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Research in Veterinary Science 85 (2008) 176–183

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Genetic characterization of Indian peste des petits ruminants

virus (PPRV) by sequencing and phylogenetic analysis of fusion
protein and nucleoprotein gene segments
N. Kerur a, M.K. Jhala a,*
, C.G. Joshi b

a
Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University,
Anand 388 001, Gujarat State, India
b
Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry,
Anand Agricultural University, Anand 388 001, Gujarat State, India

Accepted 30 July 2007

Abstract

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats, endemic in India. The study was undertaken to
characterize the local PPRV by sequencing fusion (F) protein and nucleoprotein (N) gene segments and phylogenetic analysis, so as
to focus on genetic variation in the field viruses. Selected regions of PPRV genome were amplified from clinical samples collected from
32 sheep and goats by RT-PCR and the resulting amplicons were sequenced for phylogenetic analysis. The phylogenetic tree based on the
322 bp F gene sequences of PPRV from five different locations clustered them into lineage 4 along with other Asian isolates. While the
425 bp N gene sequences revealed a different pattern of branching, yielding three distinct clusters for Nigerian, Turkey and Indian iso-
lates. Thus, classification of PPRV into lineages based on the N gene sequences appeared to yield better picture of molecular epidemi-
ology for PPRV.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Peste des petits ruminants virus; PPRV; F gene; N gene; Sequencing; Phylogeny

1. Introduction continent. However, in recent years, the disease has been

Peste des petits ruminants virus (PPRV), a member of
the genus Morbillivirus in the family Paramyxoviridae
(Gibbs et al., 1979) is the causative agent of Peste des petits
ruminants (PPR), an acute viral disease of goats and sheep,
characterized by fever, erosive stomatitis, conjunctivitis,
gastroenteritis and pneumonia. PPR is a highly contagious
disease, morbidity and mortality can be as high as 100%
and 90%, respectively (Abu-Elzein et al., 1990). Following
the first report of the disease in sheep and goats (Gargaden-
nec and Lalanne, 1942), for many years it was believed to
have remained restricted to western part of the African
*
Corresponding author. Tel.: +91 2692 261505; fax: +91 2692 261201.
E-mail address: mkjhala_2003@yahoo.co.in (M.K. Jhala).
recorded in several parts of the world, Southern Asia
including India, Pakistan and Nepal; Near East and the
Arabian Peninsula including Islamic Republic of Iran,
Iraq, Israel, Jordan, Kuwait, Lebanon, Oman, Saudi Ara-
bia, the United Arab Emirates and Yemen. In India, the
first outbreak of PPR was described by Shaila et al.
(1989), and since then, the disease has become endemic in
India, causing severe economic losses and is presently con-
sidered as one of the major threats to about 200 million
small ruminant population of the country.
PPR can be confused clinically with rinderpest, and
hence the clinical observations for both the diseases should
always be confirmed by a laboratory test. The diagnostic
techniques used in the past viz. VNT, AGID and isolation
of the virus in cell culture are time consuming and labour

