1. The development of attenuated strain of camelpox virus for use as safe live vaccine in camels or their offsprings (aged more than six months) for protection against clinical form of camelpox.

3. PREAMBLE TO THE DESCRIPTION
The product was developed at the Division of Virology, IVRI, Mukteswar 263 138, Nainital
[Distt.], Uttarakhand.
3.1 PRIOR ART
The following specifications particularly describe the invention and the manner in which it is
to be performed.
Camelpox is caused by camelpox virus (CMLV) that belongs to the genus. Orthopoxvirus of
Poxviridae family. It is an important generalized infection of camels in Africa, the Middle East and
southwest Asia as the camels are used as beast of burden and for milk. The disease mostly affects
young ones aged 2-3 years and outbreak in a herd is very often associated with weaning or poor
nutrition, with fatal severe form occasionally. Disease occurrence is accompanied with morbidity,
mortality and case fatality rates respectively of 30-90%, 1-15% and 25% (Al-Ziabi et al, imi:
Bhanuprakash et al, 2010a). Recovered animals become life-long immune to re-infection. The
camelpox virus was earlier thought as a zoonotic agent (Leese, 1909; Davies et al, 1975) as per the
documentary evidences from Somalia in smallpox unvaccinated individuals (Kriz, 1982; Jezek et
al, 1983) and recently from India in camel handlers (Bera et al, 2011). The virus is transmitted by
direct contact or indirectly through fomites. CMLV morphologically resembles variola virus
(VARV) (Baxby, 1972). The" incubation period is 4-15 days with an initial rise in temperature
followed by papules on labia, vesicles, pustules and formation of scabs. Camelpox infection is
diagnosed by clinical symptoms, isolation and electron microscopy (EM) and differentiated from
other orthopoxviruses (OPVs) by restriction enzyme analysis (REA) (Murphy et al, 1999). Various
cell lines like HeLa, GMK-AHl, BSC-1, WISH and Vero (Baxby, 1972) are used to adapt and
propagate the CMLV. , - - . - ..
Camelpox in camels can be controlled by vaccination and live vaccines are used in some parts of
the world for the purpose. However, camelpox vaccine is not available in India. The present study
related to the development of a cell culture attenuated camelpox vaccine at the Indian Veterinary
Research Institute, Mukteswar using a virus isolate (CMLV-1/97) from a natural outbreak. The
experimental studies have shown that the vaccine provides complete protection against high dose of
virulent challenge virus. It causes no adverse reaction at dose as high as lO^^'' TCID50, and confers
protection even at a low dose of 10^^ TCID50. The vaccine is administered by intra-dermal route, on
the abaxial surface of the tail in camel calves aged more than six months. The live camelpox
vaccine developed has been proved to be safe and efficacious by in-house trials.
4. DESCRIPTION (Description shall start from the next page)
4.1. Development of cell culture attenuated live camelpox vaccine
4.1.1. Identification of vaccine virus
The camelpox virus was initially isolated from an outbreak of camelpox infection that occurred in
camels at Bikaner, Rajasthan during 1997. The clinical material in the form of scab was collected
and processed in the laboratory. The virus (camelpox virus, CMLV-1/97 strain) was isolated in
Vero cells. The virus produces characteristic cytopathic effect (CPE) and the CPE starts as early as
24 hrs post infection and completes by 48-72 hrs post infection. The identity of the virus was
confirmed based on, neutralization, genus and species specific PCRs, virus specific real time PCR
and further by sequencing of B5R (envelope protein) gene amplified from viral genome. PCR based
on C18L gene amplifies 243bp in CMLV (Fig 1) TBalaraurugan et at, 2008; Balamurugan et ai,
2009; Bhanuprakash et ai, 2010b; Venkatesan et al., 2012).
4.1.2. Process of production of attenuated virus
The camelpox virus was isolated in Vero cell culture grown in Eagle's minimum essential medium
(EMEM)'supplemented with 10% bovine'calf'ye'riim'(BCS, M/s HyClone, USA). The virus was
passaged in Vero cells up to 50 passages. Briefly, fi^eze-thawed (twice) virus was inoculated in to
flasks containing 48 h old Vero cells with a confluency of 80% at 0.05 mukiplicity of infection
(MOI). A characteristic cytopathic effect was produced by the virus in the form of cell clumping
and rounding with increased refractivity and degeneration of cells and the CPE completes by 48-72
hrs post infection. 'The identity of virus at every tenth passage was confirmed by C18L gene based
PCR (Balamurugan et al., 2009). The virus at 50* cell culture passage was lyophilized and used for
evaluation of its safety, potency and immunogenicity in camelpox sero-negative camels.
