Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis)

Veterinary Microbiology 120 (2007) 50–62 www.elsevier.com/locate/vetmic

Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis) Jose´ Ju´nior F. Barros a,b, Viviana Malirat b,*, Moacyr A. Rebello a, Eliane V. Costa c, Ingrid E. Bergmann b a

Instituto de Microbiologia Prof. Paulo de Go´es, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Ilha do Funda˜o, Rio de Janeiro, Brazil b Pan American Foot-and-Mouth Disease Center (PANAFTOSA), PAHO/WHO, P.O. Box 589, CEP 20.001-970, Rio de Janeiro, RJ, Brazil c Laborato´rio de Enterovirus, Departamento to de Virologia, Instituto Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, Brazil

Received 25 September 2005; received in revised form 21 July 2006; accepted 10 October 2006

Abstract Genetic variation of foot-and-mouth disease virus (FMDV) isolates, serotype O, recovered serially over a 1-year period from persistently infected buffalos was assessed. The persistent state was established experimentally with plaque-purified FMDV, strain O1Campos, in five buffalos (Bubalus bubalis). Viral isolates collected from esophageal–pharyngeal (EP) fluids for up to 71 weeks after infection were analyzed at different times by nucleotide sequencing and T1 RNase oligonucleotide fingerprinting to assess variability in the VP1-coding region and in the complete genome, respectively. Genetic variation increased, although irregularly, with time after infection. The highest values observed for the VP1-coding region and for the whole genome were 2.5% and 1.8%, respectively. High rates of fixation of mutations were observed using both methodologies, reaching values of 0.65 substitutions per nucleotide per year (s/nt/y) and 0.44 s/nt/y for nucleotide sequencing and oligonucleotide fingerprinting, respectively, when selected samples recovered at close time periods were analyzed. The data herein indicate that complex mixtures of genotypes may arise during FMDV type O persistent infection in water buffalos, which can act as viral reservoirs and also represent a potential source of viral variants. These results fit within the quasispecies dynamics described for FMDV, in which viral populations are constituted by related, non-identical genomes that evolve independently from each other, and may predominate at a given time. # 2006 Elsevier B.V. All rights reserved. Keywords: Foot-and-mouth disease virus; Genetic variability; Water buffalo; Persistent infections; Quasi-species

* Corresponding author. Tel.: +55 21 3661 9080; fax: +55 21 3661 9001. E-mail address: [email protected] (V. Malirat). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.023

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1. Introduction Foot-and-mouth disease (FMD) is an acute, febrile highly contagious disease of domestic cloven-hoofed animals like cattle, buffalo, sheep and pig, among others (Bachrach, 1968; Pereira, 1981). Though mortality due to the disease is very low and mostly restricted to young animals, drastic reduction in productivity and working capacity of the recovered animals causes considerable losses to the livestock industry (Astudillo et al., 1990). The causative agent of FMD virus (FMDV) is distinguished immunologically in seven serotypes (O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1), which can be further subdivided into more than 65 subtypes. Immunization with inactivated whole virus of a given vaccine strain does not confer complete protection against other serotypes, nor, even in some cases, between subtypes (Pereira, 1981). The viral particle contains a single-stranded positive RNA of about 8200 nucleotides, within an icosahedric non-enveloped capsid consisting of 60 copies of each of the four proteins VP1, VP2, VP3 and VP4 (Acharya et al., 1989). RNA viruses exhibit a high mutation rate, which together with reassortment and recombination is probably the basis of the remarkable genetic and phenotypic variability and rapid evolution of these genomes (Holland et al., 1982). In FMDV one of the consequences of these high mutation rates is that populations are composed of ensembles of closely related non-identical genomes that are known as viral quasi-species (Holland et al., 1992; Domingo et al., 2001). An important aspect of FMDV is its capacity to establish persistent infection in both vaccinated and non-vaccinated ruminants exposed to the virus (van Bekkum et al., 1959; Sutmo¨ller and Gaggero, 1965; Auge´ de Mello et al., 1970). The mechanisms that mediate this persistence are unclear, but are likely to result from a dynamic equilibrium between the host immune response and the selection of viral antigenic variants at mucosae of upper respiratory tract (Gebauer et al., 1988; Salt, 1993). Currently, virus isolation from esophageal–pharyngeal (EP) fluid is the recommended method to recognize persistently infected animals, although detection of viral sequences in other tissues and fluids has been reported during FMDV persistence in cattle (Bergmann et al., 1996).

