IDENTIFICATION AND GENOTYPING OF VP 1 GENES OF FMD VIRUSES
ATIA BUKHARI
2003-VA-217
A THESIS SUBMITTED IN THE PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE
OF
DOCTOR OF PHILOSOPHY
IN
VETERINARY MICROBIOLOGY
UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE
2009
IN THE NAME OF ALLAH, THE MOST MERCIFUL, THE COMPASSIONATE
TABLE OF CONTENTS
DEDICATION
ACKNOWLEDGEMENTS
LIST OF TABLES LIST OF FIGURES
CHAPTER
NO.
CHAPTER PAGE NO.
I INTRODUCTION 1
II REVIEW OF LITERATURE 5
III MATERIALS AND METHODS
25
IV RESULTS
44
V DISCUSSION
80
VI SUMMARY
93
VII LITERATURE CITED
98
DEDICATION
To my honorable and affectionate parents And
My late beloved son Syed Ali Raza
ACKNOWLEDGEMENT On the accomplishment of the present study, I would like to take this opportunity
to extend my deepest sense of gratitude and words of appreciation towards
those, who helped me during the pursuit of study. I deem it a proud privilege and
feel immense pleasure to acknowledge all those who are directly or indirectly
involved.
I am thankful to the most Gracious, Merciful and Almighty ALLAH who gave me
the health, thoughts and opportunity to complete this work, I bow before my
compassionate endowments to HOLY PROPHET (PBUH), who I sever a torch of
guidance and knowledge for humanity as a whole.
Words are inadequate in the available lexicon to avouch the excellent guidance
given by my major advisor/ supervisor Dr.Irshad Hussain, professor,
Department of Microbiology, University of Veterinary & Animal sciences, Lahore.
His dedication to research, meticulous planning, consecutive counsel and
unreserved help served as a beacon light throughout the course of study,
research work and completion of this manuscript
I express my heart-felt gratitude to my Minor Advisor, Dr.Khushi Muhammad,
Professor/ Chairman Department of Microbiology, University of Veterinary &
Animal Sciences Lahore, for his learned counsel, innovative suggestions and
critical evaluation of my work. And providing me the lab facilities for my research
work.
I wish to express my sincere thanks and gratitude to member of advisory
committee: Dr. Azhar Maqbool Professor, Department of Parasitology,
University of Veterinary Sciences Lahore, for their valuable suggestions and
Keen interest in my work.
I owe my sincere Gratitude and thanks to Dr. Atif Hanif , Assistant professor,
WTO labs, University of Veterinary & animal sciences for his Keen interest,
sustained encouragement during this research work.
I take this opportunity in expressing my heartfelt thanks to Dr. Mahmood
Mukhtar, Assistant Professor, Department of Microbiology, for his constant help
during compilation of data for the results.
On the way to completion the friends, who have shared the moments of laughter
and sorrow can never be forgotten. I thank my colleagues, Dr. Rashid Munir, Dr.
Farhat Awan, Madam Rahat, Madam saleha and other friends for their constant
inspiration and for wholehearted co-operation.
My vocabulary utterly fails in expressing my accolade to my revered parents who
brought me to this stage, who dreamed me to perform best in life by manifesting
eternal characters and they prayed for my success every time.
Finally no words of admiration can reply the love, inspiration and encouragement
of my husband Syed Kamran Gilani and daughters- Sadaf, Muskan and
Emaan at the times of pain, hurdles and happiness ,whose support always
energized me to perform the best
I apologize for the faux pass of the persons who have extended the help in a way
or other and deserved such thanks.
(Atia Bukhari)
LIST OF TABLES
Sr. No.
Table No.
Title Page No.
1 1 Composition of reaction mixture for PCR
29
2 2 Thermocycling temperatures for PCR
30
3 3 Sequence, Virus specificity, genome location and size of PCR amplification product of various FMDV primers
32
4 4 Foot and mouth disease type O and Asia 1 VP1 sequence information used in phylogenetic analysis
38-40
5 5 Similarity between Asia 1 isolates of this study and most closely related isolates obtained from GeneBank using BLAST program
63
LIST OF FIGURES Sr.No.
Figure No.
Title Page No.
1 1 Effect of varying concentrations of MgCl2 on RT-PCR of FMD virus using universal primers
45
2 2 Effect of buffers with varying pH on amplification of FMD virus by universal primers
46
3 3 Effect of annealing temperatures on amplification of FMD virus by universal primers
47
4 4 Effect of primer concentrations on RT-PCR of FMD virus by universal primers
48
5 5 Effect of template concentrations on RT-PCR of FMD virus by universal primers
49
6 6 Effect of varying concentrations of Taq polymerase on RT-PCR of FMD virus by universal primers
50
7 7 An image of RT-PCR by primers which amplify B-actin gene
52
8 8 A representative image of RT-PCR of samples of FMD virus using FMD universal primers
53
9 9 A representative image of RT-PCR of samples of FMD virus using serotype ‘O’ specific primers (set-1)
54
10 10 A representative image of RT-PCR of samples of FMD virus using serotype ‘O’ specific primers (set-1)
55
11 11 A representative image of RT-PCR of samples of FMD virus using serotype ‘Asia 1’ specific primers (set-1)
56
12 12 A representative image of RT-PCR of samples of FMD virus using serotype ‘Asia 1’ specific primers (set-2)
57
13 13 A representative image of RT-PCR of samples of FMD virus using FMD universal primers & serotype ‘A’ specific primers
58
14 14 A representative image of RT-PCR of VP1 of serotype ‘O’ of FMD virus
60
15 15 A representative image of RT-PCR of VP1 of 61
serotype ‘Asia 1’ of FMD virus 16 16 Phylogenetic analysis of FMD virus type Asia 1
isolates (this study and previously reported) constructed using the neighbor joining method
65
17 17 A subtree of Fig.16 showing phylogenetic relationship of FMD virus type Asia 1 (this study and previously reported) isolates constructed using neighbour joining method with Mega 4 software
66
18 18 A subtree of Fig.16 showing phylogenetic relationship of FMD virus type Asia 1 (this study and previously reported) isolates constructed using neighbour joining method with MEGA 4 software
67
19 19 Sequence similarity tree showing relationship of FMD virus type O (this study and previously reported) isolates constructed using neighbour joining method with MEGA 4 software
69
20 20 A subtree of Fig.19 showing phylogenetic relationship of FMD virus type O(this study and previously reported) isolates constructed using neighbour joining method with MEGA 4 software
70
21 21 A subtree of Fig.16 showing phylogenetic relationship of FMD virus type O (this study and previously reported) isolates constructed using neighbour joining method with MEGA 4 software
71
22 22 Sequence similarity tree of FMD virus type O isolates of this study using the neighbour joining method with Mega 4 software
72
23 23 A comparison of the deduced amino acid sequences of a local vaccine against FMD serotype Asia 1 and this study isolates
74
24 24 A comparison of the deduced amino acid sequences of a local vaccine against FMD serotype O and this study isolates
75
25 25 A comparison of the deduced amino acid sequences of previously published FMD serotype O VP1 segment with thatof this study isolates
77
26 26 Agar gel precipitation test of hyperimmune sera with that of field isolates
78
27 27 A representative image depicting comparative OD value of vaccines
79
1
INTRODUCTION
Foot and mouth disease (FMD) is a severe, debilitating, highly contagious and clinically
acute disease of cloven-hoofed animals including cattle, swine, sheep and goats as well
as more than 70 species of wild animals caused by various serotypes of FMD viruses
(Alexanderson et al., 2003). The disease is prevalent in many parts of the world,
resulting in heavy economic losses in terms of reduced milk and meat production, death
of animals, weight loss and loss of draught power in animals taking typically a long
recovery period following the disease (Grubman and Baxt, 2004).
About 35 million rural population of Pakistan is associated in raising the dairy animals
which play a pivotal role in people’s day to day economy. In other words, the total
financial system of the rural population of Pakistan is directly or indirectly linked to the
livestock. Dairy animals are a major source of milk, mutton, wool, hair, bones, fats,
blood, hides and skins. In Pakistan there are 25, 28, 25, 62, and 0.7 million heads of
cattle, buffaloes, sheep, goats and camels respectively (Anonymous, 2005-2006).
These animals have great genetic potential for high milk yield but unplanned breeding, a
poor market system, a shortage of animal feed and abundance of infectious diseases
like FMD are the main obstacles in the development of the livestock sector to its
optimum capacity.
Pakistan is endemic for FMD. About 1286 FMD outbreaks were recorded during the
years 2002 and 2005 in Pakistan (Zahur et al., 2006). Although, largely sporadic, FMD
is seen almost throughout the year (Ikhwan et al., 1999, Ahmad et al., 2002) in all parts
of the country. It causes heavy economic losses every year in Pakistan. The gravity of
the economic losses due to this disease could be realized from the fact that a loss of
Rs. 4 million was estimated in only one Tehsil of District Lahore (Yamin, 1998) during
one year and Rs. 27.449 million was estimated in District Sahiwal in the year 1998
(Gorsi, 1998). Because of its associated economic impact and the difficulties in its
2
effective control, FMD ranks first in the A list of infectious diseases of animals,
published by the Office International des Epizootics (OIE).
Foot and mouth disease virus is a member of the genus Aphtho virus of the family
Picornaviridae. It is a single stranded positive sense virus with an RNA genome of
8.5Kb. The genome encodes four structural proteins and eight non structural proteins.
The genome is encapsidated by sixty copies each of the four structural proteins of
which VP1-3 are exposed outside and VP4 is completely internal. According to the
antigenic properties of the capsid proteins, FMDVs are classified into 7 serological
types, namely O, A, C, Asia-1 and SAT 1-3 (Mittal et al., 2005, Murphy, 1999).
Conventionally, primary virus diagnosis of FMD is carried out by virus isolation in cell
culture, enzyme linked immunosorbant essay (ELISA), complement fixation test (CFT),
and serum neutralization test (SNT). Advances in molecular biology have resulted in the
development of techniques such as the reverse transcription polymerase chain reaction
(RT-PCR) method for prompt detection of FMD virus (FMDV) genomic RNA in cell
culture fluids, oesopharyngeal scrapings, epithelial or other tissues such as tonsils
(Amarel-Doel et al., 1993). Moreover, in molecular epidemiology, RT-PCR can be
combined with sequence analysis to study the phylogenetic relationship between
various isolates (Knowles and Samuel, 1994).
Four antigenic sites have been defined on FMD type O virus. Two of these sites are
located on VP1, one is on VP2 and the fourth is on VP3 (Kiston et al., 1990). Critical
amino acid residues (144, 147,148,154 and 208) for the trypsin sensitive neutralizable
antigenic site 1 are located in the well known regions 133 - 160 and 200 - 213 of VP1. In
the region 43-60 of VP1, amino acid residues 43, 44, 45 and 48 are involved in
antigenic site 3. A fifth neutralizable site has been identified on residue 149 in antigenic
site 1 of VP1 and represents a conformational epitope (Tsai et al., 2000). Any
alterations at these critical residues confer antigenic specificity to FMD viral variants.
VP1 is the most frequently studied protein due to its significant roles in virus attachment,
protective immunity and serotype specificity (Junzheng et al., 2007). Since the VP1
3
gene harbors almost 3 major antigenic sites, nucleic acid sequencing of this part is likely
to reveal mutational change. Similarly, various researchers have attempted to elucidate
antigenic epitopes of FMD Asia 1 serotype. Sanyal et al.(1997), reported four
independent trypsin sensitive, neutralizing antigenic sites present on FMDV type Asia-
1.Vp1 is reported to harbor major antigenic sites as trypsin treatment of the whole virus
particle results in cleavage of VP1 with a drastic reduction in infectivity and
immunogenicity (Wild et al., 1969). The amino acid region 141-160 of VP1 forms an
antigenic site, the main target of virus neutralizing antibodies (Mateu et al.,1990)
In studies of FMDV serotype A, epitopes were also identified on the VP1 G-H loop,
together with an additional site within the VP1 H-I loop (residue 618), adjacent to the B-
C loop of VP1 (BAXT et al., 1989, LEA et al., 1994). Similarly two major antigenic sites
were indicated by cross-neutralization studies and mapping of the amino acid changes.
The first site was trypsin sensitive and included the VP1 140 to 160 sequences. The
second site was trypsin insensitive and included mainly VP3 residues. Two minor sites
were located near VP1 169 and on the C terminus of VP1 (Thomas et al., 1988).
Vaccination, although practiced in the country by the farmers, is not effective, as FMD
outbreaks have been commonly observed in vaccinated and non vaccinated herds
alike. Locally prepared FMD vaccines don’t afford complete protection against the
disease. This may be due to poor quality of vaccines, or variation in the serotypes/strain
of the FMD field viruses which might be antigenically different from the serotypes or the
strains used in the preparation of the vaccines. A recent increase in the death rate due
to FMDVs also supports the impression of mutational change in FMD viruses (Sorbino
et al.,1986) and calls for molecular typing of FMD field and vaccinal isolates to see
whether any variants of the virus are in circulation. As new strains have been emerging
constantly within each serotype owing to high mutation rate and the quasispecies nature
of this RNA virus (Tosh, 2002), molecular typing of all the serotypes prevalent in
Pakistan is needed.
4
Antigenic variation might be of adoptive value for two reasons. First, antigenic variation
generated over the course of a single viremia might act to extend or intensify a single
infection, thereby resulting in greater transmission potential from infected animals.
Second, sufficiently distinct strains might be capable of re-infecting (or more rapidly re-
infecting- in the case of waning immunity) hosts with previous experience of a related
progenitor strain, thereby effectively increasing the fraction of the host population that is
susceptible to these new strains. The occurrence of antigenic variation requires that
vaccine strains be updated periodically (Feigelstock et al., 1996; Klein et. al., 2008).
If the hypothesis of mutations in the field virus is true, this is going to have a major
impact on FMD vaccine manufacturing practices in the country. As the slaughtering of
affected animals is not possible due to the prevailing socio-economic conditions in
Pakistan, vaccination is the only way to control this disease. Proper protection by these
vaccines will minimize the economic losses due to FMD in the country. The proposed
objectives of the present study therefore were:
1. RT-PCR optimization and identification of O, A and Asia-1 types of FMD
viruses from the field outbreaks by RT-PCR using FMD universal and
serotype specific primers (Reid et al., 2000).
2. Amplification of VP1 genes of prevalent strains of FMD viruses using serotype
specific VP1 primers (Tsai et al.,2000).
3. Nucleotide sequencing and sequence similarity tree of VP1 genes of FMD
serotypes collected from field outbreaks and that of vaccinal strains used by
FMD vaccine manufacturers in the country
4. Testing of the locally available FMD vaccines for their ability to neutralize
various filed isolates of FMD viruses.
5
REVIEW OF LITERATURE
HISTORICAL BACKGROUND OF FOOT AND MOUTH DISEASE Although the first written incident of FMD outbreak was reported by an Italian monk,
Girolamo Fracastoro in 1546 (Sutmoller et al., 2003), the causative agent was identified
by Loeffler and Frosch in 1897. The agent was found to pass through very fine filters
that normally do not allow bacteria to pass pointing to the possibility of a virus. This
observation stimulated research by many scientists on FMD in animals. Since labs
involved in conducting research on FMD also used animals for other studies, these
FMD susceptible animals too often contracted infection resulting in the death of animals,
which necessitated establishment of separate FMD labs in institutions. The first
research center for FMD was established at the Insel Riems, an island in Germany in
1909 followed by similar research centers at Pirbright in England, Landholm in
Denmark in 1925 and Plum Island, North Fork of Long Island in United States of
America in 1954 (Brown, 2003).
ECONOMIC SIGNIFICANCE
FMD has worldwide economic impact which is unsurpassed by any other animal virus in
the world. FMD is on the A list of infectious diseases of animals of the Office
International des Epizooties (OIE) and has been recognized as the most important
constraint to international trade in animals and animal products (Trautwein 1927). The
astonishingly great economic losses caused by FMD are due to death of young animals,
marked reduction in milk yield, abortion at an advanced stage of pregnancy and
reduced working ability of the animals (Singh, 2003) and quality and quantity of meat,
reduction in fertility, loss of quality of semen in breeding bulls (Yadav, 2003), and loss of
productivity for a considerably longer time. The disease also restricts the possible
export of livestock and livestock products. Countries that are free of the disease have
introduced a number of measures to retain FMD free status because of the economic
outcomes resulting from its presence.
6
INCIDENCE AND DISTRIBUTION
FMD is endemic in the Middle East; in Africa, excluding South Africa, Namibia,
Zimbabwe and possibly Tunisia and Morocco, in South America excluding Chile,
southern Argentina, Uruguay, Guyana, Surinam and French Guiana and also endemic
in all other Asian countries including Pakistan, India and Afghanistan etc; Europe is free
of FMD although there have been occasional outbreaks that have quickly been
eliminated. Australia, Japan, Indonesia and Korea are free of FMD (Merck Veterinary
Manual 2000). The geographical distribution of FMD serotypes according to Fenner et
al. (1993) is as follows: Asia (A, O and Asia 1), Europe (A, O and C), Africa (A, O, SAT-
1, 2 and 3) and South America (A, O and C). Public awareness of this highly infectious
disease has significantly increased after the recent outbreaks of FMD in a number of
FMD- free countries, in particular Taiwan in 1997 and the United Kingdom in 2001.
HOST RANGE & CLINICAL SIGNS
FMD is a highly contagious disease of cloven-hoofed animals including cattle, swine,
sheep and goats and more than 70 wildlife species (Coetzer et al.,1994), including deer
(Plum Island Animal Disease centre, 1975). It is characterized by high fever, increased
salivation and appearance of vesicular lesions on the mucous membranes of the mouth,
tongue, lips, epithelia of the muzzle, interdigital space (in claws) and on the teat and
udder of infected animals (Grubman et al., 2004). The vesicles rupture to produce large
denued ulcerative lesions. Rauf et al., 1981, noted that cows were more susceptible to
FMD than buffaloes in Pakistan, but foot lesions were more prominent in buffaloes.
FMD causes low mortality but high morbidity. In younger stock, FMD causes multi-focal
myocarditis leading to acute heart failure (Sharma and Adhlakha, 1995). In sheep and
goats, the clinical signs are often mild (Callens et al., 1998, Hughes et al., 2002).
Moreover, certain strains of the virus may be of lower virulence for some species than
the others (Donadson 1998).
SEROTYPES AND STRAINS OF FMD
Foot and mouth disease is caused by seven antigenically distinct serotypes of genus
Aphthovirus in the family of Picornaviridae (Murphy et al., 1999). Initially type O and A
7
were identified (Vallee and Carre, 1922) and a few years later type C was added
(Waldmann and Trautwein, 1926). Several African field strains collected since 1931
were re-examined by Brooksby in 1948 who demonstrated a new strain from the South
African Territories (SAT 1). Two more strains from Southern Africa (SAT 2 and SAT 3)
were also identified (Brooskby, 1982). FMD type Asia 1 was identified from a sample
originating from Pakistan in 1957 (Brooksby and Roger, 1957). Within each serotype
there is considerable diversity and antisera against one strain of a serotype may not
recognize other strains of the same serotype. Isolates were classified into antigenically
related “subtype” within a serotype and each serotype contains a number of subtypes
(Buxton and Frazer, 1977).
