The the new Indian isolate SKN-11, the length

The observed
divergence between Sheimen/HVRI and NG79-11 was 2.3% at nucleotide level and
1.9% at protein level. The number of nucleotide and amino acid substitutions in
NG79-11 in comparison to CSFV Sheimen/HVRI, are listed in table 7. Nucleotide identity between NG79-11 and Sheimen/HVRI at individual gene level was 97% for
the C, E2 and NS5A genes; 98% for the Npro, Erns, E1, p7, NS2, NS3, NS4B, and
NS5B genes; and 99% for the NS4A gene.  Out
of 285 nt substitutions, maximum changes were seen in the 3’UTR region (6.14%)
followed by C (3.37%), E2 (3.7%) and NS5A (2.58%) genomic regions. Similar
comparison of NG79-11 polyprotein revealed 72
amino acid (aa) substitutions with maximum divergence in the E2 protein
followed by Npro, NS4B and C protein. NS4A and p7 proteins were identical in
both viruses.

Three isolates
NVD-11, ND-169-11, KHG-06 composed of a 373-nt
5’NTR an 11,697-nt open reading frame (ORF) encoding a polyprotein of 3,898
amino acids (aa), and a 228-nt 3′-NTR with total length of genome 12298nt
whereas the new Indian isolate SKN-11, the length of 3′-NTR was 227-nt with
total genome length 12297nts. The sequenced isolates from genotype 2.2
of this study exhibited the analogous genomic organization to others CSFV
strains. 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

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NCBI BLAST results of full genome nucleotide sequence of NVD-11, ND-169-11, KHG-06 and SKN-11 revealed homology 97%-98% with each other and 92% with Chinese recombinant virus strain 39. The isolates showed 90% homology with group 2.1 Indian isolate IND/AS/GHY/G4 and other isolates such as Padarborn, Taiwanese isolate 96TD which was followed by some other genogroup 2.1 Chinese viruses (SXYL06, GXWZ02, HEBZ, YC11WB, PC11WB) and genogroup 2.3 viruses with homology 88%. At amino acid level all four isolates (NVD-11, ND-169-11, KHG-06 and SKN-11) shared 97% homologous with each other and other Indian isolate LAL-290. Next highest 94% homology was found with strain-39. 

BLAST result of
full E2 protein of NVD-11, ND-169-11, KHG-06 and SKN-11  revealed close homology with each other and
many earlier reported Indian strains of group 2.2 and Chinese virus strain 39
(96% homology).  New Indian isolates of
genotype 2.2 are more closely related (97-99%) to other Indian isolates such as
Mathura, CSFV-UP-BD-BD-11, MIB56, CSFV-UP-BR-793-09,
and CSFV-UP-GZ-NG39-10. E2 region Showed 97% homology with isolates CSF 1059
and CSF1060 isolated in Nepal in 2011, 92% homology with
group 2.2 isolate CSF0073 which was isolated in Austria in year1990, 90%
with Sukoharjo (Indonesia) isolated in 2007, 92%
with CSF0378 which was isolated in year 1994 from Czech
Republic, 92% homology with isolate 84-KS1 which was isolated in 1995 from Taiwan, 91% CSF0573 Parma isolated in 1998 in Italy, 90% with CSF0014
Isolated from Germany. Full E2 gene of new Indian isolates showed highest
homology with LAL-290 (100%), CSF0073 (96%), CSF0073 (93%), 84-KS1 (92%), CSF0573 Parma (91%). 

Phylogenetic analysis of CSFV
isolates

Phylogenetic analysis of the CSF virus isolates
was done as per the ecommendation of European Union Diagnostic Manual on
classical swine fever, comparing 5’NTR, E2 and NS5B region of CSFV. The
nucleotide sequences database for phylogenetic analysis of CSFV isolates was
updated with new data pertaining to India. For genetic typing of new Indian
isolates, using sequence data of 5’UTR, E2 and NS5B genes, complete ORF and
whole genome sequence was carried out using MEGA 6.06 software package (Tamura et
al., 2013).  Phylogenetic analysis
was performed by aligning sequences in ClustalX and subsequent analysis by
Neighbor Joining (Tamura-Nei) method.

Phylogenetic analysis on the basis of
5’NTR sequence comparison

5’NTR (150 nucleotides) sequence
analysis of 111 virus isolates (table 8) were phylogenetically analysed from
the different geographical regions of the India included new, 45 isolates
belonged to genotype 1 and 66 isolate belonged to genotype 2. The isolates of
genotype 1.1 were collected from different places in Uttar Pradesh Tamilnadu
and NE States of India. Out 45 Indian isolates of genotype 1, total 38 fell in
genotype 1.1 and seven Indian isolates collected between year1989-2003from
Andaman & Nicobar, West Bengal, Uttar Pradesh, Madhya Pradesh and
North-eastern states belonged to genotype 1.2. Most of the new isolates from
NorthEastern states of India grouped in genotype 1.1. CSF-MZ-LUN-421,
IND-AS-GHY-G4 from Assam, CSF40-02 from NE2002, Nbe244 and NLB2-36 from MP,
Supol2009 from Bihar belongs to genotype 2.1. 
New Indian CSFV isolates including CSFV-UP-BR-757-09, CSFV-UP-BD-BD-11,
KHG-06, SKN-11, NVD-11 and ND-169-11 belonged to genotype 2.2. Most of isolates
Indian strains of sub-genogroup 2.2 are reported between 2005 to 2013 from
various geographical regions of India like Uttaranchal, Karnataka, Madhya
Pradesh, Tamil Nadu, Maharashtra, Bihar, Uttar Pradesh, Assam, Haryana, Mizoram
and Meghalaya. These new isolate shared close homology with some earlier
isolates reported from China (GXW02) and Taiwan (0406/CH/01/TWN) in Asia. Total 6 isolates collected from different
outbreak in Mizoram. Out of which 2 of the isolates are from wild pig
population of Mizoram. Phylogenetic analysis of the 5 isolates
revealed presence of CSFV belonging to 1.1 genotypes in domestic pigs and 2.2
genotypes in wild pig population of Mizoram. The isolates from wild pigs showed
close link and 99% homology with an isolate from CSF outbreak in domestic pigs
in Assam. Isolate no AIZWAL (CSF/MZ/AIZ/115) was collected from an outbreak of chronic form of swine fever which was
recorded in local pigs ZOVAWK of Mizoram.  Isolate CSF-MZ-AIZ-352 and CSD-MZ-AIZ-348
showed 99% homology with the HCLV-India (Vaccine strain). Isolate from Chennai
TNVLI belonged to genotype 1.1.