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The emergence of genetic and antigenic variants of FMDV during cytolytic and persistent infections, and their dominance through positive selection and random drift, has been extensively documented in cell cultures (Sobrino et al., 1983; de la Torre et al., 1985; Borrego et al., 1993). Genetic and antigenic heterogeneity has also been described in virus populations recovered from persistently infected cattle (Gebauer et al., 1988; Malirat et al., 1994), and genetic characterization of FMDV during persistent infection in buffalos has also been illustrated in southern Africa (Vosloo et al., 1996), where the central role played by the African buffalo (Syncerus caffer) in the epidemiological situation has been studied (Dawe et al., 1994a,b; Bastos et al., 2000, 2001). Although previous work has been published on FMDV infection in water buffalo (Bubalus bubalis) in the early and persistent stages (Gomes et al., 1997; Samara and Pinto, 1983), no reports are recorded describing the genetic variability of FMDV recovered during persistent infection in this host. In the present study we describe the extent of genetic variation of sequential FMDV isolates, type O, recovered from five persistently infected buffalos by nucleotide sequencing of the VP1-coding region and T1 RNase oligonucleotide fingerprinting, and raise discussions about the putative viral evolutionary pathways under controlled conditions.

2. Materials and methods 2.1. Animals, cells, viruses and experimental design Persistent infection of cross-bred Indian buffalo (named also water buffalo) of the Jafarabadi and Murrah races with plaque-cloned FMDV O1Campos (O1C/58) was established by intranasal inoculation of 104.0 50% infective doses (ID50) in three animals (82, 83 and 86). Two additional animals (88 and 90) were kept in the same pen to allow contact transmission. Buffalos were non-vaccinated, 10-month old, and came from a farm that had been FMDV-free for several years and were seronegative to FMDV strains O1Campos, A24 Cruzeiro, and C3 Indaial by seroneutralization assays. Paired samples of esophageal– pharyngeal (EP) fluid and sera were collected at

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weekly or bi-weekly intervals for up to 71 weeks after inoculation and were analyzed for FMDV recovery in cell culture and for the presence of specific antibodies, respectively. The prevalence of antibodies against non-capsid viral antigens was determined by enzyme-linked immunoelectrotransfer blot (EITB) assay as described previously (Neitzert et al., 1991), with minor modifications. Neutralization of infectivity was carried out in vitro by a microtiter neutralization assay essentially according to the protocol described by Ferreira (1976). EP samples obtained using a probang cup were treated for virus isolation by vigorously mixing with seven volumes of trichlorotrifluoroethane and centrifuged at 800  g for 30 min as described previously (Sutmo¨ller and Gaggero, 1965). The baby hamster kidney (BHK-21) cell (clone 13) monolayers were inoculated with the clarified supernatant. Isolation was considered negative when no cytopathic effect was observed after three passages. Plaque purification of the recovered isolates was not carried out in order to avoid arbitrary selection of any particular virus population. The buffalo number followed by the week after inoculation identifies viral isolates. The 1958 strain, O1C/58 used to inoculate the animals, corresponds to a South American reference strain, and was isolated from a diseased bovine in the county of Campos, Rio de Janeiro, Brazil in January 1958.

