3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be performed.
Goats of over 120 million population constitute an important component of Indian livestock industry, benefiting large section of rural population. Amongst a number of infections that adversely affect caprine productivity, goatpox is one of the important diseases with a serious economic impact in terms of productivity loss, mortality, hide damage and losses due to international trade restrictions. It is a highly contagious viral disease of goats characterized by fever, oculo-nasal discharges, pock lesions over the skin and mucosa of respiratory and gastro-intestinal tract. The disease caused by DNA virus (goatpox virus, Genus-Capripoxvirus) is categorized as notifiable disease of the OIE (Office International des Epizootics) reflecting the severity and economic significance of the disease.
Considering the economic impact due to goatpox, the need for an efficacious vaccine has long been realized. Hence, attempts were made for developing an inactivated vaccine in the past (Yadav et al., 1986). However owing to low level of protective immunity and short duration of immunity engendered by the killed vaccines, live vaccines are considered as the best choice as long term solution for control of goatpox. Furthermore, being a stable DNA virus, capripoxvirus infections can be effectively controlled through vaccination; this is best exemplified by use of an effective small pox vaccine for control of smallpox, which enabled global eradication of smallpox during 1980.
In this backdrop, work was initiated for developing a live modified vaccine for goatpox. For protection of goatpox, homologous vaccines based on indigenous strains are desirable for complete protection (Rao and Bandyopadhyay, 2001; Bhanuprakash et al., 2006). However no homologous vaccine was available for control of goatpox. Hence a live attenuated goatpox vaccine was developed using an indigenous strain of goatpox virus by cell culture propagation of the virus. Cell culture passaging of the virus (Urtarkashi isolate) was initiated by adaptation and propagation of an indigenous strain of goatpox virus in lamb testicle (LT) cells. Vero cells were subsequently used for cell passaging after 34 passages in LT cells till 60 serial passages.

GTPV showed evidence of attenuation when tested at 55th passage (34LT + 21Ver) in sero-negative goats. Virus was further passaged till 60th passage in order to stabilize the attenuation, when the systematic laboratory trials were under taken to test the vaccine safety, potency and protective immunity in the natural hosts i.e., goats/In house experimental trials and limited field trials have shown that the vaccine is safe, potent and immunogenic in goats. Detailed information on the vaccine is given in the following sections.
4. DESCRIPTION (description shall start from the next page)
4.1. Development of attenuated goatpox vaccine:
4.1.1. Identification of vaccine virus
Vaccine was derived-from Uttarkashi isolate of GTPV originally isolated from Naugaon block of Uttarkashi in 1978 (Das et al., 1978). The virus was initially propagated in lamb testis cells followed by in Vero cells. Virus was found to be completely nonpathogenic when tested in sero-negative goats at passage 55. Virus was further passaged till 60th passage to stabilize the attenuation and used as experimental vaccine. The virus takes about 5-7 days to produce 80-90 % cytopathic changes after infection of preformed monolayers. Virus has been confirmed by PCR assay (Heine et al., 2000) at regular passages, virus neutralization test (Kitching, 1986) using hyperimmune serum and also sequencing of highly conserved gene P32 (GenBank accession number-AY382869) amplified from the viral genome.
4.1.2. Process of production of attenuated virus
GTPV Uttarkashi isolate was initially adapted and propagated in primary lamb testis culture till 34 passages. Subsequently the virus was adapted in Vero cell line and continuously passaged till 60 passages. The virus has been plaque purified at 55th passage. It takes about 5-7 days for the virus to grow, following infection of the complete monolayers as evidenced by cytopathic effects (CPE) characterized by rounding, ballooning, increased granularity and degeneration of the cells. Virus harvested at about 80-90% of the CPE produces consistently high titres up to JO6'5'763 TCID50/ml. Although the virus was found to be avirulent at 55th passage, it was further passaged to cause stable attenuation. Presence of the virus has been tested at regular passages using PCR assay as described earlier (Heine et al., 2000). At 60th passage, virus was lyophilized and used for evaluation of the vaccine potency, safety, immunogenicity in experimental goats.
For the first time, the inventors have developed a homologous indigenous live goatpox vaccine for control of goatpox in goats. For production of the vaccine, Vero cells are employed instead of primary cultures, as use of Vero cell line enables

consistently high virus titres, cost effective production and freedom from adventitious pathogens possibly associated with primary cell cultures.
4.2 Vaccine evaluation:
4.2.1. Potency
Initially, a controlled experimental trial was carried out in experimental goats to determine the virus safety, potency and protection against virulent virus challenge. Apparently healthy goats of either sex, aged 6 to 12 months were used for testing these attributes. The OIE (Office International des Epizootics) recommended dose of vaccine in goats in 1025 TCIDso. For potency testing of the vaccine, experimental trial was carried out by inoculation of 10, 100 and 100000 TCID50 per animal. Animals were daily clinically monitored for general health status and daily rectal temperatures recorded.
Study showed that the experimental vaccine provided complete protection against high dose of virulent challenge virus. It produced no adverse reaction at the high dose
5 1
(10 TCIDso), while complete protection was recorded even at a low dose of 10 Following administration of vaccine, goats react by way of formation of local hyperemia at the site of inoculation (Figure 1, Annexure III). This is an indirect indication of viability of the virus, like in other pox viral vaccines. Marginal rise in temperature is also observed in the vaccinated goats during day 5-7 days post vaccination. These reactions are generally transient that subside gradually.
For challenge infection, GTPV Mukteswar isolate was employed by intradermal route of inoculation. All the control goats exhibited systemic pox infection characterized by typical pox lesion on the body, accompanied with high pyrexia while the vaccinated goats were completely immune to challenge (Figure 2, Annexure III)
The serum samples collected from all the immunized goats and control goats showed rise in antibody titres in the immunized goats as measured by indirect ELISA even in the group that received a dose of 10 TCIDso (Figure 3, Annexure IV). Virus neutralization test was carried out (Kitching, 1986) to determine the neutralization index (NI), which ranged between 0.5 to 1.75. No sero-conversion was detected in the control animals.
Horizontal transmission of the virus from the immunized animals to in-contact animals is not observed under experimental conditions. In the group maintained in close

