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Molecular Pathogens
MP2011, Vol.2, No.1
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virulence of virus and host tropism (Feng et al., 2004).
So far, few PKs with virulence-related research are
reported.
FMDV mutations mostly happened in the capsid
protein coding regions, of which the VP1 would be the
largest variation region, and their two antigenic sites
located on 42~60 aa (B-C loop) and 133~158 aa (G-H
loop) are a highly variable region. The antigenic
variation created by sequence differences resulted in
lack of immunologic cross-reactivity among outbreak
strains using sera from vaccinated animals (Mattion et
al., 2009). The binding sites for RGD cell receptor
located the G-H loop of
VP1
gene of O/YM/YN/2000
did not change any more (Figure 5), and phylogenetic
analysis displayed the virus cluster with the Cathay
topotype (Figure 3). Therefore, the O/YM/YN/2000
would be selected to be as a potential promising
vaccine candidate strain, which provided the genetic
information about the epitopes to play dominant roles
in vaccine-induced protection. It was recognized that
high antigenic variation in FMDV causes a major
problem in selection of vaccine strain. Comparing
sequence data generated from FMDV might be helpful
to rapidly select candidate vaccine strain for better
control the disease (Mattion et al., 2009; Parida, 2009).
However, there is extensive antigenic variations
within FMDV, the changes might be limited to very
specific regions of the viral surface. The antigenicity
of the tested vaccine candidate strain could not be
predicted only based on the strains’ topotype or
genotype but it would be enough for only selecting the
vaccine strain using these genetic information.
Therefore, vaccine strain selection might mainly rely
on serological and immunological approaches,
combining with the virus genetic analysis information.
Previously, we carried out the antigenic relationship
values (r values) between O/YM/YN/2000 (belongs to
the Cathay topotype) and two other topotype (ME-
SA and SEA), FMDV type O vaccine candidate
strains, the serological cross-protection tests showed
that the antigenicity of O/YM/YN/2000 can cover
these two topotype isolates (r>0.3) ( data not shown).
So, to select the vaccine candidate strain, particular in
selecting a broad spectrum of antigenic viruses, the
immunogenicity and production performance of
candidate strains should be considered to be known
(Mattion et al., 2009).
In conclusions, O/YM/YN/2000, potentially as a
FMDV vaccine candidate strain serotype O, belongs
to the Cathy topotype. A 4
nt deletion in S fragement
and 43
nt in FUR exist in the 5´
-
UTR of the
O/YM/YN/2000. The arginine-glycine-aspartate (RGD)
sequence located in the G-H loop of the VP1 protein
might involved in binding of FMDV to cellular
receptor. A 10
-
aa deletion in the 3A nonstructural
protein was found in the O/YM/YN/2000 strain.
Those genetic information of the O/YM/YN/2000,
combining the previous studies on serology and
pathology, might be helpful for identifying vaccine
candidate strain, although its antigenicity and immu-
nogenicity did need to be further clear.
3 Materials and Methods
3.1 Virus isolate
The O/YM/YN/2000 strain was isolated from the
animal disease monitoring station of border region of
Yunnan Province and isolate was passaged by baby
hamster kidney (BHK
-
21) monolayer cell culture.
3.2 RT-PCR and Sequencing
Viral stock was propagated as previously described
approach (Xin et al., 2009). Virus RNA was extracted
from virus stock using the Viral RNA Extract Kit
(Bio.Basic.Inc.) according to the manufacture’s
instructions. Reverse transcription was carried out
using 6 random primes and Superscript II reverse
transcriptase (Invitrogen). Seven cDNA fragments
covering the entire FMDV genome were amplified by
PCR using seven primer sets (Figure 7). The primers
showed in Table 2. PCR amplifications with Pyrobest
Figure 7 Strategies for amplifying the whole genome of FMDV
O/YM/YN/2000 strain by RT-PCR