Cell culture adapted sheeppox virus as a challenge virus for potency testing of sheeppox vaccine.

Abstract

Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use. The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2 TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its propagation and titration.

Full text

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Indian Journal of Experimental Biology
Vol. 46, October 2008, pp. 685-689






Cell culture adapted sheeppox virus as a challenge virus for potency testing of
sheeppox vaccine
M Hosamani1, V Bhanuprakash1, D J Kallesh, V Balamurugan, A Pande & R K Singh*
Division of Virology, Indian Veterinary Research Institute, Campus Mukteswar 263 138, India
Received 1 January 2008; revised 1 July 2008
Sheeppox virus from an outbreak of sheeppox that occurred in Srinagar (Jammu and Kashmir, India) in 2000 was
isolated by inoculation of susceptible sheep and further re-isolated in cell culture. The field virus, adapted to grow in lamb
testes culture, was evaluated for its potential use as challenge virus in potency testing of sheeppox vaccine currently in use.
The virus (passage 6) produced severe disease in susceptible sheep when inoculated subcutaneously with a dose of 106.2
TCID50. The virus identity was confirmed by PCR, sequencing of P32 gene and species-specific signature residues identified
in deduced aa sequence of the gene. The virus was successfully evaluated for its virulence using two batches of sheep pox
vaccines. Use of this field virus enables consistent potency experiments of sheeppox vaccines avoiding use of animals for its
propagation and titration.
Keywords: Cell culture, Potency testing, Sheeppox vaccine, Sheeppox virus, Virulent virus challenge.
Sheeppox is a notifiable disease of Office International
des Epizooties (OIE). The disease is recognized as
most fatal in sheep, characterized by formation of
dermal pox lesions and nodular lesions on the
respiratory and digestive systems. The disease causes
high morbidity (100%) and mortality up to 75% in
young and immunologically naïve animals. The
causative sheeppox virus (SPPV) is classified in the
genus Capripoxvirus along with goatpox virus (GTPV)
and lumpy skin disease (LSDV) of Poxviridae family1.
These viruses are closely related antigenically2,3. A
number of outbreaks of sheeppox have been reported in
the recent past4-7. The live attenuated sheep pox
vaccine is used for mass immunization as a preventive
measure in India. Hence, a large quantity of vaccine
needs to be produced by many public and privately
owned vaccine-manufacturing units to meet the
national requirement. Potency testing of live attenuated
sheep pox vaccine was performed in sheep using scab
suspension derived from animals experimentally
infected with virulent SPPV with a minimum dose of
skin reactive dose50 (SRID50)105.0/ml per animal where
the exact number of viral particles cannot be
ascertained.
In the present study, a field isolate of SPPV from
Srinagar district of Jammu and Kashmir, India was
isolated and characterized for its potential use as a
challenge virus in potency studies. The virus was
initially revived in sheep and subsequently adapted in
Primary lamb tests (PLT) cell culture. The virus
grown in bulk at 6th passage level was tested for
producing experimental infection in a susceptible
sheep. A major immunogenic gene (P32) of the virus
was also amplified by PCR and its sequence
determined to confirm the virus identity.

Materials and Methods
Virus isolation—Scab suspension (10%w/v) from
the outbreak was used for experimental reproduction of
the disease in a sheep. The virus is maintained in the
laboratory as a scab form through regular animal
passages. It was isolated in primary/secondary lamb
testes (SLT) cell culture grown in Glasgow’s modified
minimum essential medium (GMEM, Sigma, St Louis,
USA) supplemented with 10% newborn calf serum
(HyClone, USA). For virus isolation, sonicated scab
suspension was added on to LT cell monolayer and
allowed for viral adsorption for 1 hr, in 1ml of GMEM.
Un-adsorbed virus was then removed by washing the
monolayer thrice with GMEM. Cells were fed with
medium containing 2% serum and observed daily for
appearance of specific cytopathic effects (CPE).
PCR and sequencing—Viral DNA was extracted
from cell culture virus using AuPreP DNA Extraction
_________________________
*Correspondent author
Telphone: +91-5942-286348,
Fax: +91-5942-286347
Email: rks_virology@rediffmail.com
1Both authors share equal contribution

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INDIAN J EXP BIOL, OCTOBER 2008