0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2007.07.007
 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183
intensive. With the advent of molecular biological tech-
niques like PCR, rapid and specific diagnosis of PPR has
become possible (Forsyth and Barrett, 1995; Couacy-
Hymann et al., 2002).
PPRV is genetically grouped into four distinct lineages
(1, 2, 3 and 4) on the basis of partial sequence analysis of
fusion protein (F) gene; despite the fact that only a single
serotype of PPRV has been reported (Shaila et al., 1996;
Dhar et al., 2002; Ozkul et al., 2002). This classification
of PPRV into lineages has broadened our understanding
of the molecular epidemiology and worldwide movement
of PPR viruses. However, continuing increase in circula-
tion of the virus among the susceptible population in ende-
mic countries like India, a demand was felt to delve into the
molecular details of the field virus. The objectives of our
research were to detect the PPRV in suspected outbreaks
of PPR among sheep and goats of Gujarat (a western state
of India), by employing F gene based primers described by
Forsyth and Barrett (1995). In addition, a new pair of
nucleoprotein (N) gene based primers was developed for
specific and sensitive detection of PPRV from clinical sam-
ples. Further, the genetic relationships between these
viruses were investigated comparing the nucleotide
sequences of the PCR products amplified from F and N
genes with other PPRV sequences available in GenBank
of National Center for Biotechnology Information
(NCBI).
2. Materials and methods
2.1. Clinical samples and reference virus
Blood, nasal swabs, spleen and lymph node samples
from 32 sheep and goats with symptoms suggestive of
PPRV infection were collected. A total of 48 clinical sam-
ples from five outbreaks at different locations (Anand,
Mehsana, Banaskantha, Patan and Rajkot districts) in
Gujarat were collected. A representative sample from each
district was subsequently taken for sequencing work, where
the samples from Anand, Mehsana and Banaskantha were
from goats, while the same from Patan and Rajkot districts
were from sheep. Lyophilized freeze-dried live PPR vaccine
(Sungri isolate) obtained from the Division of Virology,
IVRI, Mukteswar, India, was used as the reference virus
during the course of study.
2.2. Oligonucleotide primers
The random hexamers used for cDNA synthesis and the
primers F1/F2 and N1/N2 used were obtained from Sigma
Genosys and MWG-Biotech AG, Germany. The primers
N1/N2 amplifying a region of N gene were designed in
house using the software GeneFisher 1.2 available online
(http://bibiserv.techfak.uni-bielefeld.de/genefisher/), based
on the published sequence information (accession Nos.
AJ563705, AY560591, X74443 and AJ849636). Details of
the primers are listed in Table 1.
177
2.3. RT-PCR for PPRV detection
RNA was extracted from the clinical samples (100 mg
tissue), reference vaccine virus (250 ll) and a blood sample
(200 ll) from an apparently healthy goat, using TRI
ReagentÒ (Sigma–Aldrich), as per the modified method
of guanidium thiocyanate-phenol-chloroform extraction
method originally described by Chomczynski and Sacchi
(1987), and the RNA was resuspended in 20 ll of nuclease
free distilled water. Five microliters of the extracted RNA
was reverse transcribed using Omniscriptä Reverse Trans-
criptase Kit (QIAGEN, Germany). The reaction was car-
ried out in 200 ll tubes with the following conditions:
1 ll of random hexamer, 0.125 mM each dNTPs, 10 units
of ribonuclease inhibitor (MBI Fermentas), two units of