4.2. Vaccine evaluation
4.2.1. Sterility: The vaccine was tested for bacterial (aerobic/anaerobic), mycoplasmal and fungal
contamination employing standard tests prescribed^in OIE Manual (OIE Manual, 2009) and the
vaccine lot found to be devoid of all these contaminants.
4.2.2. Innocuity test: The inocuity test was done using guinea pigs and mice as per OIE manual of
standards adapted for other pox vaccines with modifications. A group of two guinea pigs, each
were inoculated with 0.5ml of vaccine (50 doses) intramuscularly and another group of two guinea
pigs, each were inoculated with 0.5ml of vaccine (50 doses) intra-peritoneally. The third group
containing two guinea pigs was maintained as in-contact controls. Similarly, six mice each were
inoculated with 0.1ml (1 dose) of vaccine intra-muscularly. The animals were maintained for 14
days and observed for mortality, if any. All the animals remained healthy and none died. Thus, the
vaccine found innocuous in these laboratory animals.
4.2.3. Safety test: The safety test was done using camelpox sero-negative camel calves aged more 6
months to one year (Hafez et al, 1992). Three susceptible camel calves were selected at National
Research Centre on Camels; Bikaner and serum samples were collected. Five randomly chosen
vials of the freeze-dried vaccine were reconstituted in., 1 ml of sterile distilled water each, and
pooled. One camel calf was inoculated intra-dermally on the abaxial surface of the caudal fold with
0.5ml (10^** TCID50) of the concentrated y^ccjne. The remaining vaccine was diluted in sterile
distilled water and one camel calf was inoculated intradermally on the ventral aspect of the caudal
fold with 0.5 ml - with a dose (10 ' TCID50). The remaining one camel calf served as in contact
control. The animals were clinically examined daily and rectal temperatures were recorded. On day
28 post vaccination, the camel calves were again serum sampled. All the camel calves (n= 3
including in-contact animals were observed for clinical response and recorded during the following
14 days. The VaccJinated animals showed' Id-dfelayfcd-type hypersensitivity reaction in the form of
"take", which disappeared within 8* day post vaccination. Serum samples were collected at
different intervals and were examined for sero-conversion. Study showed that the experimental
vaccine found safe in camels and safety ranged from 10^° to 10^^'' TCID50. It produced no adverse
reaction even at high dose (10^^'* TCID50). The "takes" of peanut size (1.7 to 2.0 cm) disappeared
by 8* day post vaccination. A transient raise in temperature (l-l.S^F) was noticed in vaccinated
animals, whereas control ammiar remained ribhrial throughout the study (Fig. 2). Following
administration of vaccine, camel calves reacted initially through a way of formation of local
hyperemia at the site of inoculation implying the viability of the virus. Shedding/horizontal
transmission of the vaccine virus from the immunized animals to in-contact animals was not
observed under experimental conditions. In the camel maintained in close contact with vaccinates
did not exhibh antibody response, suggesting that the vaccine virus is not secreted from vaccinated
animals.
4.2.4. Potency test: The potency of the vaccine was done as per Hafez et al. (1992) with modifications.
Fifteen camel calves each aged more than 6 months to one year and sero-negative to CMLV were
randomly selected from National Research Centre on Camels, Bikaner, Rajasthan for the experiment. The
animals were divided in to three groups of Group I, Group II each containing six camels; and Group III with
three animals. All animals in Group I were inoculated with 10^^ TCID50 (one dose), whereas Group II
animals received 10^" TCID50 (100 doses) in 0.5ral diluent (sterile distilled water). Animals in third
group were injected with 0.5ml sterile distilled water as placebo. The animals were inoculated
intradermally on the abaxial surface of the caudal fold. All the vaccinated and control animals were
observed daily for the development of 'takes' and rise in body temperature up to 21 days post vaccination.
