Genetic typing of recent classical swine fever isolates from India.

Abstract

Seventeen classical swine fever virus (CSFV) isolates recovered during the period of 3 years (2006-2008) from India were subjected to nucleotide sequencing in the 5' untranslated region (UTR). For genetic typing, 150 nucleotides within this region were used. For better epizootiological understanding, 39 nucleotide sequences of the above region, including 13 Indian CSFV sequences, available either in the Genbank or published literature were also included in the study. Based on the phylogenetic analysis, the Indian isolates could be grouped in to two subgroups, viz., 1.1 and 2.2. The study also revealed predominance of subgroup 1.1 and involvement of viruses of more than one subgroup in an outbreak.

Full text

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Short communication
Genetic typing of recent classical swine fever isolates from India
S.S. Patil, D. Hemadri*, B.P. Shankar, A.G. Raghavendra, H. Veeresh, B. Sindhoora,
S. Chandan, K. Sreekala, M.R. Gajendragad, K. Prabhudas
Project Directorate on Animal Disease Monitoring and Surveillance, ICAR, Hebbal, Bengaluru 560024, India
1. Introduction
Classical swine fever is an economically important,
highlycontagiousviraldisease ofpigs. Thecausative agent,
classical swine fever virus (CSFV) is a member of the genus
Pestivirus within the family Flaviviridae and is closely
related to the viruses of bovine viral diarrhoea and border
disease of sheep (Wengler et al., 1995). CSFV is an
enveloped virus containing a single stranded, non-seg-
mented positive-sense RNA genome of approximately
12.3 kb in length. The viral genome contains untranslated
regions (UTR) at both 30and 50ends and encodes a single
polyprotein which is proteolytically processed to produce
12 functional viral proteins (Meyers and Thiel, 1996).
The first documented report of classical swine fever in
India dates back to 1962, where, an outbreak in a piggery
unit in Morol, a suburb of Mumbai (formerly Bombay),
which later lead to widespread outbreaks in other parts of
city has been described (Sapre et al., 1962). Although the
disease has been reported from most of the states in India,
it is most frequently reported from the North-Eastern
states due to higher density of pigs in that region. A
compilation of data from OIE website indicates that there
were 1308 outbreaks of classical swine fever in India
during 1996–2008. A map showing various states of India
where CSFV outbreaks have been recorded during 2007–
2008 is presented in Fig. 1. Because of the sporadic nature
of the disease and the lower preference for the pig
husbandry (barring North-Eastern states) in India, CSF has
not been studied systematically and therefore epidemiol-
ogy of the disease is largely unknown.
Genetic typing has been widely used to assist epide-
miological studies (Pereda et al., 2005). Three regions of
the genome, i.e., 50UTR (150nt), E2 envelope glycoprotein
gene (190 nt) and NS5B polymerase gene (490 nt), have
beenmostcommonlyusedfor classifyingCSF virusstrains/
isolates into groups and subgroups (Bjorklund et al., 1999;
Greiser-Wilkeetal.,1998;Lowingsetal.,1996;Patonetal.,
2000). Further, phylogenetic analysis of these sequences
provides invaluable information about the possible origin
of outbreaks and virus spread in the field. To our
knowledge, a similar study involving CSFV isolates drawn
from all over India has not been carried out till date. A
recent study by Singh et al. (2004) has been limited to one
field isolate from Madhya Pradesh and a lapinised vaccine
strain. Keeping this in mind, we have carried out the
nucleotide sequence analysis of 50UTR region with an aim
to create baseline epidemiological information on CSF in
India and also to create sequence database of Indian CSFV
isolates for any future use.
Veterinary Microbiology xxx (2009) xxx–xxx
A R T I C L EI N F O
Article history:
Received 15 April 2009
Received in revised form 7 September 2009
Accepted 22 September 2009
Keywords:
Classical swine fever virus
Genetic typing
Indian isolates
A B S T R A C T
Seventeen classical swine fever virus(CSFV) isolatesrecoveredduringtheperiod of 3years
(2006–2008) from India were subjected to nucleotide sequencing in the 50untranslated
region (UTR). For genetic typing, 150 nucleotides within this region were used. For better
epizootiological understanding, 39 nucleotide sequences of the above region, including 13
Indian CSFV sequences, available either in the Genbank or published literature were also
included in the study. Based on the phylogenetic analysis, the Indian isolates could be
grouped in to two subgroups, viz., 1.1 and 2.2. The study also revealed predominance of
subgroup 1.1 and involvement of viruses of more than one subgroup in an outbreak.
? 2009 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +91 80 23419576; fax: +91 80 23415329.
E-mail address: divakar.hemadri@gmail.com (D. Hemadri).
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Contents lists available at ScienceDirect
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
0378-1135/$ – see front matter ? 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2009.09.021