DESCRIPTION OF THE AGENT & GENOME ORGANIZATION
The FMD virus contains a single molecule of plus sense ssRNA and possesses a non-
enveloped, icosahederal symmetry. The capsid is composed of 60 copies of each of
four structural proteins namely VP1 (1D region in the genome), VP2 (1B), VP3 (1C) and
VP4 (1A). Virion RNA acts as an mRNA and is translated into a polyprotein molecule
which is later cleaved to yield 11 individual proteins. The crystallographic study has
shown that the particle shell of FMD virus is relatively smooth, because of the truncation
of certain loops. There is no surface canyon or pit around the pentameric apex (reported
to be present in poliovirus, a member of picornaviridae) which has been shown to permit
receptor binding for poliovirus (Acharya et al., 1989).
Antigenic sites on the surface of the FMD virion have been identified for five of the
seven serotypes of the virus except for serotypes SAT 1 and SAT 3 (Boldrini et al,
1978). At least four of the antigenic sites involve one or more of the capsid proteins.
Interestingly, three of the sites have elements located within the flexible loops which
connect the β-sheets of the viral proteins, and at least two of the sites include the C-
terminus of VP1 (Manocchio 1974). While all of the sites appear to be necessary for a
complete immunologic response to either infection or vaccination, the major antigenic
site to which most of the immune response is directed and which is common to all of the
serotypes is located within the G-H loop of VP1(Tsai et el., 2000). For serotype “O”
8
critical amino acid residues (144,147,148,154 and 208) for the trypsin sensitive
neutralizable antigenic site 1 are located in the well known regions 133-160 (the G-H
loop) and 200-213 (the C-terminus region) of VP1. In the region 43-60 of VP1, amino
acid residues 43, 44, 45 and 48 are involved in antigenic site 3. A fifth neutralizable site
has been identified on residue 149 in antigenic site 1 of VP1 and represents a
conformational epitope (Crowther et al., 1993). Any alterations at these critical residues
confer antigenic specificity to FMD viral variants.
Antigenic epitopes of other FMD serotypes are not very well understood. Researchers
have attempted to elucidate antigenic epitopes of FMD Asia 1 serotype. A peptide
consisting of amino acids 1-20 of VP1 of serotype Asia-1 was found to be antigenic but
failed to induce neutralizing antibody (Frieberg et al., 2001). In another study, a fusion
protein containing amino acids 133-158 of VP1 and amino acids 20-34 of VP4 of FMDV
type Asia 1 (Zhang et al., 2002), elicited neutralizing antibodies in guinea pigs, but did
not completely protect the animals against FMDV challenge. Wang et al., (2007)
screened for immunogenic peptides derived from VP1 and VP2 of FMDV serotype Asia
1 and found that amino acids 133-163 of VP1 induced a high level of neutralizing
antibodies in guinea pigs while another immunogenic peptide, amino acids 1-33 of VP2,
enhanced the neutralizing antibody response elicited by the VP1 peptide. Butchiah and
Morgan (1997) have characterized epitopes located within the VP1 G-H loop and also in
the B-C loop of VP2, the B-B knob of VP3 and N terminus of VP2. Sanyal et al. (1997)
reported four independent trypsin sensitive, neutralizing antigenic sites present on
FMDV type Asia-1. Vp1 is reported to harbor major antigenic sites as trypsin treatment
of the whole virus particle results in cleavage of VP1 with a drastic reduction in
infectivity and immunogenicity (Wild et al., 1969). The amino acid region 141-160 of
VP1 forms the antigenic site, the main target of virus neutralizing antibodies (Mateu et
al.,1990).
In studies of FMDV serotype A, epitopes were also identified on the VP1 G-H loop,
together with an additional site within the VP1 H-I loop (residue 618), adjacent to the B-
C loop of VP1 (BAXT et al., 1989, Lea et al., 1994). Similarly two major antigenic sites
9
were indicated by cross-neutralization studies and mapping of the amino acid changes.
The first site was trypsin sensitive and included the VP1 140 to 160 sequences. The
second site was trypsin insensitive and included mainly VP3 residues. Two minor sites
were located near VP1 169 and on the C terminus of VP1 (Thomas et al., 1988, and
Silva et al., 1993).
TRANSMISSION, SPREAD AND EXCRETION OF FMD VIRUSES
Susceptible livestock may be infected by FMD viruses as a result of direct or indirect
contact with infected animals or with an infected environment. When infected and
susceptible animals are very close to each other, the aerial transfer of droplets is
probably the most common mode of transmission (Alexanderson et el., 2003). Infection
of cattle occurs via the respiratory route by aerosolized virus. Infection can also occur
through abrasions on the skin or mucous membranes, but is very inefficient, requiring
almost 10,000 times more virus (McKercher and Gailuinas, 1969). Infected cattle also
aerosolize large amounts of virus, which can infect other cattle in addition to other
species. A number of studies have suggested that the lung or pharyngeal areas are the
sites of initial virus replication (Domanski et al., 1959) with rapid dissemination of the
virus to oral and pedal epithelial areas (Donaldson et al., 1970) possibly mediated by
cells of monocyte/macrophage origin. Vesicles develop at multiple sites, generally on
the feet and tongue, and are usually preceded by fever. Several lesions often occur in
areas subjected to trauma or physical stress, and most animals develop viremia. The
incubation period can be between 2 and 14 days, depending on the infecting dose and
route of infection (Rubino, 1946), susceptibility of the host, and strain of the virus. FMD
virus can survive in dry fecal material for 14 days in the summer, in slurry up to 6
months in the winter, in urine for 39 days, and on soil for 3 (summer) to 28 days (winter)
(Ryan et al., 2008).
FMD virus is excreted by lesion material, saliva, milk, feces, urine, semen, nasal
discharge and exhaled air (Donaldson 1983). The contagious period usually starts
about 24 hours prior to the development of clinical signs. The level of transmission
drops about 5-7 days after development of lesions, coinciding with drop in virus titres
10
and the first development of antibodies (Graves, 1971). It is often stated that pigs give
rise to highest levels of aerosolized virus, followed by cattle and sheep (Sellers et al.,
1971b). However cattle with FMD are the greatest producers of FMD virus of all
species.
VIRAL PERSISTENCE AND CARRIERS OF FMD VIRUS
Carrier animals are defined as those from which live virus can be isolated at 28 days or
later after infection. Some ruminant animals exposed to FMDV become carriers,
irrespective of whether they are fully susceptible or immune (i.e., they are protected
from disease as a result of vaccination or recovery from infection). The percentage of
animals that become carriers under experimental conditions is variable but averages
about 50% (Alexanderson et al., 2003). The infectivity titre of virus in oesophageal
pharyngeal (OP) samples from carriers is usually low (10-100 TCID50/ml); excretion is
also intermittent and the titre declines over time. Both the animal species and strain of
the virus appear to be determinants in the development and persistence of the carrier
state. The maximum reported duration of the carrier state in different species is as
follows: Cattle, 3.5 years; sheep, 9 months; goat, 4 months; African buffalo, 5 years;
and water buffalo, 2 months. The information about water buffalo is limited it may be at
least 6 weeks (Moussa et al., 1979). Although virus isolation from esophageal-
pharyngeal fluids is the most sensitive and reliable method to detect carrier animals,
reverse transcription-PCR (RT-PCR) assays can also be used for this purpose.
Murphy et al., (1994) explored possible sites of FMDV persistence during the carrier
state. Tissue samples taken from experimentally infected animals at different times post
infection were examined by the conventional viral isolation and the polymerase chain
reaction technique. The analysis of samples from several organs taken from 17 bovines
between 3 and 270 days post infection (p.i) allowed the following conclusions: (1) Virus
present in oesophageal pharyngeal fluid (OPF) during the carrier state originates in the
pharynx as shown by the detection of antisense FMDV RNA by RT-PCR, (2) RT-PCR is
more sensitive than standard isolation techniques and may be used for the rapid
11
detection of FMD virus in specimens obtained during the acute stage of FMD and for
identification of persistently infected cattle.
HIGHLY MUTABLE RNA VIRUSES OF FMD
Genetic variation in FMD Viruses is of interest for at least two reasons. First, changes to
the genes encoding capsid proteins results in antigenic variation and affect vaccine
efficiency and effectiveness of vaccination programs, second, genetic changes can lead
to important insights into the transport of virus between countries, regions, herds and
even possibly individuals (Haydon et al., 2001). Commonly cited mutation rates for RNA
viruses lie in the range10-3 to 10-5 mutations per nucleotide site per genome replication
(Domingo and Holland, 1988; Drake, 1999). At these rates of mutation, replicated FMD
Viral genomes would differ from their parental strands at an average of between 0.1 and
10 base positions. Such high error rates have led to the development of the quasi-
species concept (as reviewed by Nowak and Eigen, 1993) to describe viral genetic
heterogeneity within the host. FMD is a notoriously variable virus. Genetic variants
accumulate rapidly in the field and co-circulate (Martinez et al., 1991).
Antigenic variation might be of adoptive value for two reasons. First, antigenic variation
generated over the course of a single viremia might act to extend or intensify a single
infection, thereby resulting in greater transmission potential from infected animals.
Second, sufficiently distinct strains might be capable of re-infecting (or more rapidly re-
infecting- in the case of waning immunity) hosts with previous experience of a related
progenitor strain, thereby effectively increasing the fraction of the host population that is
susceptible to these new strains. The occurrence of antigenic variation requires that
vaccine strains be updated periodically (Feigelstock et al., 1996).
Domingo et al. (1997) described that RNA viruses exploit all known mechanisms of
genetic variation to ensure their survival. Distinctive features of RNA virus replication
include high mutation rates, high yields, and short replication times. As a consequence,
RNA viruses replicate as complex and dynamic mutant swarms called viral
12
quasispecies. Mutation rates at defined genomic sites are affected by the nucleotide
sequence context on the template molecule as well as by environmental factors. In vitro
hypermutation reactions offer a means to explore the functional sequence space of
nucleic acids and proteins. The evolution of a viral quasispecies is extremely dependent
on the population size of the virus that is involved in the infections. Repeated bottleneck
events lead to average fitness losses, with viruses that harbor unusual, deleterious
mutations. In contrast, large population passages result in rapid fitness gains, much
larger than those so far scored for cellular organisms. Fitness gains in one environment
often lead to fitness losses in an alternative environment. An important challenge in
RNA virus evolution research is the assignment of phenotypic traits to specific
mutations. Different constellations of mutations may be associated with a similar
biological behavior. In addition, recent evidence suggests the existence of critical
thresholds for the expression of phenotypic traits. Epidemiological as well as functional
and structural studies suggest that RNA viruses can tolerate restricted types and
numbers of mutations during any specific time point during their evolution. Such limited
tolerance to mutations may open new avenues for combating viral infections.
Propagation of FMD Virus in Animals and Cell Culture
The isolation of FMD virus has been attempted in various animal host species, and
cultivation of the virus has been accomplished by inoculation of scarified areas of the
buccal mucosa and the tongue of calves or by foot- pad inoculation of guinea pigs.
Mice, chick embryos, and various tissue cultures have also been used for isolating FMD
virus (Lennete and Schmidt., 1964).
In FMD virus, a highly immunogenic loop between the G and H beta strands of VP1
(Acharya et al., 1989; Logan et al., 1993) contains a conserved Arg-Gly-Asp (RGD)
sequence that has been implicated in cell interaction, since RGD-containing synthetic
peptides inhibit virus binding to cells (Baxt and Becker 1990; Fox et al., 1989). Moreover
the RGD sequence of type A12 virus is required for cell binding and infectivity, since
viruses with mutations (Mason et al., 1994) or deletions (Mckenna et al.,1995) of this
sequence are non-infectious for cells in culture. In addition, type A12 viruses with
13
deletions in this sequence are avirulent in cattle (McKenna et al., 1995) even when
administered in high doses (Rieder et al., 1996). The essential role of the RGD
sequence in infection by FMDV was also supported by studies showing that antibodies
to the RGD-binding integrin, alpha-v Beta-3, can inhibit binding of FMDV type A12 to
cells in culture (Berinstein et al., 1995).
Ferris and Dawson, (1988) reported that the quantities of clinical samples are often
insufficient for strain characterization; therefore samples have to be inoculated on cell
cultures for the production of sufficient antigen, which may require several days before
typing can be undertaken.
The quasispecies nature of the FMD Virus genome allows for the rapid adaptation to a
new environment. Adaptation is accompanied by the acquisition of ability to bind
heparin, strongly suggesting that the selected viruses utilize cell surface heparin
sulphate to gain entry to these cells. The adaptation to this new receptor appears to
alter plaque morphology and host range in vitro, even though the heparin binding
viruses still appear to be able to bind to the integrin. Adaptation that accompanies serial
passage in baby hamster kidney (BHK) cells (a cell line typically used to propagate this
virus) occurs more slowly and appears to result from the selection of viruses that can
compete more effectively in cell culture as a result of their affinity for heparin.
Interestingly, heparin binding viruses are dramatically attenuated in bovines, indicating
that the acquisition of binding to a new receptor can abrogate disease by sequestering
the virus to sites that are not favorable for replication (Daniel et al., 1997).
DIAGNOSIS OF FMD
Rapid diagnosis of FMD is of paramount importance, especially in countries that are
usually free of infection so that eradication can proceed as quickly as possible. Since
three other viruses can produce clinically indistinguishable lesions in domestic animals,
confirmation by lab diagnosis is essential, although the history of the disease and
involvement of different species can be valuable pointers to the diagnosis.
14
A range of serological tests are available for diagnosis of FMD virus including virus
neutralization, complement fixation test and ELISA etc. Cell cultures, suckling mice and
occasionally cattle are used to isolate virus when the concentration of virus in the
vesicular epithelium or fluid is low. Cell cultures are generally used to isolate virus from
other tissues, blood, esophageal and pharyngeal fluids. The isolated virus is identified
by any of the serological tests.
Remond et al. (2002) reviewed FMD diagnostic methods. As the presence of clinical
signs alone was inconclusive, laboratory diagnosis therefore, should always be carried
out. The presence of FMDV could be demonstrated by cell culture isolation,
complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA) or the
more recent polymerase chain reaction (PCR) method. Serological diagnosis was also a
valuable tool. The virus neutralization (VN) test has been replaced by ELISA and the
antibody response to some viral non-structural proteins allowed discrimination between
vaccinated and infected animals on a herd basis.
Virus Neutralization test (VNT)
OIE has categorized VNT as a prescribed test for international trade. Rweyemamu
(1984) reviewed that the neutralization reaction is the most appropriate in-vitro
reference test system for assessing intra-type antigenic variation as it involves the
antigenic determinant responsible for virus strain specifically and evoking protective
antibody. Antigenic relationships determined in different neutralization test systems
were independent of the system used and were assumed to truly reflect antigenic
variation. The two-dimensional micro-neutralization test was found to be appropriate for
FMD virus strain differentiation.
RT-PCR for Diagnosis of FMD
The conventional diagnostic techniques described previously cover detection and
limited virus characterization but go only a little way towards supporting epidemiological
investigations. In addition, these techniques are considered by many as slow and
laborious, requiring unacceptable use of animals, and, above all, they are expensive.
15
The development of molecular biological diagnostic techniques in recent years has
added to our increasing knowledge of the molecular basis of the foot and mouth
disease, and has led many workers to investigate the possibility that diagnosis could be
achieved in a single test. The name of the test is RT-PCR.
Use of various sets of primers for RT-PCR of FMD Vi rus
Different sets of primers in different combinations were used by researchers to amplify
various parts of the FMD viral genome. Locher et al. (1995) used reverse transcription
with PCR for the detection of foot and mouth disease virus serotypes A, C and O in
organ extracts from experimentally infected cattle. Primers were selected from
conserved sequences flanking the genome region coding for the major antigenic site of
the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of
the capsid is highly variable its coding sequence is considered to be the most
appropriate for the characterization of virus isolates and, therefore, for the determination
of the epidemiological relationship between viruses of the same serotype. The results
showed that RT-PCR followed by restriction enzyme analysis and /or nucleotide
sequence analysis of the PCR products is useful for the rapid detection and
differentiation of FMDV.
Vangrysperre and deClerk (1995) used RT-PCR for the detection of FMDV, regardless
of the serotype. A primer set corresponding to a highly conserved region of the 2B
sequence was selected. In a similar way, FMD Virus types O, A, C, SAT 2 and Asia 1
could be identified and differentiated, using primers selected from the 1D (VP1) genome
region. All results were confirmed by direct sequencing of the PCR product. The very
fast RNA extraction, reverse transcription and PCR product results within three hours
after arrival of the samples, is of great importance in case of an FMDV suspicion.
Furthermore a very high sensitivity was achieved (less than one plaque forming unit,
pfu).
Callens and Clercq (1997) observed that the multi-primer mixes of P33-P (A-C-O-Asia
1) and P1-P (SAT 1-2-3) are capable of detecting their serotypes respectively. The
16
serotype specific primers are selected to correspond to regions of the genomes coding
for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus
group.
Pattnaik et al.,(1997) used eight oligonucleotide primers in 7 different combinations to
amplify 3D gene sequences of foot and mouth disease virus by RT-PCR. Six of the
primers were designed at their laboratory (Central laboratory, IVRI- Nainital, India). All
the primer combinations could specifically amplify 3D gene sequences of FMDV
serotypes O, A and C. The largest fragment amplified was of 1,393 bp and the smallest
was of 208 bp in size.
Reid et al. (1999) extensively evaluated multiple primers designed from the 1D and 2AB
regions of FMD viral genome for the detection of all the seven serotypes of the virus by
reverse transcription polymerase chain reaction (RT-PCR) OIE/FAO World reference
laboratory for FMD, Pirbright. The primers had been shown to identify and differentiate
all seven serotypes of FMD virus. Each of the serotype specific primers in selected RT-
PCR protocols demonstrated suitable specificity and detected cell cultures passaged
isolates with some success but were not adequate for the serotyping of the suspensions
prepared from clinical samples of epithelium.
Reid et al. (2000) used the primer pair IF and IR which was designed with reference to
the conserved sections of the 5׳UTR of the FMD virus genome and was intended for the
diagnosis of all seven serotypes. The primers ASR4, A-1C562 and C-1C536 when used
individually with the reverse primer NK-61 were designed for the theoretical detection of
types O, A and C respectively. They found that the universal primer (IF/IR) located in
the 5 UTR of the FMD virus genome successfully detected serotypes O, A, Asia-1 and
C in clinical samples. The other examined universal and serotype specific primer sets,
located principally in the P1 capsid coding regions, which were generally inferior to the
5UTR universal primer sets.
Sensitivity of RT-PCR for Identification of FMDV
17
Various researchers compared different tests to find out the efficiency for detection of
FMDV. Rodriguez et al. (1993) evaluated PCR assays for the detection and
characterization of FMD Virus serotypes A, O and C. This assay allowed the detection
of around 15 pfu, being 500 fold more sensitive than a conventional indirect ELISA.
Similarly, for fast and accurate detection of FMDV and finding out the source of
outbreak, RT-PCR with sequence analysis of VP1 gene plays a major role (Stram et al.,
1995). Reid et al, 2002 compared the use of fluorogenic and conventional RT-PCR
procedures and reported that fluoregenic method can detect a concentration of 107
TCID per ml of serotype O, Asia 1 and SAT 3 FMD viruses which is equivalent to
approximately 1010–1011 RNA molecules per ml.
Reid et al. (1997) compared RT-PCR with virus isolation in cell culture and antigen
detection ELISA for primary diagnosis of FMD in 166 clinical samples from the field. He
found that RT-PCR can rapidly facilitate the molecular analysis of field isolates if it is to
be used in conjunction with current procedures like cell culture and antigen detection
ELISA. RT-PCR should be used with nucleotide sequence analysis for facilitating
molecular analysis and providing information regarding source of outbreak (Reid et al.,
1998).