Taq DNA Polymerase and the primers referred above, flanking the VP1-coding region. The thermal profile for all reactions was 42 8C for 60 min, 70 8C for 15 min (RT), and 30 cycles of 94 8C for 5 min, 94 8C for 1 min, 60 8C for 0.45 min, 72 8C for 2 min and a final hold step of 72 8C for 5 min (PCR). Amplification products of approximately 0.8 kb were purified through affinity columns. Amplicons were sequenced by a modified dideoxi-mediated chain-termination method (Sanger et al., 1977) using fluorescent dye dideoxiterminators and fragments were analyzed in an automated sequencer. 2.3. Phylogenetic analysis Alignment of multiple sequences corresponding to the 639 nucleotides coding for the VP1 protein was carried out with the Clustal W program as implemented in the BioEdit package (Hall, 1999). All pairwise comparisons of complete sequences were performed giving each base substitution equal statistical weight. A binary tree was then constructed according to sequence relatedness using the neighborjoining algorithm (Saitou and Nei, 1987) and confidence levels on the tree branches were assessed by bootstrap resampling analysis as implemented in the MEGA 3.0 phylogeny reconstruction software package (Kumar et al., 2004). 2.4. T1 RNase oligonucleotide fingerprinting

2.2. RT-PCR and nucleotide sequencing When needed, viruses were passaged in BHK cell monolayer cultures to the minimal extent needed to provide RNA for analysis, in most cases, this involved three passages from EP fluid material. RNA extraction, reverse transcription of viral RNA, polymerase chain reaction (PCR) as well as primer pairs and cycle sequencing protocols using an automated sequencer were all performed as described previously (Malirat and Bergmann, 2003). Total RNA was extracted from infected cell culture supernatants by using a phenol-derived reagent in a modified Chomczynski and Sacchi (1987) method. Reverse transcription of viral RNAs (about 3–5 mg) was carried out with random hexamers using MMLVreverse transcriptase. In vitro amplification was performed in a programmable thermocycler using

Preparation of 32P-labelled RNA from infected cells and extraction of RNA were carried out as described previously (Costa Giomi et al., 1984). The method of separation of oligonucleotides resistant to RNase T1 used was a modification of earlier techniques, and was performed as described previously (Auge´ de Mello et al., 1986; Bergmann et al., 1989).

3. Results 3.1. Variability of the VP1-coding region in FMDV variants isolated from persistently infected buffalos After recovery from acute infection induced by inoculation or contact exposure to FMDV O1C/58,

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Fig. 1. Persistent infection established in five buffalos inoculated with FMDV O1Campos/Bra/58. Esophageal–pharyngeal (EP) and serum samples were obtained at weekly or bi-weekly intervals. FMDV isolation was attempted from clarified EP fluids as described in Section 2. VI: positive viral isolation in BHK-21 cell cultures from EP fluid samples (solid circles); EITB: presence of antibodies against non-capsid proteins assessed by enzyme-linked immunoelectrotransfer blot assay (empty circles); SN: presence of antibodies against capsid proteins analyzed by microneutralization assays (stars); FP: viral samples analyzed by T1 RNase oligonucleotide fingerprinting (solid triangles); Seq: viral samples analyzed by nucleotide sequencing of VP1-coding region (solid squares); ND: not determined.

establishment of a persistent state was confirmed in all buffalos by isolation of infective FMDV from EP fluid in cell culture (Fig. 1). Prevalence of antibodies against FMDV capsid and non-capsid proteins, as measured by neutralization assay, and enzyme-linked immunoeletrectrotransfer blot assay, respectively, was also demonstrated. In general, viral isolation was observed continuously during the early stages of infection (up to 19–29 weeks post-infection, wpi). After that period, viral recovery became rather intermittent, probably explained by the self-progress of the infection with time, in which a diminished viral replication is expected, with the concomitant decrease in viral excretion by the host. The last positive isolation was registered at 51 wpi for animals 82, 88 and 90, and at 27 and at 39 wpi for animals 83 and 86, respectively. However, no correlation could be established between the duration of the persistence state and the infection route. To determine the extent and patterns of viral variation occurring during the persistent state,

