GENOME CHARACTERIZATION AND IMMUNO BASED DIAGNOSIS USING SYNTHETIC PEPTIDES OF LEEK YELLOW STRIPE VIRUS (LYSV)
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Date
2017
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DIVISION OF PLANT PATHOLOGY ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
Leek yellow stripe virus (LYSV), a member of the genus Potyvirus (family
Potyviridae) infects garlic (Allium sativum) worldwide including India.
Morphologically, it is a flexuous filamentous particle of 815-820 X 11-13 nm
dimensions containing ssRNA of approx 10.2 kb and transmitted by aphids in non
persistent manner. On the basis of serology, immunosorbent electron microscopy
(ISEM), reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing,
we report for the first time, the association of LYSV in garlic plants in India. CP of
two LYSV isolates was sequenced and deposited in the GenBank database with the
accession number KF724857 and KP168262 corresponding to the garlic cultivar AC-
50 and PGS-14, respectively. The complete genome of an isolate of Leek yellow stripe
virus from India was sequenced from garlic cultivar AC-50 and the predicted amino
acid (aa) sequence was analyzed. The LYSV RNA genome was 10,131 nucleotide (nt)
long excluding the poly(A) tail. The genome had a large open reading frame (ORF)
encoding a putative polyprotein of 3152 aa, containing conserved motifs typical to the
genus Potyvirus and potentially cleaved into 10 mature proteins similar to other
potyviruses. An additional small overlapping ORF designated as pretty interesting
Potyviridae ORF (PIPO) was deduced in the P3 gene. The complete genome sequence
of LYSV “isolate INDIA” was deposited in the GenBank database with the accession
number KP168261. The LYSV “isolate INDIA” shared maximum nt and aa sequence
identity of 79.9% and 87.2% with isolate from Australia (HQ258895) at full genome
level and polyprotein level respectively and clustered with the isolates of clade II
from China, Mexico, Australia, Brazil and Spain. P1 gene in Indian isolate was highly
variable and showed identity in the range of 48.9-70.9% with other isolates. P1 region
of Indian isolate had two large deletions (105 and 99 nt) compared to Japanese and
Australian isolates. The ratio of non synonymous (dN) and synonymous (dS)
polymorphic sites suggested that purifying selection dominates in the evolution of
LYSV. The dN/dS ratio for gene P1 was highest which indicated the higher selection
pressure on P1 gene. The complete genome sequence analysis indicated that this is a
genetically divergent isolate of LYSV from garlic in India.
CP of LYSV-AC-50 was over-expressed in vitro as recombinant fusion
protein in pET 28a+ system and used as immunogen for the production of polyclonal
antisera. Antisera to LYSV (titre 1:2000) detected the LYSV in DAC-ELISA in 16
out of 21 different garlic accessions. The antiserum specifically reacted with LYSV
virions in immunosorbent electron microscopy, Dot immunobinding assay (DIBA)
and direct antigen coated-enzyme linked immunosorbent assay (DAC-ELISA). The
LYSV antiserum (1:2000) was successfully used in DAC-ELISA for specific
detection of LYSV infection in field samples. To further simplify the methodology of
antigen preparation, synthetic peptides representing antigenic epitopes were identified
and used for production of polyclonal antibodies to LYSV. Two linear epitopes were
identified at N and C-terminal of CP of LYSV (pep-I and pep-II), synthesized and
used for polyclonal antiserum production. The antisera produced didn’t react against
the purified CP in both western blotting and DAC-ELISA. The result suggested that
both the epitopes are not surface exposed and which may either be a cryptotope
(surface buried) or a conformational epitope.
Description
T-9589
Keywords
GENOME ; CHARACTERIZATION ; IMMUNO BASED DIAGNOSIS ; SYNTHETIC PEPTIDES ; LEEK YELLOW STRIPE VIRUS (LYSV)