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Sequencing and Translational Analysis Revealed Huge Mutation in the N-terminus End of Leader Proteinase (lpro) Gene of Foot and Mouth Disease Viruses Isolated from Cattle in Bangladesh

Islam M S
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Ruba T
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Habib M A
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Rima U K
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Hossain M Z
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Saha PC
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Das PM
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

*Khan MAHNA
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Abstract/ Executive Summary:

Foot and Mouth Disease virus (FMDV) comprises four structural and ten nonstructural proteins in its genome. The Leader proteinase (Lpro) is structurally and functionally related to papain-like cysteine proteinase with catalytic cystein and histidine residues which is the first functional component of the Aphthoviral polyprotein. Complete Lpro genes of six Bangladeshi isolates of FMDV of three different serotypes were sequenced and compared with each other as well as with those sequences available in the GenBank to evaluate the extent of mutation in this gene. Out of six isolates investigated a serotype O viruses (BD_SI_5_2013) showed highest level of amino acid (aa) substitution with a critical substitution at L10 by V10. The Lpro gene of the investigated viruses showed mutation in 9% (≤) nucleotide and substitution of aa in 11.4% position. A total of 55% variability of aa was seen in the N terminus end between first two conserved initiation codons at 1st and 29th aa positions of Lpro sequences. Methionine at position 1, 29, 126 and 132 are conserved in the Lpro sequences. The Lab form of the Lpro was found more variable than Lb form. Conserved KRLK/R sequence was found at Lpro/VP4 cleavage site at the C terminus end of Lpro in all the isolates. The invariant motif, the catalytic triad and other critical amino acids were totally conserved. Specific clustering of viruses on the basis of serotype as well as geographical origin was not found in the phylogenetic trees constructed but the viruses had lineage specific signature clustered together in Maximum Likelihood (ML) tree.

Keywords: Clustering, FMDV, Lpro gene, Sequencing, Substitution of amino acid

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Cattle, Virus

Objectives:

Therefore, to analyze the extent of mutation present in Lpro genes and as well as to evaluate phylogenetic position of Bangladeshi FMDV, six FMDV isolates were sequenced and the sequenced data were compared with other Lpro sequences available in GenBank.

Methodology:

2.1 Viruses Six virus isolates collected from cattle of FMD outbreak areas (3 O, 2 Asia1 and 1 A type) were used in this study. A number (N= 18) of Lpro sequences available in GenBank were included in this study for comparison and analyses.

2.2 RNA Extraction and RT-PCR The total RNA was extracted from the oral/padal epithelial tissue homogenates using Viral Nucleic Acid Extraction Kit II (Geneaid Biotech Ltd., Taiwan) as per manufacturer’s instructions. The purity and concentration of extracted viral RNA were measured by using spectophotometry (A260/A280). Extracted RNA was then subjected to RT-PCR amplification of full length Lpro gene of FMDV using designed primer pairs. FMD LproF (5’-cttctacgcctgaataagcg-3’) and FMD LproR (5’-gatgatacttcccgtgttgc-3’) primers were designed using the sequence downloaded from GenBank. The RT-PCR was conducted by using SuperScript III one step RT-PCR kit with Platinum Taq (Invitrogen). The RT-PCR was carried out in 50µl volume containing of 2X reaction mixture, forward and reverse primers (20pmol in each), Taq polymerase (1 µl), RNAse out (1 µl), nuclease free water (16 µl) and l RNA template (150-200ng in 5 µl/reaction). The reverse transcription (RT) was carried out in a thermocycler (eppendorf, Germany) at 45°c for 45 minutes. A total of 35 cycle of PCR amplification was carried out with an initial denaturation at 94⁰c for 5 minutes. The cycling condition consisting of denaturation at 94°c for 1 minute, annealing at 52°c for 1 minute and extension at 72°c for 2 minutes. After final extension at 72°c for 7 minutes, the reaction was hold at 4°c. The PCR products were electrophoresed on 1.5% agarose gel containing ethidium bromide with a transilluminator (Alpha imager, USA).

2.3 Sequencing of PCR products The amplified PCR products/gel of Lpro segment were cleaned with Wizard SV gel and PCR clean-up system (Promega). The gene cleaned PCR products were then sequenced commercially from 1st Base, Malaysia.

2.4 Sequence Analysis The raw sequence data were first checked for its quality and then edited and assembled with the programmes Chromas Lite, EditSeq and MegAlign. Edited sequences were aligned with MegAlign or MEGA6 programmes. Multiple alignments were done with Clustal W algorithm and Neighbour-Joining as well as Maximum Likelihood phylogenetic trees were constructed with MEGA6 programme. The stability of the nodes in the phylogenetic trees was tested by bootstrapping with 1000 replications. Translational analysis was carried out using MegAlign program to evaluate the level of mutation in Lpro genes and for comparison.

Source of Information: IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 9, Issue 2 Ver. I (Feb. 2016), PP 24-30 www.iosrjournals.org

Article Link (Online Journal): DOI: 10.9790/2380-09212430

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

L pro isoforms have indistinguishable activities and specificities and played a role in virulence through the regulation of host interferon responses. The L pro gene also contributed in the phylogenetic analysis of the viruses. There are two methionines in the L pro sequence of the virus (aa position 1 and 29) are invariant, indicating that two methionine in L pro isoforms are significant for aspects of FMD viral biology. The catalytic triad formed by cysteine, histidine and aspartic acid residues at the 51st, 148th and 163rd amino acid sites respectively is critical for Lpro activity and found conserved. Results of position wise aa substitution in Lpro gene of Bangladeshi isolates showed that 22 out of 40 substitutions occurred in between 1st and 29th aa positions. Highest substitutions (N=12) was seen in BD_SI_5_2013 among six isolates. The aa residues N46, D49, N54 and D164 were conserved in the Lpro sequences in the studied viruses. Cysteine at 6th, 133rd, 153rd and histidine at 109th, 138th, 148th aa position was also conserved among all isolates. These data indicate that despite a wide range aa substitution in L pro sequence in the FMDV isolates examined, functional elements such as the catalytic sites and cleavage sites and previously described motifs are invariant. The variability observed at the N and C termini of L pro sequence may affect specific host-virus interactions, including ribosomal recognition of alternative start codons and virulence of the viruses, required further study.

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