Majority of the animals developed peanut sized "takes" at the site of inoculation except the controls. Sera
from all the animals were collected periodically on day 0, 7,14,21,28 post vaccinations (dpv). On 28* dpv,
animals in each group were challenged, with lO" TCIDjo/ml pf low cell culture passaged virulent camelpox
virus (CMLV-2,passage 2), in 0.5 ml dose intra-dermally at two sites on the ventral surface of the caudal
fold. Then, challenged animals were observed daily for clinical parameters namely thermal response and
erythematous skin lesions. Again the animals were sera sampled on 10, 14 and 21 day post challenge for
sero-monitoring. Both the groups, which received 1 dose and 100 doses, did not manifest any skin lesions
after challenge with virulent virus. But, the control group that received only sterile distilled water showed
reddening, swelling and erythematous skin lesions at the site of challenge (Fig. 3). Further, control group
showed a rise in body temperature on day 4 to 8 post-challenge which was not observed in vaccinated
animals (Fig. 4). When the sera samples were screened for the presence of CMLV specific antibodies using
serum neutralization test (SNT)^ vabcinated aniirikl^'hykhbv/h gradual rise in SNT titre up to 21" day post
vaccination and a drastic rise in serum antibody titers after challenging with virulent virus. This seroconversion
was not observed with control animals, which shown slight change in antibody titre on 10* day
post challenge (Fig. 5).
4.2.5. Humoral immune response: Experimental vaccine induced virus neufralizing antibodies in serum as
detected by serum neufralization test.'Ris^'Jri antibody response was detectable by day 7 post
immunization. The kinetics of antibody response of vaccinated and post challenged sera is depicted
(Fig. 5).
nini;il'; hrul s i .—
8
4.2.6. Dose, route, age and species of vaccination: The recommended dose of the vaccine in camel is
10^" TCID50 in 0.5 ml diluents (sterile distilled water). The vaccine is administered intra-dermally,
on the ventral aspect of the caudal fold and minimum age of vaccination in camels is six months.
4.3. Method of Manufacture
The following methodology describes the production of live camelpox vaccine using the vaccine
seed virus developed by the inventors;
4.3.1. Cells and culture medium: Vero cells free from bacterial (aerobic/anaerobic), mycoplasmal, fungal
and viral contamination have been used for virus propagation and production. Eagle's minimum
essential medium (EMEM) with sodium bicarbonate at a concentration of 2.2g/L is used for culture
of Vero cells. For cell growth and maintenance of grown monolayer, concentrations of 10% and 2%
bovine calf serum respectively;(BCS,\!M/s!HyGiloiieviUSA), was used.
A
4.3.2 Propagation of vaccine virus for primary seed or working seed: Freeze-dried camelpox virus
(CMLV-1/97) at 49* passage (seed virus) is used for subsequent propagation. To produce vaccine
lots, preformed monolayer of Vero cells grown in tissue culture flasks to log phase of growth (48
hrs old monolayer) were infected.
Vero cells were seeded to roller culture bottles to allow formation of monolayer, and then it was
infected with the seed virus at 49* passage to grow vaccine virus at passage 50. The inoculum was
added to the monolayer of Vero cells at a multiplicity of infection (m.o.i) of 0.05 and allowed for
virus adsorption by incubation at 37°C for 1 hr. Culture vessels were incubated at 37°C after adding
maintenance niediiim contairiiri^'2%'BCS Whh'k"finie of cell monolayer. After the appearance of
complete CPE, infected culture was subjected to freeze-thawing to make lysate. Lysates from
several infected culture vessels is pooled to make a vaccine batch. After clarification of infected
lysate, virus supernatant was subjected to titration. Virus was adjusted to contain 10 TCID50 per
dose and a vial containing 100 doses is provided in lyophilized form. Vaccine virus stocks
equivalent to 100 doses were diluted in the Hank's balanced salt solution (pH 7.3) in the presence
of lactalbumin hydrolysate and sucrose at final concentration of 2.5 and 5% (w/v) respectively and
1^ I )V :i >niii ii f HIM.
lyophilized. For reconstitution of vaccine, the fi-eeze-dried contents of each virus vial were added
with distilled water and used for immunization in animals.
4.4. Vaccine Quality Control
4.4.1. Identity: The identity of the camelpox virus was confirmed based on neutralization test, genus and
species specific PCRs, real time PCR and cloning and sequencing of B5R (envelope protein) gene
as described in section 4.1.1..
4.4.2. Sterility: The vaccine was tested for bacterial (aerobic/anaerobic), mycoplasmal and fungal
contamination by employing standard tests.
4.4.3. Virus infectivity titration: Seed virus and final vaccine were titrated in tissue culture tubes or
microtitre plates. Vaccine samples must be examined for the presence of adventitious viruses. The
vaccine virus produced in several lots can be pooled and held at -80°C until all sterility tests and
titrations are completed to make a batch. After this, virus was fi-eeze-dried in 1 ml volume in glass
vials to contain 100 doses. The vaccine virus diluted with lactalbumin hydrolysate and sucrose to
have a titre of log 10^" TCIDsoper ml after fi-eeze-drying, equivalent to 100 field doses.
