MOLECULAR CHARACTERIZATION OF THE VIRUS AND HOST FACTORS IN RESPECT TO FOOT AND MOUTH DISEASE INFECTION IN CATTLE
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Date
2011
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
In India Foot and mouth disease (FMD) is endemic since many centuries. It is
present almost in all parts of the country and occurs round the year. India is losing Rs
18,000 crore annually due to the dreaded FMD in cattle and livestock (Economic Times,
2011). Out of the possible seven serotypes of the foot and mouth disease virus (FMDV) ,
only four serotypes, viz, ‘O’, ‘A’, ‘C’ and Asia 1 were ever recorded in India. The
molecular epidemiological studies have established that the Pan-Asian strain is the major
cause of outbreak of FMD involving serotype ‘O’ in India . Since 1996, type C outbreaks
have not been recorded in India.
The FMDV has a single stranded positive sense RNA genome. The viral genome
is translated as a single polyprotein, which is post-translationally cleaved by viral
proteases into four structural proteins (VP1, VP2, VP3 and VP4). Among the 4 structural
polypeptides, VP1 is the most immunogenic protein of FMDV. During the course of
outbreaks, the high rate of mutation in the replicating virus population can lead to the
accumulation of genomic changes and eventually to the emergence of immunogenic
variants. This poses serious threat to the FMD control campaigns in endemic countries.
The innate immune system comprises the cells and mechanisms that defend the
host from infection by other organisms, in a non-specific manner. Induction of the
antiviral innate immune response depends on recognition of viral components by host
pattern- recognition receptors; one of them is the Toll-like receptors (TLRs). To date, 10
TLRs have been identified in cattle. Pathogen-associated molecular patterns (PAMPs)
specific to viruses are recognized by four TLR family members (TLR 3, 7,8 and 9).
TLRs play essential roles in the production of type I interferons (IFNs) and
proinflammatory cytokines. The interferon(IFN) are a family of proteins that have
antiviral properties and role in immunoregulatory actions. One of the IFNs, interferongamma
(IFN-γ) is a dimerized soluble cytokine that is the only member of the type II
class of interferons. The importance of IFN-γ in the immune system stems in part from
its ability to inhibit viral replication directly, but, most important, derives from its
immunostimulatory and immunomodulatory effects.
On the other hand, interleukin-10 (IL10) is an anti-inflammatory cytokine and
has pleiotropic effects in immunoregulation and inflammation. It down-regulates the
expression of Th1 cytokines (IFN-γ).
Most of the differences in the genome of the individuals of the same species are
due to single base substitution polymorphisms, popularly known as single nucleotide
polymorphisms (SNPs). SNP has been correlated to disease severity in human subjects.
The TLR-3 mRNA expression was not found to be affected by FMDV infection.
With the above facts, it was hypothesized that viral isolates causing FMDV
infection among the cattle from Assam and Ranchi may have serotypic and genotypic
diversity which may correlate with the disease severity. We also hypothesized that
certain host factor molecules involved in innate immunity of mammals may also be
associated with the severity of the disease. To that direction an effort was made to
examine the expression or detection of certain host factors such as TLR3 (with its SNP
analysis), status of IFN-γ and IL10 and finally to correlate the observations with severity
of the disease. Therefore, the present research programme was undertaken to analyze the
serotype and genotype of the FMD viral isolates from affected animals; to analyze the
expression of TLR3 in healthy and infected cattle; to detect important mutation(s) (if
any) in the TLR3 gene in affected cases and to correlate TLR3 expression and
polymorphism with immunomodulation in FMD infected cases.
APPROACH:
In the present study, a total of 52 clinical materials were collected out of which
40 were from the FMD affected animals from Assam and Ranchi and12 numbers of
tongue epithelial samples were collected as control samples from cattle slaughter
houses. The clinical materials were in the form of tongue/feet epithelium and sera
samples.The tissue and the sera samples were stored in -200C.
The degree of severity of the infected sample was based on the visual observation
of the severity of the manifestation of the symptoms. They were graded as ‘no disease’,
‘less severe’ and ‘more severe’ and were depicted as ‘-’, ‘++’ and ‘+++’ respectively.
In order to confirm the serotype of the isolates collected, samples were tested by
sandwich ELISA as per the bench protocol of Project Directorate on Foot -and-Mouth
disease, IVRI, campus, Mukteswar, Uttarakhand.