sequencing of VP1-coding region (1D) and oligonucleotide fingerprinting analysis was carried out in selected isolates, as depicted in Fig. 1. Nucleotide sequences of each individual isolate were edited manually, aligned and compared with the one used for inoculation (O1C/58). Only point mutations were observed, not being registered nucleotide insertions or deletions. As shown in Fig. 2, most changes found (60%) were concentrated in two variable regions of the VP1 gene (nucleotide positions 129–174 and 398–467). Silent mutations corresponded to approximately 60% of the recorded changes for all isolates, and of the total base changes, 90% were transitions and 10%, transversions. Overall, there was a direct relationship between the degree of variation and the time elapsed after infection (Table 1). The nucleotide sequence recorded for VP1-coding region of each isolate differed from that of the initial virus in an extent that varied from 0.0% (isolates 82/5 and 88/5) to 2.5% (88/49), revealing homology values ranging from

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Fig. 2. Nucleotide sequences of the complete VP1-coding region of the indicated FMDV variants recovered from persistently infected buffalos. Only positions that differ from that of the initial strain O1C/58 (printed in full) are depicted. Shadowed boxes delimit the two hipervariable regions within VP1 gene.

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Table 1 Genetic variation recorded for FMDV variants isolated from persistently infected buffalos Isolates

82/5 82/9 82/33 82/37 83/19 83/25 83/27 86/33 86/35 88/5 88/13 88/19 88/23 88/27 88/43 88/45 88/49 90/29 90/37 90/41 90/51

VP 1-coding region

Genome

Nucleotide variation (%)

S/nt/y

0.0 0.6 0.9 1.3 ND 1.1 ND 0.8 2.0 0.0 0.6 0.9 1.1 1.1 2.2 1.9 2.5 0.8 2.0 1.1 2.3

– 3.4  10 1.4  10 1.7  10

2 2 2

Amino acid variation (%)

Variation (%)

S/nt/y

– 0.9 1.4 1.4

0.3 ND 1.2 1.4 0.9 1.1 1.0 1.5 1.8 0.9 ND ND ND ND 1.7 1.8 ND 1.3 ND 1.7 ND

3.0  10

2

2.0  10 2.1  10 2.5  10 2.3  10 1.9  10 2.5  10 2.8  10 9.2  10

2

2.1  10 2.1  10

2

2.4  10

2

2.1  10

2

2.3  10

2

1.9

1.1  10 3.0  10 – 2.4  10 2.4  10 2.2  10 2.1  10 2.7  10 2.0  10 2.6  10 1.2  10 2.8  10 1.2  10 2.3  10

2

1.4 2.3 – 0.5 1.4 1.9 1.4 2.3 1.9 2.3 1.9 1.9 1.4 1.9

2

2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2

2

S/nt/y: substitutions per nucleotide site per year and ND: not determined.

97.5% to 100%. The rate of fixation of substitutions defined as the number of mutations per unit time, calculated for the 639 nucleotides sequenced, ranged from 1.1  10 2 substitutions per nucleotide site per year (s/nt/y) for isolate 86/33 to 3.4  10 2 s/nt/y for variant 82/9. Consecutive isolates recovered from the same animal showed that substitutions were not always sequentially maintained. Pairwise comparisons indicated that percentage of variation found between two sequences did not always increase when samples isolated at increasing time intervals were compared. Rate of fixation of substitutions was higher when isolates recovered at close time points were compared, as can be seen between isolates 90/37 and 90/41 (4.0  10 1 s/nt/y) and isolates 86/33 and 86/35 (6.5  10 1 s/nt/y), suggesting a coexistence of related non-identical FMD viral variants evolving independently. In order to better explore this finding, a phylogenetic analysis was performed by neighbor-joining algorithm with viral sequences obtained from animal 88, as indicated in Section 2. As can be seen in the topology of the resulting tree, three clusters differing