After lyophilization, five randomly chosen vials of the fi-eeze-dried preparation were reconstituted
into a total of 5 ml sterile water and titrated in micro titre plates to confirm the titre. A serial logio
dilution of the virus (10"' to 10"'°) is made in EMEM with 2% bovine calf serum (BCS). Vero cells
in EMEM containing 10% BCS were grown to confluency ( ^ 8 hrs) in a microtitre 96 well cell
culture plate. The media from 96 wells cell culture plate was discarded and the wells of the plate
were washed with EMEM. 100 yA of each dilution of the camelpox virus in triplicates was added on
to the pre-formed Vero cell monolayer. Cell control was also maintained in triplicates with addition
of EMEM containing 2% BCS; Media'wa^''cH^^W' every alternative day and the cell monolayer
was observed for CPE changes. The infectivity titer was determined and expressed as TCIDso/ml
(Reed and Muench, 1938).
10
4.4.4. Virus neutralization assay: A test serum can either be titrated against a constant titer of camelpox
virus [100 TCID50 [50% tissue culture infective dose] or a standard virus strain can be titrated
against a constant dilution of test serum in order to calculate a neutralization index. The test is
described using 96-well flat-bottomed tissue culture grade microtitre plates, but it can be performed
equally well in tissue culture tubes with the appropriate changes to the volumes used.
Procedure
• Test sera including a negative and a positive control are diluted 1/5 in Eagle's/HEPES (N-2-
hydroxyethylpiperazine, N-2-ethanesulphonic acid) after inactivating at 56°C for 30 minutes.
• 50 \il of the first inactivated serum is added to columns 1 and 2, rows A to H of the microtitre plate.
The second serum is placed in columns 3 and 4, the third in columns 5 and 6, the positive control
serum is placed in columns 7 and 8, the negative control serum is placed in columns 9 and 10, and
50^1 of Eagle's/HEPES without serum is placed in columns 11 and 12 and to all wells of row H.
• Vaccine strain known to grow'well in tissue cilifiire' is^serially diluted in EMEM.
• Starting with row G and the most diluted virus preparation, 50^1 of virus is added to each well in
that row. This is repeated with each virus dilution, the highest titre virus dilution being placed in
row A. The plates are covered and incubated for 1 hour ai 37°C.
• Vero cells are prepared fi-om pre-grown monolayers as a suspension of 10^ cells/ml in Eagle's
medium containing antibiotics and 2% BCS. Following incubation of the microtitre plates, 100^1 of
cell suspension is added to all the wells, except wells HI 1 and H12, which serve as control wells
for the medium. The remaining wells' of row H are cell and serum toxicity controls. The microtitre
plates are covered and incubated at 37°C for 42-72hrs.
• The monolayers are examined daily under an inverted microscope starting 24 hrs post incubation
for evidence of CPE. There should be no CPE in the cells of row H. The final reading is taken on
day3,andthetitreofvirusirieachduplicate1ib^tt6rf1S'calculated(Karber, 1931).
Interpretation of the results: The neutralization index is the log titre difference between the titre of
the virus in the negative serum and ih the test serum.
4.5. Advantages of this technology
1. The camelpox vaccine developed for camel is safe, potent, and efficacious.
2. The vaccination with this attenuated strain is the most cost-effective way to control disease in
endemically affected farm.
: ' . • l U n i t U U l I'.
11
References
Al-Ziabi, 0., Nishikawa, H. and Meyer, H. (2007). The first outbreak of camelpox in Syria. J. Vet. Med.
&/., 69(5): 541-543.
Balamurugan, V., Bhanuprakash, V., Hosamani, M., Kallesh, D. J., Venkatesan, G., Bina Chauhan, Singh,
R. K. (2009). A polymerase chain reaction strategy for the diagnosis of camelpox J. Vet. Diagn.
Invest. 21:231-231.
Balamurugan, V., Bhanuprakash, V., Hosamani, M., Srinivasan, V. A. and Singh, R. K. (2008).
Comparative sequence analyses of B5R gene of Indian Camelpox virus isolates with other
orthopoxviruses./«fi?w«^ Virol, 19(2): 168-172.
Baxby, D. (1972). Smallpox viruses from camels in Iran. Lancet, 17%: 1063-1065.
Bera, B. C, Shanmugasundaram, K., Barua, S., Venkatesan, G., Virmani, N., Riyesh, T., Gulati, B. R.,
Bhanuprakash, V., Vaid, R. K., Kakker, N. K, Malik, P., Bansal, M., Gadvi, S., Singh, R. V.,
Yadav, V., Sardarilal, Nagarajan, G., Balamurugan, V., Hosamani, M., Pathak, K. M., Singh, R.
