Asia1 Foot-and-mouth Disease Virus Research Articles

Phylogenomics and molecular evolution of foot-and-mouth disease virus.

This report describes the use of Bayesian methods to analyze polyprotein coding region sequences (n = 217) obtained from GenBank to define the genome-wide phylogeny of foot and mouth disease virus (FMDV). The results strongly supported the monophyly of five FMDV serotypes, O, A, Asia 1, C, and SAT 3, while sequences for the two remaining FMDV serotypes, SAT 1 and SAT 2 did not separate into entirely distinct clades. The phylogenomic tree revealed three sister-group relationships, serotype O + Asia 1, A + C, and SAT 1 + 3 + 2, with a new branching pattern: {[(O, Asia 1), (A, C)], (SAT 1, 2, 3)}. Within each serotype, there was no apparent periodic, geographic, or host species influence on the evolution of global FMDVs. Analysis of the polyprotein coding region of these sequences provided evidence for the influence of purifying selection on the evolution of FMDV. Using a Bayesian coalescent approach, the evolutionary rate of FMDV isolates that circulated during the years 1932-2007 was estimated to be 1.46 × 10(-3) substitutions/site/year, and the most recent common ancestor of the virus existed approximately 481 years ago. Bayesian skyline plot revealed a population expansion in the early 20(th) century that was followed by a rapid decline in population size from the late 20(th) century to the present day. These findings provide new insights into the mechanisms that impact on the evolution of this important livestock pathogen.

Absence of Heat Intolerance (Panting) Syndrome in Foot-and-Mouth Disease-Affected Indian Cattle (Bos indicus) is Associated with Intact Thyroid Gland Function

Foot-and-mouth disease (FMD) is a highly contagious and economically important viral disease with high morbidity and reduced productivity of affected animals. We studied the heat intolerance (HI) (panting) syndrome and the effect of FMD virus (FMDV) infection on thyroid gland function in Indian cattle (Bos indicus). Experimental infection with FMDV Asia 1 resulted in a mild form of disease with superficial lesions. Heat intolerance syndrome and its signs were not observed among the recovered animals. Subtle changes in the serum level of thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄) were observed. However, there were no distinct histological changes in the thyroid gland, and FMDV antigens were not detected in the thyroid tissues. Our results thus suggest that the absence of panting syndrome in FMD-affected Bos indicus cattle may be associated with intact thyroid gland function.

Identification of a conformational epitope on the VP1 G-H Loop of type Asia1 foot-and-mouth disease virus defined by a protective monoclonal antibody

Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT 50 of the MAb 3E11 completely protected suckling mice from a dose of 10 LD 50 of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide 141SXRGXLXXLXRR 152. These results provide additional insights into the virus–MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.

The relationship between cellular immune response to foot-and-mouth disease virus Asia 1 and viral persistence in Indian cattle (Bos indicus)

Foot-and-mouth disease (FMD), the most contagious animal disease, is associated with persistent viral infection in ruminants, despite the induction of systemic immune response. The present study was performed to decipher the relation between the persistent FMD virus (FMDV) infection and cellular immune response in Indian cattle (Bosindicus) following experimental inoculation of FMDV Asia 1. Persistent viral infection (carriers) was detected by antigen capture RT-PCR on the oesophageal-pharyngeal fluid. Viral excretion was found to be intermittent and strongly variable among the persistently infected Indian cattle. Lymphocyte proliferative (LP) response, assessed as reactivity of peripheral blood mononuclear cells to FMDV Asia 1 antigen (Ag) was of low magnitude indicating a weak primary cellular immune response following infection. LP response to FMDV Ag was higher among the non-carriers than carriers of FMDV Asia 1. An enhanced LP response was associated with the lack of virus shedding in the OPF. The findings of this study are suggestive of relationship between cellular immune response and virus excretion during persistence of FMDV Asia 1 in infected cattle.

Phylogenetic analysis of Indian serotype Asia1 foot-and-mouth-disease virus isolates revealed emergence and reemergence of different genetic lineages

Foot-and-mouth-disease virus (FMDV) serotype Asia1 causes significant number of disease outbreaks in India. Indian Asia1 virus isolates were shown to be genetically heterogeneous and of the two lineages (lineage B and lineage C) described in India, lineage C caused majority of the outbreaks. Emergence of a novel divergent lineage (lineage D) within lineage C has been described in 2001. In the present report, the complete VP1 genomic region of 41 FMDV Asia1 field isolates collected between 2003 and 2008 was sequenced. Phylogenetic analysis revealed reemergence of lineage C since 2005 following exclusive dominance of lineage D in the period between 2002 and 2004. At many positions lineage specific signature residues were identified. The antigenic relationship of the field isolates with the currently used vaccine strain IND63/72 was also determined, which reflects antigenic stability of serotype Asia1 in-spite of genetic heterogeneity.