from original virus could be distinguished, suggesting three different genetic differentiation pathways (Fig. 3). Percentages of variation recorded values of approximately 0.3–0.6% for isolates within the same cluster. Among clusters, genetic distances showed average values of 1.6–2.3%, confirming the existence of different subpopulations evolving independently from each other and arising within the same animal during the persistent infection of buffalos exposed to FMDV. Moreover, different patterns of variation were also observed among animals. Comparisons of variants 82/ 33 and 86/33, and of isolates 82/37 and 90/37, recovered at the same day post-inoculation showed genetic distances of 1.4% and 3.3%, respectively. To determine the degree to which genetic diversity is reflected in the VP1 protein, alignment and comparison of the deduced amino acid sequences of isolates was carried out (Fig. 4). It was observed that 40% of nucleotide mutations led to amino acid substitutions. Of the total amino acid changes, only 18% occurred within rather conserved regions, while most substitutions fell within the two recognized

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Fig. 3. Phylogenetic analysis of viral variants recovered from persistently infected buffalo 88. The dendogram was constructed using neighborjoining algorithm as described in Section 2, from the complete VP1 sequences of the referred isolates.

hypervariable locations (amino acid positions 43–58 and 133–156). The latter one accumulated approximately 47% of the amino acid substitutions, although mostly conserving the chemical groups. In this region, no amino acid substitutions were registered neither in the tripeptide motif (residues arginine–glycine–aspartic acid at positions 145, 146 and 147) involved with integrin binding during internalization of viral particle, nor at position 149, which constitutes the fifth antigenic site, in all the viral isolates studied. 3.2. Genomic variation of FMDV isolated from persistently infected buffalo Patterns of genomic changes were also assessed for selected isolates using oligonucleotide fingerprinting as described in Section 2. Oligonucleotide spot patterns of each sample studied were compared to that of the initial virus O1C/58, and a composite map of the conserved and variant T1 oligonucleotides was constructed for each buffalo (Fig. 5). Values deduced from Fig. 5, calculated as reported previously (Nakajima et al., 1978), indicated that the genetic homology for whole genome was over 98%, falling within the expected range in which T1 map differences of various RNA (s) may be compared (Aaronson et al., 1982). From the 64 oligonucleotides considered for analysis in strain O1C/ 58, 88% remained unchanged in all isolates from animals 82 and 83. The values for animals 86, 88 and 90 were 81%, 78% and 75%, respectively. Analysis of the T1 RNase oligonucleotides on 2D gels also revealed that genomes clearly did not undergo massive mutations during persistence. Again, and as it was observed through nucleotide sequencing for VP1-coding region, a direct although irregular

relationship between the degree of variation and time after infection could be noted (Table 1). The extent of variation ranged from 0.3 to 1.4% (buffalo 82) and from 0.9 to 1.8% (buffalo 88) with respect to initial strain and the rate of fixation of substitutions mutations varied between 2.0  10 2 s/nt/y (buffalo 82/33) and 9.2  10 2 s/nt/y (buffalo 88/5). Analysis of patterns of mutations revealed unique spot changes (additional or missing spots occurring in only one of the sequential isolates studied) recorded for 19%, 55%, 56%, 37% and 76% of the total modified oligonucleotides registered in animals 82, 83, 86, 88 and 90, respectively. Some changes appeared repeatedly, although only rarely in consecutive samples. Similarly to the results obtained for the VP1 gene, the s/nt/y in the whole genome were extremely high when quantitation was performed between two variants isolated at close time points. For example, values of 3.9  10 1 and 4.4  10 1 s/nt/y were reached when samples 86/33 and 86/35, and 83/25 and 83/27 were compared, respectively, and the former represented the largest value registered in this work, even when the time elapsed between those two isolates was of only 14 days. In summary, no predictable patterns of changes were observed in the five experimentally infected animals, suggesting once again the randomness of FMDV genome variation during persistent infection.