Moss et al. (1999) compared the sensitivity of plaque test and reverse transcription
nested PCR for the diagnosis of FMD. They concluded that FMD Virus could be
detected by both tests before the onset of clinical symptoms. However after two weeks,
FMDV was only detected routinely in the probing samples. Examination of nasal swabs
revealed a higher number of infected animals using RT-nPCR than by the use of plaque
test. Similarly, in another comparison of RT-PCR and cell culture in five European
reference labs Ferris et al., (2006) found that RT-PCR is the best test that gave
comparable results while the sensitivity of cell culture was variable.
CONTROL OF FMD
The control strategy of FMD comprises of four components namely: disease monitoring
and surveillance, information and education, animal movement management and
18
vaccination. In countries where FMD is endemic, protection, particularly of high yielding
of dairy cattle is by a combination of vaccination and prevention of FMD virus entering
the dairy premises. It can be difficult if prevalence of FMD in the unvaccinated
population is high and climatic conditions are suitable for aerosol transmission of the
virus. Randolph et al (2002) recommended that the key elements of the control strategy
were to increase the monitoring and surveillance of the disease and of the areas in
which eradication had been achieved, to enhance the control of animal movement
through the introduction of checkpoints, modify the vaccination from “blanket”
vaccination to “strategic” ( in which 80% population immunity is achieved only within the
high risk population, designated as within 3Km on both sides of a national highway, a 5
Km radius from the infected premises or zone, and a 3Km radius from affected
slaughterhouses and markets). No vaccination is conducted in areas designated as
FMD-free.
The occurrence of FMD in countries previously free of the disease can have a major
effect on local and international trade arrangements. Many countries free of FMD have
a policy of slaughtering of all affected and in contact susceptible animals and have strict
restrictions on movement of animals and vehicles around infected premises. After
slaughter the carcasses are either burned or buried on or close to the premises and the
buildings are thoroughly washed and disinfected with mild acid or alkali by fumigation
(Merck Veterinary Manual 2000). Tracing is done to identify the source of the outbreak
and premises to which FMD virus could have already been transmitted by infected
animals or animal products, by contaminated vehicles or people, or by aerosol.
VACCINES
FMD vaccines are killed virus preparations and at best afford good protection against
challenge for 4-6 months. However the antigenic diversity of strains of FMD virus within
each of the serotypes is an additional complication so it is necessary to ensure that
vaccines contain strains antigenically similar to the potential outbreak strains otherwise
the duration of immunity provided by vaccines containing dissimilar strains may be very
short. FMD vaccines for ruminants require an oil or aluminum hydroxide/saponin
19
adjuvant. There are currently no alternatives to vaccine antigens derived from whole
virus grown in tissue culture and then chemically inactivated. The diversity of FMD virus,
its transmissibility and the currently available diagnostics and vaccines make it highly
improbable that the virus could ever be eliminated from the poorer countries of Africa or
Asia (Kitching et al., 2006).
GENOTYPING AND PHYLOGENETIC ANALYSIS OF FMD Virus
Marquardt et al. (1997) worked on a large part of the capsid protein VP1 coding
sequence of FMDV, isolated between 1993 and 1996 in Europe and with some non-
European virus isolates. The products were sequenced, and the sequences aligned.
The alignment comprised sequences of the types A, O and Asia 1. Although the origin
of virus introduced to Europe remains unknown, genetic relation to some other isolates
was indicated. Several genotypes of the virus were found to circulate in the field since
years.
Researchers conducted experiments to explore antigenic variants of FMD Virus
serotype Asia-1. Tulasiram et al. (1997) studied antigenic variation in Foot and Mouth
disease virus type Asia 1 isolates circulated during 1993-95 in India. They collected
FMDV isolates from vaccinated and unvaccinated animals from different parts of the
country and compared their relationship with Asia 1 vaccine virus. The immunogenic,
hypervariable region of viral protein (VP1) gene was amplified by RT-PCR and
sequenced. Similarly, complete nucleotide sequences of the 1D (VP1- encoding) gene
of 61 foot and mouth disease serotype Asia 1 virus isolates recovered from different
outbreaks in India between 1985 and 1999 including two vaccine strains used currently
were determined by Gurumurthy et al. (2001). The sequences were compared with
each other and those from other Asian countries. Mohapatra et al. (2002) determined
the nucleotide sequence of the L (603 nt) and VP1 (633 nt) genes of 27 FMDV serotype
Asia 1 isolates recovered from different outbreaks in India and compared with each
other and vaccine strain, IND 63/72, used in the country. Independent phylogenetic
analyses on both the aligned gene sequences identified two major lineages (designated
A&B) in the Asia 1 isolates. Both L and VP1 based trees were congruent with respect to
20
the major branching pattern of the isolates. The annual rate of evolution in L and VP1
genes was found similar and estimated to be 4 x10-3 and 3.8 x 10-3 substitutions per
nucleotide, respectively.
Hemadri et al. (2000) performed genetic analysis of FMD virus type O. They determined
partial nucleotide sequence at the 3/ end of 1D (VP1 – encoding) gene of 90 foot and
mouth disease virus type O isolates recovered from field outbreaks in India between
1993-1999. The sequences were compared with each other and reference viruses. The
published sequences of 15 type O isolates recovered from different parts of Asia and
one isolate from Europe, and one from Egypt (O1/ Sharquia/ Egypt/72) were also
included in the analysis for comparison. On the basis of phylogenetic analysis, the
viruses could be grouped into four distinct genotypes (genotypes 1-1V). The present
study reveals the occurrence of viruses belonging to multiple genetic groups over a
short period of time and persistence of single genetic group in the same geographical
area over several years. This was consistent with the endemic nature of the disease in
the country.
Tsai et al. (2000) studied molecular epidemiology of FMD type O Taiwan viruses from
the 1997 epidemic. They amplified VP1 gene from 49 clinical specimens during an
explosive foot and mouth disease outbreak in Taiwan for assessing their sequence
diversity. Type O Taiwan viruses were genetically highly homogeneous, as seen by the
minute divergence of 0.2%-0.9% revealed in 20 variants. Comparison of deduced amino
acid sequences around the main neutralizable antigenic sites on the VP1 polypeptide
showed no significant antigenic variation. However, one variant had an alternative
critical residue at position 43 in antigenic site 3, which may be due to selective pressure
in the field. Two vaccine production strains (O1/Manisa/Turkey/69 and
O1/Campos/Brazil/71) probably provide partial heterologous protection for swine
against O Taiwan viruses. The O/Hong Kong/9/94 and O/1685/Moscow/Russia/95
viruses in sublineage A2 are closely related to the O Taiwan variants. The causative
agent for the 1997 epidemic presumably originated from a single common source of
type O FMD viruses prevalent in neighboring areas (Tsai et al. (2000).
21
Adam et al. (2002), described a RT-PCR assay that amplifies the genes encoding the
capsid proteins VP1-3 of at least three evolutionary lineages each of the foot and mouth
virus type O, A and Asia-1. Most of these lineages are circulating at present in Asia and
Africa. This method is not only suitable to confirm suspected outbreaks of FMD, but also
describes the modulation of major and minor antigenic sites in the course of an
epizootic by nucleotide sequence determination of the obtained RT-PCR products.
Hemadri et al. (2002) determined complete 1D gene sequences of 13 Indian FMD virus
type C field isolates and a vaccine strain. All the field isolates showed a greater genetic
homogeneity (95-100%) among themselves and were 19.7-21.2% divergent from the
vaccine strain. In the phylogenetic analysis, the Indian field isolates formed a separate
lineage different from the previously identified six lineages in type C. The vaccine strain
was grouped with European lineage (lineage II). Comparison of the deduced amino acid
sequences of antigenic sites A and C of field isolates showed no significant variation
from the vaccine strain.
Knowles et al. (2002) described the results of phylogenetic analysis carried out on the
VP1 coding sequences of viruses isolated from different outbreaks. They concluded that
PanAsian strain of FMD virus type O continued to be widespread throughout Asia,
although other type O strains are also co-circulating in many countries. A new type O
strain had been detected in India and the Middle East. Apparently new strains of types
A and Asia-1 had been found in Iran and their possible origin was being investigated.
Oem et al (2002) recorded 16 FMD outbreaks due to the serotype O virus, Pan Asia
strain in Korea. The viruses were identified by ELISA, RT-PCR and sequence analysis.
The overall nucleotide sequence divergence of the VP1 region among the four isolates
in 2002 was 0 and 1.4%. Phylogenetic analysis with the same known strains from the
East Asian countries showed that the 4 Korean isolates in 2002 formed one distinct
cluster, which is different from clusters of Korean isolates in 2000. Deduced amino acid
sequences around neutralizable antigenic site on VP1 site of O/SKR/2002 viruses were
22
aligned and compared with other strains. At the antigenic site 1, the replacements of the
critical amino acid residues at position 144 from V to L and at position 152 from A to T
were observed in O/SKR/2002 viruses. For antigenic site 2 and 4, there were no
significant variations in general. At the antigenic site 3, the substitutions of amino acid
residues were present at positions 54 and 56 in O/SKR/2002 isolates and an alternative
residue 1 at position 54 were observed only at the sequence of O/SKR/AS/2002 virus.
Feng et al. (2003) determined and compared the nucleotide sequences of the VP1
coding region of FMD virus strain HKN/2002 isolated from a disease outbreak occurring
in Hong Kong in February 2002 with the sequences of other FMDVs. Comparison of
the amino acid sequence of the major immunogenic region of HKN/2002 with that of the
serotype O vaccine strain, O1/ Manisa/ Turkey/69 reveals significant similarity,
indicating that current serotype O vaccines may offer some degree of protection against
HKN/2002.
Biswas et al. (2005) compared the sequences of the large fragment of the 5/
untranslated region among FMD virus of serotype Asia-1.They analyzed twenty field
isolates, of which 13 isolates were from the previously identified lineages/genotypes
circulating in the country including the recently emergent divergent viruses. For the
remaining seven isolates, VP1 sequences were generated in the present study and
these isolates were found to cluster phylogenetically into the previously identified
lineages/ genotypes. Sequence analysis revealed that phylogenetic grouping of type
Asia 1 field isolates on the basis of the large fragment of the 5/ untranslated region was
quite similar to that based on the sequences of the capsid coding (VP1) region of the
same viruses.
Knowles et al. (2005) documented that a particular genetic lineage of FMD virus of
serotype O (PanAsia strain) was responsible for an explosive pandemic in Asia and
extended to parts of Africa and Europe from 1998 to 2001. In 2000 and 2001, this virus
strain caused outbreaks in the Republic of Korea, Japan, Russia, Mongolia, South
Africa, the United Kingdom, Republic of Ireland, France and the Netherlands, countries
23
which last experienced FMD outbreaks decades before. A pandemic such as this is a
rare phenomenon but demonstrates the ability of newly emerging FMD Viral strains to
spread rapidly throughout a wide region and invade countries previously free from the
disease.
Mittal et al. (2005) analyzed the capsid coding (P1) and 3A regions of FMD virus type A
field isolates including two vaccine strains collected during 1977-2000. In the
phylogenies, the isolates were distributed into two previously identified genotypes VI
and VII, with multiple sub genotypes that are temporally clustered. Comparison of the
antigenic relationship of field isolates with two vaccine strains (IND 17/77 and IND
490/97) and the reference strains of the genotypes VI (IND 233/99) and VII (IND 40/00)
indicated two broad antigenic groups that correlate with the phylogenetic groupings
(genotypes VI and VII), and were highly divergent from the vaccine strains.
Klein et al. (2007) worked on a new subtype of FMD Virus serotype A which was
detected in Iran in 2005, designated as A/IRN/2005, rapidly spread throughout Iran and
moved westwards into Saudi Arabia and Turkey where it was initially detected from
August 2005 and subsequently caused major disease problems in the spring of 2006.
The same subtype reached Jordan in 2007. As part of an ongoing project, they detected
this subtype in Pakistan with the first positive samples detected in April 2006. For
characterization of this subtype in detail, they sequenced and analyzed the complete
coding sequence of three subtype A/IRN/2005 isolates collected in Pakistan in 2006.
They concluded that potential recombination events had been detected in parts of the
genome region coding for the non structural proteins of FMDV. In addition, amino acid
substitutions had been detected in parts of the genome coding for the non structural
proteins of FMD. Indications of differential susceptibility for developing a sub clinical
course of disease between Asian buffaloes and cattle have been detected.
Wadsworth et al. (2007) received samples from FMD outbreaks, typed by ELISA and
viruses isolated in cell culture. The genome region encoding the VP1 capsid protein was
amplified by RT-PCR using specific primers and the resultant amplicons were directly
24
sequenced. Vaccine matching was performed using virus neutralization or ELISA
employing antisera raised in cattle against a range of vaccine virus strains. Phylogenetic
analyses revealed that recent FMD virus isolates from Iran, Pakistan, Saudi Arabia and
Turkey formed single genetic lineage distinct from previous virus strains present in the
region including A-Iran-96 and A-Iran-99. They concluded that monitoring the
emergence of new strains of FMD Virus in the Middle East is important to enable
appropriate vaccines to be selected and control measures to be implemented as rapidly
as possible. They proposed that the new Middle East strain be named as A-Iran-05.
25
MATERIALS & METHODS
Summary of Research Plan
PHASE I: Sample Collection and Optimization of RT-P CR
1) Collection of samples from FMD outbreaks
2) Extraction of RNA from FMD virus suspension/ vesicular fluid/ vesicular
epithelium or cell culture
3) Reverse transcription of the extracted RNA into cDNA
4) Optimization of PCR
5) PCR of cDNA for simultaneous identification of FMD serotypes
6) Analysis of PCR products
PHASE II: Amplification, Sequencing and Phylogeneti c Analysis of VP1
genes
1) Amplification of VP1 genes of various serotypes of FMD by RT-PCR
2) Sequencing of VP1 amplicons of field and vaccinal viruses
3) Sequence similarity tree of VP1 sequences of field and vaccinal viruses with
previously published GenBank sequnces
PHASE III: Raising of Antisera against Vaccines an d Virus Neutralization
Test
1) Raising of antisera against locally available FMD vaccines in rabbits
2) Virus neutralization test with randomly selected field isolates
26
PHASE 1: Sample Collection and Optimization of RT-P CR
1. Collection of samples from FMD outbreaks
A total of sixty clinical samples were collected from animals during various FMD
outbreaks in Punjab. Samples included preferably fluids from blisters (buccal cavity) and
mucosal epithelium. However, some animals were approached at a time when
secondary infection had already started. Samples were collected in the transport
medium containing 0.04 M phosphate buffer (pH 7.2-7.6), 1% phenol red, antibiotics
(penicillin 100 U/ml and streptomycin 100 µg/ml), and equal volume of glycerol and
stored at -40oC until processed. Samples were collected from different parts of Punjab
including Lahore, Faisalabad, Jhang, Toba, Multan, Rahim Yar Khan, Sialkot, Okara,
Sahiwal, Pattoki, Muridkey, Hafizabad and Sheikhupura over a period of about two
years. Annually, two high-seasons for FMD outbreaks are encountered in Punjab: first in
October-November, and the second in April-May. Most of the sampling was performed
in these months of the year. Reference viruses of FMD type O, A and Asia 1 were
obtained from Institute of Animal Health, Pirbright Lab, UK and were included for RT-
PCR as positive controls to validate the assays.
2. Extraction of RNA from FMD Virus Containing Samp les
Viral RNA was extracted from each sample with TRIZOL® reagent (Gibco, UK)
according to the manufacturer’s instructions. The protocol for RNA extraction included
various steps as follows:
i. A volume of 200 µl of the sample was added to 800µl of TRIZOL® reagent
in an eppendorf tube and the mixture was shaken.
ii. After 5 minutes of incubation at room temperature, 200 µl chloroform was
added and mixed vigorously for 15 seconds. The sample was centrifuged
at 12,000 x g for 15 minutes at 2 to 8oC. The mixture separated into a
lower red phenol- chloroform phase, an interphase, and a colorless upper
aqueous phase. RNA remains exclusively in the aqueous phase.
iii. The aqueous phase was transferred to a new tube. The RNA was
27
precipitated from the aqueous phase by mixing with 500 µl Isopropyl
alcohol. The RNA precipitated and formed a gel-like pellet on the side and
bottom of the tube.
iv. The pellet was washed with 75% ethanol.
v. The RNA was resuspended in 25 µl RNase-free water and 3-5 µl of this
RNA was used for reverse transcription.
3. Reverse Transcription of extracted RNA into cDNA
Sterile thin-walled DNAse- and RNAse-free 0.2 ml microfuge tubes were used for
making cDNAs. The cDNAs were made from the extracted RNA with the help of first
strand cDNA synthesis kit (Revert AidTM Cat No. K1662. Fermentas). The kit contained
reaction buffer, random hexamers, 10mM dNTP’s and reverse transcriptase enzyme.
The cDNAs were made according to the manufacturer’s instructions as outlined below:
i. Briefly, 4 µl DEPC water was taken into a tube and 1µl of random hexamers
(0.2 µg/µl) was added followed by addition of 5µl extracted RNA.
ii. The mixture was incubated at 70oC for 5 minutes, chilled on ice and spun
briefly to collect all the fluid at the bottom of the tube.
iii. While the tube still on ice, 4 µl of 5X reaction buffer, 1 µl Ribolock (20U/µl)
and 2 µl 10mM dNTP’s mix were added, mixed gently and the tube incubated
at 25oC for 5 minutes.
iv. Next, 1µl RevertAidTM M-MulV Reverse transcriptase (200U/µl) along with 2 µl
DEPC water was added to make the final volume as 20 µl.
v. The mixture was briefly vortexed for mixing and was incubated at 25oC for 10
minutes followed by incubation at 42oC for 60 minutes.
vi. The reaction was stopped by heating at 70oC for 10 minutes, chilled on ice
and stored at -20 oC until further use.
4. Optimization of PCR
RT-PCR was optimized for each primer set used in the study. The PCR was optimized
for pH of the reaction buffer, primer concentrations, temperature profile, MgCl2, Taq
28
polymerase and template concentrations. The buffers contained 200mM (NH4)2SO4,
750mM Tris HCl, and 0.1%Tween 20.
PCR was also optimized for hot start, where all ingredients of the reaction were mixed
except the Taq polymerase which was added following denaturation of the templates for
5 minutes. Temperature optimization included annealing of primers at 48 oC, 52 oC,
56oC, 58 oC, 62 oC. Moreover, gradient PCR and touchdown PCR were also used with
the denaturation and extension temperatures as 94 oC and 72 oC respectively, whereas
the annealing temperatures were lowered by 1 oC from 62 to 53 oC in the first 10 cycles
followed by 52 oC for the rest of the 20 cycles. In the gradient PCR, there was 1 degree
temperature increment in each column of the block of the thermal cycler and 12 different
reactions (51 oC to 62 oC) were tried during the same run of the PCR on the machine.
Different concentrations of the primers were also optimized for the reaction which
included 50 nM, 100 nM, 200 nM, 250 nM and 300 nM. Template concentrations of 1 µl,
1.5 µl, 2 µl, 2.5 µl and 3 µl were used for optimization process. Table 1 on next page
describes the composition of reaction mixtures used in the PCR in this study and Table
2 denotes conditions of PCR cycles.
