Antigenic characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies

Foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates (n = 100) were compared using a panel of 11 monoclonal antibodies (Mab) in sandwich ELISA. The majority (over 89%) of the isolates showed either homologous (76% and above reactivity) or reduced affinity (20-75% reactivity) for the Mabs 2A, 13, 40, 34 and 81, suggesting that these Mab binding epitopes are conserved, whereas a more variable reactivity was observed for the Mabs B3, 1A, 24, 72, 82 and 89. Polyclonal relationship ('r' value) of the field isolates in liquid phase blocking (LPB) ELISA was examined, and the mean 'r' value was 0.62 relative to vaccine virus IND 63/72. Some of the field isolates (n = 34) were tested in virus neutralization test (VNT) and showed an 'r' value of >0.40. Although a minor antigenic difference was observed in the Mab profiling study, there has not been large antigenic divergence between reference virus and field viruses, thereby providing evidence of wide antigenic coverage of the vaccine strain.     

Antigenic relationship among field isolates of Tritrichomonas foetus from cattle

Analysis of protein and antigen profiles of Tritrichomonas foetus isolates from cattle from 5 western states was accomplished by sodium dodecyl sulfate polyacrylamide-gel electrophoresis, immunoblot, immunoprecipitation, and fluorography techniques. Total protein profiles of all isolates were compared by Coomassie brilliant blue staining of T foetus protein samples prepared by 4 protein-extraction methods. Antigenic tritrichomonas proteins were identified by immunoblot assay with polyclonal bovine or rabbit anti-T foetus serum. Additionally, [14C]glucosamine-labeled T foetus was used for total and antigenic glycoprotein analyses. Detectable differences in the composition of total proteins or antigenic tritrichomonal proteins were not observed among all isolates. However, intensity differences in some antigenic protein bands were apparent. Bovine and rabbit sera from immunized animals possessed antibodies to the same antigenic tritrichomonal proteins. Each T foetus isolate contained 4 to 7 molecular weight size classes of glycoprotein, which were labeled by [14C]glucosamine; however, only 3 to 4 glycoproteins were identified as antigens by bovine or rabbit antiserum.     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

A serological and biochemical study of new field isolates of foot-and-mouth disease virus type A in Peru, 1975 to 1981

Three foot-and-mouth disease virus type A isolates recovered from field outbreaks in the Department of San Martin, Peru, during the period 1975 to 1981 were compared with each other, and the South American vaccine strains A24 and A27, by complement fixation (CF), virus neutralization (VN) and polyacrylamide gel electrophoresis (PAGE). Complement fixation and VN tests gave comparable results distinguishing the field isolates from each other and from the vaccine strains. Analysis of the structural polypeptides by PAGE also showed clear differences between all the viruses examined. Samples from tissue culture passaged and mouse adapted strains of one of the field isolates gave identical patterns in PAGE, but differences were observed in the polypeptide pattern of the A24/BRA/55 strain and the Peru vaccine strain, which were serologically indistinguishable. Results illustrate a continued antigenic variation in an endemic area where vaccination has been used; however, asymmetric serological reactions between the A24 vaccine strain and the most recent field isolate indicated that a vaccine incorporating A24 should still give adequate protection.     

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.     

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

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 O(1)Campos, 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 T(1) 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 0.44s/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 quasi-species 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.     

Variation in foot-and-mouth disease virus isolates in Kenya: an examination of field isolates by T1 oligonucleotide fingerprinting

Ribonuclease T1 oligonucleotide maps of strains of 4 of the endemic serotypes of foot-and-mouth disease virus isolated in Kenya between 1964 and 1982 have been compared with data obtained in complement-fixation and neutralization tests. There was a continual change in the oligonucleotide maps obtained for all the serotypes examined. This genetic heterogeneity was generally associated with antigenic variation. Viruses isolated during the 12-month course of an epidemic of the SAT 1 serotype showed few changes in their oligonucleotide fingerprints, and were serologically related. These maps form a data base that will be useful in future epidemiological studies on the maintenance and spread of foot-and-mouth disease virus in this region.     

Sequence variability in the structural protein-encoding region of foot-and-mouth disease virus serotype Asia1 field isolates

A total of 30 field isolates of foot-and-mouth disease virus (FMDV) serotype Asia1 belonging to two different lineages and five isolates belonging to a divergent group as delineated earlier in 1D (encodingVP1 protein) gene-based phylogeny were sequenced in the structural protein (P1) coding region. Phylogenetic comparison of these isolates along with some of the published exotic sequences revealed the presence of five different lineages around the world. Similar grouping pattern was observed for the P1 region and 1D gene-based phylogeny, where the Indian isolates were clustered in two genetic lineages. The recently identified divergent group of virus falls into a separate sub-cluster. Similar grouping was also observed in L gene-based phylogeny. Comparison of amino acid sequences identified lineage-specific signature residues in all the structural proteins. Comparison of Asia1 field isolates at the identified key residues of other FMD viruses involved in the formation of the heparan sulfate-binding ligand confirmed many of them to be conserved and the presence of VP3(56) Arg suggested their cell culture adaptation. Although a considerable genetic variation was observed among the isolates of present study, all of them tested in micro-neutralization test were serologically related to the vaccine strain.     

