Sequence Analysis of the Foot and Mouth Disease Virus Type O/IRN/2007 VP1 Gene from Iranian isolate
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Abstract:
The foot and mouth disease virus (FMDV) causes a vesicular and contagious disease of cloven-hoofed animals. In this study, the virus was isolated from vesicles of the infected cattle using cell culture and serotyped by ELISA test. The extracted RNA from the infected cells was reverse transcribed and amplified using VP1 gene-specific primer pairs by means of one-step RT-PCR. The purified VP1 gene was sub-cloned into the uniqe KpnI and BamHI cloning sites of the pcDNA3.1+ vector. The DH5α strain of E. coli was transformed by the vector. The sequences of sub-cloned FMDV type O/IRN/2007 VP1 were aligned with FMDV type O/UKG/2001 VP1 using MegAlign software. Nucleotide sequence comparisons were made using the BLAST software available from the NCBI website. The amino acid sequences of three sub-cloned FMDV type O/IRN/2007 VP1 were also aligned with three other similar sequences using MegAlign software. Nineteen of the most similar VP1 nucleotide sequences (by BLASTN program), FMDV O/IRN/2007 VP1 sequence, twenty isolates of FMDV-O VP1 in Iran and eight topotypes of FMDV type O were aligned by Mega5 to create a FMDV-O VP1-based sequence similarity tree. The nucleotide sequence comparison indicated that FMDV O/ IRN/2007 VP1 had the greatest nucleotide sequence similarity to the VP1 gene of FMDV O1/Manisa/Turkey/69 (99%), FMDV O1/Manisa/Netherlands (98%) and FMDV O1/Manisa/iso87/Turkey (98%). It was also observed that the highest identity between FMDV O/IRN/2007 VP1 sequence and other nucleotide sequences of FMDV type O VP1 genes isolated in Iran during 1997-2004 was about 91%.Keywords:
Cloning (programming)
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Aphthovirus
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Viral Interference
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Summary: The infectivity curve of the virus of foot-and-mouth disease was studied in cultures of suspended trypsinized cattle-tongue epithelial cells. By this technique a known number of cells could be exposed to a known amount of virus. The pattern of multiplication was that adsorption of the virus by the cells occurred rapidly and, except with low concentrations of virus, was complete in 15–30 min. A latent period of about 2·5 hr. followed during which the virus was closely associated with the cells, was protected from the neutralizing action of antiserum, was not readily extractable but retained its infectivity. At the end of the latent period the infected cells became producers of virus at a rate estimated to be between 102 and 103 ID 50/15 min. At about 12 hr. the infectivity of the culture began to decline because of death of virus-producing cells and thermal inactivation of the virus.
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Growth pattern of foot-and-mouth disease virus types 0 and Asia 1 in BHK-21 Razi cells was compared; while type 0 virus grew in high titre, Asia 1 virus was produced in low titre. Inhibition of host protein synthesis in type 0 virus-infected cells was more pronounced than in Asia 1 virus-infected cells. Foot-and-mouth disease virus type 0 infected cells showed higher lactic dehydrogenase activity when compared to Asia 1 virus. A significant decrease in virus yield was observed when Actinomycin D had been added at 50 micrograms/ml to infected cells.
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The effect of sodium and magnesium chloride in 1 and 2 m concentration at temperatures of 37 and 50 C on type C, strain 149, foot-and-mouth disease virus during storage for 6 days was studied. The exclusively passaged cattle strain and its tissue culture-adapted line were compared. Preparations of the various chemicals and their concentrations were made directly in suspensions of the virus, which, together with untreated control virus suspensions, were stored at indicated temperatures and tested daily for concentration of virus present. Both 1 and 2 m concentrations of Mg markedly slowed the degradation of the bovine-passaged virus, as compared with untreated virus stored at 37 or 50 C. Such was not the case with 1 and 2 m concentrations of Na at 37 and 50 C, in which instance the treated virus was degraded faster than the untreated controls at 37 C, and but slightly influenced at 50 C. The tissue culture-adapted virus at the 25th passage was not stabilized by any concentration of chemical additive either at 37 or 50 C, except for 1 and 2 m concentrations of Na at 37 C, which partially retarded degradation of the virus. After 91 passages of the virus in tissue culture, only a suggestion of the influence of 1 and 2 m concentrations of Na at 37 C remained to show a stabilizing effect. These responses tend to separate the bovine-passaged virus from the tissue culture-adapted virus under the conditions of this study.
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The process of penetration and subsequent early stages of replication of Foot and Mouth Disease Virus (FMDV) in BHK21 cell cultures have been studied in order to obtain further data about the infectious cycle of this virus. Results suggest that FMDV penetrates BHK21 cells by way of pinocytic vesicles. Studies of lysosomal (LF) and supernatant (SF) fractions of homogenized suspension of infected cells were carried out to learn the percentage of possible non-specific absorption of infectious virus particles. Furthermore, analysis of intracellular virus shows that LF-associated virus is not completely degraded by the enzymatic process expressed by cellular proteases and RNAses and can be potentially useful for genetic expression. Data suggest that: i) only a part of LF-associated virus replicates; ii) the remaining LF-associated virus may be a reservoir of potentially active virus.
Penetration (warfare)
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Antibody-dependent enhancement
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Guanidine
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Aphthovirus
Antibody-dependent enhancement
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