FMDV Strains Research Articles

Genetic characterization of a new pandemic Southeast Asia topotype strain of serotype O foot-and-mouth disease virus isolated in China during 2010

The full-length nucleotide sequence of the foot-and-mouth disease virus O/BY/CHA/2010 strain, Mya-98 lineage of Southeast Asia (SEA) topotype, was determined and compared with O/HKN/20/2010 and other known FMDV strains. Homology analysis indicated >98.0% nucleotide identity between O/BY/CHA/2010 and the epidemic strains, O/HKN/20/2010, and O/VN/2009. However, with the exception of the VP4, 2A, and 3BCD regions, O/BY/CHA/2010 showed a lower similarity with SEA topotype strains, O/VN/2006, and HLJOC12/03. A comparison of O/BY/CHA/2010 with non-SEA topotype strains showed the highest level of homology (97.4-100%) with UKG/7B/2007, Akesu/58, and the PanAsia strains in the 2A, P2, and 3CD regions, which suggested the presence of similar characteristics among these strains. Phylogenetic analysis revealed that O/BY/CHA/2010 is clustered in the Mya-98 lineage of the SEA topotype and is linked to four other isolates: HKN/20/2010, O/VN/2009, O/VN/2006, and HLJOC12/03. The VP1-based phylogenetic tree was divided into distinct clusters according to the different topotypes, while other gene-based phylogenetic trees exhibited some degree of intercrossing among topotypes. Furthermore, sequence analysis of the Lpro gene revealed a single amino acid insertion in O/HKN/20/2010 and a single amino acid deletion in O/BY/CHA/2010, in addition to a 70-nucleotide deletion within the 5'-untranslated region of O/HKN/20/2010. The majority of strains were shown to be homologous in the pseudoknots region although some exceptions were noted. This study provides a comprehensive genetic characterization of a novel FMDV isolate of the Mya-98 lineage.

Development of one-step multiplex RT-PCR method for simultaneous detection and differentiation of foot-and-mouth disease virus serotypes O, A, and Asia 1 circulating in Vietnam

A one-step multiplex RT-PCR method using new primers was developed for the simultaneous detection and differentiation of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The RT-PCR method used a cocktail of one universal minus-sense primer designed in the 2B gene and three serotype-specific plus-sense primers designed in the hypervariable regions of the capsid VP1 coding gene of FMDV. These serotype-specific primer pairs amplified 658, 535, and 427bp PCR products corresponding to FMDV serotypes O, Asia 1, and A, respectively. In this study, six well-characterized FMDV strains belonging to serotypes O, A, and Asia 1 were used as reference strains for validation tests. Among these six FMDV strains were three vaccine strains for type O (O1/Manisa), A (A22/Iraq), and Asia 1 (As1/Shamir/89). The other reference strains included one pandemic strain of FMDV serotype Asia 1 (Asia1/MOG/05) and two pandemic strains of FMDV serotype O (O/UKG/34/2001 and O/SKR/2000). For field application, 37 positive-clinical samples and 18 cell culture-adapted viruses belonging to serotypes O, A, and Asia 1, as confirmed previously by antigen ELISA for FMDV detection, were used. The present method showed high sensitivity and specificity and can be adapted for detection and typing of FMDV serotypes O, A, and Asia 1 circulating in Vietnam.

Development of a Foot-and-Mouth Disease Infection Model in Severe Combined Immunodeficient Mice for the Preliminary Evaluation of Antiviral Drugs

Recent European guidelines facilitate the use of emergency vaccines during outbreaks of foot-and-mouth disease. Antiviral drugs could be used as a complementary measure. This study aimed at developing a small animal model to assess the in vivo activity of early antiviral lead molecules with anti-foot-and-mouth disease virus (FMDV) activity in vitro. In a first attempt, several FMDV strains were titrated in Balb/c mice. Inoculations with O₁ Manisa or C₁ Noville did not induce clinical disease, whereas Asia1 Shamir induced death too rapidly [i.e. within 4 days post-inoculation (dpi)]. Therefore, we switched to severe combined immunodeficient mice which are frequently used as a model for viral infections and experimental therapeutics. Strain O₁ Manisa did not induce clinical disease, but titrations with A₂₂ Iraq, C₁ Noville or Asia1 Shamir resulted in virus-induced morbidity (including respiratory problems and weight loss) with subsequent mortality. Inoculations with strain A₂₂ Iraq resulted in a reproducible mean time of death of 6 dpi (this was shorter for the other strains). In this newly developed rodent model, strain A₂₂ Iraq seems the most suited to assess the in vivo anti-FMDV activity of selective inhibitors of FMDV.