29
Table 1. Composition of Reaction Mixture for PCR
Components Final
Concentration
Volume/reaction
1. 10X Taq buffer without
MgCl2
1X 5µl
2. MgCl2 0.5- 2.5 mM 1µl
3. dNTP mix 2 mM 5µl
4. Primer 1 50-300 nM 1.5µl
5. Primer 2 50-300 nM 1.5µl
6. Template DNA Undiluted cDNA 1- 3µl
7. DEPC H2O --- Variable
Total volume 50µl
30
Table 2. Thermocycling Parameters for PCR
Step Temperature Time
1. Denaturation 94oC 60 sec
2. Annealing 56oC 60 sec
3. Extension 72oC 90 sec
4. Final Extension 72oC 7 min
31
5. Identification of Various FMD Serotypes by RT-PC R
The samples were first subjected to RT-PCR using universal primers, IF and IR, which
identified whether the virus belonged to FMD group or not. The primer pair IF and IR
were selected with reference to the conserved sections of the 5′UTR of FMD virus
genome and was intended for identification of all seven serotypes which amplifies a 328
bp fragment (Reid et al., 2000). After initial confirmation of FMD virus by universal
primers, serotype specific primers were used for serotype O (NK61 and ARS4, named
here as primer set 1, yielding 1301 bp fragment; NK61 and 1C-283, named primer set 2
yielding 1142 bp fragment), serotype Asia-1(P33, P75, primer set 1, ) serotype A
(NK61- A 1C 562) amplifications. The name of specific primers of each serotype along
with universal primers, their nucleotide sequences, locations in the genome and
expected sizes of amplification products are mentioned in Table 3. The Table also
shows primers for VP1 sections. The known positive and negative controls were
included during the extraction, reverse transcription and PCR amplification phases.
32
Table 3. Primer Sequence, Virus Specificity, Genom e Location and Size of PCR Amplification Products for FMD Viruses
Sr.
No.
Prime
r
Primer Sequence(5 /-3/) Serotype
specificity
Location Amplic
on
size(bp)
Reference
1. BA1 GAGAAGCTGTGCTACGT
CCGC
Control
primers
β-actin
gene
275 Collins et al
(1996)
2. BA2 CCAGACACGCACTGTGT
TGGC
3. IF GCCTGGTCTTTCCAGGT
CT
All
serotypes
5/UTR
328
Reid et al
(2000)
4. IR CCAGTCCCCTTCTCAGAT
C
All
serotypes
5/UTR
5. NK61 GACATGTCCTCCTGCATC
TG
All
serotypes
2B
6. ARS4 ACCAACCTCCTTGATGTG
GCT
O 1C 1301
7. A-
1C562
TACCAAATTACACACGGG
AA
A 1C 863-866 Reid et
al.,(2000)
8. 1C283 GCCCAGTACTACACACA
GTACAG
O 1C 1142 Knowles et
al., (2005)
9. P33 AGCTTGTACCAGGGTTTG
GC Asia 1
2B
292
Reid et al.,
2001
10. P75 GACACCACCCAGGACCG
CCG
VP1
11. EUR-
2B52
R
GACATGTCCTCCTGCATC
TGGTTGAT
All
Serotypes
2B Knowles et al.
(2005)
12. F1
(Forw
TTTGAGCTGCGTCTGCCA
CTTGA
VP1 (O) VP3 (1C) 869 Oem et al.
(2002)
33
ard)
13. F1
(Reve
rse)
GAGGGCCCAGGGTTGGA
CTC
VP1(O) 2A
14. 1C530 CCACAAGTGTGCAGGGA
TGGGT
VP1
(Asia 1)
VP3 (1C) 959 Knowles et al.
(2005)
15. 1C505 TACACTGCTTCTGACGTG
GC
VP1
(Asia 1)
VP3 (1C) 911 Gurumurthy et
al (2001)
34
5. Analysis of PCR products
For analyzing the PCR products, 6X loading dye (10mM Tris- HCl, pH 7.6, 0.03%
bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol and 60mM EDTA) was used.
An aliquot of 6 µl of the product was taken, mixed with 1µl of the 6X loading dye, loaded
along with various DNA ladders or markers on a 1.5% agarose gel. The agarose gel
was run in 1X TBE buffer (89mM Tris base, 89 mM Boric Acid, 2 mM EDTA, pH 8.3) at
100 V for 35-45 minutes in an electrophoresis tank. The gel was placed in staining
solution of ethidium bromide (2-3 drops of ethidium bromide 0.07% in 150 ml de-ionized
water). After staining for 25-30 minutes, the results were visualized under UV light and
were recorded by a gel documentation system (BioRad, USA).
35
PHASE II: Amplification of VP1 and Its Sequencing
1. Amplification of VP1 gene by PCR
After initial confirmation of the FMD virus and its serotype (O, Asia-1, and A) by PCR,
the isolate was subjected to VP1 amplification by using VP1 specific primers (Table 3).
VP1 specific primers for serotype ‘O’ were F1 (Forward) and F1 (reverse), and for
serotype Asia 1, two separate sets of primers - NK61- Asia-1C 505 and EUR-2B52R-
Asi-1C530 - were used. The primer pair NK61-A 1C562 was used both for serotype A
identification as well as for its VP1 amplification but sequence data could not be
validated indicating some problem with the amplicons or sequencing process, hence the
data was excluded from the final analysis.
The above mentioned primers amplified specific products containing all the important
VP1 nucleotide sequences that code for critical amino acid residues. For VP1
amplification of serotype “O”, a nested PCR was used.
2. Sequencing of the amplified VP1 amplicons
After elution from the gel, the amplicons containing the VP1 gene were sent to
Macrogen® (Seoµl, Korea) and Centre of Excellence for Molecµlar Biology, Pakistan
(CEMB) for sequencing adhering to the guidelines of the vendors with respect to
minimum concentration of the amplicons and the primers. Employing Sanger’s method,
the PCR products were sequenced bidirectionally using strand specific primers by the
vendors. Each run offered about 650-700 of clean bases unidirectionally.
Sequence identity and multiple sequence alignment of molecular sequences (nucleotide
and amino acids) were performed with Clustal W algorithm (Thompson et al., 1994).
Neighbour joining trees were constructed by using MEGA version 4.0 (Kumar et al.,
2004). Nucleotide distance matrices were computed by Kimura two parameter
algorithms based on the total nucleotide substitutions, and evolutionary trees for VP1
were constructed.
36
3. Analysis of VP1 sequences of locally available v accines with field isolates
FMD viruses were eluted from three FMD commercial oil-based and gel-based vaccines
according to the protocol of Milles et al., 2004 and Hutcheon et al., 2006.
(a) PROTOCOL FOR VIRAL GENOME ELUTION FROM OIL BASE D VACCINES
i. A volume of 200µl of benzyl alcohol was added to 1.0 ml of vaccine.
ii. After vortexing for 5 minutes, the mixture was transferred to the
microcentrifuge tube.
iii. The mixture was centrifuged at 2500 x g for 20 minutes which got
separated into three distinct layers.
iv. The middle aqueous layer was carefully obtained from the three phase
system for RNA extraction.
(b) PROTOCOL FOR VIRAL GENOME ELUTION FROM GEL-BASED (ALUMINIUM HYDROXIDE ADJUVANT) VACCINES
i. Ultra pure guanidine HCl (0.5ml of 8M, Sigma) and 10µl of 0.85% H3PO4
(final pH of ~ 1) were added to 0.5 ml of the vaccine samples so that the
samples were diluted two fold to a final elution condition of 4M GnHCl.
ii. Elution of the vaccine samples and controls was performed for 48 hours at
2-8oC using slow rocking samples.
iii. Samples were checked frequently to ensure that a minimum rocking
speed (one to one half inversions per second) was maintained to prevent
aluminium hydroxide particle settling (aggressive mixing of samples was
found to cause protein fragmentation).
iv. After rocking, all tubes were centrifuged in a microcentrifuge for 10
minutes at 16,000 x g.
v. The supernatant was removed for RNA extraction.
RNA extraction, cDNA synthesis and PCR with universal as well as serotype specific
and VP1 specific primers was performed in the same way as described previously.
37
4. Comparison of VP1 sequences of locally prevalent strains of FMD with previously published Pakistani, Indian, Iranian, an d Afghanistani strains
For FMDV serotype ‘O’ sequence similarity tree, 14 VP1 sequences from various field
isolates were compared with some previously published Pakistani FMD O type VP1
specific sequences available with GenBank along with some recently published VP1
sequences reported by countries bordering with Pakistan including India, Iran and
Afghanistan (Table 4). Similarly, 12 VP 1 sequences of FMDV serotype Asia-1 isolates
of this study were compared with previously published sequences (Table 4) and their
phylogenetic relationship was established. However, the sequencing results of serotype
A were inconclusive and were not included for phylogenetic analysis.
38
Table 4: Foot-and-mouth disease type O and Asia 1 VP sequence information used in sequence similarity tree
Isolate of serotype O Origin and year of isolation GenBank Accession # VP1 Sequences of FMD Serotype O
O/JPN/2000 Japan, 2000 AB079061
O/SKR/2000 South Korea, 2000 AF377945
China/1/99(Tibet) China, 1999 AF506822
O/IRN/9/99 Iran, 1999 AJ318838
O/PAK/1/98 Pakistan, 1998 *AJ318848
TAW/2/99 TC Taiwan, 1999 AJ539136
TAW/2/99 BOV Taiwan, 1999 AJ539137
Tibet/CHA/99 China, 1999 AJ539138
SKR/2000 South Korea, 2000 AJ539139
SAR/19/2000 South Africa AJ539140
FRA/1/2001 France, 2001 AJ633821
O/SKR/2000 South Korea, 2000 AY312586S2
O/NY00 China, 2000 AY333431
01SKR iso85 South Korea, 2000 AY593824
UK2001x iso84 United Kingdom, 2001 AY593836
O5/IND/1/62 India, no information DQ164890 O/IRN/16/2001 Iran, 2001 DQ164893 O/IRN/58/2001 Iran, 2001 DQ164895 O/IRN/67/2001 Iran, 2001 DQ164897
O/PAK/53/2003 Pakistan, 2003 DQ164943
O/TAI/9/99 Thailand, 1999 DQ164978
O/UAE/3/2000 United Arab Emirates 2000
DQ164996
O/VIT/9/2002 Viet Nam, 2002 DQ165022
O/AFG/16/2003 Afghanistan 2003 DQ165035 O/AFG/50/2003 Afghanistan, 2003 DQ165036 O/IRN/2/2003 Iran, 2003 DQ165048 O/IRN/16/2003 Iran, 2003 DQ165052 O/IRN/8/2004 Iran, 2004 DQ165054 O/IRN/15/2004 Iran, 2004 DQ165055
39
O/PAK/1/2003 Pakistan, 2003 DQ165065
O/PAK/12/2003 Pakistan, 2003 *DQ165066
O/PAK/14/2003 Pakistan, 2003 *DQ165067
O/PAK/16/2003 Pakistan, 2003 *DQ165068
O/PAK/17/2003 Pakistan, 2003 *DQ165069
O/PAK/73/2003 Pakistan, 2003 *DQ165070
O/Konya/TUR/512/10/99 Turkey, 1999 DQ296515
UKG/14603/2001 United Kingdom, 2001 DQ404159
UKG/173/2001 United Kingdom, 2001 DQ404175
UKG/127/2001 United Kingdom, 2001 DQ404178
UKG/11/2001 United Kingdom, 2001 DQ404180
Ir_07 Iran, no information DQ767863 O/AFG/120/2004 Afg 2004 *EF457984 O/AFG/201/2004 Afghanistan, 2004 *EF457985 O/AFG/210/2004 Afg, 2004 EF457986 type O isolate 24.1_8 Pak, 2006 *EF494493
PAK02_2006 Pakistan, 2006 *EF494499
type O isolate PAK04_2006 Pak, 2006 *EF494500 type O isolate PAK06_2006 Pakistan, 2006 *EF494501 O isolate PAK08_2006 Pakistan, 2006 *EF494502 Type O Pak_2006 Pakistan, 2006 *EF494503
PAK12_2006 Pakistan, 2006 *EF494505
PAK14_2006 Pak, 2006 *EF494506 O/APV/42/04 India, 2004 EU109774 O/APV/84/04 India, 2004 EU109775 O/APWg/52/04 India, 2004 EU109777 O/APKu/91/04 India, 2004 EU109779 O/APMb/67/04/2 India, 2004 EU109780 O/APP/98/04/1 India, 2004 EU109781 O/APP/96/04/2 India, 2004 EU109787
VP1 Sequences of FMD Serotype Asia 1 IND/14/95 India, 1995 AF390678 IND/82/96 India, 1996 AF390705 IND 388-04 India, 2004 *DQ101235 BHU/34/2002 Bhutan, 2002 DQ121112 IRN/10/2004 Iran, 2004 *DQ121119 IRN/31/2004 Iran, 2004 *DQ121121 IRN/58/99 Iran, 1999 DQ121122 PAK/30/2002 VP1 Pakistan, 2002 *DQ121124 PAK/31/2002 VP1 Pakistan, 2002 *DQ121125 PAK/20/2002 VP1 Pakistan, 2003 DQ121126
40
PAK/69/2003 VP1 Pakistan, 2003 *DQ121127 PAK/1/2004 VP1 Pakistan, 2004 *DQ121128 IND 97-03 India, 2002 DQ989323 As/AFG/22/2003 Afghanistan, 2003 *EF457987 As/AFG/24/2003 Afghanistan, 2003 *EF457988 As/AFG/26/2003 Afghanistan, 2003 *EF457989 As/AFG/33/2003 Afghanistan, 2003 *EF457990 As/AFG/44/2003 Afghanistan, 2003 *EF457992 As/AFG/116/2004 Afghanistan, 2004 *EF457993 As/AFG/138/2004 Afghanistan, 2004 *EF457994 Asia1/IRN/1/73 Iran, 1973 EU553912 PAK/2/98 Pakistan, 1998 EU553914 AFG/2/2001 Afghanistan, 2001 FJ785226 AFG/3/2001 Afghanistan, 2001 FJ785227 IND/16/76 India, 1976 FJ785242 IRN/11/2001 Iran, 2001 FJ785243 IRN/25/2001 Iran, 2001 FJ785244 IRN/63/2001 Iran, 2001 FJ785245 IRN/30/2004 Iran, 2004 *FJ785246 PAK/33/2002 Pakistan, 2002 *FJ785262 PAK/34/2002 VP1 Pakistan, 2002 *FJ785263 PAK/2/2004 VP1 Pakistan, 2004 *FJ785264 PAK/19/2005 VP1 Pakistan, 2005 FJ785265 PAK/22/2005 VP1 Pakistan, 2005 FJ785266 TAJ/1/2003 Tajikistan, 2003 *FJ785270 TAJ/2/2003 Tajikistan, 2003 *FJ785271 IND 180/02 India, 2001 FJ785291 IND 763/2003 India, 2003 FJ785292 IND 147/2004 India, 2004 FJ785294 IND 150/2004 India, 2004 FJ785295 IND 153/2004 India, 2003 FJ785296 IND 158/2004 India, 2004 FJ785298 IND 325/2004 India, 2004 FJ785301 IND 327/2004 India, 2004 FJ785302 IND 328/2004 India, 2004 *FJ785303 IND 389/2004 India, 2004 *FJ785304
Note: Sequences marked with the asterisk had >85% nucleotide similarity with the sequences of this study.
41
PHASE III: Raising of antisera against commercial v accines and testing neutralizing ability against FMDV isolates
1. Raising of antisera
Twelve rabbits were purchased from the local market and divided randomly into 4
groups each containing 3 rabbits. Three commercial vaccines were separately injected
into 3 groups with boosters at 2 weeks apart. The blood samples from all the rabbits
were collected on day 0, 13 and 21 post-vaccination. The sera were expressed from
blood, pooled (from each group separately) and stored at -20oC until processed for
calculation of antibody titers. The presence of anti- FMD virus antibodies in the serum
samples were determined by an agar gel precipitation test (AGPT) and serum
neutralization test as described in the OIE FMD manual (2004).
1.1 PROTOCOL FOR AGAR GEL PRECIPITATION TEST
a) PREPARATION OF AGAR GEL PLATE
Noble agar gel was prepared by adding 0.9 grams agar, 8.0g of sodium chloride and
0.01g of sodium azide to 100 ml of distilled water. All these ingredients were mixed
in the distilled water and then heated to boil until a uniform suspension was
obtained. The uniform suspension of the agar gel was then cooled to 45oC and
poured in a 4 inch diameter Petri dish. The plate after pouring was kept at room
temperature until the gel solidified. The solidified gel was transferred to a refrigerator
(4oC) until use. Wells of size approximately 5 mm were punched by using a gel
borer, sealed with molten agar. The wells were charged with various concentrations
(2-fold dilutions) of antigens and antibodies. On the basis of results of this
experiment, an optimum concentration of antigens was chosen for subsequent
experiments. The serum samples (2-fold dilutions) were added in the peripheral six
wells and the known FMD antigens (O, A, Asia-1) in the central wells.
In the second attempt, the central well contained serum samples and peripheral
wells contained different antigens (field isolates) of serotype O, A and Asia-1.The
plates were incubated for 48-72h in a humidified chamber. Development of a
42
precipitin line between known antigen and unknown serum confirmed the presence
of anti FMD antibodies in the samples.
2. Virus neutralization test with randomly selected field isolates
After confirming the presence of FMD specific antibodies in the rabbit sera, the sera
were used for their ability to neutralize various FMD field isolates using serum
neutralization test as described below:
2.1 Protocol for serum neutralization test
i. The BHK-21 cell line in large sized cell culture plates was cultivated until a
confluent monolayer was formed.
ii. BHK-21 cell line suspension was harvested, washed and counted
aseptically. Finally the cell suspension was prepared in growth medium
(MEM-199) containing 10% fetal calf serum at the rate of 106 cells /ml. A
volume of 50µl of the cell suspension was added to each well of the flat
bottomed 96 well culture plate.
iii. The culture plate was covered with a lid and sealed with tape and
incubated at 37oC for 72 hours until a complete monolayer of the cells was
formed.
iv. Each serum sample was diluted two fold starting from 1:2 to 1:256 in
separate eppendorf tubes and 50 µl of the antigen in each dilution was
added, incubated for 30 minutes. Each sample was added in their
respective wells up to 8th well.
v. Controls included the standard negative serum (fetal calf serum), a cell
control with growth medium and above mentioned virus control (50 µl of
each) was added in the well number 10, 11 and 12 respectively.
vi. The cell culture plate was covered with the lid and incubated at 37oC for
48 hours.
vii. The plate was examined under an inverted microscope for cytopathic
effects (CPE). The wells containing highest dilution of serum and showing
normal cells in each row were recorded. Similarly, status of the cells in
43
wells having each control was also recorded.
viii. After proper examination, cells in each well of the plate were stained by
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay.
2.2 PROTOCOL FOR MTT ASSAY
i. After 48 hours incubation, cell culture microtiter plates were centrifuged at
90g for 10 minutes and the medium removed by rapidly inverting the plates
with a firm flick.
ii. 100µl of MTT (1 mg/ml in tissue culture medium without phenol red) was
added and plates incubated for a further 3-4 hrs. The plates were centrifuged
and medium removed as before.
iii. 100µl of isopropyl alcohol was added to each well to solubilize the formazan
dye.
iv. The plates were read on an ELISA reader at wavelength 570nm.
v. A graph of the relevant serotypes versus optical density as taken by ELISA
reader was plotted.
