Whole different isolates submitted to the GenBank and acquired

Whole Genome
amplification and sequence analysis of a Classical swine fever Virus isolates

4.1 Annotation and Analysis of Sequence

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The
sequences of different overlapping fragments of CSFV genome thus generated were
identified by NCBI-BLAST program
(http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) using existing CSFV sequence
database. The whole genome amplification was done by using different
overlapping set of primers and these fragments (contigs) were sequenced (Fig
gel pic full genome). Sequence data obtained from commercial firm were checked
for CSFV genes using NCBI-BLAST and EditSeq program. The sequenced fragments
were assembled and aligned to generate whole genome sequences of different
isolates. Result of BLAST confirmed the gene or region specific for CSFV. The
whole genome sequence was annotated from all sequences by EditSeq program of
DNASTAR software. The annotated full length genome sequences thus generated was
submitted to NCBI-GenBank database. The complete genome sequences of six
classical swine fever strains (CSFVs) from different genotypes which are
circulating in Indian field were determined in this study using various
overlapping fragments. The whole genome sequences of different isolates
submitted to the GenBank and acquired the accession number along with length
and genotype showed in table 4.

 

Table 4. The
list of isolates sequenced along with accession number, length and genotype in
this study

Sr no

 Isolate name

Total
nucleotide  length

Polyprotein
length

 Accession number

Genogroup

1.              
 

CSFV-PK15C-NG79-11

12302

3898

KC503764

1.1

2.              
 

CSFV212L-13

12300

3898

KY860615

1.1

3.              
 

CSFV-UP-GZ-NVD-11

12298

3898

JQ861548

2.2

4.              
 

CSFV-UP-BR-KHG-06

12297

3898

KC533775

2.2

5.              
 

CSFV-UP-BD-SKN-11

12297

3898

KC533776

2.2

6.              
 

CSFV-UP-ND-169-11 

12297

3898

KC533793

2.2

 

 

4.1.2 Nucleotide/amino acid substitutions and genetic
heterogeneity analysis:

NCBI-BLAST result was
analysed for all six isolates sequenced in this study namely,
CSFV-PK15C-NG79-11(NG79-11), CSFV212L-13(212L-13), CSFV-UP-GZ-NVD-11(NVD-11),
CSFV-UP-BD-SKN-11(SKN-11), CSFV-UP-ND-169-11(ND-169-11) and
CSFV-UP-BR-KHG-06(KHG-06) (fig –fig). Blast result of sequenced isolates
NG79-11 and CSFV212L-13 showed highest homology with isolates of genogroup 1 while
NVD-11, KHG-06, SKN-11 and ND-169-11 showed homology with genogroup 2 isolates.
The table 5 showing blast result of sequenced isolates.

Table 5.
Showing the percent similarity (identity) analysis on the basis of BLAST result

 

Sr.  no

Isolate name

Maximum identity at nucleotide level

Maximum
identity at Amino acid level

1

NG79-11

VB-131(99%), Sheimen/HVRI
(98%), CSFV-GZ-2009(98%)

VB-131(99%),
Sheimen/HVRI (98%), cF114 (98%), JL1 (06) (98%)

2

212L-13

LOM (99%), Alfort/187(99%), Thiverwal (99%),

LOM(99%),
Alfort/187 (99%) Sheimen/HVRI (98%)

3

NVD-11

KHG-06 (99%), SKN-11(98%), LAL-290(97%), Strain
39(93%), Bergen (92%)

SKN-11(99%),
KHG-06(98%) Strain 39(96%), Bergen (95%)

4

KHG-06

NVD-11(98%), SKN-11(98%), LAL-290 (97%), Strain
39(93%), Bergen (92%)

NVD-11(98%),
SKN-11(98%), Strain39 (95%), GD53/2011(95%) Bergen (92%)

5

SKN-11

NVD-11(98%) KHG-06 (98%), LAL-290(97%), Strain
39(93%), Bergen (92%)