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2. Materials and methods
Seventeen CSFV isolates recovered from field out-
breaks in various parts of India, stored in the virology
laboratory of Project Directorate on Animal Disease
Monitoring and Surveillance (PD_ADMAS) were selected
for genetic analysis. Details of the isolates are given in
Table 1. After retrieval from the liquid nitrogen, the
Fig. 1. Map showing the various Indian states (shaded in grey) which have reported CSF outbreaks during 2007–2008.
Table 1
History of CSFV isolates used in the study.
S. No.Isolate Acc. No.Place of outbreakYear of outbreakGenbank Acc. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
CSFV/IND-163/07
CSFV/IND-173/08
CSFV/IND-174/08
CSFV/IND-175/08
CSFV/IND-187/08
CSFV/IND-239/08
CSFV/IND-240/08
CSFV/IND-243/08
CSFV/IND-268/08
CSFV/IND-270/08
CSFV/IND-271/08
CSFV/IND-272/08
CSFV/IND-273/08
CSFV/IND-294/08
CSFV/IND-296/08
CSFV/IND-298/08
CSFV/IND-299/08
Karnataka
Madhya Pradesh
Kerala
Kerala
Kerala
Maharashtra
Maharashtra
Andhra Pradesh
Andhra Pradesh
Punjab
Punjab
Punjab
Punjab
Karnataka
Karnataka
Karnataka
Karnataka
December 2007
November 2007
November 2007
November 2007
March 2008
May 2007
February 2006
July 2008
July 2008
2008
2008
2008
2008
August 2008
August 2008
August 2008
August 2008
EU446419
FJ183444
FJ183445
FJ183446
FJ183448
FJ183449
FJ183450
FJ183452
FJ183453
FJ183454
FJ183455
FJ183456
FJ183457
FJ183461
FJ183458
FJ183459
FJ183460
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isolates were passaged once in porcine kidney (PK-15)
cells.
The total RNA was extracted from the cell culture
supernatant of the infected PK-15 cells, using QIAmp Viral
RNA mini-kit (Qiagen, Germany). Reverse transcription-
PCR (RT-PCR) was performed on the so obtained RNA
template using the previously published primer pairs
(CSFV-UP1: 50-ctagccatgcccwyagtagg-30and CSFV-UP2: 50-
cagcttcarygttgattgt-30; Paton et al., 2000) and one-step RT-
PCR kit (Qiagen, Germany). Briefly, reverse transcription
was performed at 50 8C for 30min, followed by initial
enzyme activation step at 958C for 15min. Subsequent
PCR amplification was conducted with 35 cycles of
denaturation at 948C for 1 min, annealing at 50 8C for
1 min, extension at 72 8C for 1 min followed by final
extension for 10 min at 728C. An expected amplicon of
approximately 421 bp was obtained for all the isolates
whose identity was confirmed by digestion with restric-
tion enzyme PstI, which yielded two fragments of 188bp
and 233bp size.
After gel purification, all the 17 amplicons were sent for
nucleotide sequencing of both strands to a commercial
service (Bioserve India Pvt. Ltd., Hyderabad). Phylogenetic
trees were calculated as described previously (Greiser-
Wilke et al., 2000). Briefly, 150 nucleotides (nt positions
corresponding to 200–349 of Riems, GenBank Acc. No.
AY259122) of the 50UTR region were aligned using the
Clustal X, Ver. 1.8.3 programme (Thompson et al., 1997)
and edited manually using GeneDoc (Nicholas and
Nicholas, 1997). Nucleotide divergence/similarity was
calculated using Megalign module of Lasergene package
(DNASTAR Inc., USA). The transition/transversion (Ts/Tv)
rate was estimated from the dataset using TreePuzzle, Ver.
5.2 (Strimmer and von Haeseler, 1996, 1997), and was
found to be 4.12. The bootstrap values were calculated
with the modules SEQBOOT, DNADIST, NEIGHBOUR and
CONSENSE of the PHYLIP Ver. 3.59 package (Felsenstein,
1989). The phylogenetic trees, calculated by using the
neighbour-joining method, were computed with the
DNADIST, NEIGHBOUR modules with the same para-
meters. Trees were visualised using the programme,
TREEVIEW Ver. 1.6.6 (Page, 1996). The nomenclature of
the groups and subgroups was as per Paton et al. (2000).
3. Results and discussion
Seventeen CSFV isolates collected from 10 outbreaks in
6 states of India during 2006–2008 were nucleotide
sequenced in the 50UTR region. Alignment of 150 nucleo-
tidesofthisregiontothoseof39otherstrains,including13
Indian CSFV isolates, available either in GenBank or
published literature, is given in Fig. 2, while, phylogenetic
tree constructed from the above aligned sequences is
shown in Fig. 3. These above two analyses could segregate
the strains into three major groups (groups 1–3) with
various subgroups. On the phylogenetic tree, majority of
Indian isolates have clustered in subgroup 1.1 within the
group 1. These Indian isolates are0–1% and 3–4% divergent
respectively from the members of the subgroups 1.1 and
1.2. On the phylogenetic tree, the Indian isolates are placed
separately from the other members of the subgroup 1.1
from Europe, and China. Slightly different genetic make up
of Indian cluster can be seen from the fact that all the
isolates of this cluster have T in place of C at nt position,
332 (Fig. 2). In addition, the Indian branch at subgroup 1.1
is further subdivided into two sub-branches (Fig. 3)
indicating the minor genetic differences. Isolates of the
larger sub-branch differ by A! G substitution (nt position
251, Fig. 2) from those of smaller sub-branch. Thus it is
clear that there were at least two populations of subgroup
1.1-viruses involved in the outbreaks during 2006–2008 in
the country.
Interestingly, lone isolate (IND-294/08) from our study
belongs to subgroup 2.2 and has formed a small cluster
with eight other Indian isolates within the same subgroup