K.(2011). Zoonotic cases of camelpox infection in India. Vet Microbiol., 152(1-2): 29-38.
Bhanuprakash, V., Prabhu, M., Venkatesan, G., Balamurugan, V., Hosamani, M., Pathak, K.M.L., Singh, R.
K. (2010a). Camelpox: epidemiology, diagnosis and control measures. Expert Rev Anti Infect Ther.,
8(10): 1187-1201.
Bhanuprakash, V., Balamurugan, V., Hosamani, M., Venkatesan, G., Chauhan,B., Srinivasan, V. A.,
Chauhan, R. S., Pathak, K. M. L. and Singh, R. K. (2010b). Isolation and Characterization of
Indian isolates qf camel pox^vii:qji?.s.;J]!-p^,,/^n,/%:^(?a^^^ 42(6): 1271-1275.
Davies, F.G., Mungai, J. N. and Shaw, T. (1975). Characteristics of a Kenyan camelpox virus. J. Hyg., 7,
381-385.
Hafez, S. M., Al-Sukayran, A., Dela Cruz, D., Mazloum, K. S., Al-Bokmy, A. M., Al-Mukayyel, A.,
Amjad, A. M. (1992). Development of a live, cell culltqre camelpox vaccine. Vaccine, 8(10): 533-
539.
Jezek, Z., Kriz, B. and Rothbaur, V. (1983). Camelpox and its risk to the human population. J. Hyg.
Epidemiol. Microbiol. Immuno., 27:29 -r.42.r,.,.,.
Karber, G. (1931). Beitrag. zur kollektiven.behandlungipharmakologischer reihenversuche. Archive ftir
Experimentelle Pathologic Pharmakologie, 162,480-483.
Kriz, B. (1982). A study of camelpox in Somalia. J. Comp. Pathol, 92:1-8.
Leese, S. (1909). Two diseases of young camels. J. Trop. Vet. ScL, 4:1.
Murphy, F. A., Gibbs, E. P. J., Horzinek, M. C. and Studdert, M. J. (1999). Veterinary Virology, III Edn.
(Academic Press, San Diego, CA, USA), pp. 282.
OIE Manual (2008). Manual of diagnostic tests and vaccines for terrestrial animals. VI Edition, Vol 2,
Chapter 2.7.14, OIE, France.
Reed, L. J., Muench, H. A. (1938). Simple method of estimation of fifty percent end points. Am. J. Hyg, 27:
493-497.
Venkatesana, G., Bhanuprakasha, V., Balamurugan, V., Prabhua, M. and Pandey, A. B. (2012). TaqMan
hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin
scabs. ^ F/w/.Me//iod!y, 181(2012): 192-196.
•::-":-''!'^.-n poiinndluni'. DW:,-
->. -T.\ , o \ ,T i^V ^^^^ .
Table 1: Salient features of live attenuated camelpox vaccine
SALIENT FEATURES OF LIVE ATTENUATED CAMELPOX VACCINE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Name ofthe virus isolate
Place of Origin
Year of isolation
Phytogeny
Current passage level of the
seed virus
Titre
Passaging method
Type of CPE
Time required/passage
Reversion
Protective dose
Recommended dose
Safety
Potency
Duration of Immunity
Vaccine presentation
Stabilizers used
Diluents
Dose
Route of inoculation
Thermo-stability
Camelpox virus isolate (CMLV-1/97)
Bikaner, Rajasthan [India]
1997
Closely related to other orthopoxviruses
50 passages in Vero cells
10*""to iC'tCIDjo/ml
Pre-adsorption
Rounding, increased refractility, clumping and detachment of cells
4 to 5 days
Not studied
lOOOTCIDso
lO'tCIDjo
Innocuous in rodents
No clinical reactions
No horizontal transmission/shedding
Protected vaccinated animals against virulent challenge and induces
neutralizing antibodies
Not studied
100 Doses
2.5% Lactalbumin hydrolysate (LAH) & 5 % Sucrose
Distilled water
1000 TCID50 in 0.5 ml single dose
Intra-dermal on the ventral aspect ofthe caudal fold
'"••"• 11),
Stable
N(^: •ai'iii.jd

CLAIMS:
We claim:
1. The development of attenuated strain of camelpox virus for use as safe live vaccine in camels or their offsprings
(aged more than six months) for protection against clinical form of camelpox.