44
RESULTS
A. Optimization of PCR
Polymerase chain reaction was optimized with respect to MgCl2, buffer pH, annealing
temperature, primer concentration, template concentration, and Taq polymerase. A
concentration of 2.5 mM of MgCl2 resulted in the best amplification of the target
sequences (Figure 1). The buffer with pH 8.8 yielded the best results (Figure 2)
Although, the suggested annealing temperatures for various primers (of various
serotypes) ranged from 48 oC to 63 oC, however, a temperature of 56 oC was found to
be the best with all sets of primers (Figure 3). The highest intensity bands of DNA were
observed with 0.3 µM concentration of the primers (Figure 4). Moreover, the best cDNA
template concentration giving optimum amplification was found to be 3.0 µl per reaction
(Figure 5). Lastly, a concentration of 0.5 U of Taq polymerase was not sufficient for
amplification of cDNAs, however, 1.0 U of enzyme was found to yield better
amplification (Figure 6).
45
Figure 1. Effect of varying concentrations of MgCl2 on RT-PCR of FMD virus using
universal primers. RT-PCR of a known FMD virus sample was performed using varying
concentrations of MgCl2. Amplicon size is 328 bp. Lane 2 to 5 contained respectively 1.
0, 1.5, 2.0 and 2.5 mM of MgCl2. Lane 1 is a DNA ladder. PCR reaction mixture was
prepared by adding 5 µl reaction buffer (pH- 8.8), MgCl2 (5 µl- variable concentrations) ,
2 mM dNTPs mix (5 µl), 300 nM each primer (1.5 µl), cDNA template (3 µl), 1U Taq
polymerase (1 µl) and 29 µl DEPC water to make a final volume of 50 µl. Thirty five
cycles were used in amplifying the target molecules. The PCR product was subjected to
electrophoresis in 1.5% agarose.
328bp
1 2 3 4 5
46
Figure 2. Effect of buffers with varying pH on the amplification of FMD virus by
universal primers. RT-PCR of a known FMD virus sample was performed using buffers
of different pH values. Amplicon size is 328bp. Lanes 2 to 5 contained buffers of pH 9.2,
8.8, 8.4, and 8.0, respectively. Lane 6 contained a DNA ladder, although a bit faint. PCR
reaction mixture was prepared by adding 5 µl reaction buffer (pH, Variable), 2.5 mM
MgCl2 (5 µl), 2 mM dNTPs mix (5 µl), 300 nM each primer(1.5µl), cDNA template (3 µl),
1U Taq polymerase (1 µl) and 29 µl DEPC water to make a final volume of 50 µl. Thirty
five cycles were used in amplifying the target molecules. The PCR product was
subjected to electrophoresis in 1.5% agarose.
1 2 3 4 5
328bp
6
47
Figure 3. Effect of annealing temperatures on RT-PCR of FMD virus with universal
primers. Amplicon size is 328bp. Lane 2 to 6 contained amplicons with annealing
temperatures of 48oC, 52oC, 54oC, 56oC, and 58oC respectively. Lane 1 contained
DNA ladder. PCR reaction mixture was prepared by adding 5 µl reaction buffer (pH-
8.8), 2.5 mM MgCl2 (5 µl) , 2 mM dNTPs mix (5 µl),300 nM each primer (1.5 µl), cDNA
template(3 µl), 1U Taq polymerase(1 µl) and 29 µl DEPC water to make a final volume
of 50 µl. Thirty five cycles were used in amplifying the target molecules. The PCR
product was subjected to electrophoresis in 1.5% agarose.
1 2 3 4 5 6
328bp
48
Figure 4. Effect of primer concentrations on RT-PCR of FMD virus using universal
primers. Amplicon size is 328 bp. Lane 1 to 5 contained RT-PCR products with 50, 100,
200, 250, and 300 nM primer concentrations respectivly. Lane 6 had DNA ladder. PCR
reaction mixture was prepared by adding 5 µl reaction buffer (8.8), 2.5 mM MgCl2 (5 µl) ,
2mM dNTPs mix (5 µl), primer (variable), template (3 µl), 1U Taq polymerase(1 µl) and
29 µl DEPC water to make a final volume of 50 µl. Thirty five cycles were used in
amplifying the target molecules. The PCR product was subjected to electrophoresis in
1.5% agarose.
1 2 3 4 5 6
328bp
49
Figure 5. Effect of template concentration on RT-PCR of FMD virus using universal
primers. Amplicon size is 328bp. Lane 1 to 5 contained cDNA volumes of 1, 1.5, 2.0,
2.5, and 3.0 µl respectively. Lane 6 is DNA ladder. PCR reaction mixture was prepared
by adding 5 µl reaction buffer (8.8), 2.5 mM MgCl2 (5 µl) , 2mM dNTPs mix (5 µl), 300
nM each primer(variable), template(variable volumes), 1U Taq polymerase(1 µl) and
DEPC water to make a final volume of 50 µl. Thirty five cycles were used in amplifying
the target molecules. The PCR product was subjected to electrophoresis in 1.5%
agarose.
1 2 3 4 5 6
328bp
50
Figure 6. Effect of various concentrations of Taq Polymerase on RT-PCR of FMD virus
using universal primers. Amplicon size is 328 bp. Lane 2 to 4 contained 0.5, 1. and 1.5
units of Taq Polymerase, respectively. PCR reaction mixture was prepared by adding 5
µl reaction buffer (8.8), 2.5 mM MgCl2 (5 µl) , 2 mM dNTPs mix (5 µl), 300 nM each
primer (1.5 µl), cDNA template (3 µl), Taq polymerase (variable) and 29 µl DEPC water
to make a final volume of 50 µl. Thirty five cycles were used in amplifying the target
molecules. The PCR product was subjected to electrophoresis in 1.5% agarose.
1 2 3 4
328bp
51
RT-PCR Amplification of FMD viruses using FMD unive rsal and serotype specific
primers
A total of sixty field samples from clinically FMD virus infected buffaloes were collected
from various districts of Punjab and were subjected to RT-PCR amplification by
employing various FMD specific primers. Quality of RNA extraction from epithelial
samples was monitored by simultaneous amplification of β-actin gene (Figure 7). FMD
universal primers identified only forty eight samples as FMD positive. The rest of the
samples could not be amplified by the universal FMD primers, hence were not further
processed. Universal primers target a portion of FMD virus genome yielding an
amplicon of 328bp (Figure 8). Of 48 FMD positive samples, 24 were identified as
serotype O using two different sets of serotype O specific primers (Figure 9 and 10), 16
as Asia 1, again using two different sets of primers (Figure 11 and 12) and another 8 as
serotype A of FMD virus (Figure13). Samples were run on agarose gel yielding various
bands specific for their respective sets of primers. The amplicons of 328 bp, 1301 bp,
1142bp, 292bp, 911bp and 863bp were seen corresponding to universal primers,
serotype “O” specific primers set 1, serotype “O” specific primers set 2, serotype Asia-1
specific primers set 1 and set 2, and serotype A specific primers, respectively (Fig 8-
13).
52
β- actin fragment 275bp
Figure 7. A representative image of RT-PCR by primers that amplify β-actin gene from
RNA extracted from epithelial cells infected with FMD viruses. The amplicon of 275 bp
was obtained. PCR reaction mixture was prepared by adding 5 µl reaction buffer (pH-
8.8), 2.5 mM MgCl2 (5 µl) , 2 mM dNTPs mix(5 µl), 300 nM each primer(1.5 µl), cDNA
template (3 µl), 1U Taq polymerase (1 µl) and 29 µl DEPC water to make a final volume
of 50 µl. Thirty five cycles were used in amplifying the target molecules. The PCR
product was subjected to electrophoresis in 1.5% agarose.
53
Figure 8. A representative image of RT-PCR of samples of FMD virus using FMD
universal primers. The amplicon of 328 bp was obtained. PCR reaction mixture was
prepared by adding 5 µl reaction buffer (pH- 8.8), 2.5 mM MgCl2 (5 µl) , 2 mM dNTPs
mix (5 µl), 300 nM each primer(1.5 µl), cDNA template(3 µl), 1U Taq polymerase (1 µl)
and 29 µl DEPC water to make a final volume of 50 µl. Thirty five cycles were used in
amplifying the target molecules. The PCR product was subjected to electrophoresis in
1.5% agarose. Lanes next to the marker are negative and positive controls,
respectively.
328bp
54
Figure 9. A representative image of RT-PCR of samples of FMD virus using serotype
‘O’ specific primers (set 1, NK61 and ARS4) and universal primers. Lane 1 and 2 had
‘O’ specific primers yielding a 1301 bp amplicon. Lane 4 contained FMD universal
primers with 328 bp product. Lane 5 is a ladder. PCR reaction mixture was prepared by
adding 5 µl reaction buffer (pH- 8.8), 2.5 mM MgCl2 (5 µl) , 2mM dNTPs mix (5 µl), 300
nM each primer(1.5 µl), cDNA template(3 µl), 1U Taq polymerase(1 µl) and 29 µl DEPC
water. Thirty five cycles were used in amplifying the target molecules. The PCR product
was subjected to electrophoresis in 1.5% agarose.
1 2 3 4 5
1301 bp
55
Figure 10. A representative image of RT-PCR of samples of FMD virus using serotype
‘O’ specific primers (set 2, NK61 and 1C-283). Amplicon size is 1142 bp. PCR reaction
mixture was prepared by adding 5µl reaction buffer (pH- 8.8), 2.5 mM MgCl2 (5µl) , 2
mM dNTPs mix (5µl), 300 nM each primer (1.5µl), cDNA template (3 µl), 1U Taq
polymerase (1 µl) and 29µl DEPC water to make a final volume of 50 µl. Thirty five
cycles were used in amplifying the target molecules. The PCR product was subjected to
electrophoresis in 1.5% agarose.
1142bp
56
Figure 11. A representative image of RT-PCR of samples of FMD virus using serotype
Asia 1 specific primers (set 1, P33, P75). Amplicon size is 292 bp. PCR reaction mixture
was prepared by adding 5 ul reaction buffer (pH- 8.8), 2.5 mM MgCl2 (5 ul) , 2 mM 4
dNTP mix (5ul), 300 nM each primer (1.5ul), cDNA template (3ul), 1U Taq polymerase
(1ul) and 29ul DEPC water to make a final volume of 50 ul. Thirty five cycles were used
in amplifying the target molecules. The PCR product was subjected to electrophoresis in
1.5% agarose.
292bp
57
Figure 12. A representative image of RT-PCR of samples of FMD virus using serotype
Asia 1 specific primers (set 2,). Amplicon size is 911bp. PCR reaction mixture was
prepared by adding 5ul reaction buffer (pH- 8.8), 2.5mM MgCl2 (5ul) , 2mM 4dNTP
mix(5ul), 300 nM each primer (1.5ul), cDNA template (3 ul), 1U Taq polymerase(1 ul)
and 29 ul DEPC water to make a final volume of 50 ul. Thirty five cycles were used in
amplifying the target molecules. The PCR product was subjected to electrophoresis in
1.5% agarose.
911bp
58
Figure 13. A representative image of RT-PCR of FMD virus samples using FMD
universal primers and serotype ‘ A’ specific primers. Lanes 1 and 3 (universal primers-
328bp), Lane 2 (serotype A primers – 863bp). Lane 4- ladder (ranging from 100 bp- 3
Kb fragment). PCR reaction mixture was prepared by adding 5 µl reaction buffer (pH-
8.8), 2.5mM MgCl2 (5µl), 2mM dNTPs mix (5µl), 300 nM each primer (1.5µl), cDNA
template (3µl), 1U Taq polymerase (1µl) and 29 µl DEPC water. Thirty five cycles were
used in’ amplifying the target molecules. The PCR product was subjected to
electrophoresis in 1.5% agarose.
1 2 3 4
59
Amplification of VP1 using VP1 specific primers for serotype O, Asia-1 and A
After initial confirmation of the FMD virus and its serotypes prevalent in Pakistan (O,
Asia-1 and A), positive samples were subjected to VP1 amplification by using VP1
specific primers. These primers amplified the fragments of 869bp, 911bp, 959bp and
863bp for serotype O, Asia-1 (two sets) and A respectively (Figures 12-15). The primer
pair NK61-A 1C562 was used both for serotype A identification as well as for its VP1
amplification. For amplification of VP1 gene of serotype ‘O’, the amplified product of
serotype O was used as the template for nested PCR. Although, attempts were made to
amplify VP1 gene of serotype O using the original sample, but no product was seen.
60
Figure 14. A representative image of RT-PCR of VP1 of serotype O of FMD virus.
Amplicon size is 869 bp. PCR reaction mixture was prepared by adding 5 µl reaction
buffer (pH- 8.8), 2.5 mM MgCl2 (5µl) , 2 mM dNTPs mix (5µl), 300 nM each primer
(1.5µl), cDNA template (3µl), 1U Taq polymerase (1µl) and 29 µl DEPC water to make a
final volume of 50 µl. Thirty five cycles were used in amplifying the target molecules.
The PCR product was subjected to electrophoresis in 1.5% agarose.
869bp
61
Figure 15. A representative image of RT-PCR of VP1 of serotype Asia-1 specific primer
(set 3). Amplicon size is 959bp. PCR reaction mixture was prepared by adding 5µl
reaction buffer (pH- 8.8), 2.5mM MgCl2 (5µl), 2mM dNTPs mix (5µl), 300 nM each
primer (1.5µl), cDNA template (3µl), 1U Taq polymerase (1µl) and 29µl DEPC water to
make a final volume of 50 µl. Thirty five cycles were used in amplifying the target
molecules. The PCR product was subjected to electrophoresis in 1.5% agarose.
959bp
62
Sequencing of VP1 gene of various FMD serotypes and their phylogenetic Analyses
The specific amplicons were eluted from the agarose gel by using DNA Extraction Kit
(#K0513, Fermentas) and sent to Macrogen® (Seoul, Korea) and/or Centre of
Excellence for Molecular Biology (CEMB), Pakistan for sequencing. Although, all
positive samples for various serotypes were included for VP1 amplification and
subsequent sequencing, some sequences could not be resolved and were excluded
from the final analyses.
FMD viruses from independent outbreaks in different parts of Punjab were collected
from clinically infected cattle and buffaloes over a period of about two years. The
samples were initially tested for the presence of FMD virus by RT-PCR using FMD
universal primers followed by serotype specific primers to classify them into various
serotypes. Total RNA was extracted from samples and VP1 gene was amplified using
various sets of primers. In this study, VP 1 gene sequences of 12 FMD serotype Asia 1
isolates along with two vaccinal strains were compared with each other and serotype
Asia 1, VP 1 sequences obtained from Genbank belonging to previously published
Pakistani, Indian, Iranian, Afghani etc. isolates.
Sequence similarity tree of VP 1 of FMDV Serotype A sia 1 A nucleotide sequence comparison conducted using the BLAST program with default
search parameters indicated that VP1 of FMDV Asia 1 of isolates from Toba, Hafizabad
and Lodhran had the greatest sequence similarity (86-97%) to some of FMDV isolates
of Pakistan, India, Afghanistan and Iran from year 2002 to 2005 (Table 5). Sequence
similarity index for isolates of Lahore and Kasur ranged from 90 to 95 % with the
isolates obtained from GenBank database whereas isolates of Okara, Pattoki and
Faisalabad had a similarity percentage of 89 to 94 when compared with GenBank
database sequences. Compared with the 21 isolates of GenBank, the nucleotide
identities of Bahawalpur, Rahim Yar Khan and Multan ranged from 86% to 92% (Table
5). Similarly, when VP1 sequences of FMDV serotype O of this study were compared
63
with those of GenBank sequences (see Figure 16 for Accession Nos) using BLAST
program, a sequence similarity of more than 85 % was observed (data not shown).
Table 5. Similarity between Asia 1 isolates of this study and the most closely related isolates obtained from GenBank using the BLAST program with default search parameters
GenBank Accession
No.
Year Location Toba, Hafizabad, Lodhran
Kasur, Lahore
Okara, Pakpattan, Faisalabad
Bahawalpur, Rahim Yar
Khan, Multan
Percent Similarity DQ101235 2004 India 97 * * * DQ101237 2004 India 94 * * * DQ121119 2004 Iran 87 90 90 88 DQ121121 2004 Iran * 90 89 86 DQ121124 2002 Pakistan 86 92 92 89 DQ121125 2002 Pakistan 86 93 92 90 DQ121126 2003 Pakistan 87 91 91 89 DQ121127 2003 Pakistan 87 95 94 92 DQ121128 2004 Pakistan 87 95 94 92 DQ989323 2002 India 94 * * * EF457988 2003 Afghanistan * 94 93 91 EF457989 2003 Afghanistan 86 94 94 91 EF457990 2003 Afghanistan * 94 93 91 EF457992 2003 Afghanistan * 94 93 91 EF457993 2004 Afghanistan * 94 93 91 EF457994 2004 Afghanistan 86 95 94 92 EU553914 1998 Pakistan 88 90 91 89 FJ785246 2004 Iran 87 90 90 86 FJ785261 1998 Pakistan 87 90 90 88 FJ785262 2002 Pakistan 86 93 92 90 FJ785263 2002 Pakistan 86 95 92 90 FJ785264 2004 Pakistan 86 95 94 92 FJ785265 2005 Pakistan 88 90 91 89 FJ785266 2005 Pakistan 88 90 91 89 FJ785296 2003 India 94 * * * FJ785303 2004 India 97 * * * FJ785304 2004 India 97 * * *
* Match not found in the first 101 sequence search of BLAST
64
When their phylogenetic relationships with previously reported isolates were evaluated,
FMD Asia 1 isolates of this study were found to be scattered into two distinct groups
(Figure 16). Group one consisted of isolates of Lodhran, Toba and Hafizabad that were
morel closely related to Indian isolates sharing more than 98% identity with each other
and more than 94 % sequence identity with isolates of Indian 2001 to 2004 (Table 5 and
Figures 16 and 17). However, they shared more than 86% sequence similarity with
Pakistani isolates of 2002-2005 (Table 5). Group two comprised of isolates of kasur,
Lahore, Pakpattan, Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and
multan alongwith vaccine A and B (Figure 16). The isolates of group 2 were found to be
closely associated with previously published isolates of Pakistani and Afghani origin of
year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall 70%
sequence identity with each other. However, isolates of Bahawalpur, Rahim Yar Khan
and Multan shared more than 98% similarity with each other, a measurement of close
relationship denoting a likely common origin as one clan or clade. Similarly, isolates of
Pakpatan, Faisalabad, Okara, Kasur, and Lahore shared 88% sequence identity with
each other and qualified as one clan.
65
Figure 16. Sequence similarity tree showing relationship of FMD virus type Asia 1 (this study and previously reported) isolates constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates; blue square, vaccines.