(Fig. 3). From the phylogenetic tree (Fig. 3) as well as from
aligned nt sequences (Fig. 2), it can be seen that members
of this cluster show higher divergence among themselves
compared to another Indian cluster within subgroup1.1.
The isolate, IND-294/08, which was collected from an
outbreak in Harohalli, Karnataka, differs from other
isolates (IND-296/08, IND-298/08 and IND-299/08) of
the same outbreak. Grouping of this isolate within the
subgroup 2.2 has held true, even when the phylogenetic
trees were constructed by maximum likelihood and
UPGMA methods (data not shown). It is about 2.6%
nucleotide divergent from the other isolates of the same
subgroup from Europe. Comparison of 50UTR and other
regions of the genome from a large pool of subgroup 2.2
isolates may be needed to determine the origin of this
isolate. Nevertheless, grouping of this isolate within
subgroup 2.2 indicates that Harohalli outbreak was caused
by viruses of two different lineages. Though slightly
surprising, it was laterfound that new stocks were brought
to this farm from different sources and outbreak involving
two different strains was a mere coincidence rather than a
sequencing error.
Based on aligned nt sequences and results of the
phylogenetic analysis, it can also be speculated that the
subgroup 1.1 CSF viruses in India have probably originated
from European isolates. It can be noted that the cross
breeding programmes of early 20th century to uplift the
genetic make up of indigenous pigs using European
counterparts correlate well with the first documented
report of CSF in India (Sapre et al., 1962). However, it is not
clear whether hog cholera was present in India much
before that date, particularly during the colonial period. It
is also noteworthy that lapinized vaccine strain (HCLV
India), currently used in India was originally obtained from
CVL Weybridge, UK (Singh et al., 2004) and the larger sub-
branch of subgroup 1.1-Indian isolates and vaccine strains
from Europe, India and China (Figs. 2 and 3) still share a
substitution from A ! G (nt position 251, Fig. 2). Therefore
it is plausible that subgroup 1.1 Indian CSF viruses have
originated from European isolates, which over the years,
after their establishment in Indian pig population, have
evolved to become slightly divergent.
In Europe, a shift from group 1 viruses, which
predominated in the 1940s and 1950s, to group 2 in the
1980s has been reported (Paton et al., 2000). Although, the
later outbreaks in Basevalde, Belgium in the year 1989 and
Ukrine (1990s) have been caused by group 1 viruses, they
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Fig. 2. Alignment nucleotide sequence of CSFV isolates corresponding to positions 200–349 of the vaccine strain, Riems (Genbank Acc. No. AY259122). Dot
(.) indicates sequence identity, asterisk (*) region not sequenced, and dash (-) represents gaps. Substitution specific to the Indian cluster is shaded.
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Fig. 2. (Continued).
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were speculated to be either due to reintroductions or
vaccine escapes (Paton et al., 2000). Recent reports from
China (Tu et al., 2001), Columbia (Sabogal et al., 2006),
Taiwan (Deng et al., 2005) are also suggestive of similar
shift. As a consequence, group 2 and 3 viruses are now
predominantly involved in CSF outbreaks world over. In
this context, predominance of group 1 viruses in CSF
outbreaks in India is slightly surprising. We consider the
following possibility to explain the said situation: since pig
husbandry in India is mainly limited to North-Eastern
Fig. 3. Phylogenetic tree based on the analysis of 150 nucleotides within 50UTR. The tree is outgrouped to the corresponding sequence of the Kanagawa/74
(GenBank Acc. No. AB252185) isolate and constructed using the neighbour-joining method. Numbers at the node represent the bootstrap values after 100
replicates. Genbank accession numbers are given in the parenthesis. Indian cluster on the phylogenetic tree is encircled. *Sequences from Greiser-Wilke
et al. (1998).
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states and few pockets in other states, classical swine fever
never assumed epidemic proportions and thus was
restricted to few sporadic outbreaks every year. With
the decline of cross breeding programme, which peaked
during 1960–1980s, the probable new virus (groups 2 and
3) introductions from outside the country also came down
and as a result, group 1 virus, which was present in larger
proportions due to repeated introductions, continues to
dominate the outbreaks. However, frequent detection/
isolation of group 2 viruses in the recent years, particularly
fromtheNorth-Easternstatesmightbe anindicationofthe
things to come.
Finally, phylogenetic analysis conducted in this study
proves to be useful epidemiological investigation of the
CSF outbreaks in India. Large scale study involving other
regions of the genome in the future may be useful for
further resolving the groups as well as the movement and
spread of the disease in our country.
Acknowledgements
Authors are grateful to the Indian Council of Agricul-
tural Research for providing funds and necessary facilities.
We are also thankful to the Directors and the scientists of
various state biological institutes and veterinary colleges.
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