NVD-11(99%),
KHG-06 (98%), ND-169-11(97%) Strain 39(93%), Bergen (92%)

6

ND-169-11 

LAL-290(98%), SKN-11(98%) NVD-11(98%) KHG-06 (98%),
Strain 39(92%), Bergen (91%)

SKN-11(97%)
NVD-11(97%) KHG-06 (97%), Strain 39(94%), Bergen (94%)

 

Two isolate
NG79-11 and 212L-13 belongs to genotype 1. NG79-11 isolate showed full genome
of 12302 nucleotide length which includes 373 nucleotide length of 5´UTR, 11697
nucleotides long ORF (open reading frame) and 227 nucleotides length of 3´UTR.
The ORF of the NG79-11 genome encodes a polyprotein consisting of 3898 amino
acids (aa). Isolate 212L-13 showed full genome of 12300 nucleotide length which
includes 373 nucleotide length of 5´UTR, 11697 nucleotides long ORF (open
reading frame) and 225 nucleotides length of 3´UTR. The ORF of the 212L-13
genome encodes a polyprotein consisting of 3898 amino acids (aa). A comparative
analysis showed that the individual gene/region and viral proteins of Sequenced
isolates were identical or very similar in size to those from all reference
CSFV strains/isolates (Table-6).

 Table-6: Gene-wise details of sequenced
isolates NG79-11 and 212L-13 in genome

Gene name

Genome location

Amino
Acid location

5’UTR

1-373

N-Pro

374-877

1-168

Capsid
C

878-1174

169-267

ERNS

1175-1855

268-494

E1

1856-2341

495-656

E2

2342-3559

657-1062

P7

3560-3769

1063-1132

NS2-3

3770-7183

1133-2270

NS4A

7184-7381

2271-2336

NS4B

7382-8428

2337-2685

NS5A

8429-9913

2686-3180

NS5B

9914-12070

3181-3898

3’UTR

12071-12302

 

Further, the
nucleotide sequence of NG79-11 shared 90–98% similarity with group 1 viruses,
85–86% with group 2 viruses, and 85% with group 3 viruses. In comparison to Sheimen/HVRI,
the 3’UTR of NG79-11 had a six nucleotide insertion (TTTTTT) at position 12133
and deletion of 1 nt at position 12,225.
The single large open reading frame (ORF) (11,697 nts) was capable of coding
for a polyprotein of 3,898 amino acids. Table: 7. Nucleotide and amino acid
substitutions in NG79-11 in comparison to CSFV Sheimen/HVRI.

BLAST
analysis on the basis of full genome of NG79-11 revealed the highest homology
(99%) with the other Indian field isolate CSFV-IVRI-VB-131.
NG-79-11 showed high homology with historical strains of genogroup 1.1 (fig).
BLAST analysis of full length E2 gene sequence (1219 nts) of NG79-11 indicated
99% identity with Indian field isolates CSFV-IVRI-VB-131, isolates  from Assam, and the lapinized vaccine-India
(Weybridge, AY422081) strain. BLAST analysis of the 409 nts from the NS5B
region of NG79-11 showed high sequence identity (99%) with some Indian isolates
such as VB-131, BHD/03, Chaygaon, Baksa, Boko, Goalpara, Khanapara, Jagiroad,
Dhemaji, Hoflong, IND/TRI-AGR/2009-1/MUKT/MDCK-P10, CSF/MZ/AIZ/87, NFP/MN-1 and
CSF/MZ/KOL/73.

BLAST analysis
of the full genome and complete polyprotein of 212L-13 showed, nucleotide
sequence shared 99–92% similarity with group 1 viruses, 90–85% with group 2
viruses, and 85% with group 3 viruses. Isolate 212L-13 showed highest
similarities (99%) with the vaccine strain LOM. 212L-13 also showed close
homology with Alfort/187 and Thiverwal (99%).