4. Discussion This is the first report of the long-term persistent infection of FMDV established in water buffalo.

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Fig. 4. Deduced amino acid sequences of VP1 capsid protein as predicted from the nucleotide sequence (213 residues) of FMDVisolates. Filled-line boxes (residues 43–58 and 133–156) and dotted-line boxes (residues 43–48 and 144–154) correspond to hypervariable regions and antigenic sites of VP1, respectively. Residues of critical amino acids within them (Barnett et al., 1989; Crowther et al., 1993; Kitson et al., 1990) are represented by stars. The tripeptide motif RGD (residues 145–147), located inside second hypervariable region (Fox et al., 1989), is also displayed.

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Fig. 5. T1 RNase oligonucleotide maps of selected FMDV isolates recovered from persistently infected water buffalos. Autoradiograms of the T1 resistant oligonucleotides of the indicated viral samples, together with diagrams displaying the comparison of each viral fingerprint with that of the original strain O1C/58. When required, the identity of spots in different isolates was confirmed by co-electrophoresis of mixtures containing equivalent amounts of T1 RNase digests. The black line marks an arbitrary limit between the small and large oligonucleotides, the latter (64 in the original strain O1C/58) used for comparisons. Numbered solid circles represent invariant oligonucleotides present in all isolates; numbered circles correspond to oligonucleotides present in the strain O1C/58 that disappeared in at least one isolate. Uncircled numbers denote newly appearing oligonucleotides. Arbitrary numbers were assigned to each oligonucleotide for identification. Criteria for the analysis were as described in Costa Giomi et al. (1984).

Variability and patterns of mutation of viral isolates studied during a period of almost 1 year indicated an irregular increase in genomic variation of isolates when compared to the strain used to establish the persistent infection. The rate of fixation of mutations was extremely high when quantitation was based on comparisons between two isolates recovered at close time points. These observations, together with the non-cumulative changes observed in consecutively isolated viruses, which are in agreement with previous studies with FMDV (Costa Giomi et al., 1988;

Gebauer et al., 1988; Malirat et al., 1994; Vosloo et al., 1996), strongly suggest the presence of a mixture of variants evolving independently from each other. No predictable patterns of virus variation could be distinguished for the five experimentally infected animals, either by direct inoculation or by contact transmission, although each one received an identical inoculum. This suggests that at least for the O serotype, a complex mix of genotypes may be generated during the development of the persistent

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state. These results differ from the data reported for parallel persistent infections with visna virus, in which similar patterns of variation were observed (Clements et al., 1982), but are in agreement with those obtained for equine infectious anemia virus (Payne et al., 1987), HIV (Hahan et al., 1986) and for FMDV when infection occurs in other hosts (Malirat et al., 1994; Vosloo et al., 1996). Studies on genetic relationships among viruses recovered from the same animal through phylogenetic analysis revealed at least three different variation pathways. Furthermore, genetic differences were also observed in isolates recovered from two different animals at the same day post-infection, suggesting that viral evolution follow different pathways also among animals, strengthening the observation that at any time during persistent infection a variant virus can gain upper hand from a pool of viral mutants. The high percentage of variation registered shortly after establishment of infection (variants 82/5 and 88/ 5) when compared to the initial inoculum through fingerprint, but not through nucleotide sequencing of the VP1 region, suggests that rapid adaptation of the virus to the new host (buffalo) probably involves regions outside VP1. Substitutions in the third codon position were more frequent (57%) than in the first and second ones. Similar results were observed in field samples (Malirat et al., 1994). These findings suggest the existence of strong constraints to the fixation of non-synonymous mutations due to the need of maintaining a functional capsid structure, especially for changes in the VP1 gene, which can cause negative selection (Hughes and Nei, 1988, 1989). This finding has been observed for FMDV serotype C (Martı´nez et al., 1992), and many other evolutionary systems (Kimura, 1983; Nei, 1983; Buonagurio et al., 1985). A comparison of the VP1 primary structure of all variants studied show that changes concentrated essentially in two highly variable regions within the VP1 protein, falling between amino acid positions 43 and 58 and between positions 133 and 156, the second one carrying the antigenic site I of type O virus. Within this site, amino acid residues 144, 148, 154 and 208, positions critical for eliciting neutralizing antibodies (Xie et al., 1987; Kitson et al., 1990; Aggarwal and Barnett, 2002), and position 149 of the antigenic site V (Crowther et al., 1993), were highly

