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PubMed2005

A novel genetic lineage differentiating RT-PCR as a useful tool in molecular epidemiology of foot-and-mouth disease in India.

Comparison of nucleotide sequences at the VP1 coding region of foot-and-mouth disease serotype Asia1 viruses from India has revealed two genetic lineages with emergence of a genetically divergent group in recent years. In this study a simple, fast, relatively costeffective multi-primer RT-PCR assay to differentiate genetic lineages of type Asia1 viruses was developed. Efforts were made in the design of novel lineage-specific primers and in optimization of the multi-primer assay protocol in conjunction with the use of the serotype specific primer for confirmation of serotype Asia1 virus. This assay promises to be an effective tool in molecular epidemiological investigation of FMD in the country.

Capsid Proteins↗
PubMed2006

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus.

Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

Animals↗
PubMed2006

Comparison of SAT-1 foot-and-mouth disease virus isolates obtained from East Africa between 1971 and 2000 with viruses from the rest of sub-Saharan Africa.

Foot-and-mouth disease serotype SAT-1 seems to be endemic in many sub-Saharan African countries. Phylogenetic analysis using the 1D gene of 51 SAT-1 isolates from East, West and southern Africa indicated the presence of at least 6 lineages and 11 genotypes with linkages between various geographical regions of the subcontinent. Differences were observed between countries in East Africa, the main focus of this study, with individual countries suffering outbreaks from isolates belonging to various genotypes, which is evidence of reintroduction of strains and long-term circulation of outbreak viruses. The amount of variation observed has significant implications for disease control on the subcontinent.

Africa South of the Sahara↗
PubMed2001

Diagnosis of foot-and-mouth disease by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples.

The results of type-specific RT-PCR diagnostic assays on foot-and-mouth disease (FMD) viruses in clinical samples were mapped onto serotype-specific dendrograms representing the degree of nucleotide sequence variation between the FMD virus isolates. This novel approach assisted the selection of suitable PCR primer sets for the diagnosis of FMD virus isolates belonging to different topotypes within each serotype. These interpretations were qualified by using a universal (FMD virus group) specific primer to confirm that FMD virus RNA had been extracted from the samples under investigation. The analyses showed that the design of primer sets for the detection of FMD virus serotypes O, A, Asia 1, SAT 1 and SAT 3 were generally satisfactory, as most virus isolates within the major virus sub-groupings were successfully detected. However, the FMD virus serotype C and SAT 2 specific primers were less efficient as certain virus sub-groups were not detected. This identified the need for additional or alternative primers to improve RT-PCR procedures for more comprehensive detection of divergent virus strains within these serotypes. There were some examples where not all virus isolates from the same outbreak reacted with particular type-specific primers which suggested that either further minor refinements may be necessary in the primer design or that there were shortcomings in the RT-PCR methodology.

Animals↗
PubMed2001

Genetic heterogeneity of SAT-1 type foot-and-mouth disease viruses in southern Africa.

Genetic relationships of 50 SAT-1 type foot-and-mouth disease viruses were determined by phylogenetic analysis of an homologous 417 nucleotide region encoding the C-terminal half of the VP1 gene and part of the 2A segment. Viruses obtained from persistently-infected African buffalo populations were selected in order to assess the regional genetic variation within the host species and compared with ten viruses recovered from recent and historical cases of clinical infection. Phylogenetic reconstructions identified three independently evolving buffalo virus lineages within southern Africa, that correspond with the following discrete geographic localities: (1) South Africa and southern Zimbabwe, (2) Namibia, Botswana and western Zimbabwe, and (3) Zambia, Malawi and northern Zimbabwe. This strict geographic grouping of viruses derived from buffalo was shown to be useful for determining the origin of recent SAT-1 epizootics in livestock. The percentage of conserved amino acid sites across the 50 SAT-1 viruses compared in this study was 50%. Most mutations were clustered within three discrete hypervariable regions, which coincide with the immunogenic G-H loop, H-1 loop and C-terminus region of the protein. Despite the high levels of variation within the primary sequence, secondary structural features appear to be conserved.

Africa South of the Sahara↗
PubMed2002

Phylogenetic analysis of serotype A foot-and-mouth disease virus isolated in India between 1977 and 2000.