For genotyping, total RNA was isolated from the tissue sample by following
standard protocol. cDNA was prepared using standard PCR (Eppendorf Germany)
protocol. The cDNA thus prepared was stored at -200C and was used for PCR
amplification of 3D gene.
The 3D gene was amplified by using the cDNA. 3D specific primer were used in
the process. Both positive and negative controls were run parallely along with the test
samples. The 3D gene amplicons were sent to Macrogen® (Seoμl, Korea) for sequencing
adhering to the guidelines of the vendors with respect to minimum concentration of the
amplicons and the primers. The sequences received were subjected for Phylogenetic
analysis of FMDV isolates using Molecular Evolutionary genetic Analysis (MEGA 4.1)
software.
The expression of TLR3 gene was studied by semi-quantitative reverse
transcriptase polymerase chain reaction (RT-PCR) as well as validated by Real Time
PCR. Total RNA was isolated from the tissues using the standard TRIZOL method.
cDNA was prepared as stated above by employing standard protocol. Semiquantitaive
rtPCR was performed for TLR3 using β-actin as internal controls.
Expression of the RNA transcripts of TLR3 and β-actin, isolated from the
epithelial tissue samples from FMDV infected samples was determined by Real Time
RT-PCR by using SYBR Green Fluorescent Dye. The mRNA levels of the target genes
were normalized to the transcript level of the housekeeping gene β -actin. For relative
quantification, the expression of mRNA transcripts of the target genes from normal
tissue was also determined.
Primers were validated on an Applied Biosystems machine by using serial
dilutions of total RNA with endogenous control and target primers, whose values were
plotted as the log input amount versus ΔCT values (target CT − endogenous CT) for
relative efficiency. Primers with a slope of less than 0.1 were used, due to similar
amplification efficiencies as the endogenous control.
For TLR3 SNP polymorphism, the genomic DNA from the freshly collected
tissue samples was isolated by standard Proteinase-K digestion and phenol/ chloroform
extraction procedure. The quantity and quality of DNA was measured by using
Nanodrop spectrophotometer (GE NanoVue plus). Exon 3 of the TLR3 gene was
amplified through PCR in an gradient thermocycler (Epphendroff) by using specific
primers.
The amplicon product was sent to Macrogen, Korea for sequencing. The
variations in the nucleotide sequences were verified by comparing with the sequences
reported in the National Centre for Biotechnology Information (NCBI) database and
were analyzed using the Clustal X software output (Rosalind Franklin Centre for
genomics Research; http://www. hgmp. mcc. ac.uk).
The IFN-γ level was detected in the serum samples from infected and control
samples using the Bovine IFN–γ ELISA kit (BioSource Bovine IFN–γ EASIA, Belgium)
following the manufacturers instructions. Samples yielding mean OD below the Assay
Cut off was considered as negative and samples yielding mean OD above the Assay Cut
off was considered as positive for IFN- γ.
Expression of the RNA transcripts of IL10 and β-actin, isolated from the
epithelial tissue samples from FMDV infected samples was determined by Real Time
RT-PCR using SYBR Green Fluorescent Dye. The mRNA levels of the target genes
were normalized to the transcript level of the housekeeping gene β-actin. For relative
quantification, the expression of mRNA transcripts of the target genes from normal
tissue was also determined.
Primers were validated on an Applied Biosystems machine by using serial
dilutions of total RNA with endogenous control and target primers, whose values were
plotted as the log input amount versus ΔCT values (target CT − endogenous CT) for
relative efficiency. Primers with a slope of less than 0.1 were used, due to similar
amplification efficiencies as the endogenous control.
From the real time PCR plots ΔCt, ΔΔCt and 2 -ΔΔCt values were calculated and
tabulated. Expression of the target gene normalized to the reference gene and relative to
the calibrator = 2 -ΔΔCt indicates the fold change in expression of the target gene
compared to that in the normal (reference). 2 -ΔΔCt values indicated fold changes in IL10
mRNA expression.
All analysis was performed as per the method described by Snedecor and
Cochran (1994) followed by the statistical package for social science, version 13.0
(SPSS, Chicago, IL) software.
Description
MOLECULAR CHARACTERIZATION OF THE VIRUS AND HOST FACTORS IN RESPECT TO FOOT AND MOUTH DISEASE INFECTION IN CATTLE
Keywords
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