Molecular epidemiology of foot-and-mouth disease virus type A in South America

A databank of 78 VP(1) complete sequences of type A foot-and-mouth disease virus (FMDV) from South American isolates was constructed. Forty-nine samples corresponded to FMDV that circulated between the years 1999-2008, mainly in Venezuela, where most type A outbreaks have occurred lately and twenty-nine to strains historically relevant for the continent. The phylogenetic analysis showed that all South American FMDV belonged to the Euro-SA topotype. Sixteen subgenotypes could be identified, based on a 15% nucleotide divergence cut-off criterion: eight are extinguished, three were active until the year 2002 and the remaining five circulated in Venezuela during the years 2001-2007, illustrating the potential for FMDV diversification under appropriate selective pressure. The last emergencies reported in already-free areas of Colombia in 2004 and 2008 were closely related to isolates acting in Venzuela. Evidence of positive selection over codon 170, within the immunogenic site 4 of VP1 protein, was recorded. A codon deletion in amino acid position 142, within the G-H loop, was found in some isolates within subgenotypes 14, 15 and 16. Conversely amino acid deletion 197 was restricted to all isolates within a particular genetic cluster. The present work is the first comprehensive phylogenetic analysis of FMDV type A in South America, filling a gap of knowledge with respect to both, historical and acting viruses. The results provided evidence that supports the ecosystem dynamics in the region, and also served as an input to establish genetic links of emergencies in already-declared free areas, highlighting the need for strengthening control activities.     

Molecular characterisation of Victorian Newcastle disease virus isolates from 1976 to 1999

OBJECTIVE: To characterise Newcastle disease virus isolates obtained in Victoria from 1976 to 1999 and identify the diversity of FO cleavage signal. DESIGN: RT-PCR using viral RNA extracted from positive NDV allantoic fluid was performed to amplify a segment of the NDV F and HN genes. Molecular characterisation of the nucleotide and amino acid sequences within the FO cleavage site was undertaken. RESULTS: All isolates contained 'avirulent FO cleavage signal sequence of varied amino acid composition. CONCLUSIONS: Molecular characterisation of past and present NDV FO cleavage signal sequences will provide valuable epidemiological information and assist in understanding the genetic origins and relationships of outbreaks.     

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.     

Sequence analysis of the non-structural 3A and 3C protein-coding regions of foot-and-mouth disease virus serotype Asia1 field isolates from an endemic country

A total of 18 foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates belonging to two different lineages (including the divergent group) as delineated earlier in VP1-based phylogeny were sequenced in the non-structural 3A and 3C protein-coding regions. The phylogenetic trees representing the regions coding for the non-structural proteins were very similar to that of the structural VP1 protein-coding region. Phylogenetic comparison at 3C region revealed clustering of Asia1 viruses with the isolates of serotypes O, A and C in the previously identified clade. Comparison of amino acid sequences identified lineage-specific signature residues in both the non-structural proteins. Overall analysis of the amino acid substitutions revealed that the 3A coding region was more prone to amino acid alterations than 3C region.     

The generation and persistence of genetic variation in foot-and-mouth disease virus

Genetic variation in foot-and-mouth disease virus (FMDV) is of interest for at least two reasons. First, changes to the genes encoding capsid proteins results in antigenic variation, and affects vaccine efficiency and effectiveness of vaccination programs; second, genetic changes can lead to important insights into the transport of virus between countries, regions, herds, and even possibly individuals. Current estimates of RNA virus mutation rates suggest that an average of about one base mis-incorporation is likely to occur each time a single FMDV genome replicates. This should result in the introduction of every possible 1-step mutation from the progenitor genotype into the viraemia of a single infected animal many times a day. In the absence of purifying selection, a single infected animal should therefore generate a genetically very diverse population of virus.Viral-capsid sequences obtained from infected animals sampled over long-term FMDV epidemics suggest that these genetic changes accrue in a remarkably linear 'clock-like' fashion and at rates of around 1% change per year. While such a rate is generally regarded as quite high, it is actually somewhat lower than one might expect based on the rate at which viral diversity could be generated within a single animal. The difference might be explained in a variety of possible ways: (1) the mutation rate has been overestimated; (2) purifying selection is stronger than predicted; (3) only a restricted subset of excreted virus is actually infectious; (4) infected animals only excrete virus from a small partitioned subset of amplified virus, and that most of the generated viral diversity is unable to exit the animal; or (5) only a small fraction of all infected animals participate in the actual disease-transmission process.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.