The genetic diversity among the Indian serotype A Foot-and-mouth disease virus (FMDV) isolates sampled over a period of 24 years (1977-2000) was studied by sequencing the VP1 gene. In the phylogenetic tree, constructed from 83 Indian and 37 other available sequences, the FMDV type A isolates were distributed into 10 major genotypes (designated as I-X). The Indian isolates were distributed in 4 genotypes (I, IV, VI and VII), and co-circulation of at least 2 genotypes (VI and VII) in different states of the country in recent years is evident from the result. The study also revealed differential geographic distribution of genotypes, for example, some (genotypes I and VII) were recovered from large geographical areas, some time even across the continents, suggesting the spread of the viruses beyond continental barriers. Localization of genotypes restricting to a particular country (genotypes III and X) was also observed in the study. The genetic diversity in the field isolates has been discussed in relation to the amino acid substitutions at the known antigenic sites. This work provides valuable insights into the epidemiological situation of type A FMDV in India and necessary information in the selection of suitable vaccine strain(s), if required, for the National FMD control program.

Amino Acid Sequence↗
PubMed2001

[Detection of p53 mutation in mouth mucosa smears of patients with oral squamous epithelial carcinoma].

Tumour-suppressor gene p53 encodes for an important cell-cycle regulatory protein and is therefore probably important for the development of many malignant diseases, e.g. squamous cell carcinoma of the mouth. This gene has mutated most frequently in connection with the development of cancer, so it has been well explored. Hence we chose it to find out whether swabs of the oral mucosa are suitable for supplying material for the detection of mutations in a gene that is connected with the development of oral squamous cell carcinomas, because swabs are easier to obtain than biopsies. We examined biopsies, swabs from the tumour, and swabs from mucosa that appeared healthy from 32 patients with oral squamous cell carcinoma and mucosal swabs from 35 healthy persons with polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TGGE). Fourteen of the 32 patients with a tumour showed mutations of p53, and in all cases the mutation could be demonstrated both in the biopsy and in the tumour swab. In four cases the mutation was also found in the swab of normal mucosa. Our investigations revealed that swabs are a suitable method for obtaining material for the detection of gene mutations in oral squamous cell carcinomas.

Adult↗
PubMed2005

A novel mucosal vaccine against foot-and-mouth disease virus induces protection in mice and swine.

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6xHis-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1-hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1-hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10(5) FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1-hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10(6.5) FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV.

Adjuvants, Immunologic↗
PubMed2006

Molecular characterization of foot-and-mouth disease virus in Hong Kong during 2001-2002.

Most of the molecular epidemiological studies of foot-and-mouth disease virus (FMDV) are based on comparison of VP1 gene sequence. In this report, The nucleotide sequences of the VP1 coding region of FMDV type O strains O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02, isolated from the disease outbreak that occurred in Hong Kong Special Administrative Region (Hong Kong SAR) of China during 2001-2002, were determined and compared with the sequences of other FMDVs. The results revealed that the VP1 gene of the five isolates had the same nucleotide (nt) sequences (639 nt), coding for 213 amino acids, and no changes were found either at the critical amino acid sites 144 (Val), 148 (Leu), 154 (Lys) and 208 (Pro) within the VP1 protein epitope (amino acids 140-160, 200-213), or in the amino acids 145-147 comprising the arginine-glycine-aspartic acid (RGD) sequence that is involved in the adsorption of virus to host cell. Analysis of the VP1 gene nucleotide sequence revealed that the five isolates examined were most closely related to FMDVs found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Furthermore, although the critical amino acids on the antigen epitope of the prevalent Hong Kong isolates and the serotype O vaccine strain, O1/Manisa/Turkey/69, showed relative conservativeness, they were distantly related genetically, which showed that there existed variation between the prevalent Hong Kong FMDV strains and the vaccine strain.

Amino Acid Sequence↗
PubMed2006

Plus- and minus-stranded foot-and-mouth disease virus RNA quantified simultaneously using a novel real-time RT-PCR.

Even though tagged RT-PCR and rTth RT-PCR have been developed to improve strand-specific detection of RNA virus, these assays are not quantitative. In this study, a novel real-time RT-PCR assay, which combines the benefits of both the tagged and rTth RT-PCR has been developed to quantify the strand-specific RNA of foot-and-mouth disease virus (FMDV). The tagged-primers plus a TaqMan probe located within the highly conserved viral 3D region were used. The in vitro synthesized minus-and plus-stranded RNA templates were used as a dual control along with the copy number of viral RNA molecules, which is more reliable than reported RT-PCR employing a DNA-based standard. This assay was used to quantify FMDV RNA from 10(9) to 10(1) copies with a maximum sensitivity of between ten and five copies and was shown to be highly reproducible with low intra-and inter-assay variation. Coefficients of variation (CV) values were 0.70-1.39% and 0.98-2.1%, respectively. Importantly, the method was applied to simultaneously quantify both plus-stranded and minus-stranded FMDV RNA using tagged-RT and tagged-FP primer during a high-temperature reverse transcription. Highly sensitive and strand-specific real-time RT-PCR assay has been established. We tested the ratio of viral plus-stranded to minus-stranded RNA in acutely infected and persistently infected BHK-21 cells, for which the values ranged from 22/1 to 143/1 and from 287/1 to 334/1, respectively, suggesting different replication patterns of plus-and minus-stranded RNA in acutely infected and persistently infected cells. This value ranged from 83/1 to 93/1 in enriched FMDV virions, indicating that FMDV encapsidation is highly specific for plus-stranded RNAs. In addition, the method was applied to surveille the FMDV replication at animal level.