OmniscriptÒ reverse transcriptase and the buffer provided
by the manufacturer in a total volume of 20 ll. The reac-
tion was carried out at 37 °C for 60 min and stopped by
incubation at 95 °C for 10 min.
Three microliters of the RT product was used as tem-
plate for the PCR, carried out in 200 ll thin walled PCR
tubes, in a final reaction volume of 25 ll with following
reagents: 12.5 ll of PCR Master mix (MBI Fermentas)
containing 0.05U/ll TaqDNA polymerase (recombinant)
in reaction buffer, MgCl2 (4 mM) and dNTPS (0.4 mM of
each), 1 ll of forward and reverse primers (10 pmoles of
each primer) and 7.5 ll of deionized water. In case of F1/
F2 primers, 35 cycles of amplification was carried out using
PCR reaction conditions as described by Dhar et al. (2002).
The reaction conditions for N1/N2 primers were as fol-
lows: 94 °C for 10 min, followed by 35 cycles at 94 °C for
30 s, 50 °C for 30 s and 72 °C for 45 s, and a final extension
step at 72 °C for 10 min. Ten microliters of the PCR prod-
ucts were resolved on a 2% agarose gel stained with 0.5 lg/
ml ethidium bromide.
2.4. Cloning and sequencing of PCR products
The PCR products (a representative sample from each
location) specific to F and N genes of PPRV amplified
using the primer pairs F1/F2 and N1/N2, respectively,
were ligated into pTZ57R/T vector supplied with InsT/
Acloneä PCR product cloning kit (MBI Fermentas) fol-
lowing the manufacturer’s instructions. Three microliters
of ligation product was used to transform Escherichia coli
DH5-a made competent, as per the method described by
Sambrook and Russel (2001). The bacteria containing the
recombinant plasmids were screened by standard aplha
complementation method using X-gal and IPTG on LB
agar plates containing ampicillin (50 lg/ml). The white col-
onies containing desired inserts were screened by colony
PCR using previously described conditions for each set of
primers. The bacterial colonies containing desired insert
were cultured in LB broth containing appropriate antibi-
otic and the recombinant plasmid was purified from 3 ml
of culture using QIAprepÒ Spin Miniprep Kit (QIAGEN,
Germany) following manufacturer’s instructions. The pres-
178 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183
Table 1
Details of the primers used for detecting PPRV by PCR
Sr. no. Primer Primer sequence
1 F1 (F) 50 ATC ACA GTG TTA AAG CCT GTA GAG G 3 0
2 F2 (R) 50 GAG ACT GAG TTT GTG ACC TAC AAG C 3 0
3 N1 (F) 50 GAT GGT CAG AAG ATC TGC A 3 0
4 N2 (R) 50 CTT GTC GTT GTA GAC CTG A 3 0
a
Position of the primers is as per the numbering of Genbank sequence X74443.
ence of appropriate insert was once again confirmed by car-
rying out PCR using the purified recombinant plasmid as
template. The inserts in the recombinant plasmids were
sequenced by cycle sequencing using ABI PRISMÒ 310
Genetic Analyzer (Applied Biosystems, USA) and Big-
DyeÒ Terminator v3.1 Cycle sequencing kit (Applied Bio-
systems, USA).
2.5. Sequence analyses
The nucleotide sequences of 322 bp and 425 bp seg-
ments amplified from F and N genes, respectively were
aligned with corresponding sequences available in Gen-
Bank using Clustal W (Thompson et al., 1994). Phyloge-
netic analysis of F and N gene segments of PPRV was
performed with the DNADIST and FITCH programmes
of PHYLIP 3.65 software (Felsenstein, 1989). The phylo-
genetic tree was generated as follows: A set of 500 aligned
sequences was generated by the bootstrap method (Seq-
boot programme). Genetic distances were calculated for
each set of aligned sequence with the DNADIST pro-
gramme, and the trees were constructed by the FITCH
