Kingdom. The polyprotein of ERAV/ON/05 had a 96 % nucleotide and amino acid sequence identity to reported ERAVs, and it appears that, despite the high error rate of RNA-dependent RNA polymerase, this isolate has retained high sequence identity to the strain first described by Plummer in 1962.

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

Equine rhinitis B virus: a new serotype.

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the family PICORNAVIRIDAE: The sequence from the 5' poly(C) tract to the 3' poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genus Erbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.

Type-independent detection of foot-and-mouth disease virus by monoclonal antibodies that bind to amino-terminal residues of capsid protein VP2.

The characterization of monoclonal antibodies raised against the foot-and-mouth disease virus isolates A22 Iraq/1964, Asia1 Shamir-Israel/1989, and SAT1 Zimbabwe/1989 with regard to neutralizing activity and sensitivity of their epitopes for treatment with trypsin, resulted in the identification of one non-neutralizing antibody in each panel that binds to a trypsin-sensitive epitope. Furthermore, each of these antibodies recognized 27 isolates of different provenance, representative of six serotypes. These antibodies are recommended for type-independent antigen detection by ELISA. The epitopes for these antibodies reside at the intertypically conserved N-terminus of capsid protein VP2. The two are specified by the lysines at positions two and three, but differ from each other as indicated by the variable heavy chain sequences of their antibodies.

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus A22-India.

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus (FMDV) A22-India 17/77 was determined using non-radioisotopic technique. Comparison of nucleotide and deduced amino acid sequence with A22-Iraq 24/64 revealed 175 synonymous (silent) and 42 non-synonymous nucleotide changes resulting in 34 amino acid substitutions along the capsid proteins (VP1-VP4). Out of the 4 structural proteins VP4 is highly conserved. The highly variable and immunodominant protein VP1 showed 47% of the total amino acid substitutions. VP2 and VP3 contain 38.2% and 14.7% of the amino acid substitutions, respectively. The VP1-based phylogenetic analysis of 18 different type A viruses including A22-India 17/77 divided them in to two broad genetic groups (Asian and European/South American), and each group is further subdivided in to two separate genotypes. A22-India 17/77, A22-Iraq 24/64 and A22-Azerbaijan/65 formed one genotype and the 4 Chinese strains formed a separate genotype in the Asian group of viruses. In the European/South American group, A-Argentina/87 represents one genotype and the remaining 10 strains formed the second genotype in this group.

Molecular epidemiological studies on foot-and-mouth disease type O Taiwan viruses from the 1997 epidemic.

Sequence diversity was assessed of the complete VP1 gene directly amplified from 49 clinical specimens during an explosive foot-and-mouth disease (FMD) outbreak in Taiwan. Type O Taiwan FMD viruses are genetically highly homogenous, as seen by the minute divergence of 0.2-0.9% revealed in 20 variants. The O/HCP-0314/TW/97 and O/TCP-022/TW/97 viral variants dominated FMD outbreaks and were prevalent in most affected pig-raising areas. Comparison of deduced amino acid sequences around the main neutralizable antigenic sites on the VP1 polypeptide showed no significant antigenic variation. However, the O/CHP-158/TW/97 variant had an alternative critical residue at position 43 in antigenic site 3, which may be due to selective pressure in the field. Two vaccine production strains (O1/Manisa/Turkey/69 and O1/Campos/Brazil/71) probably provide partial heterologous protection of swine against O Taiwan viruses. The type O Taiwan variants clustered in sublineage A1 of four main lineages in the phylogenetic tree. The O/Hong Kong/9/94 and O/1685/Moscow/Russia/95 viruses in sublineage A2 are closely related to the O Taiwan variants. The causative agent for the 1997 epidemic presumably originated from a single common source of type O FMD viruses prevalent in neighboring areas.

Genetic heterogeneity of Indian field isolates of foot-and-mouth disease virus serotype O as revealed by partial sequencing of 1D gene.

The sequence of 165 nucleotides at the 3' end of the 1D gene, determined from RT PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987 1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/ field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3 20.6%) from the exotic type O strains of O1BFS, O1K, and O1Campos. The type O strains analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 71% divergent (7.3-15.2%) from the field strains which revealed significant ( > 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of India, the viruses/ sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149-->E and another from P208-->L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogeneous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it.

A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase.

The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analysis have been used to classify the L proteinases as papain-like cysteine proteinases. However, sequence identity within the L proteinases themselves is low (between 18% and 32%) and only 14% between the L proteinases and papain. Secondary structure predictions, sequence alignments that take into account the positions of the essential catalytic residues, and structural considerations have been used in this study to investigate more closely the relationships between the L proteinases and papain. In spite of the low sequence identities, the analyses strongly suggest that the L proteinases of foot-and-mouth disease virus and of equine rhinovirus 1 have a similar overall fold to that of papain. Regions in the L proteinases corresponding to all five alpha-helices and seven beta-sheets of papain could be identified. Further comparisons with the proteinase bleomycin hydrolase, which also displays a papain topology in spite of important differences in size and amino acid sequence, support these conclusions and suggest how a C-terminal extension, present in all three L proteinases, and predicted to be an alpha-helix, might enable C-terminal self-processing to occur.

Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction.

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A,C,O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

One-tube and one-buffer system of RT-PCR amplification of 1D gene of foot-and-mouth disease virus field isolates.

A method of reverse transcription (RT) and polymerase chain reaction (PCR) amplification of 1D (VP1) gene of foot-and-mouth disease (FMD) virus using one reaction mixture containing both avian myeloblastosis virus (AMV) reverse transcriptase (RTase) and Tfl DNA polymerase is described. The procedure was time saving, made use of a single buffer for both RT and subsequent amplification and performed better than the two-step procedure usually conducted with Moloney murine leukemia virus (MMLV) RTase and Taq DNA polymerase for amplification of the VP1 gene of field isolates of FMD virus serotypes O,-A, C and Asia 1. The failure to amplify the VP1 gene of many type O and Asia 1 viruses using MMLV RTase-Taq polymerase enzyme system could be overcome by performing RT of the viral genome at a higher temperature (48 degrees C) with AMV RTase which is not possible with MMLV RTase.

Molecular analysis of foot-and-mouth disease type O viruses isolated in Saudi Arabia between 1983 and 1995.

Partial nucleotide sequence of the capsid polypeptide coding gene 1D (VP1) was determined for 68 serotype O foot-and-mouth disease viruses isolated between 1983 and 1995 from outbreaks occurring in Saudi Arabia. The sequences were compared with previously published sequences: 14 viruses of Middle Eastern origin (isolated between 1987 and 1991); and with four vaccine virus strain sequences, three originating from the Middle East (O1/Turkey/Manisa/69, O1/Sharquia/Egypt/72 and O1/Israel/2/85) and one from Europe (O1/BFS 1860/UK/67). The virus isolates from Saudi Arabia and the Middle East vaccine virus strains formed a related genetic group distinct from the European O1 virus. Within this large group 12 distinct genetic sublineages were observed.

The performance of southern African territories serotypes of foot and mouth disease antigen in oil-adjuvanted vaccines.

The performance of selected oil adjuvants containing Southern African Territories (SAT) serotypes of foot and mouth disease virus was assayed by testing antibody levels elicited in cattle, sheep and goats, and by testing protection of cattle on challenge. Various oil adjuvant formulations were tested initially in cattle and guinea pigs, and compared with a standard alhydrogel and saponin-based (AS) vaccine. A commercial double oil emulsion vaccine elicited higher antibody titres and a more prolonged antibody response than the conventional AS vaccine, than a commercial single emulsion oil formulation or an incomplete Freund's adjuvant formulation. Incomplete Freund's adjuvant was selected for the formulation of a trivalent vaccine which was assayed in cattle, sheep and goats for antibody response and local reactions. The double oil emulsion elicited a high level of antibodies, which was maintained for at least six months after a single inoculation. The product was easily injectable and did not cause local or systemic reactions.

The solution structure of the immunodominant and cell receptor binding regions of foot-and-mouth disease virus serotype A, variant A.

Abstract: The solution structure of a 20 amino acid long peptide corresponding to the region 141-160 of the envelope protein Vp1 from foot-and-mouth disease virus (FMDV) serotype A, variant A, has been determined by a combination of NMR experiments and computer calculations. The peptide contains both the immunodominant epitope as well as the sequence (RGD) used by the virus to bind the cell receptor in the initial stages of infection. These two sites have been shown to partially overlap. One hundred and thirty-five NMR distance constraints were used to obtain a set of 11 structures by distance geometry, minimization and molecular dynamics simulations. These structures were divided into two homogeneous families based upon backbone superimposition. The first and most populated family was characterized by a backbone RMS of 1.5 +/- 0.4 A, the second by a backbone RMS of 0.8 +/- 0.2 A. The two families had similar structural features and differed mainly in the backbone angles of G149. In the larger of the two families these angles favoured the formation of a loop comprising residues 147 to 152 and stabilized by a H-bond between NH of D147 and the CO of A152. In the second family, where this bond was absent, the peptide adopted in this region the shape of an irregular helix. The C-terminal half of the peptide (152-159) was similar in both families and largely helical. Similar structural features were also found within the VRGDS sequence (144-148) which was assigned to a beta-turn type IV. The features of the two families of structures were found to be different from those of the recently published X-ray structure of the antigenic loop of a chemically modified form of FMDV. Proposals accounting for these differences are provided which take into account the dual activity of the 141-160 sequence (i.e. antibody binding and cell invasion through receptor binding).

The solution conformational features of two highly homologous antigenic peptides of foot-and-mouth disease virus serotype A, variant A and USA, correlate with their serological properties.