FJ785298 India 2004
FJ785296/India 2003
FJ785294/India 2004
FJ785295 India 2004
FJ785291 India 2001
DQ989323/India 2002
DQ121112/Bhutan 2002
FJ785301 India 2004
FJ785302/India 2004
FJ785304 India 2004
FJ785303/Asia 1/India 2004
DQ101235/India 2004
Asia 1/Lodhran 1
Asia 1/Toba 1
Asia 1/Hafizabad 1
FJ785292 India 2003
FJ785242 India 1976
EU553912 Iran 1973
FJ785245 Iran 2001
FJ785227 Afg 2001
FJ785226 Afg 2001
FJ785244 Iran 2001
FJ785243 Iran 2001
AF390705/India 1996
AF390678/India 1995
DQ121122/Iran 1999
DQ121126 Pak 2003
EU553914/Pak 1998
FJ785265 Pak 2005
FJ785266 Pak 2005
DQ121119/Iran 2004
DQ121121/Iran 2004
FJ785246 Iran 2004
DQ121125 Pak 2002
FJ785263 Pak 2002
FJ785262/Pak 2002
DQ121124 PAK 2002
Asia 1/Kasur 1
Asia 1/Lahore 1
Vaccine B
Asia 1/Pakpattan 1
Asia 1/Okara 1
Asia 1/Faisalabad 1
Asia 1/Jhang 1
Vaccine A
Asia 1/Rahim Yar Khan 1
Asia 1/Bahawalpur 1
Asia 1/Multan 1
DQ121128 Pak 2004
FJ785264 Pak 2004
EF457994 Afg 2004
DQ121127 Pak 2003
EF457993 Afg 2004
EF457989/AFG 2003
FJ785271/Tajikistan 2003
FJ785270/Tajikistan 2003
EF457992 Afg 2003
EF457988/AFG 2003
EF457987 Afg 2003
EF457990/AFG 2003
33
100
94
43
58
100
31
100
100
36
100
100
97
50
100
100
53
99
83
71
99
48
61
41
42
90
99
99
75
99
53
91
31
89
90
80
78
66
96
30
89
100
70
100
89
90
95
85
92
97
47
69
69
45
62
85
33
66
FJ785298 India 2004
FJ785296/India 2003
FJ785294/India 2004
FJ785295 India 2004
FJ785291 India 2001
DQ989323/India 2002
DQ121112/Bhutan 2002
FJ785301 India 2004
FJ785302/India 2004
FJ785304 India 2004
FJ785303/Asia 1/India 2004
DQ101235/India 2004
Asia 1/Lodhran 1
Asia 1/Toba 1
Asia 1/Hafizabad 1
FJ785292 India 2003
FJ785242 India 1976
EU553912 Iran 1973
FJ785245 Iran 2001
FJ785227 Afg 2001
FJ785226 Afg 2001
FJ785244 Iran 2001
FJ785243 Iran 2001
83
71
99
48
61
41
100
42
97
31
100
50
100
90
99
94
43
58
100
53
99
Figure 17. A subtree of Figure 16 showing phylogenetic relationship of FMD virus type Asia 1 (this study and previously reported) isolates constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates.
67
DQ121119/Iran 2004
DQ121121/Iran 2004
FJ785246 Iran 2004
DQ121125 Pak 2002
FJ785263 Pak 2002
FJ785262/Pak 2002
DQ121124 PAK 2002
Asia 1/Kasur 1
Asia 1/Lahore 1
Vaccine B
Asia 1/Pakpattan 1
Asia 1/Okara 1
Asia 1/Faisalabad 1
Asia 1/Jhang 1
Vaccine A
Asia 1/Rahim Yar Khan 1
Asia 1/Bahawalpur 1
Asia 1/Multan 1
DQ121128 Pak 2004
FJ785264 Pak 2004
EF457994 Afg 2004
DQ121127 Pak 2003
EF457993 Afg 2004
EF457989/AFG 2003
FJ785271/Tajikistan 2003
FJ785270/Tajikistan 2003
EF457992 Afg 2003
EF457988/AFG 2003
EF457987 Afg 2003
EF457990/AFG 2003
75
99
30
89
100
100
53
36
100
96
100
70
33
100
89
33
85
62
69
90
95
85
92
47
69
45
97
100
Figure 18. A subtree of Figure 16 showing phylogenetic relationship of FMD virus type Asia 1 (this study and previously reported) isolates constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates; blue square, vaccines.
68
Sequence similarity tree of VP 1 of Serotype O of F MD Virus
FMD viruses from independent outbreaks in different parts of Punjab were collected
from clinically infected cattle and buffaloes over a period of about two years. The
samples were initially tested for the presence of FMD virus by RT-PCR using FMD
universal primers, and, if positive, were then followed by serotype specific primers to
classify them into various serotypes. Total RNA was extracted from samples and VP1
gene was amplified using various sets of primers. In this study, VP 1 gene sequences of
14 serotype O isolates along with one vaccinal strain were compared with each other
and with various serotype O sequences obtained from Genbank belonging to previously
published Pakistani, Indian, Iranian, Afghani and some other sequences. When
compared, the isolates of this study shared more than 85% sequence identity with
previously published Pakistani and neighboring country’s isolates reported in the
GenBank. Serotype O isolates of this study distributed themselves into two distinct
clusters (Figure 19). First cluster comprised of Sheikhupura 1 and 2, Muridkey 1,
Raiwind 1, Nankana 1, Gujranwala 1 and Gujrat 1 isolates (Figures 19 and 20),
whereas the second cluster included Depalpur 1, Sahiwal 1, Okara 1, Multan 1, Toba 1,
Faisalabad 1 and Pattoki 1 isolates (Figures 19 and 21). The first cluster was found to
be associated with previously published Pakistani isolates of 2006 mostly. However, it
also showed association with Afghanistan’s isolates of 2004 (Figure 20). The second
cluster seemed to be mostly related to previously published Pakistani isolates of 2003
(Figure 21). The overall grouping of the 14 sequences, when compared with each other,
depicted a three clustered phylogram (Figure 22). Serotype O isolates from Depalpur,
Sahiwal, Okara, Multan, Pattoki, Toba Tek Singh and Faisalabad grouped together into
a clan and had more than 85% sequence similarity with each other. The second cluster
consisted of isolates of Sheikhupura, Nankana, Raiwind and Muridkey. These
sequences had more than 86% similarity with each other. The third cluster consisted of
only two isolates which were 100 % similar to each other. However the third cluster had
only 74 % sequence similarity to cluster 1 and 73 % sequence similarity when
compared with cluster 2.
69
Figure 19. Sequence similarity tree showing relationship of FMD virus type O (this study and previously reported) isolates constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates; blue square, vaccine A
DQ404180/UK/2001
DQ404175/UK/2001
AJ633821/UK/2001
DQ404178/UK/2001
DQ404159/UK/2001
AY593836/ouk2001xIso84(PanAsia)
AJ539140/S Africa 2000
AB079061/Japan/2000
AY312587/S Korea/2000
AJ539139/S Korea 2000
AY593824/S Korea 2000
AY333431/China 2000
DQ164996/O/UAE/3/2000
DQ164978/O/TAI/9/99
DQ165022/O/VIT/9/2002
AJ539137/Taiwan 1999
AJ539136/Taiwan 1999
DQ296515/O/Konya/TUR/512/10/99
AJ318838/O/IRN/9/99
AF506822/China 1999
AJ539138/China 1999
DQ165052 Iran 2003
DQ165048 Iran 2003
DQ164895 Iran 2001
AF377945/S Korea 2000
DQ165036 Afg 2003
DQ165035 Afg 2003
DQ164893 Iran 2001
DQ164893/O/IRN/16/2001
EF457986 Afg 2004
DQ164897 Iran 2001
DQ165054 Iran 2004
O/Sheikhupura 2
O/Muridkey 1
O/Raiwind 1
O/Sheikhupura 1
O/Nankan 1
Vaccine A
O/Gujranwala 1
O/Gujrat 1
EF457984 O/AFG/120/2004
EF457984/O/AFG/120/2004
EF457985 Afg 04 Caprine
EF457985/O/AFG/20/2004
EF494506 Pak 14 2006
EF494499/O/PAK 02/2006
EF494506/O/PAK 14/2006
EF494505/O/PAK 12/2006
EF494493 isolate 24.1 8
Lahore O type Vaccine
EF494503 Pak 2006
EF494500/O/PAK 04/2006
EF494500 Pak04 2006
EF494502/O/PAK 08/2006
EF494501 PAK06 2006
EF494503/O/PAK 10/2006
EF494502 PAK08 2006
EF494501/O/PAK 06/2006
DQ165055 Iran 2004
DQ165065/O/PAK/1/2003
DQ164943/O/PAK/53/2003
DQ164890 India
O/Depalpur 1
O/Sahiwal 1
O/Okara 1
O/Multan 1
O/Toba Tek 1
O/Faisalabad 1
O/Pattoki 1
DQ165070/O/PAK/73/2003
DQ165068/O/PAK/16/2003
DQ165069/O/PAK/17/2003
DQ165067/O/PAK/14/2003
DQ165066/O/PAK/12/2003
AJ318848/O/PAK/1/98
EU109780 India 2004
EU109787 India 2004
EU109781 India 2004
EU109779 India 2004
EU109777 India 2004
EU109775 India 2004
EU109774 India 2004
DQ767863 Iran
99
95
99
99
57
99
33
99
99
99
99
33
99
99
99
95
66
60
79
94
81
79
99
99
99
9999
99
35
99
20
18
66
46
63
8
8
2
0
0
2
64
99
99
99
49
88
16
46
19
28
53
97
56
31
99
30
4653
51
92
16
12
57
45
57
77
42
20
30
10
42
66
23
13
11
10
4
13
12
70
O/Sheikhupura 2
O/Muridkey 1
O/Raiwind 1
O/Sheikhupura 1
O/Nankan 1
Vaccine A
O/Gujranwala 1
O/Gujrat 1
EF457984 O/AFG/120/2004
EF457984/O/AFG/120/2004
EF457985 Afg 04 Caprine
EF457985/O/AFG/20/2004
EF494506 Pak 14 2006
EF494499/O/PAK 02/2006
EF494506/O/PAK 14/2006
EF494505/O/PAK 12/2006
EF494493 isolate 24.1 8
Lahore O type Vaccine
EF494503 Pak 2006
EF494500/O/PAK 04/2006
EF494500 Pak04 2006
EF494502/O/PAK 08/2006
EF494501 PAK06 2006
EF494503/O/PAK 10/2006
EF494502 PAK08 2006
EF494501/O/PAK 06/2006
33
99
99
99
99
99
16
99
99
20
18
66
46
63
8
2
8
2
0
0
64
99
49
88
Figure 20. A subtree of Figure 16 showing phylogenetic relationship of some of serotype O isolates of this study with previously published ones, constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates; blue square, vaccine A.
71
O/Depalpur 1
O/Sahiwal 1
O/Okara 1
O/Multan 1
O/Toba Tek 1
O/Faisalabad 1
O/Pattoki 1
DQ165070/O/PAK/73/2003
DQ165068/O/PAK/16/2003
DQ165069/O/PAK/17/2003
DQ165067/O/PAK/14/2003
DQ165066/O/PAK/12/2003
AJ318848/O/PAK/1/98
99
99
95
33
99
94
66
60
79
81
79
Figure 21. A subtree of Figure 16 showing phylogenetic relationship of some of serotype O isolates of this study with previously published ones, constructed using the neighbor joining method with Mega 4 software. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. Red dots represent this study isolates; blue square, vaccine A.
72
Figure 22. Sequence similarity tree of FMD virus type O isolates of this study constructed using the neighbor joining method with Mega 4 software. The bar represents the genetic distance. Numbers on the nodes indicate bootstrap values calculated using 1000 replicates. The isolates made three distinct clusters.
O/Depal 1
O/Sahiwal 1
O/Okara 1
O/Multan 1
O/Faisalabad 1
O/Pattoki 1
O/Toba 1
O/Gujranwala 1
O/Gujrat 1
O/Sheikhupura 1
O/Nankana Sahib 1
O/Raiwind 1
O/Sheikhupura 2
O/Muridkey 1
100
100
100
100
100
100
79
100
100
0.02
73
Comparison of Deduced Amino Acid Sequences of FMDV Serotype Asia 1, O and the Vaccines A comparison of the deduced amino acid sequences in the critical VP1 region, a βG-βH
loop structure forming the main immunogenic epitope of FMDV (Brown et. al., 1999; Fox
et. al. 1989), of FMD serotype Asia 1 revealed that most of this study isolates shared a
rather high homology with the sequences of Vaccine A. However, the sequences of
isolates of Lodhran, Hafizabad, and Toba did not match much with those of either
vaccines, A or B (Figure 23).
When the amino acid sequences of Asia-1 isolates were aligned for comparison with the
amino acid sequences of the Vaccine A, the first common amino acid change was
observed at position 13 where Vaccine A had a “K, lysine” which was replaced by a “T,
threonine” in the sequences of most of the isolates (Figure 23). Next substitution in most
of the isolates was a “P, proline” for “A, alanine” at position 27, a K (lysine) for R
(arginine) at 31, an L (leucine) for M (methionine) at 32, and R (arginine) for G (glycine)
at position 36 in comparison to the Vaccine A.
A comparison of the deduced amino acids of FMD serotype O isolates also exhibited
such scattered changes when compared the sequences of the vaccinal virus (Figure
24). Overall, sequences of Sheikhupura, Muridkey, Raiwind and Nankana while closely
resembling with each other were very different from those of the other isolates and the
vaccine. Most of the rest of the isolates of this study had a “G, glycine” replacing “E,
glutamic acid” at position 14 and 15 when compared with the sequences of the vaccine
Next, a “P, proline” replaced an “H, histidine” at position 16, a “V, valine” replaced a “T,
threonine” at position 17, a “K, lysine” substituted an “N, asparagine” at position 19, an
“R, arginine” replaced a “K, lysine” at position 33 and an “A, alanine” replaced “T,
threonine” at position 34 with the sequences of the vaccine (Figure 24).
74
!Title Comparison of Deduced Amino Acid Sequences o f Asia 1; [ 1234567890 1234567890 1234567890 1234567890 #Vaccine_A PHRVLATVYN GKKTYGETTA RRGDMAALAQ RMSGGLPTSF #Asia_1/Jhang_1 .......... .......... .......... .......... #Asia_1/Faisalabad_1 .......... ..T....... .......... KL..R..... #Asia_1/Okara_1 .......... ..T....... .......... KL..R..... #Asia_1/Pakpattan_1 .......... ..T....... .......... KL..R..... #Asia_1/Bahawalpur_1 .......... ..T....... ......P... .L..R..S.. #Asia_1/Rahim_Yar_Khan_1 .......... ..T....... ......P... .L..R..S.. #Asia_1/Multan_1 .......... ..T....... ......P... .L..R..S.. #Asia_1/Lodhran_1 .TVCWQQCTT .RRRT.MQPH GAVTWHH.HR DLAS.C.PPS #Asia_1/Hafizabad_1 .TVCWQQCTT .RRRT.MQPH GAVTWHH.HR DLAS.C.PPS #Asia_1/Toba_1 .TVCWQQCTT .RRRT.MQPH GAVTWHH.HR DLAS.C.PPS #Vaccine_B .LA....... ..T.VR.N-- N.*AWQHGGP CTKN*WAAAH #Asia_1/Lahore_1 .......... ..T...K... .......... KLR.R..... #Asia_1/Kasur_1 .......... ..T...K... .......... KLR.R..... #FJ785227_Afg_2001 .......... ..TA..AE.P ....L..I.. .VNSS..... #FJ785226_Afg_2001 .......... ..TA..AE.P ....L..I.. .VNSS..... #EF457994_Afg_2004 .......... ..T....... .......... .L..R..... #EF457993_Afg_2004 .......... ..T....... .......... .L..R..... #EF457992_Afg_2003 .......... ..T......E .......... .L..R..... #EF457987_Afg_2003 .......... ..T....... .......... .LG.R..... #FJ785304_India_2004 .......... ..TA..DAAP .......... .L.ER...F. #FJ785301_India_2004 .......... ..TA....PS .......... .L.ER..... #FJ785298_India_2004 .......... ..T.....PS .......... .LGER..... #FJ785295_India_2004 .......... ..T.....PS .......... .LGER..... #FJ785292_India_2003 .......... ..T.....PS .......... .L.ER..... #FJ785291_India_2001 .......... ..T.....PS ........T. .L.ER..... #FJ785242_India_1976 .....S.... .MT....EPS ....L....R .VNNR..... #FJ785246_Iran_2004 .......... ..T......S .......... .L..R..... #FJ785245_Iran_2001 .......... ..TA..AE.P ....L..I.. .VNSS..... #FJ785244_Iran_2001 .......... ..TA..AE.P ....L..I.. .VNSS..... #FJ785243_Iran_2001 .......... ..TA..AE.P ....L..I.. .VNSS..... #EU553912_Iran_1973 .......... ..T....E.S ....L..IV. .L.NR..... #DQ121124_PAK_2002 .......... ..T....... ....T..... .L.ER..... #DQ121125_Pak_2002 .......... ..T....... ....TV.... .L.ER..... Figure 23. A comparison of the deduced amino acid sequences of a local vaccine against FMD serotype Asia 1 and isolates from this study. Some of other isolates (from GenBank) have also been included to emphasize the level of variation in amino acid residues. The highlighted area in fluorescent green above indicates βG-βH loop structure forming the main immunogenic epitope of FMDV (Brown et. al., 1999; Fox et. al. 1989).
75
!Title Serotype O Amino Acid Alignment with Vaccine ; [ 1234567890 1234567890 1234567890 1234567890 #Vaccine_A(2) VLATVYNGNC KYGESHTTNV RGDLQVLAQK AAKTLPTSFN #O/Depalpur_1 .......... ...GGPV.K. ....ASVGP. GG..A.PLLQ #O/Sahiwal_1 .......... ...GGPV.K. ....ASVGP. GG..A.PLLQ #O/Okara_1 .......... ...GGPV... .......... ..RA..P... #O/Multan_1 .......... ...GGPV... .......... ..RA..P... #O/Toba_Tek_1 .......... ...GGPV.K. .......D.. .GRA...A.. #O/Faisalabad_1 .......... ...GGPV.K. .......D.. .GRA...A.. #O/Pattoki_1 .......... ...GGPV.K. .......D.. .GRA...A.. #DQ165070/O/PAK/73/2003 .......... ...GGPV... .......D.. ..RA...... #DQ165068/O/PAK/16/2003 .......... ...GGPV... .......D.. ..RA...... #DQ165069/O/PAK/17/2003 .......... ....GPV... .......... ..RA...... #DQ165067/O/PAK/14/2003 ..S....... ...GGPV... .......... ..RA...... #DQ165066/O/PAK/12/2003 ..?....... ...GGPV... .......... ..RA...... #Vaccine_A .......... .......... .......... .......... #O/Sheikhupura_2 CWLPFTT.TA ST.RALQPM. .....CWPR. RQERS.PPSI #O/Muridkey_1 CWLPFTT.TA ST.RALQPM. .....CWPR. RQERS.PPSI #O/Raiwind_1 CWLPFTT.TA ST.RALQPM. .....CWPR. RQERS.PPSI #O/Sheikhupura_1 SWLPYTT.TA STARAPQPT. ....KCCPK. RRERF....K #O/Nankan_1 SWLPYTT.TA STARAPQPT. ....KCCPK. RRERF....K #EF494501/O/PAK_06/2006 .......... .......... .......... ..R....... #EF457986_Afg_2004 .......... .....PV... .......... ..R....... #EU109787_India_2004 .MG.G...V. L.SQNPR..L .......P.. V.RA...... #EU109781_India_2004 .MG.G...V. L.SQNPR..L .......P.. V.RA...... #EU109780_India_2004 .M..G...V. L.CQ.PI..L .......P.. G.R....... #EU109779_India_2004 .MT.G...V. L.SPNPR..L ....RA.P.. V.R...S..K #EU109777_India_2004 .MR.G.H.V. L.SPNPIS.. .......P.. VSR....... #EU109775_India_2004 .MR.G.H.V. L.SPNPI... ....R..PP. VVR....... #EU109774_India_2004 .MR.G.H.V. L.SPNPI... ....R..PP. VVR....... #DQ164890_India .......... ..ADGPVA.. .......... ..RA...... #DQ165036_Afg_2003 .......... .....PA... .......... ..R....... #DQ165035_Afg_2003 .......... .....PA... .......... ..R....... #DQ165055_Iran_2004 .......... ...D.PV... .......... ..R....... #DQ165054_Iran_2004 .......... ...G.PV... .......... ..R....... #DQ165052_Iran_2003 .......... R..T.PVA.. .......T.. ..R....... #DQ164897_Iran_2001 .......... .....PV... .......... ..R....... #DQ164895_Iran_2001 .......... .....PV... .......... ..R....... #DQ164893_Iran_2001 .......... .....PV... .......... ..R....... #DQ404180/UK/2001 .......... .....PV... .......... ..R....... #DQ404159/UK/2001 .......... .....PV... .......... ..R....... #DQ404175/UK/2001 .......... .....PV... .......... ..R....... #DQ404178/UK/2001 .......... .....PV... .......... ..R....... #AJ633821/UK/2001 .......... .....PV... .......... ..R....... Figure 24. A comparison of the deduced amino acid sequences of a local vaccine of FMD serotype O and isolates from this study. Some previously published sequences (GenBank) have also been included to emphasize the level of variation in amino acid residues. The highlighted area in fluorescent green above indicates βG-βH loop structure forming the main immunogenic epitope of FMDV (Brown et. al., 1999; Fox et. al. 1989).