programme using corresponding sequence of Rinderpest
virus (RPV) as an out group. Finally, the consensus tree
for 29 (F gene sequences) and nine (N gene) sequences
of representative isolates was generated using the Con-
sense programme.
Further, Phylogenetic network analysis was carried out
using the software Network 4.111 (available for download
from website www.fluxus-engineering.com). Both F and N
gene sequence data were processed by star contraction
algorithm (Forster et al., 2001), followed by processing
with median-joining (MJ) network algorithm (Bandelt
et al., 1999) to draw separate phylogenetic network for
each gene sequence, depicting the points of variation
among the sequences under comparison.
3. Results
3.1. Detection of PPRV by F and N gene based RT-PCR
Amplification with primers F1/F2 yielded an expected
amplicon of 372 bp in reference vaccine virus as well as
24 (50.00%) samples consisting of 12 blood, 11 nasal swabs
and one lymph node. The remaining 24 (50.00%) samples
Target gene Expected amplicon Reference
and position size
F gene 777-801 372 bp Forsyth and Barrett (1995)
F gene 1124-1148
N gene 1208-1226a 463 bp Designed during the
N gene 1670-1652a experiment
including 13 blood, 10 nasal swab and one spleen samples
as well as the blood sample taken from apparently healthy
goat failed to produce the targeted amplification.
A total of 18 representative samples, half of which were
negative and the other half positive by F gene based RT-
PCR, were selected for detection of PPRV by the newly
designed primers N1/N2. Care was taken to include at least
two samples from each of the five locations (Anand, Meh-
sana, Banaskantha, Patan and Rajkot districts). Primers
N1/N2 could produce the desired amplicons (463 bp) in
12 of the 18 samples tested.
3.2. Sequence analyses
The analysis of the 322 bp F gene sequences (accession
Nos. DQ267183, DQ267184, DQ267185, DQ267186,
DQ267187) revealed that, the homology among the five
field isolates (Ana/Guj/05, Ptn/Guj/05, Meh/Guj/05, Bsk/
Guj/05 and Rjk/Guj/05) of present study ranged between
96% (for Bsk/Guj/05 and Ptn/Guj/05) and 99% (for Ana/
Guj/05 and Rjk/Guj/05). None of the field isolates had
homology less than 96% with any of the Indian isolates
described previously. Further, none of the field isolates
had identities less than 97% with the reference vaccine virus
sequenced. All the field isolates and vaccine virus shared
comparatively lesser homology (92–93%) with the Nigerian
isolate Nigeria/75/1.
Similarly, analysis of 425 bp sequences of N gene (acces-
sion Nos. DQ267188, DQ267189, DQ267190, DQ267191,
DQ267192) of field PPRV isolates (Ana/Guj/05, Ptn/Guj/
05, Meh/Guj/05, Bsk/Guj/05 and Rjk/Guj/05) revealed
that all the isolates from the present study and Indian iso-
lates described earlier were closely related to each other
with sequence identity of 98–99%. Homology of all the field
isolates with Nigeria/75/1, isolates of Turkey (Turkey-2000
and Turkey/00) and Sungri/96 ranged between 88–89%,
94–95% and 98–99%, respectively.
The Phylogenetic tree (Fig. 1) based on the 322 bp
sequence of F gene clustered all the isolates of India includ-
ing the isolates from the present study, isolates of turkey,
an isolate of Pakistan and Iran into a separate branch from
Nigerian isolate. While the Phylogenetic tree based on the
425 bp N gene sequences out-rooted the Nigerian isolate,
while the Turkey and Indian isolates were grouped into
two clusters, although with low bootstrap values (Fig. 2).
 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183 179