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Kit [M/s Life Technologies (India) Pvt Ltd, New Delhi,
India] as per manufacturer’s protocol. Diagnostic PCR
was carried out using primers-B68 and B698. Full-
length P32 gene was also amplified using the A95 and
B7 primers8. PCR amplicon was purified using
MinElute gel extraction kit (QIAGEN, Hilden,
Germany) as per manufacturer’s instructions, ligated to
pGEMT-Easy vector (Promega, USA) and transformed
into competent E.coli strain (TOP10, Invitrogen, USA).
Sequencing of the PCR amplicon was carried out using
an automated DNA sequencer (ABI PRISM 3100,
Perkin Elmer) in either direction.
Sequence analysis—Nucleotide and deduced amino
acid sequences were
representative capripoxvirus isolates from different
regions and sequence identity (%) was determined
using Clustal W method9.
Animal inoculation and virus confirmation—Virus
titrated10 in SLT culture was inoculated into susceptible
sheep. Animal was inoculated subcutaneously with 106.2
TCID50 of virus (passage 6) in 1.0 ml dose at multiple
sites @ 0.1 ml/site. Animal was observed daily for rectal
temperature and appearance of systemic or local
responses. After experimental infection, virus was
confirmed by detection of viral nucleic acid in the scab
samples collected from the affected animals using PCR8.
Validation of the virus for challenge studies—Two
batches of sheep pox vaccines, made using Rumanian
Fanar from Veterinary Biologicals, Lucknow and
Srinagar strain from the Division of Virology were
tested using this challenge virus as per the prescribed
protocol11. Two groups each of 6 sheep, which were
sero-negative to SPPV antibodies, were administered
with a recommended field dose (102.5 TCID50 in 0.2
ml) of each vaccine at the abaxial surface of the tail. A
compared with other
third group of two sheep was maintained as in-contact
control. Temperature of all the animals was recorded
for a period of 14 days following vaccination.
Appearance of clinical symptoms was also observed
regularly. Then, all the animals were challenged with
virulent SPPV (Srinagar 38/00 isolate, 6th passage) at
106.5 TCID50/animal intradermally at lower abdomen
on day 28 post immunization. The appearance of
clinical symptoms and temperature response were
recorded daily for a period of 14 days.

Results
Virus isolation—Virus induced cytopathic effects
(CPE) were characterized by rounding, ballooning of
cells, increased refractility and detachment of PLT
cells. CPE was initiated by day 2-3 post infection (PI)
and progressed to 80% by day 5-6. Virus was
confirmed by capripox virus-specific PCR, which
amplified a product of 390 bp (Fig. 1).
PCR and Sequence analysis of P32 gene—PCR
using the primer pair A95 and B7 yielded a product of
≈1000 bp. Sequence of the gene showed a open
reading frame (ORF) of 972 bp (GenBank acc#
EU835938). The gene showed 100% sequence
identity with other SPPV, followed by 98.5% and
97.7% identity with LSDV and GTPV respectively at
nt level. Similarly, the deduced aa sequence identity
was >99%, 97.2% and 95.4% with SPPV, LSDV and
GTPV, respectively (Table 1).
Animal observations—At 6th passage, virus was
inoculated into susceptible sheep for producing the
experimental infection. Animal started signs of
disease as early as 2 days with moderate rise in rectal
temperature. From 4th day onwards, local sites of
inoculation showed hyperaemia accompanied with

Fig. 1—(a) Cytopathic changes of sheeppox virus (Srinagar) in primary lamb testes. Healthy mock infected secondary cell culture (10x);
(b) SPPV (Srinagar) infected cells showing characteristic rounding, ballooning and syncytia with detachment on day 4 post-infection
(10x); (c) PCR products from SPPV sample; Lane M: 100 bp DNA molecular size marker, Lane 1: SPPV (Srinagar 38/00) isolate, Lane 2
& 3, Vero cell control.

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HOSAMANI et al.: CELL CULTURE ADAPTED SHEEPPOX VIRUS FOR CHALLENGE STUDIES


high rise in rectal temperature (106°C). The severity
of the local lesions increased gradually over the next
3-4 days and generalized subcutaneous redness was
noticed on day 6-post infection. Animal was pyrexic
(106.8°C) (Fig. 2) and off fed from day 5 onwards.
Nodular lesions were found on the face, eyelids and
groin, though generalized hyperaemia was more
prominent than the nodular type of dermal lesions
(Figs 3a and 3b). Animal was treated with broad-
spectrum antibiotics and analgesics to reduce the
clinical severity. Scab samples collected were
confirmed by PCR amplification.
Table 1⎯Percent sequence identity of P32 gene of SPPV-
Srinagar 38/00 isolate with other capripoxviruses at the nucleotide
(nt) and amino acid (aa) levels.