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conserved among all variants, suggesting that the alterations would not modify the behavior of antigenic site I that includes the bG–bH loop (residues 144–154) and the carboxy terminal region (residue 208) of protein VP1. Furthermore, most mutations which led to changes in chemical groups within this region (amino acids 133–142) did not represent determinants necessary for serospecificity, positions 144–159 (Pfaff et al., 1982) and positions 144–160 (Bittle et al., 1982). Conversely, an amino acid change was registered, only for isolate 88/161, in position 43, considered critical inside the antigenic site III, bB–bC loop of VP1 (Kitson et al., 1990; Aggarwal and Barnett, 2002), inside the first variable region (residues 43–58). The RGD triplet (Fox et al., 1989) was also maintained highly conserved among all isolates analyzed, probably reflecting again constraints to substitutions affecting functional domains. This is expected because structural and functional studies have shown that the RGD, and in particular the Asp residue, is critical for both the interaction with integrins (Leippert et al., 1997; Neff et al., 1998) and the interaction with site A-specific neutralizing antibodies (Verdaguer et al., 1998). Significant restrictions to amino acid changes also seem to exist for this variable region, since changes maintaining the same functional amino acid groups were larger than those observed in the first variable region (positions 43–58). This is in agreement with previous reports of FMDV serotype C suggesting that antigenic diversity for field samples recovered during a 60-year period arose as a consequence of alternation of a very limited number of amino acid at a reduced number of positions (Martı´nez et al., 1992). It has been described that mutant viruses with chemically conserved amino acid substitutions, prone to behave in a quasi-neutral fashion, are more likely to be represented in a viral quasi-species than variants with mutations that adversely affect fitness even if viable, as documented with phage Qb (Domingo et al., 1978). This fact may be due to a severe limitation in this region of the residues whose substitution is possible, as reflected in the lack of accumulation of amino acid replacements over time. Despite the fact that variations obtained for the VP1-coding region and for the whole genome of

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FMDV isolates confirm the genetic heterogeneity observed for FMD viral populations, a parallel serological analysis of viral isolates would be required for the precise confirmation of the presence of immunogenic variants. However, it can be speculated that, the extent of antigenic changes resulting from genome variation in FMDV isolates studied in this work would be insufficient to circumvent the immune response induced by a potent vaccine including the original virus strain, as had been reported for FMDV isolated, type O recovered from persistently infected cattle (Malirat et al., 1994). In this report, the long-term persistent infection of FMDV in water buffalo described suggests that besides acting as viral reservoirs, this persistently infected host also represent a potential source of viral variants. However, long-term carriage of FMDV does not imply efficient transmission of disease. Transmission of infective FMDV from persistently infected animals, often referred to as carriers, to susceptible individuals they come in contact with, has raised controversial debates for many years. There has been no experimental data, and only limited or anecdotal records on viral transmission from persistently infected cattle to susceptible animals (Sutmo¨ller and Barteling, 2004), but there is circumstantial evidence that although such transmission is inefficient it can eventually occur from infected African buffalos (Hedger and Condy, 1985; Bengis et al., 1986; Gainaru et al., 1986; Dawe et al., 1994a,b; Vosloo et al., 1996). The results present in this report are consistent with the complexity and dynamics of FMDV quasi-species in vivo, but the probability of these viral variants present in persistently infected water buffalos to be transmitted to susceptible animals, their fitness relative to the parental population, and their potential to contribute to the antigenic diversity of the virus in vivo remains to be studied.

Acknowledgments The authors thank Drs. Paulo Auge´ de Melo and Ivo Gomes for providing the experimental animal model. J.J.F.B. Barros PhD fellowship was supported by a grant from CNPq (Conselho Nacional de Desenvolvimento cientı´fico e Tecnolo´gico).

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