76
Comparison of deduced amino acid sequences of FMDV serotype ’O’ with
Published Sequences
Deduced amino acid sequences of the major VP1 protein antigen epitopes (spanning
RGD sequences) for various FMD serotype O were aligned and compared with other
field and published sequences (Figure 25). The comparison revealed that the
substitutions were scattered along the entire length of the sequence, however, RGD
sequence (underlined with a thick black line in Figure 25) was conserved in all the
isolates. This sequence is part of the viral ligand that binds to the cell receptor, an
important interaction required for viral entry and infection in the host cells. Although,
overall amino acid sequence similarity of our isolates was not strikingly different from
that of the published isolates, however, amino acid substitutions with dissimilar
properties were found with a scattered pattern of distribution. For example, 15th amino
acid residue which is hydrophilic in the previously published isolates had a substitution
with a hydrophobic amino acid residue in our three isolates namely Sheikhupura 2,
Muridkey 1 and Raiwind 1. Similarly, 14th amino acid residue which is hydrophobic in
nature was found to be replaced with a hydrophilic one in our last five isolates. Amino
acid residue number 13 had a substitution with a hydrophobic residue in some of our
isolates etc. etc. It is interesting to note that such substitutions with amino acids having
dissimilar properties have also been found, albeit at lower rate, in previously published
sequences by many researchers (Figure 25).
77
Figure 25. A comparison of the deduced amino acid sequences of previously published FMD serotype O VP 1 segment with sequences from this study. Some of amino acid residues have been underlined with colored dashes to indicate various properties of amino acids: Red dash underlines a negatively charged amino acid; pink dash, a hydrophobic amino acid; blue dash, a hydrophilic amino acid; and green dash, a positively charged amino acid. A thick black line at the top underlines three critical amino acids (RGD) that form part of FMD receptor. Letters such as EF, EU and DQ followed by a number depict Accession Numbers of isolates from GenBank.
78
PHASE III: Virus Neutralization Test
Estimation of Antibody titers by agar gel precipita tion test
Of the three hyper immune sera, raised in rabbits against three different
commercial vaccines, only one vaccine induced a measureable immune
response yielding a good precipitation line against various FMD virus antigens
(Figure 26). A maximum titer of 1:64 was observed with AGPT.
Figure 26. A representative image of agar gel precipitation test with various
dilutions of a hyperimmune serum showing precipitation line against FMD
serotype O antigen. Purple colored arrow showed precipitation line of vaccine A,
Red colored arrows pointed to wells with no precipitation lines
79
Virus Neutralization Test
BHK-21 cell monolayer was tested for its ability to support replication of various
serotypes of FMD viruses in the presence or absence of serum containing
antibodies to the virus. MTT assay was used to asses the viability of cells. In
other words, CPE was inversely correlated with OD values. As is seen in Figure
27, vaccine A was able to neutralize all three serotypes of FMD viruses tested in
the experiment and the viability of cells was equivalent to that of the controls.
Other vaccines exhibited very little or no protection/virus neutralizing capacity
against the viruses.
Figure 27 . MTT assay was performed on the BHK 21 cell line with sera raised against various vaccines and their efficiency to neutralize the virus was tested. Results represent one of 3 experiments done with various isolates selected randomly for VN test.
Comparative OD values of vaccines
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Vaccine A Vaccine B Vaccine C Control
OD
val
ues O
A
ASIA-1
80
DISCUSSION
FMD is a highly contagious viral disease of cloven hoofed animals such as
buffalo, cattle, goat, sheep, camel, and some wild animal species. This acute and
fast-spreading disease is characterized by vesicle formation on the dorsum of the
tongue, dental pad, interior of lips, interdigital space, coronary bands, and
occasionally teats (Lennete and Smith, 1964; Sharma and Adhlakha, 1995). Both
the speed with which the virus spreads and the ability to change its antigenic
identity make the virus very threatening to the beef and dairy industries across
the world (Stram et. al., 1994). In the present study, vesicular epithelial tissues
from the affected animals were collected and tested by RT-PCR using universal
primers for FMD. The positive ones were amplified with VP-1 specific primers,
and their amplicons were sequenced for establishing phylogenetic relationships
among them and with the previously published FMD sequences of countries
bordering with Pakistan were established.
Early detection of infected animals prior to the appearance of clinical signs is
essential for effective control of FMD viruses and requires a rapid and sensitive
method of diagnosis. Recent advances in molecular biology have resulted in the
development of a technique known as RT-PCR for the detection of FMD virus
genomic RNA in cell culture fluids, oesophageal pharyngeal scrapings, epithelial
or other tissues such as tonsils (Amarel-Doel et al., 1993). In the first part of this
study, RT-PCR for identification of FMD viruses was perfected and optimized
(Figures 1-6), a pre-requisite for subsequent VP 1 sequencing activity.
The fidelity of amplification by PCR is dependent on several factors:
annealing/extension time, annealing temperature, dNTP concentration, MgCl2
concentration, and the type of DNA polymerase used. In general, the rate of
misincorporation may be reduced by minimizing the annealing/extension time,
81
maximizing the annealing temperature, and minimizing the dNTP and MgCl2
concentration (Eckert and kunkel, 1991; Ellsworth, et al., 1993). Since PCR is
sensitive to a number of parameters, the reaction conditions were optimized to to
obtain the best reproducibility with maximum specificity and sensitivity and to
avoid non-specific amplification products like primer-dimers or fragments of
heterogeneous sizes. The selection of PCR primers in most cases is made with
the help of a computer program. These programs are designed to indicate
conditions concerning salt concentration and annealing temperature, at which
PCR will perform well. However, in most cases these suggestions are not
optimal, and further optimization is necessary. In most reports, the
concentrations of the single compounds in the PCR buffer mix are basically the
same such as 200mM (NH4)2 SO4, 750mM Tris- Hcl (pH 8.8), 2.5mM MgCl2, 300
nM of each primer, 2 mM of each mononucleotide and 1.5 units /100ul of Taq-
polymerase enzyme (Saiki et al. 1988).
In the present study, the initial composition of PCR mixture was the same as
described above. However, various components were tested for their optimum
results. The most straightforward method of optimizing a PCR with a given primer
pair is to change the concentration of MgCl2 or the annealing temperature. The
Mg ion concentration affect primary annealing, strand dissociation temperature of
both of templates and PCR product, product specificity, formation of primer-dimer
artifacts and enzyme activity, and fidelity (Innis and Gelfand 1990). The starting
concentration of MgCl2 was 1.5 mM in the reaction mixture. Higher concentration
of MgCl2 increased the yield of non-specific products including primer dimmers
and lowers the fidelity of synthesis, whereas lower concentration of MgCl2
decreases the yield of PCR product. Mg ions make complexes with dNTPs,
primers and DNA templates, thus the optimal concentration of MgCl2 has to be
selected for each given experiment. The MgCl2 optimization results of this study
showed that a final concentration of 2.5 mM of the salt had the optimal
performance (Figure 1).
82
Likewise, selection of a suitable pH is very important for the optimal performance
of the reaction especially the polymerase enzyme. As a rule, a higher pH of
buffer gives the DNA strands greater protection against depurination and nicking
during thermal cycling (Cheng et al. 1994). Therefore buffers with higher pH are
recommended for longer fragments of DNA. Theoretically the required pH for
serotype specific primers should be high enough because their amplicon sizes
were relatively longer (1301, 959, 863 bp) but practically all primer sets worked
effectively at the theoretically calculated optimum pH value for universal set of
primers. The optimized pH was 8.8 and other conditions for the reactions were
optimized at this pH (Figure 2).
Although the theoretical annealing temperatures for various primers used in the
study for universal and serotype specific primers ranged from 48oC to 63oC, all
the primers worked best at 56oC (Figure 3). The annealing temperature was
calculated on the basis of G, C, T and A contents of the primers. The literature
recommended annealing temperature of IF, IR primers was 52oC but the intensity
was very poor at this temperature. The hot start technique prevents pre-PCR
mispriming and primer dimerization, and it is useful in low copy number
amplification (Innis and Gelfand 1990; Birch, 1996). The complete denaturation
of the DNA template at the start of the PCR reaction is of key importance.
Incomplete denaturation of DNA results in inefficient utilization of template in the
first amplification cycles and in a poor yield of PCR product. This initial
denaturation period is recommended for 5-10 minutes at 94oC depending upon
the GC contents of the template. The higher the GC contents, the longer the time
required for denaturation. In the present study the initial denaturation
temperature was subjected to variation for 5 minutes at 94oC which yielded good
results.
The primer concentrations should be optimized between 0.1 to 0.5 µM in the
100ul reaction volume. A higher primer concentration can cause mis-priming and
non-specific products, whereas a low concentration can cause low efficiency of
83
the reaction. During this study, weak product signals were detected below 0.3 µM
concentration of primers, however, a concentration of 0.3 µM yielded better
intensity DNA bands (Figure 4). Too high as well as too low a concentration of
Taq polymerase reduced the product yield. In this study, 1.5 U of Taq
polymerase showed an optimal performance.
It is very important to have an equal concentration of each dNTP (dATP, dGTP,
dCTP, dTTP), as inaccuracy of concentration of a single dNTP dramatically
increases the mis-incorporation level. The final concentration should be between
2mM of each dNTP (Bruce, 2005). Lower concentrations can improve fidelity,
whereas higher concentrations can support multiplex reactions (i.e. simultaneous
amplification of multiple DNA sequences using multiple primer pairs). This study
utilized 0.3 mM dNTPs which resulted in optimum amplification of PCR products.
In Pakistan, outbreaks of FMD are attributed to serotypes O, A, Asia 1 and C
(Klein et. al, 2008). The disease occurs round the year and in all parts of the
country. During recent years, the serotype Asia 1 has emerged as the second
most important cause of outbreaks next to serotype O. Though regular
vaccination is practiced in some pockets of the country, failure of successful
control of the disease in Pakistan is mainly due to: (i) large population of
susceptible animals; (ii) absence of restriction on animal movement; (iii) limited
availability of vaccines; and (iv) other socio-economic conditions. Maximum
outbreaks of FMD in the province of Punjab, Pakistan, are observed in the winter
season; hence most of the samples were collected in the winter season. The
probable reason for the season specificity might be underfeeding or poor quality
feeding of the animals as the fodder in winter becomes scarce and animals are
usually fed on dry roughages. The stress due to adverse temperature and
underfeeding might lead to lower immunity making animals more prone to the
diseases. Secondly the roughages might cause laceration in the buccal cavity
making the local environment more favorable for the virus to cause lesions. Also,
84
the virus may remain intact and infectious for a longer time during the winter
which may contribute towards increased incidence of the disease.
Of sixty samples collected, only 48 were successfully amplified by RT-PCR, and
of these, only 26 samples were found to be sequencing. Being RNA in nature,
FMD genome is very sensitive to degradation by RNAses and other degradative
enzymes. If a sample is collected during the early phase of the infection
(especially from vesicles), chances of viral amplification by RT-PCR are higher,
but if the lesions get invaded by bacteria, or lesions start healing, the probability
of obtaining the intact viruses from samples decreases drastically. Some
samples were collected in the late phase of infection and the viral genome may
have been degraded by bacterial RNAses and other degradative enzymes
resulting in either weak or no detectable signals by RT-PCR. Moreover, we may
have missed detection of FMD viruses in some of the samples as conventional
PCR is less sensitive than the real-time PCR that makes use of fluorescence
dyes for detection (Reid et al., 2002).
Despite significant knowledge of how foot-and-mouth disease (FMD) spreads,
the availability of vaccines and a host of diagnostic tests, FMD remains one of
the most important challenges for raising livestock. The recent epizootic in the
UK in the year 2001, the control of which called for the destruction of millions of
animals from over 2000 infected premises, demonstrates the economic impact
that FMD may cause.
RT-PCR can be combined with sequence analysis to study phylogenetic
relationships between various isolates of FMD (Knowles and Samuel, 1994).
Primers, IF and IR (Universal set of primers for all seven types of FMD virus),
were used for the identification of all seven serotypes of the virus. These primers
have been designed from highly conserved regions of the 5/ untranslated
region(5/ UTR), which is less susceptible region to nucleotide changes than
85
capsid coding regions which are subjected to immunological pressure and host
selection.
Over the last few years, there has been an explosion in the use of molecular
epidemiology to study the spread of FMD and other infectious diseases of plants,
animals and man. In the case of foot-and-mouth disease virus (FMDV), its rapid
rate of evolution, which results from a polymerase without proof-reading activity
and the ability of the genome to accommodate considerable amounts of
mutations, has made this pathogen particularly amenable to epidemiological
studies through comparisons of the nucleic acid sequences of the viral genome
(Samuel & Knowles, 2001). The capsid of FMD virus is composed of 60 copies of
each of four structural proteins namely VP1 (1D region in the genome), VP2 (1B),
VP3 (1C) and VP4 (1A) which make various antigenic and viral attachment sites.
While all of the sites appear to be necessary for a complete immunologic
response to either infection or vaccination, the major antigenic site to which most
of the immune response is directed (Acharya et al., 1989) and which is common
to all of the serotypes is located within the G-H loop of VP1(Tsai et el., 2000).
Therefore, VP1 is the most frequently studied protein due to its significant roles in
virus attachment, protective immunity and serotype specificity (Junzheng et al.,
2007). Since VP1 gene harbors almost 3 major antigenic sites, nucleic acid
sequencing of this part is likely to reveal mutational changes. Consequently, the
differences in VP1 sequences had been exploited for developing RT-PCR tests
to identify different serotypes (Rodriguez et al., 1992) and to establish
phylogenetic relationships among FMDV isolates. Various sets of primers
published previously by epidemiologists were chosen for this study to identify
FMD serotypes and to amplify their VP1 gene sequences for establishing
sequence similarity trees.
A nucleotide sequence comparison conducted using the BLAST program with
default search parameters indicated that VP1 of FMDV Asia 1 of isolates from
Toba, Hafizabad and Lodhran had the greatest sequence similarity (86-97%) to
86
some of FMDV isolates of Pakistan, India, Afghanistan and Iran from year 2002
to 2005 (Table 5). Sequence similarity index for isolates of Lahore and Kasur
ranged from 90 to 95 % with the previously published isolates of India, Iran and
Pakistan obtained from GenBank database, whereas isolates of Okara, Pattoki
and Faisalabad had a similarity percentage of 89 to 94 when compared with
GenBank database sequences. Compared with the 21 isolates of GenBank, the
nucleotide identities of Bahawalpur, Rahim Yar Khan and Multan ranged from
86% to 92% (Table 5). Similarly, when VP1 sequences of FMDV serotype O of
this study were compared with those of GenBank sequences (see Figure 16 for
Accession Nos) using BLAST program, a sequence similarity of more than 85 %
was observed (data not shown).
When their phylogenetic relationships with previously reported isolates were
evaluated, FMD Asia 1 isolates (12) of this study were found to be scattered into
two distinct groups (Figure 16). Group one consisted of isolates of Lodhran,
Toba and Hafizabad that were morel closely related to Indian isolates sharing
more than 98% identity with each other and more than 94 % sequence identity
with isolates of Indian 2001 to 2004 (Table 5 and Figures 16 and 17). However,
they shared more than 86% sequence similarity with Pakistani isolates of 2002-
2005 (Table 5). Group two comprised of isolates of kasur, Lahore, Pakpattan,
Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and multan alongwith
vaccine A and B (Figure 16). The isolates of group 2 were found to be closely
associated with previously published isolates of Pakistani and Afghani origin of
year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall
70% sequence identity with each other. However, isolates of Bahawalpur, Rahim
Yar Khan and Multan shared more than 98% similarity with each other, a
measurement of close relationship denoting a likely common origin as one clan
or clade. Similarly, isolates of Pakpatan, Faisalabad, Okara, Kasur, and Lahore
shared 88% sequence identity with each other and qualified as one clan.
87
Additionally, when 14 FMD serotype O isolates of this study along with a vaccinal
strain were analyzed phylogenetically, the isolates of this study shared more than
85% sequence identity with previously published Pakistani and neighboring
country’s isolates reported in the GenBank. Serotype O isolates of this study
distributed themselves into two distinct clusters (Figure 19). The first cluster
comprised of Sheikhupura 1 and 2, Muridkey 1, Raiwind 1, Nankana 1,
Gujranwala 1 and Gujrat 1 isolates (Figures 19 and 20), whereas the second
cluster included Depalpur 1, Sahiwal 1, Okara 1, Multan 1, Toba 1, Faisalabad 1
and Pattoki 1 isolates (Figures 19 and 21). The first cluster was found to be
associated with previously published Pakistani isolates of 2006 mostly. However,
it also showed association with Afghanistan’s isolates of 2004 (Figure 20). The
second cluster seemed to be mostly related to previously published Pakistani
isolates of 2003 (Figure 21). However, the overall grouping of the 14 sequences,
when compared with each other, depicted a three clustered phylogram (Figure
22). Serotype O isolates from Depalpur, Sahiwal, Okara, Multan, Pattoki, Toba
Tek Singh and Faisalabad grouped together into a clan and had more than 85%
sequence similarity with each other. The second cluster consisted of isolates of
Sheikhupura, Nankana, Raiwind and Muridkey. These sequences had more than
86% similarity with each other. The third cluster consisted of only two isolates
which were 100 % similar to each other. However the third cluster had only 74 %
sequence similarity to cluster 1 and 73 % sequence similarity when compared
with cluster 2.
The analysis of nucleotide sequences of the VP1 gene showed a high degree of
overall identity among the local serotype O and Asia 1 isolates. Furthermore, the
genetic similarity of various clans of our local isolates with those of the bordering
countries such as India, Iran and Afghanistan suggested common sources of
their origins, although identification of those sources could only be guessed by
indirect evidence. The fact that there are no restrictions on the movement of
animals and animal related products within the country or even across the
88
bordering countries, sequence similarity of the isolates of this study and those of
previously published bordering countries is not surprising.
When outbreaks occur year round and in all parts of the country, unrestricted
animal movement could introduce new strains into areas where they were not
quite prevalent and these new strains could dominate over strains that are
currently prevalent at that place (Gurumurthy et al., 2002). The endemicity of the
disease and the quasispecies nature of the causative virus contribute to the
continuous evolution of FMD viruses in the subcontinent.