B cell epitopes within VP1 of type O foot-and-mouth disease virus for detection of viral antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 (epitope1), tandem repeat 200-213 (epitope2 (+2)) and the combination of two epitopes (epitope1-2) was genetically cloned into the prokaryotic expression vector pP(RO)ExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 (+2) and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 (+2), GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Variation analysis of theVP1and3ABCgenes from yellow cattle isolates persistently infected byFoot-and-mouth disease virus

AbstractThe potential relationship between the establishment ofFoot-and-mouth disease virus(FMDV) persistent infection and gene variation was identified by investigating the variation ofVP1and3ABCgenes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×104ID50/ml of FMDV O/Akesu/58 strain. After displaying clinical or subclinical signs, they probably became asymptomatic carriers. Oesophageal–pharyngeal fluids were collected monthly from the carriers with a probang and inoculated into a baby hamster kidney cell line (BHK-21); 12 FMDV isolates were obtained. TheVP1and3ABCgenes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of theVP1nucleotide and amino-acid sequence of all the isolates was above 98%, with no base deletion or insertion. When compared with the O/Akesu/58 FMDV strain, the homology of theVP1nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in theVP1gene of the isolates, including 16 consistent nucleotide mutations, with only two of them leading to a change in amino acid (I56→T, A210→E). Moreover, it was found that four nucleotide points and three amino-acid points had transversions among all isolates. The3ABCgene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in theVP1and3ABCgenes, and was probably related to other structural protein gene and key factors.

FMD Vaccines: Reflections on Quality Aspects for Applicability in European Disease Control Policy

Most foot-and-mouth disease (FMD) vaccines used around the world are inactivated vaccines for prophylactic or emergency use, generally manufactured by the same basic methodology outlined in the OIE Manual and, for Europe, in the European Pharmacopoeia, and for the EU Member States in compliance with Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products as amended by Directive 2004/28/EC. Most of the requirements that apply to all immunological veterinary medicinal products apply equally to FMD vaccines. There are, however, some unique features of the disease and vaccines used against it that require a different approach to fulfil the requirements of the relevant legislation, if a vaccinate-to-live policy will be applied with 'authorized' vaccines. Several aspects of vaccine efficacy and safety are elaborated with emphasis on quality assurance/quality control (QA/QC). The purity of the vaccine in respect of the presence of non-structural protein antibodies could be checked indirectly by serology after vaccination. The viability of a vaccine bank approach was greatly aided by the principle of storing inactivated concentrated FMD viral antigen (Ag) over liquid nitrogen for subsequent formulation into vaccine. A worldwide Ag bank network might be an option for the far future and a solution to the problem of covering many different FMDV serotypes and strains. The producers should respect the strict FMD biosecurity rules worked out by the FAO EUFMD and described in Council Directive 2003/85/EC. Making the experience related to vaccine QA/QC available to all countries will reduce the risk of an FMD outbreak within these countries and consequently will reduce the FMD risk around the world.

Genome comparison of a novel foot-and-mouth disease virus with other FMDV strains

The genome of a novel foot-and-mouth disease virus, HKN/2002, was 8104 nucleotides (nt) in length (excluding the poly(C) tract and poly(A) tail) and was composed of a 1042-nt 5′-untranslated region (UTR), a 6966-nt open reading frame, and a 93-nt 3′-UTR. Genome sequences of HKN/2002 and other known FMDV strains were compared. The VP1, VP2, and VP3-based neighbor-joining (NJ) trees were divided into distinct clusters according to different serotypes, while other region-based NJ trees exhibited some degree of intercross among serotypes. Mutations in HKN/2002 were revealed, including frequent deletions and insertions in the G–H loop of VP1, and deletion involving 10 amino acid residues in the 3A protein. An evolutionary relationship of HKN/2002 with an Asian FMDV lineage isolated from a Hong Kong swine host in 1970 was postulated. A 43-nt deletion identified in the 5′-UTR of HKN/2002 possibly contributed to the loss of one pseudo-knot domain.

Molecular epidemiology of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is the most economically important veterinary pathogen due to its highly infectious nature, ability to cause persistent infections and long term effects on the condition and productivity of the many animal species it affects. Countries which have the disease have many trade restrictions placed upon them. In the last 15 years there have been significant advances in the understanding of FMD epidemiology. These have largely been due to the application of the molecular biological techniques of polymerase chain-reaction amplification and nucleotide sequencing. In the World Reference Laboratory for FMD (Pirbright, UK), a large sequence database has been built up. This database has been used to aid in the global tracing of virus movements. It has been possible to genetically group many FMDV's based on their geographic origin and this has led to their being referred to as topotypes. The implications of this are that inter-regional spread of viruses can often be easily recognised and any evolutionary changes which subsequently occur can be monitored. Using these techniques, for the first time, we have been able to unequivocally show the recent pandemic spread of a FMDV type O strain through the whole of Asia and into Africa and Europe. This type of surveillance will become increasingly important as further globalisation of markets occurs. An increased understanding of how FMDV strains move between geographic regions will play a pivotal role in the development of future disease control strategies.