Sl. No Virus strain nt level aa level
687
Validation of the virus for challenge studies—
When two batches of vaccines were tested, the
vaccinated animals remained normal without showing
any disease specific clinical signs, whereas, in contact
healthy sheep succumbed to the disease showing rise
in body temperature and dermal lesions (Figs 3c and
3d) with similar clinical course as described.

Discussion
In the present study, isolation and characterization of
a field isolate of SPPV has been carried out in order to
employ a cell culture adapted virulent field sheeppox
virus as challenge virus. The virus was associated with
a severe outbreak of sheeppox in Srinagar district of

Fig. 3⎯(a) Sheep showing dermal lesions on the lower abdomen at the site of inoculation on day 4 post infection; (b) and on the abaxial
surface of the tail with profuse erythematous lesions in subcutaneous tissue on day 5 post infection on experimental infection with SPPV-
SRIN 38/00 (passage 6); (c) In contact sheep showing dermal lesions on the lower abdomen at the site of inoculation on day 4 post
infection; (d) No takes were observed in sheep vaccinated and challenged on 28th day post vaccination .
1 GPPV-MU
(AY159333)
GPPV-G20
(AY077836)
SPPV-RF
(AY368684)
SPPV-Niskhi
(AY077834)
LSDV-58
(AF325528)
LSDV-99
(AF409137)
97.7 95.4
2 97.9 96.3
3 99.8 99.1
4 100 99.7
5 98.2 97.2
6 98.5 97.2
Figures in parentheses are GenBank accession number

Fig. 2⎯Mean rectal temperature of the sheep following
experimental infection with SPPV

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INDIAN J EXP BIOL, OCTOBER 2008


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Jammu and Kashmir during August 2000. Virus
isolation was carried out in lamb testes cell culture and
the virus produced visible CPE in the second cell
culture passage. Virus harvested at 80-90% cytopathic
changes was further confirmed by diagnostic capripox-
specific PCR. Virus was passaged till 6 passages and
subsequently used to produce experimental infection in
sheep.

Nucleotide sequence of the P32 gene amplicon
showed an ORF of 972 bp like in other SPPV, which is
969 bp in GTPV. Members of capripoxviruses are
highly related among themselves antigenically while
phylogenetically they are distinct12. Molecular methods
such as duplex PCR13 and identification of signature
residues in the sequence of immunogenic gene are
often used for differentiation14. Many signature
residues that are unique to SPPV including L62F,
S132L and I134T were identified in the deduced amino
acid sequence of the protein. The virus showed high
sequence identity with SPPV having 99.8% and
>99.1% identity at the nt and aa level, respectively.

The cell culture adapted virus at passage 6 was used
to produce experimental infection in sheep by dermal
inoculation. Initially, animal remained apparently
healthy for the first three days, despite moderate rise in
rectal temperature as early as day 2 PI and no local
reactions were evident at the site of inoculation.
Generalized hyperaemia was observed from 4-5 day of
infection, as evident from subcutaneous redness,
although external raised dermal lesions were
moderately observed. Animal showed only little
improvement in spite of rehabilitation, initiated on day
7-post infection. Similar results were obtained in in-
contact animals when two batches of sheeppox
vaccines were tested. The vaccinated animal remained
apparently normal. Broad-spectrum antibiotic and
analgesics were administered to reduce the animal
suffering.

Though protection induced upon sheep pox
vaccination can be confirmed either by assessing cell
mediated and/or humoral immune responses, challenge
study is the conclusive to ascertain the protection15,16.
Therefore, it is required to maintain virulent challenge
virus in the laboratory for vaccine efficacy testing. The
present study showed that the cell culture adapted virus
(6th passage) retained
characteristic capripox disease on experimental
infection in sheep and may be used in potency testing
of sheep pox vaccine. Its application should overcome
the disadvantages of using of homologous animals for
its virulence showing
propagation and titration of virulent challenge
sheeppox virus. The virus thus characterized has the
potential application as challenge virus for the potency
testing of various live attenuated sheeppox vaccines
currently used in India.

Acknowledgement
Thanks are due to the Director, Indian Veterinary
Research Institute (IVRI) and to the Ministry of
Environment and Forests, Government of India, New
Delhi for funding the project under AICOPTAX.

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