The solution structure of a peptide corresponding to the VP1 region 141-160 of foot-and-mouth disease virus (FMDV) serotype A variant USA has been studied by NMR and computer calculations and compared with the results from a study on a highly homologous peptide deriving from serotype A, variant A. The two peptides differ in their serological behavior and contain the immunodominant epitope of the virus which partly overlaps with its receptor binding region. Distance constraints, derived both from 2D and 3D homonuclear NMR and 2D-heteronuclear NMR experiments, were combined with DG calculations to yield 50 structures. After refinement through EM and restrained molecular dynamics simulations the selected structures shared several general features. In particular the 151-158 region was a helix in all cases while a large loop similar to that found in peptide A but comprising less residues and stabilized by an H-bond between the side chains of D147 and S150 was found in the majority of structures. A further loop, common to all structures, was identified around the RGD sequence (145-147). This was different from that found in the corresponding region of peptide A as were the conformations of the individual residues within the RGDX sequence. The different structural features shown by the two peptides were rationalized in terms of the S148 (peptide A) to F148 (peptide USA) mutation. The second mutation, that at position 153 (L in A, P in USA) did not appear to affect the structure of the peptide significantly although the different dimensions of the loop in the central region and the type of H-bond stabilizing it could be potentially ascribed to this second mutation. All criteria used pointed to different structural features for the two peptides consistent with their serological behaviour.

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Antibodies raised in a natural host and monoclonal antibodies recognize similar antigenic features of foot-and-mouth disease virus.

Swine polyclonal antibodies directed against a major antigenic site (site A) of foot-and-mouth disease virus (FMDV) of serotype C, and monoclonal antibodies (MAbs) which recognize different epitopes within this site, have been compared with regard to reactivity with a panel of synthetic peptides. The peptides used represent different segments or variant sequences of site A, and their reactivities reflect differences in antigenic specificity. The results indicate a remarkable immunochemical similarity between the site A epitopes defined by murine MAbs and those recognized by antibodies elicited in a natural host of FMDV. This similarity further validates previous conclusions, based on analyses with MAbs, on the relevance of amino acid substitutions at a few critical positions on the intratypic antigenic variation of FMDV in the field. They also give further support to a dual function of the Arg-Gly-Asp motif of the G-H loop in cell attachment and in the recognition by host antibodies, as recently documented with the elucidation of the three-dimensional structure of an antigen-antibody complex of FMDV. In addition, the results encourage the use of extended panels of well-characterized MAbs for a precise molecular analysis of the antigenic variation of FMDV, and of other viruses, in the field.

Nucleotide sequence of the P1 region of serotype Asia1 foot-and-mouth disease virus.

Differences in the amino acid sequence of foot-and-mouth disease virus (FMDV) virion proteins (VP) among the various FMDV serotypes, particularly in the VP1 polypeptide, are the basis for antigenic diversity of this virus group. This phenomenon provides the basis for type diagnosis of FMDV by the polymerase chain reaction (PCR). In order to specifically identify the Asia1 FMDV serotype by PCR, the nucleotide sequence of its P1-coding region was determined. The sequence exhibited over 70% homology with the P1 gene segment of type O1k. The deduced amino acid sequence shares 79% homology with that of the P1 region of serotype O1k.

The structure of an immunodominant loop on foot and mouth disease virus, serotype O1, determined under reducing conditions.

Residues 136-159 of VPI of foot and mouth disease virus (FMDV) comprise the G-H loop of the protein and form a prominent feature on the surface of virus particles. This sequence contains an immunodominant neutralizing epitope, which can be mimicked with synthetic peptides, and includes an Arg, Gly, Asp motif which has been implicated in the binding of the virus to cellular receptors. Crystallographic analysis of native virus particles failed to resolve the structure of this region due to its disordered state. However, reduction of a disulphide bond between cysteine residues 134 of VP1 and 130 of VP2 caused the G-H loop to collapse onto the surface of the virus particle and allowed its conformation to be determined.

Amino acid changes outside the G-H loop of capsid protein VP1 of type O foot-and-mouth disease virus confer resistance to neutralization by antipeptide G-H serum.

Antiserum to a peptide corresponding to the 135-154 sequence of capsid protein VP1 of the foot-and-mouth disease virus O1 Kaufbeuren was raised in a pig. Although this serum contained neutralizing antibodies, the pig showed clinical symptoms after challenge. Virus isolated from this pig was identified as a mutant, with changes at positions 50, 198 and 211 of VP1 and at position 209 of VP2. This mutant, as well as a plaque isolate of it, differing from the challenge virus at positions 198 on VP1 (alanine being substituted for glutamic acid) and 209 on VP2 (histidine being substituted for tyrosine) resisted neutralization by the anti-peptide serum also in vitro. The same was observed with the O1 Kaufbeuren-related strain O1 Burgwedel, isolated from cattle in the field. It had substitutions only at positions 43 and 101 on VP1. The results show that neutralization epitopes flanking positions 145-147 on VP1 are modulated by other capsid protein parts. These parts seem to be important for neutralization escape in natural FMDV host species.