Antigenic analysis of type O foot-and-mouth disease virus in the persistently infected bovine

The antigenic profiles of serotype O strains of FMDV collected from the oropharynx of persistently infected cattle were defined with a panel of monoclonal antibodies (mAb's) in an indirect antigen-trapping ELISA. The mAb profiling showed no significant loss of reactivity in two neutralising antigenic sites of persistent FMDV isolates collected over a period of eight months. Early and late serum taken from a carrier animal showed similar neutralising activity against early and late carrier isolates. The antigenic stability of serotype O strains of FMDV collected during the carrier state suggests that antigenic variation facilitated by pressure from the host humoral immune response is not critical to the establishment or maintenance of a persistent infection with FMDV.

Immune response of calves to foot-and-mouth disease virus vaccine emulsified with oil adjuvant. Strategies of vaccination

Calves born to vaccinated cows under the regular annual vaccination programme were vaccinated at different ages using commercial quadrivalent (01, A79, A87 and C85 FMDV strains) vaccine emulsified in oil adjuvant. The antibody responses of vaccinated calves were evaluated using liquid-phase blocking sandwich ELISA. All calves 20, 30 and 40 days old having high maternal antibody titres responded well to vaccination. Moreover, 25–57% of vaccinated calves showed protective antibody titres both at 90 and 120 days post-vaccination (d.p.v.), whereas none of the non-vaccinated animals achieved these levels. Calves aged 3–4 months with non-protective levels of colostral-derived antibodies responded with high antibody titres to vaccination which persisted for at least 4 months. In both groups of calves a certain degree of suppression of postvaccinal response was observed which was related to colostral antibody titres. Our results suggest that in order to reduce the proportion of calves susceptible to infection it is advisable to immunize calves as young as 20 days old to induce acceptable antibody titres for the following 4 months. In addition, a second vaccination 60 d.p.v. ensures high antibody levels in high disease risk areas.

Recognition of B and T Cell Epitopes by Cattle Immunized with a Synthetic Peptide Containing the Major Immunogenic Site of VP1 FMDV 01 Campos

The precise location of B and T cell epitopes have been established in a peptide containing the major immunogenic site (residues 135-160) of FMDV strain 01 Campos (01C) VP1. The peptide (p135-160), administered free or conjugated to bovine serum albumin, induced complete protection in guinea pigs and a strong neutralizing antibody (NAb) response in cattle. Using a set of partially overlapping peptides it was shown that although several B cell epitopes were distributed along the p135-160, the residues responsible for the induction of NAb were restricted to the amino acids 135-144. In addition, bovines immunized with the p135-160 showed a strong proliferative response to this peptide but a very poor response against purified virus. Two T-epitopes were located in the 135-160 sequence; one on the region 135-144 and the other in the region 150-160. The immunodominance of these two T cell epitopes was confirmed in cattle immunized with inactivated virus vaccines.

Characterization of Foot-and-Mouth Disease Virus by Monoclonal Antibodies

Monoclonal antibodies (MAbs) were produced against foot-and-mouth disease (FMD) virus types O1 Campos Br1/58, A24 Cruzeiro Br1/55, and C3 Indaial Br1/71, which are the strains used for production of FMD vaccines in the majority of South American countries. Within the library of MAbs produced, a group was selected on the basis of their neutralizing titer in cell culture, protective titer in suckling mice, sensitivity to trypsin, and specificity for virus structural proteins. The MAbs were utilized in an ELISA test format to compare European and South American representative field isolates with vaccine production strains in their r1 relationship as obtained by 50% complement fixation (CF50) with polyclonal antibodies (PAb) and their virus neutralization (VN) relationship obtained with sera from one-time-vaccinated and from revaccinated cattle, respectively. The MAbs selected varied in their reactivity against the different strains and, therefore, enabled us to compare field FMDV strains to those against which the MAbs were produced, with definite advantages over the r1 and VN ratios. Thus, panels of MAb produced with the vaccine strains and appropriately selected are significantly useful for the FMD-control programs because they serve to provide guidance on the immunological coverage provided by the vaccines against FMDV strains circulating in the field. The MAbs are also useful for the differentiation of FMD virus strains.