Fig. 1. Phylogenetic relationship of PPRVs of Gujarat to other virus isolates, based on the 322 bp partial sequence of fusion (F) protein gene. All the
Asian isolates (lineage 4) including the isolates from the present study clustered into a separate branch from the Nigerian isolate. The lone isolate Bsk/Guj/
05 clustered (C2) with previously described kcch2000 isolate, while the other four isolates clustered along with vaccine virus in C1.

The network analysis based on 322 bp F gene sequences
resulted in a large central node URI99 on the torso, with
three identical isolates Uri99, Izat94 and Sung96 in it
(Fig. 3). The torso was formed by central node URI99
and LAXM99. Izat98 along with Chirg98 formed a second
node (IZAT98), outside the torso. The five isolates of the
present study differentiated into five minor taxa. The iso-
late Rjk/Guj/05 was the closest to the central node
URI99 with a single mutation (at position 263) disregard-
ing the torso. The isolate of Banaskantha district (Bsk/
Guj/05) with six mutations was the most distant taxon
from the central node. The other isolates of Gujarat,
Ana//Guj/05, Meh/Guj/05 and Ptn/Guj/05 showed, three,
three and two mutations, respectively disregarding the
torso.
Similarly, the Network analysis for the 425 bp sequence
of N gene showed the isolate Bsk/Guj/05 in the torso
(Fig. 4) and the four other isolates of the present study
showed comparatively fewer mutations disregarding the
torso. Among the isolates of Gujarat, comparatively more
number of mutations (4) were seen in Meh/Guj/05, while
the isolate Rjk/Guj/05 showed only one mutation. The iso-
lates Sungri/96, Ana//Guj/05 and Ptn//Guj/05 showed two
mutations each.
180 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183
Fig. 2. Phylogenetic relationship of PPRVs of Gujarat to other virus
isolates, based on the 425 bp partial sequence of nucleoprotein (N) gene.
C1, C2 and C3 clusters include the isolates from Nigeria, Turkey and
India, respectively, showing better geographical localization of the
isolates.
4. Discussion
The F gene based RT-PCR developed by Forsyth and
Barrett (1995) has gained much popularity, though PCR
based on other gene targets have become available only
recently. The PPR specific primers F1 and F2 developed
by aforementioned workers give an amplification of a
372 bp region between positions 777 to 1148 nucleotides
of PPRV F gene. In the present study, PPRV could be
detected in 24 (50%) of the 48 clinical samples tested, which
confirmed PPRV in all the five outbreaks at different loca-
tions, with highest number (18) of animals affected in
Northern Gujarat region.
Although, the complete history of the current PPR out-
breaks were not available, there were reports of six PPR
outbreaks in Rajkot district (western part of Gujarat) in
1997–98, where 545 animals (both sheep and goats) were
affected, of which, 400 died (ADMAS Annual Report,
2003–04). The same report has also detailed one outbreak
in sheep of Banaskantha district (northern part of Guja-
rat), where eight animals died in 2002. No incidence was
reported earlier from Anand (central Gujarat), Mehsana
and Patan districts (both in northern Gujarat). However,
incidences in seven other districts belonging to northern,
central and western parts of Gujarat, with one or two out-
breaks were reported from 1996 to 2002. The majority of
these outbreaks occurred between November and February
(winter months), and a few in March and April. This shows
that PPR outbreaks did not occur every month, but they
appeared every year in Gujarat state, making the disease
endemic.
Recently, there have been a couple of attempts with
promising results, targeting N gene for PCR based detec-
tion of PPRV (Couacy-Hymann et al., 2002; George,
2002). The N gene based primers, despite being proved to
offer higher sensitivity, they are yet to gain wide acceptance
for the diagnosis of PPRV. The primer pair N1/N2 was
designed in-house based on the published sequences in
Genbank. N gene codes for an internal structural protein
and also mRNAs of N gene are the most abundant tran-
scripts of the virus, making it attractive target for develop-
ment of a highly sensitive diagnostic assay. Indeed, due to
lack of proof reading function of their polymerases, RNA
viruses are known to contain high rate of nucleotide substi-
tution error frequency (Steinhauer and Holland, 1986).
Therefore, to ensure efficient amplification and detection
of such viruses, the primers have to be designed by identi-
fying conserved region in the published sequences of the
viral isolates from different geographical region; this strat-
egy minimizes the risk of false negative result due to primer
template mismatch. In order to design PCR primers that
would selectively amplify PPRV genome without cross
reacting with RPV, the N gene sequences of PPRV and
RPV available in the Genbank were aligned with Clustal
W software to identify a region unique to PPRV among
all PPRV strains. Hence, the terminal 1/3rd region of
PPRV mRNA at 3 0 end, which shares low homology with
other Morbilliviruses (Diallo et al., 1994) was chosen as a
target for designing the primers. The alignment of N1/N2
with PPRV N gene sequences showed 100% homology in
all the strains, except for a mismatch at positions 4 base
from 3 0 end of N1 in case of Sungri/96 (accession No.
AY560591) strain. Whereas, the alignment with N2 showed
one mismatch at position 15 from 3 0 end in case of Nigeria/
75/1 (accession No. X74443). Both the primers (N1and N2)
satisfied the minimal homology requirement for PCR, as
defined by Sommer and Tautz (1989), i.e. the first three
nucleotides at the 3 0 ends are conserved in all the N gene
sequences of PPRV available to date in Genbank. The
available sequences were from four different strains, Nige-
ria/75/1, Sungri/96, Turkey2000 and Turkey/00. The align-
ment of primers N1/N2 shows sufficient mismatches with
RPV ensuring that they do not amplify the RPV genome.
Primers N1/N2 detected PPRV in a greater number of
samples than F gene based primers. Three samples negative
 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183 181