Immune Response and Viral Persistence in Indian Buffaloes ( Bubalus bubalis ) Infected with Foot-and-Mouth Disease Virus Serotype Asia 1

Despite their potential role in the spread of foot-and-mouth disease (FMD), the immune response and viral persistence in FMD virus (FMDV)-infected Indian buffaloes (Bubalus bubalis) have been unexplored. We found similar kinetics of neutralizing antibody responses in the sera and secretory fluids of buffaloes following experimental FMDV Asia 1 infection, but the lymphocyte-proliferative response in infected buffaloes was of low magnitude. Despite inducing a significant systemic and secretory immune response, viral persistence seems to be a common outcome in buffaloes following FMDV Asia 1 infection, which is associated with a weak cellular immune response.

Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.

Molecular Design and Immunogenicity of a Multiple-epitope FMDV Antigen and DNA Vaccination

This article reports the design and construction of a multiple-epitope foot and mouth disease virus (FMDV) antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-γ and cytotoxic activity from mice immunized with pA were significantly increased. pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asia1 FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.

Immune response in cattle inoculated with the recombinant complete polyprotein of foot-and-mouth disease virus from Bombyx mori larvae.

The intact open reading frame (ORF) of foot-and-mouth disease virus (FMDV) Asia I/XJ strain was amplified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. Bm-N cells were transfected with pVL-ORF and linearized Bm-BacPAK6 DNA, and the recombinant silkworm baculovirus Bm-ORF containing the full ORF of FMDV was obtained. The results of indirect immunofluorescence assay (IFA) showed that Bm-ORF could be expressed efficiently in Bm-N cell. After inoculating the early 5th instar larvae of silkworm, the polyprotein of FMDV could be detected by sandwich ELISA and empty capsid-like particles could be observed under the electron microscope. Expression products from silkworm were used as the antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. The immunized cattle were challenged with the virulent FMDV Asia I/XJ strain, two of the four cattle were completely protected and clinical symptoms were alleviated and delayed in the others. The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.

Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals

Non-structural protein (NSP) 3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals. An indirect ELISA was established, using purified His-tagged 3ABC fusion protein as antigen, for detection of the antibody response to FMDV NSP 3ABC in different animal species. The method was validated by simultaneous detection of the early antibody responses to NSP and structural protein (SP) in FMDV Asia 1 infected animals. The performance of the method was also validated by detection of antibody in reference sera from the FMD World Reference Laboratory (WRL) in Pirbright, UK, and comparison with two commercial NSP ELISA kits. The results showed that the antibody response to SP developed more quickly than that to NSP 3ABC in FMDV infected animals. In contact-infected cattle, the antibody response to NSP 3ABC was significantly delayed compared with that to SP antibody. The early antibody responses to SP and NSP 3ABC in FMDV inoculated cattle and contact-infected or inoculated sheep and pigs were generally consistent. In pigs, 3ABC antibody was linked to the presence of clinical signs; however, in sheep, subclinical infection was detected by the development of 3ABC antibodies. Therefore, the antibody responses to 3ABC varied between host species. Eight out of 10 positive serum samples from FMD WRL were tested to be positive at cutoff value of 0.2. The rate of agreement with the ceditest ® FMDV-NS and the UBI ® NSP ELISA were 98.05% (302/308) and 93.2% (287/308), respectively. The prevalence of 3ABC antibodies reached 71.4% in some diseased cattle herds. The further work is required to evaluation the performance of this method in different animal species and different field situations.

Neutralization antigenic sites on type Asia-1 foot-and-mouth disease virus defined by monoclonal antibody-resistant variants

Seven neutralizing monoclonal antibodies (nMAbs) produced against serotype Asia-1 foot-and-mouth disease virus (FMDV) were used to select neutralization-resistant variants. Seven single and six multiple antibody-resistant variants were selected to identify neutralization antigenic sites on FMDV Asia-1. The variants no longer reacted with nMAbs which were used to select them when tested by microneutralization test (MNT), radioimmunoassay (RIA) and agar gel immunodiffusion (AGID) assay. Based on the binding and neutralization patterns of the variants, the nMAbs could be divided into discrete groups indicating the presence of three independent antigenic sites with evidence for occurrence of possibly a fourth site on the virus surface. Site 1 was present on 140S, 12Sps and VP1 and thus was conformation-independent. Sites 2 and 3 were restricted to the intact virion (140S) and thus were more conformation-dependent. Site 4 present on 140S virions and 12S protein subunits was less conformation-dependent. The site 3 nMAbs neutralized the infectivity of all the ten different Asia-1 virus isolates tested indicating that this site is conserved in Asia-1 virus serotype. Both cross-neutralization of different Asia-1 viruses with the nMAbs and cross-inhibition assays between MAbs demonstrated that the nMAbs recognized at least six different epitopes on Asia-1 virus.

Neutralizing activity in bovine secretions against foot-and-mouth disease virus.

Neutralizing activity in bovine secretions against foot-and-mouth disease virus.

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.

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.