Experimental infection of eland ( Taurotrages oryx), sable antelope ( Ozanna grandicomis) and buffalo ( Syncerus caffer) with foot-and-mouth disease virus

The course of experimental infection of a type SAT 1 FMDV strain was studied in buffalo, sable antelope and eland following tongue inoculation and contact and has been compared with that in cattle. All species became infected, although disease was less severe in the game animals and larger amounts of virus were required to infect game animals than cattle. Neutralizing antibody titres were high and were maintained for an extended period in buffalo, sable antelope and eland. The carrier state was demonstrated in buffalo for the longest period. Cattle carried virus for up to 56 days. Virus persistence in sable antelope was transitory and did not occur in eland.

Neutralization sites of type 01 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants

Monoclonal antibodies (MAbs) were derived from mice infected with foot-and-mouth disease virus type 01 Brugge (FMDV 01B) or immunized with inactivated virions (140 S) or viral subunits (12 S). A total of 19 neutralizing MAbs were characterized of which 17 recognized conformationally determined epitopes and two recognized amino acid sequences on isolated VP1. Neutralizing MAbs were used to select antigenic variants of FMDV 01B. Based on cross-neutralization and binding assays with MAbs the variants were divided into discrete groups demonstrating the presence of three unique neutralization sites on FMDV 01B. One site was present only on intact 140 S virions, a second was present on both 140 S virions and 12 S subunits, and the third was present on 140 S virions, 12 S subunits, and isolated VP1. Comparison of the deduced nucleic acid sequence of parental FMDV 01 B with those of the 01B variants demonstrated that the epitope recognized by the VP1-reactive, neutralizing MAbs included amino acid residues 138, 144, and 148. Cross-neutralization assays demonstrated that these three neutralization sites of FMDV O1B function on other type 01 strains of FMDV.

Nucleotide sequence of the VP1 gene of the foot-and-mouth disease virus strain A Venceslau

The VP1 coat protein of FMDV strain A Venceslau (Aven) consists of 213 amino acid residues. Serum neutralization tests demonstrated that strain Aven is closely related to strain A Argentina/79 (A 79) but significantly different from strain A 24 Cruzeiro (A 24). There is a strong correlation between the amino acid sequences and the serological data. Nucleotide and amino acid sequence analyses of VP1 showed that serologically related viruses (Aven and A 79) differ less in this region of the genome than those of serologically distinct viruses (Aven vs. A 24). The most significant variation between Aven and A 24 occurs at amino acid positions 43 to 46, in which all four residues are different.

The scale‐up from 0.1 to 100 liter of a unit process system based on 3‐mm‐diameter glass spheres for the production of four strains of FMDV from BHK monolayer cells

AbstractThis paper describes the scale‐up from 0.1 to 100 liter of the unit process based on 3‐mm‐diameter glass spheres for the growth of BHK monolayer cells. The production of four strains of FMD virus at the 0.1‐, 10‐, and 100‐liter scales was examined. Cell growth was estimated from measurements of the concentration of glucose in the growth medium, while the release of virus was inferred from measuring the concentration of LDH in the culture supernatant fluid. The yields of virus at 0.1‐, 1‐, and 10‐liter scales were similar but that from the 100‐liter version was somewhat lower. The reason for this lower yield and the method used to overcome it over outlined.

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus.

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.

Susceptibility of various FMDV strains to urea

Resistance to urea in vitro at 37 degrees C varied for each FMDV strain analysed. The urea marker did not correlate with other markers such as resistance to acid, resistance to acidity or size of plaques under agar on BHK21/13 cells. The resistance to urea of subtypes A24 Cruzeiro, O1 Caseros and C3 Resende varied in accordance with their antigenic potency when administered to swine as a trivalent water-in-oil emulsion type vaccine.

Incubation at raised temperature or enrichment media, combined with secondary enrichment in Rappaport's medium, for the isolation of salmonellas from sewage.

Two FMDV strains which had been previously differentiated by complement-fixation were compared by guinea-pig protection test, kinetic neutralization and micro-neutralization tests. It was found that these tests, which have not been previously applied by the methods described, were all capable of FMDV STRAIN DIFFERENTIATION. Similar differences were found by all methods, which suggests that comparisons made by cross-CF, cross-neutr-alization or cross-protection involve measurement of the same antigen/antibody interactions.

A comparison of some immunological methods for the differentiation of strains of foot-and-mouth disease virus

Two FMDV strains which had been previously differentiated by complement-fixation were compared by guinea-pig protection test, kinetic neutralization and micro-neutralization tests. It was found that these tests, which have not been previously applied by the methods described, were all capable of FMDV strain differentiation. Similar differences were found by all methods, which suggests that comparisons made by cross-CF, cross-neutralization or cross-protection involve measurement of the same antigen/antibody interactions.