Fig. 3. Phylogenetic network analysis based on the partial fusion (F) protein gene sequence of field PPRVs with corresponding sequences published in
GenBank. The five isolates from the present study differentiated into five minor taxa showing mutations ranging from one to six. The Nigerian isolate
showed maximum number (18) of mutations.

Fig. 4. Phylogenetic network analysis based on the partial nucleoprotein (N) gene sequence of field PPRVs with corresponding sequences published in
GenBank, showing the isolate Bsk/Guj/05 in the torso. The other four isolates of the present study showed one to four mutations. Isolates from Turkey
and Nigeria varied greatly with 18 and 48 mutations, respectively.

by F gene based RT-PCR were positive by N gene based the N gene based primers were more sensitive than F gene
RT-PCR, however, no sample positive by F gene based based primers. The higher sensitivity of N1/N2 can be
RT-PCR was negative by N gene based RT-PCR. Thus, attributed to abundance of PPRV N gene transcripts than
182 N. Kerur et al. / Research in Veterinary Science 85 (2008) 176–183
Table 2
Sequence identity of partial F and N gene sequences between isolates of different geographical locations
Sr. no Geographical location
1 Indian isolate (lineage 4)
2 Indian isolate (lineage 4)
3 Turkey isolates (lineage 4)
4 Indian isolate (lineage 4)
5 Turkey isolates (lineage 4)
the F gene (Ghosh et al., 1995), thus making N gene suit-
able target for improving the sensitivity of RT-PCR for
detection of PPRV from clinical samples.
To assess the genetic similarity and divergence among
the field PPRVs of Gujarat as well as their relatedness to
some of the previously described isolates, the partial F
and N gene sequence data were analysed in silico by
employing software tools viz. Clustal W, PHYLIP and
Network 4.111. Sequence analysis of F and N gene
sequence data revealed that the PPRVs from distant geo-
graphical regions vary to a greater extent in their N gene
sequences than in their F gene sequences (Table 2).
A consensus Phylogenetic tree based on the lineage spe-
cific 322 bp F gene sequence was constructed with help of
PHYLIP programmes. In accordance with the previous
studies (Shaila et al., 1996; Dhar et al., 2002; Ozkul
et al., 2002), all the Asian isolates including the isolates
of turkey, clustered together into a separate branch from
the Nigerian isolate, and the branching was supported by
a bootstrap values of 500 (100%). Since the sequence data
for the African isolates belonging to lineages 2 and 3 were
not available in the Genbank, they were not included in the
Phylogenetic analysis. Thus, from the Phylogenetic tree
(Fig. 1), it is evident that all the isolates of Gujarat clus-
tered together with the other Asian isolates of lineage 4.
Similarly, a Phylogenetic tree (Fig. 2) was generated for
the 425 bp sequence of N gene, which clustered the Nige-
rian isolate into a separate cluster. The isolates from Tur-
key and India (including the five field isolates of the
present study and Sungri/96), were grouped into two clus-
ters, although in a non-significant way. This needs to be
substantiated by including more length of N gene sequence,
so as to arrive at a proper conclusion.
Network analysis for the 322 bp F gene sequences
revealed that branching of Bsk/Guj/05, Avik99, PPRCBE,
Kcch2000 and Hyd99 was in conformity with the branching
pattern shown by PHYLIP programme for the correspond-
ing sequences. Other taxa diverging from the torso corre-
sponded to all the members of C1. As expected, the
Nigerian isolate showed maximum number (18) of muta-
tions disregarding the torso. Similarly, network analysis
for the 425 bp N gene sequences revealed the isolates of Tur-
key and Nigeria varying greatly from the torso with 18 and
48 mutations, respectively. Thus, the Network analysis was
in concordance with the branching pattern shown by the
consensus tree. The phylogenetic network analysis based
Sequence identity
F gene N gene
Turkey isolates (lineage 4) 96–98% 94–95%
Nigerian isolate (lineage 1) 92–93% 88–89%
Nigerian isolate (lineage 1) 92% 90%
Indian isolate (lineage 4) 96–99% 98–99%
Turkey isolates (lineage 4) 98% 99%
on the 322 bp F gene and 425 bp N gene segments by Net-
work 4.111 software, allowed identification of the number
and position of the variation in the nucleotide sequences
by depicting the varying position in each sequence in a pic-
torial format. Further, network analysis clustered the iden-
tical isolates into nodes and non-identical isolates formed
separate taxa branching from the central torso.
In summary, relative to the conventional F gene target,
the N gene target offered better sensitivity for detection of
PPRV from clinical samples. The newly designed primers
N1/N2 proved to be more efficient for this purpose and
can be used widely in PCR diagnosis of PPR. The partial
F gene sequence based classification of PPRV into lineages
placed the local isolates into lineage 4. Classification of
PPRV into different lineages based on N gene sequence
appeared to group the viruses in a better way, thus, giving
better epidemiological picture about PPRV. However,
given the ability for PPRV to mutate, it is desirable to have
more than one set of primer for diagnosis and F and N
genes are the most suitable candidates.
Acknowledgement
The authors are thankful to Dr. R.K. Singh, Head,
Division of Virology, IVRI, Mukteswar, India, for provid-
ing PPR vaccine.
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