Animals↗
PubMed2002

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1.

Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia I virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I-IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 x 10(-2) synonymous substitutions per nucleotide per year.

Amino Acid Sequence↗
PubMed1986

Predictive value of tumor thickness in squamous carcinoma confined to the tongue and floor of the mouth.

In this review of 105 consecutive patients who underwent operation for previously untreated, N0 squamous carcinomas arising in the oral tongue or the floor of the mouth, 86 percent of the determinate patients remained alive and well 2 years after treatment. Included were 48 patients, 49 patients, and 8 patients who had T1, T2, and T3 tumors respectively. Elective cervical lymphadenectomy was performed in about a third, but tumor staging did not facilitate selection of those who were most likely to have occult metastases. For this reason, we retrospectively assessed the impact of tumor thickness using an optical micrometer to measure the thickness in millimeters of the excised tumors in routinely prepared paraffin sections. Disease-related death appears to be unusual when oral tumors are thin (2 mm or less), regardless of the tumor stage. Multivariate analysis confirms that increasing tumor thickness, rather than tumor stage, had the best correlation with treatment failure and survival. These findings need to be verified in prospective studies involving a larger patient population and other head and neck sites, but they strongly suggest that measurement of tumor thickness may be a better way to select those oral cancer patients who are most likely to benefit from elective treatment of the N0 neck.

Carcinoma, Squamous Cell↗
PubMed1986

Relation of thickness of floor of mouth stage I and II cancers to regional metastasis.

We have reviewed the clinical course and histologic findings in 84 patients with stage I and II squamous carcinoma of the mouth floor. We concluded that lesion thickness may offer a useful method for predicting the probability of cervical metastasis in node negative (N0) patients. Moreover, surface area of the lesion did not correlate with subsequent nodal disease, whereas thickness did. Elective node dissection appears to be strongly indicated in any patient with a N0 lesion measuring more than 1.5 mm in thickness.

Adult↗
PubMed1989

Surgical management of squamous cell carcinoma of the floor of the mouth.

Treatment failure and survival in 209 patients with squamous cell carcinoma of the floor of the mouth treated with surgery as the single curative modality are reported. Fifty percent of the patients had stage III and IV disease. The primary tumor was excised with 1 to 2 cm margins and the mandible was resected in 73 percent of the patients; 77 percent underwent radical neck dissection. No cures were observed in 11 patients with involved surgical margins on permanent section. For 198 patients with uninvolved margins, determinate survival at 5 years for all stages was 49 percent and 69, 64, 46, and 26 percent for stages I through IV (p less than 0.01). The most common sites of initial and ultimate treatment failure were the neck (42 of 72 patients) and distant metastases (33 of 53 patients), respectively. Treatment of the neck is identified as an unresolved problem in the management of early stage disease. Recent improvements in survival for stage III and IV disease are accounted for, in part, by adequate surgical resection.

Adult↗
PubMed1975

Dermoid cyst of the floor of the mouth. Report of a case.

Dermoid cysts of the floor of the mouth comprise aproximately 0.01 per cent of all oral cystic lesions. This case is presented because of its large size, its unusual location, and the difficulty encountered in removing the mass.

Adult↗
PubMed1978

Squamous-cell carcinoma of the floor of the mouth.

This study was based on the analysis of 100 cases of squamous-cell carcinoma of the floor of the mouth. The male/female ratio was 4.25:1. The peak incidence in women was in the 50 to 59 year age group; in men there was equal frequency in each age group above 50. The five-year survival rate decreased from 86 per cent to 0 as the stage of disease progressed from I to IV. The five-year survival for all stages of disease was 52.7 per cent. Thirty-three per cent developed new primary malignancies; 22 per cent were of the upper alimentary and respiratory tracts. Distant metastases were present in 21 per cent. Fifty per cent were heavy smokers, 33 per cent were heavy drinkers, 28 per cent were both heavy smokers and heavy drinkers, and 21 per cent were nonsmokers and nondrinkers.

Adult↗