A comparison of the deduced amino acid sequences of the critical VP 1 region of
FMD serotype Asia 1 vaccines and the isolates of this study revealed that most
of this study isolates shared very high homology with sequences of Vaccine A.
However, the sequences of isolates of Lodhran, Hafizabad and Toba did not
match much with that of either vaccines, A or B (Figure 23). Sequences of
Vaccine A had a “K” which seemed to be replaced by a “T” in the sequences of
most of the isolates. Considering the properties of various amino acids, this
change does not signify a major shift in the three dimensional picture of the
protein as K is a lysine, a positively charged amino acid, whereas a T is
threonine, a hydrophilic amino acid in nature. Next substitution in most of the
isolates is a “P” for “A” in comparison to the vaccines. Again, it is not a significant
change as both P and A share the same property, hydorphobicity. Similarly a K
with an R can be substituted without much change in the overall shape of the
protein molecule. Next amino acid substitution is a leucine instead of methionine.
Again both are hydrophobic in nature; hence their impact on the overall picture is
minute, if at all. However, glycine and arginine are two very different amino
acids; the former is a hydrophobic amino acid whereas the latter is positively
charged one. Such amino acid substitutions may have the potential to make a
major impact in terms of the epitopic differences in the capsids of vaccinal and
field viruses. A comparison of the deduced amino acids of FMD serotype O
isolates also exhibited such changes with the vaccinal virus (Figure 24).
89
Furthermore, the comparison of deduced amino acids of this study isolates (Asia
1 and O) suggested that RGD sequences were conserved in most of the isolates
except three of Asia 1 isolates. This sequence is part of viral ligand that binds to
the cell receptor, an important interaction required for viral entry and infection in
the host cells. Epitopes are determined by the primary sequences of amino acid
residues in a protein molecule. Generally speaking, amino acid substitutions with
residues having similar properties do not result in major changes in the overall
three dimensional shape of a protein molecule. For example if a hydrophobic
amino acid is substituted with another hydrophobic amino acid in a primary
sequence of a protein, the overall three dimensional shape of the protein remains
more or less conserved. However, if a hydrophobic amino acid residue is
replaced with a hydrophilic amino acid residue, this kind of substitution results in
a major change in the overall three dimensional structure or shape of a protein
molecule impacting the epitopes. Although, overall amino acid sequence
similarity of our isolates was not strikingly different from that of the published
isolates, however, amino acid substitutions with dissimilar properties were found
with a scattered pattern of distribution. For example, 15th amino acid residue
which is hydrophilic in the previously published isolates had a substitution with a
hydrophobic amino acid residue in our three isolates namely Sheikhupura 2,
Muridkey 1 and Raiwind 1 (Figure 25). Similarly, 14th amino acid residue which is
hydrophobic in nature was found to be replaced with a hydrophilic one in our last
five isolates. Amino acid residue number 13 (Figure 25) had a substitution with a
hydrophobic residue in some of our isolates etc. etc. It is interesting to note that
such substitutions with amino acids having dissimilar properties have also been
found, albeit at lower rate, in previously published sequences by many
researchers (Figure 25). The implications of these changes in the critical region
of VP-1 could be serious as these changes may mean escape of the virus from
the established immunity. A relatively recent paper by Tosh et al, 2002 is of
particular interest. While reporting on the phylogenetic analysis of serotype A
foot-and-mouth disease virus isolated in India between 1977 and 2000, Tosh et.
90
al., 2002 described changes in the critical regions of VP-1 that are believed to
help the virus to escape previously established immunity in animals (Baxt et al.,
1989; Bolewell et al, 1989). A similar recommendation as to the use of a
matching strain for vaccination has been recommended by Klein et. al., 2008.
While comparing amino acid antigenic sites of serotype Asia 1, very few
substitutions were noticed in our field isolates which is in accordance with the
findings of Lieppert et al., 1997, Gurumurthy et al., 2002 and Knowles and
Samuel, 2003, who documented that serotype Asia 1 is genetically and
antigenically the least variable of the seven recognized serotypes of FMDV and
is normally found only in Asia. However, the Asia 1 serotype has previously
spread into Europe, most recently causing outbreaks in Turkey and Greece in
1999 and 2000 respectively.
While the comparison of vaccine A with respect to major immunogenic
sequences that form βG-βH loop from residues 135 to 160 in FMDV serotypes
revealed very high degree of amino acid sequence similarity with most of this
study isolates, it is likely that a host immune response generated against this
vaccine may afford some degree of protection against most field isolates of
serotype Asia 1 of FMDV. However, the amino acid sequence data of vaccine B
of Asia 1 (Figure 23) was highly variable with respect to sequences of most of the
isolates. This may be an error or an artifact introduced by the extraction protocol
that was used for obtaining the virus from oil-based inactivated FMD commercial
vaccines. The problem with the extraction of RNA from oil-based inactivated
vaccines seems highly likely as the procedure did not work well with any of the
vaccines and we were not able to sequence any of the commercially available
inactivated vaccines except where we had a direct access to the vaccinal virus
used for the preparation of the vaccines.
In order to circumvent this difficulty and to correlate the efficacy of commercially
available killed vaccines against the field isolates of serotype Asia 1 and O in
91
affording immune protection, hyperimmune sera were raised using commercially
available killed FMDV vaccines and resultant sera were used to test their ability
to neutralize randomly selected FMDV field isolates. Of three commercial
vaccines tested, only one was able to neutralize various field isolates of serotype
Asia 1 and O (Figure 27), a fact suggestive of some reality with the amino acid
residue variability data of vaccine B of Asia 1 compared with field isolates in
Figure 23. Although, there are a number of monovalent or polyvalent vaccines
available against FMD, their efficacies for affording protection against the
disease are highly questionable as no rules exist to regulate or monitor the
vaccine manufacturing practices in the country.
Serotype A of FMDV has also been reported in Pakistan (Zahur et al., 2006, and
Klein et al., 2007). Attempts were also made to amplify FMDV serotype A from
the field samples using serotype ‘A’ specific primers. Some success was noted at
least to the level of amplification with few field samples. However, their
sequencing data was poorly resolved and serotype A was not considered further.
Additionally, time constraint did not allow reprocessing of these samples.
It may be noted that variation in the amino acid sequences noted in this study
may not be real for couple of reasons. Firstly, PCR is prone to errors and these
variations may in part be attributed to errors introduced by Taq polymerase
during the reaction. Secondly, since there is no pressure for these sequences to
be actualized in the form of capsid assembly (while the virus is replicating in the
cells), these might be real mutations which could only be corroborated when a
real capsid is assembled using these sequences in vitro. Since epithelial samples
may contain unassembled RNA of the virus, which inherently might have
undergone true mutational events, possibility of such (mis-sense or untrue)
mutational events may be real. Lastly, cloning of VP-1 amplicons could have
been a better option for getting better and cleaner sequence data with less
variability, a step which we could not utilized because of some inevitable
circumstances during the carrying out of this project.
92
In summary, RT-PCR for diagnosis of serotypes A, O and Asia 1 of FMDV was
optimized and could be used for prompt and precise diagnosis of FMD in the
country. Although, RT-PCR data pertains to bovines in the current project, but
PCR optimization parameters are equally applicable to FMDV infections in other
FMD susceptible animal species such as sheep and goat. The combination of
PCR and sequencing of the VP1 gene to detect and analyze FMDV in disease
outbreaks is fast (less than 6 hours for PCR and about 24 hours for sequencing),
and it can give an accurate immunologic characterization of the virus, thus
providing a rational basis for choice of vaccine. In fact, the molecular
epidemiology of field isolates is a powerful tool to monitor the circulation of
viruses (Saiz et al., 1993).
Secondly, various isolates of serotypes O and Asia 1 were sequenced along with
some vaccinal strains. Sequence similarity tree analysis indicated that most of
our isolates were closely related to previously reported Pakistani isolates and to
those of neighboring countries such as India, Afghanistan and Iran. Additionally,
amino acid sequence similarity data of major immunogenic site that forms βG-βH
loop in FMDV serotypes revealed that serotype Asia 1 vaccinal strain and Asia 1
isolates of this study possessed high degree of similarity suggesting a likely host
immune response against the vaccine that may afford some protection against
most field isolates of serotype Asia 1 type. Lastly, of three vaccines tested, only
one was found to afford protection against field isolates of FMDV suggesting
more work on vaccine issue in the country.
In conclusion, given the mutation prone nature of the virus, frequent
epidemiological surveillance endeavors for monitoring genomic alterations in the
FMDV serotypes in the country be undertaken. The seed for FMDV vaccines
should be selected or updated in the light of data gathered through these
surveillance maneuvers in order to be able to tailor the immune response of
vaccines against the most currently circulating field strains or serotypes of FMDV
93
in the country. Such recommendations for vaccine improvement have also been
suggested previously by Klein at al., 2007 and 2008, a meticulous
epidemiological work on FMD in Pakistan. Secondly, although, there are a
number of monovalent or polyvalent vaccines available against FMD, their
efficacy for affording protection against the disease is highly questionable as no
rules exist to regulate or monitor the vaccine manufacturing practices in the
country. Therefore, rules should be implemented to strictly regulate and monitor
vaccine manufacturing practices in the country. Thirdly, the lack of biosecurity
awareness by general masses and uncontrolled movement of animals especially
during “Animal Mandies and Eid ul Azha”, make it very fitting for the FMD to
remain endemic in Pakistan. Appropriate measures should be adopted to
increase biosecurity awareness regarding various contagious diseases in general
and FMD in particular with some sort of restrictions on free animal movements in
the country to limit the spread of this economically important disease especially
during its high season.
Acknowledgements/Endorsements
This project was funded by Higher Education Commission, Islamabad under
National Research Program for Universities scheme.
94
SUMMARY
Within two decades after its first report in 1954 from Pakistan, Foot and mouth
disease has become endemic in the country and poses a serious threat to large
as well as small ruminant population. Foot and Mouth Disease (FMD) is
prevailing in cattle and buffaloes and is caused by either O, A, Asia-1 serotype of
the FMD virus in Pakistan. The present study was undertaken to study the
mutation rate of FMD virus and also molecular typing of the strains prevalent in
Pakistan was done.
A total of 60 samples from buffalo and cattle were collected from five districts of
Punjab including Lahore, Faisalabad, Sialkot, Okara and Sheikhupura. Soon
after extraction of their RNA, all of them were reverse transcribed and then
subjected to amplification by using different sets of the primers including
universal as well as serotype specific primers. Then their VP1 portions were
amplified by using VP1 specific primers. Among 60 samples, 48 were positive
with universal primers. Other 12 samples were not amplified with these primers
hence not processed.
Among 48 FMD positive samples, 24 were positive with serotype O specific
primers, 16 with serotype Asia-1 and remaining 8 were positive with serotype A
specific primers. After their amplification, the amplicons were run on the gel.
These amplicons were extracted by using DNA extraction kit. After their
purification, they were sent to Macrogen® (Seoµl, Korea) and Centre of
Excellence for Molecµlar Biology, Pakistan (CEMB) for sequencing. Each
amplicon was sequenced thrice and the consensus sequence was established
eliminating sequencing errors.
Sequence identity and multiple sequence alignment of molecular sequences
(nucleotide and amino acids) were performed with Clustal W algorithm
(Thompson et al., 1994). Neighbour joining trees were constructed by using
95
MEGA version 4.0 (Kumar et al., 2004). Nucleotide distance matrices were
computed by Kimura two parameter algorithm based on the total nucleotide
substitutions and evolutionary trees for VP1 genes were constructed.
For FMDV serotype ‘O’ phylogenetic analysis, 14 VP1 sequences from various
field isolates were compared with some previously published Pakistani FMD O
type VP1 specific sequences available with GeneBank and some recently
published VP1 sequences reported by countries bordering with Pakistan
including India, Iran and Afghanistan Similarly, 12 VP 1 sequences of FMDV
serotype Asia-1 isolates of this study were compared with previously published
sequences and their phylogenetic relationship was established. However, the
sequencing results of serotype A were inconclusive and were not included for
phylogenetic analysis. Three sequences of three locally available FMD vaccines
were also studied and compared with the outbreak strains.
Polymerase chain reaction was optimized with respect to MgCl2, buffer Ph,
annealing temperature, primer concentration, template concentration, and Taq
polymerase. A concentration of 2.5 Mm of MgCl2 resulted in the best
amplification of the target sequences (Figure 1). The buffer with Ph 8.8 yielded
the best results (Figure 2) Although, the suggested annealing temperatures for
various primers (of various serotypes) ranged from 48 oC to 63 oC, however, a
temperature of 56 oC was found to be the best with all sets of primers (Figure 3).
The best intensity DNA bands were observed with 0.3 µM concentration of the
primers (Figure 4). Moreover, the best Cdna template concentration giving
optimum amplification was found to be 3.0 µl per reaction (Figure 5). Lastly, a
concentration of 0.5 U of Taq polymerase was not sufficient for amplification of
cDNAs, however, 1.0 U of enzyme was found to yield better amplification (Figure
6).
VP 1 DNA sequences of six previously published Pakistani FMD serotype O
strains were analyzed phylogenetically with VP 1 DNA sequences of 14 isolates
of the study. Serotype O isolates of this study distributed themselves into two
96
distinct clusters (Figure 19). First cluster comprised of Sheikhupura 1 and 2,
Muridkey 1, Raiwind 1, Nankana 1, Gujranwala 1 and Gujrat 1 isolates (Figures
19 and 20), whereas the second cluster included Depalpur 1, Sahiwal 1, Okara 1,
Multan 1, Toba 1, Faisalabad 1 and Pattoki 1 isolates (Figures 19 and 21). The
first cluster was found to be associated with previously published Pakistani
isolates of 2006 mostly. However, it also showed association with Afghanistan’s
isolates of 2004 (Figure 20). The second cluster seemed to be mostly related to
previously published Pakistani isolates of 2003 (Figure 21). The overall grouping
of the 14 sequences, when compared with each other, depicted a three clustered
phylogram (Figure 22). Serotype O isolates from Depalpur, Sahiwal, Okara,
Multan, Pattoki, Toba Tek Singh and Faisalabad grouped together into a clan
and had more than 85% sequence similarity with each other. The second cluster
consisted of isolates of Sheikhupura, Nankana, Raiwind and Muridkey. These
sequences had more than 86% similarity with each other. The third cluster
consisted of only two isolates which were 100 % similar to each other. However
the third cluster had only 74 % sequence similarity to cluster 1 and 73 %
sequence similarity when compared with cluster 2.
When the phylogenetic relationships with previously reported isolates of Asia 1
was evaluated, FMD Asia 1 isolates of this study were found to be scattered into
two distinct groups (Figure 16). Group one consisted of isolates of Lodhran,
Toba and Hafizabad that were more closely related to Indian isolates sharing
more than 98% identity with each other and more than 94 % sequence identity
with isolates of Indian 2001 to 2004 (Table 5 and Figures 16 and 17). However,
they shared more than 86% sequence similarity with Pakistani isolates of 2002-
2005 (Table 5). Group two comprised of isolates of kasur, Lahore, Pakpattan,
Okara, Faisalabad, Jhang, Rahim Yar Khan, Bahawalpur and multan alongwith
vaccine A and B (Figure 16). The isolates of group 2 were found to be closely
associated with previously published isolates of Pakistani and Afghani origin of
year 2003 and 2004 (Figures 16 and 18). Collectively, they shared an overall
70% sequence identity with each other. However, isolates of Bahawalpur, Rahim
97
Yar Khan and Multan shared more than 98% similarity with each other, a
measurement of close relationship denoting a likely common origin as one clan
or clade. Similarly, isolates of Pakpatan, Faisalabad, Okara, Kasur, and Lahore
shared 88% sequence identity with each other and qualified as one clade.
Although, overall amino acid sequence similarity of our isolates was not strikingly
different from that of the published isolates, however, amino acid substitutions
with dissimilar properties were found with a scattered pattern of distribution. For
example, 15th amino acid residue which is hydrophilic in the previously published
isolates had a substitution with a hydrophobic amino acid residue in our three
isolates namely Sheikhupura 2, Muridkey 1 and Raiwind 1 (Figure 25). Similarly,
14th amino acid residue which is hydrophobic in nature was found to be replaced
with a hydrophilic one in our last five isolates. Amino acid residue number 13
(Figure 25) had a substitution with a hydrophobic residue in some of our isolates
etc. etc. It is interesting to note that such substitutions with amino acids having
dissimilar properties have also been found, albeit at lower rate, in previously
published sequences by many researchers (Figure 25).
A comparison of the deduced amino acid sequences in the critical VP 1 region of
FMD serotype Asia 1 revealed that most of this study isolates shared very high
homology with sequences of Vaccine A. However, the sequences of isolates of
Lodhran, Hafizabad and Toba did not match much with that of either vaccines, A
or B (Figure 23). Sequences of Vaccine A had a “K” which seemed to be
replaced by a “T” in the sequences of most of the isolates. Considering the
properties of various amino acids, this change does not signify a major shift in
the three dimensional picture of the protein as K is a lysine, a positively charged
amino acid, whereas a T is threonine, a hydrophilic amino acid in nature. Next
substitution in most of the isolates is a “P” for “A” in comparison to the vaccines.
Again, it is not a significant change as both P and A share the same property,
hydorphobicity. Similarly a K with an R can be substituted without much change
in the overall shape of the protein molecule. Next amino acid substitution is a
98
leucine instead of methionine. Again both are hydrophobic in nature; hence their
impact on the overall picture is minute, if at all. However, glycine and arginine
are two very different amino acids; the former is a hydrophobic amino acid
whereas the latter is positively charged one. Such amino acid substitutions may
have the potential to make a major impact in terms of the epitopic differences in
the capsids of vaccinal and field viruses. A comparison of the deduced amino
acids of FMD serotype O isolates also exhibited such changes with the vaccinal
virus (Figure 24).
Of the three hyper immune sera raised against three different vaccines in rabbits,
only one vaccine induced a measureable immune response yielding good
precipitation line against various FMD virus antigens.
In summary, RT-PCR for diagnosis of serotypes A, O and Asia 1 of FMDV was
optimized and could be used for prompt and precise diagnosis of FMD in the
country. Although, RT-PCR data pertains to bovines in the current project, but
PCR optimization parameters are equally applicable to FMDV infections in other
FMD susceptible animal species such as sheep and goat. The combination of
PCR and sequencing of the VP1 gene to detect and analyze FMDV in disease
outbreaks is fast (less than 6 hours for PCR and about 24 hours for sequencing),
and it can give an accurate immunologic characterization of the virus, thus
providing a rational basis for choice of vaccine. In fact, the molecular
epidemiology of field isolates is a powerful tool to monitor the circulation of
viruses (Saiz et al., 1993).
Secondly, various isolates of serotypes O and Asia 1 were sequenced along with
some vaccinal strains. Sequence similarity tree analysis indicated that most of
our isolates were closely related to previously reported Pakistani isolates and to
those of neighboring countries such as India, Afghanistan and Iran. Additionally,
amino acid sequence similarity data of major immunogenic site that forms Βg-Βh
loop in FMDV serotypes revealed that serotype Asia 1 vaccinal strain and Asia 1
99
isolates of this study possessed high degree of similarity suggesting a likely host
immune response against the vaccine that may afford some protection against
most field isolates of serotype Asia 1 type. Lastly, of three vaccines tested, only
one was found to afford protection against field isolates of FMDV suggesting
more work on vaccine issue in the country.
100
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