contact with the vaccinates, no thermal response and no rise in antibody response or protection on challenge infection have been evidenced, suggesting that the vaccine virus is not secreted following immunization
4.2.2. Duration of immunity
Duration of immunity following goatpox vaccination has been studied in goats. Kids aged >6 months of age were immunized intra-dermally with 103 TCIDso of vaccine for determining the duration of immunity induced by the vaccine. Challenge experiment showed that vaccine provides complete protection against virulent virus challenge, up to 26 months of post-vaccinational study. Further work is in progress to determine the longevity of protective immunity.
4.2.3. Cross immunity of the vaccine against sheep pox
Efficacy of the goatpox vaccine to cross protect against virulent sheep poxvirus was studied in sheep. Vaccine when administered intradermally @ 104TCID5o in the caudal skin fold, was found to be safe and immunogenic in sheep. However, it conferred partial protection against virulent sheep poxvirus challenge. Vaccinated animals showed mild to moderate local erythema at the site of inoculation without signs of pyrexia and generalized disease, while, control unvaccinated animals showed severe local reaction, generalized disease accompanied with high temperature (106.4°F)
4.2.4. Sterility
The vaccine was tested for mycoplasmal, bacterial and fungal contamination by employing standard specific tests recommended by the OIE and found to be devoid of these contaminants.
4.2.5. Vaccine safety and reversion to virulence studies
Safety testing was done in goats as per the procedures recommended in OIE manual of standards.
A possible reversion of the vaccine virus to virulence was studied by back-passaging the vaccine virus in goats for.4 successive times. The goats were clinically

observed for specific clinical reactions. None of the vaccinated animals that received the virus back passages showed signs of illness and adverse reactions. Tissues collected from the vaccinated animals did not show presence of virus by capripox-specific PCR. This study indicated stable attenuationof the -virus ruling out the chances of reversion to virulence.
Safety levels of the experimental vaccine were also evaluated in the pregnancy. Preliminary studies carried out in pregnant goats (n=10) that were in different stages of gestation showed that the vaccine is safe at the recommended dose of io25"30 TCID50 of vaccine. However, further controlled experimental studies on goats with synchronized pregnancy showed that the vaccine at doses higher than 103 can cause abortion in about 20% of the pregnant animals. Even though very higher doses of 107TCID5o vaccine virus were found to be safe in the pregnant does without any apparent signs of illness, the risk of abortion cannot be ruled out. In view of this, the vaccine is not recommended for use in pregnancy. Field vaccination is currently advocated only in non-pregnant animals aged more than 3-4 months. These observations were also justified by similar possible adverse reactions associated with human pox vaccines during gestation. For this reason, small pox vaccine (Vaccinia virus) is generally contraindicated in pregnant women as there is potential of vertical transfer of the virus leading to death of off springs (Benning and Hassette, 2004; Hassett, 2003). There are about 50 documented cases in the literature of Vaccinia virus during pregnancy, most of which resulted in the death of the affected offspring. This condition (vaccinia fetalis) was first described by Lynch in 1932 in USA (Green et al. 1966).
Abortions following vaccination of Asian elephants with a highly attenuated and safe, vaccinia virus-modified vaccinia Ankara (MVA) vaccine has also been recently reported (Wisser a al., 2001). A pregnant elephant vaccinated on day 293 and 322 days of pregnancy gave rise to still born male calf with generalized pox, while the mother or the other vaccinated elephants remained apparently healthy.
The present goatpox vaccine has been however plaque purified at 55* and released for field vaccination in order to minimize the chances of adverse reaction following accidental vaccination of pregnant goats.

4.2.6. Field evaluation of vaccine
A limited number of field trials were taken up in a number of states including Maharashtra, Andhra Pradesh, Orissa, West Bengal, Karnataka, Rajasthan and Tamil Nadu. Trials were carried out in the organized farms, flocks owned by small-scale farmers, state animal husbandry goat farms. So far 31000 doses have been used across the country without any adverse reactions when used in the healthy no-pregnant goats aged >4 months.
4.3. Method of manufacture
The following method describes the production of goatpox vaccine using the vaccine seed virus developed by the applicants.
4.3.1. Cells and culture medium
Vero cells (ATCC, CCL81) is used for growth of virus. Eagle's minimum essential medium (autoclavable EMEM, Sigma-Automod, Cat# M0769) containing glutamine 2mM, sodium bicarbonate and HEPES (lOmM) supplemented with defined calf serum (Hyclone, USA) is used for culture of Vero cells. Serum is added to the medium at a concentration 10% and 2% for growth of Vero and subsequent maintenance of cells during infection phase, respectively. Vero cells are used at split ratio of 1:4. Further, about 95% complete growing monolayers are best suited for infection with goatpox vaccine virus.
4.3.2. Propagation of vaccine virus for primary seed or working seed
Lyophilized or liquid phase virus (stored at -80°C) at 59th Vero passage is used for production of primary seed virus for subsequent propagation. The virus can be grown in plastic tissue culture flasks and roller bottles (surface area of 1700 cm2).

Vero cells are seeded to culture flasks at a split ratio of 1:4 and about 95% complete, preferably day old or two days old monolayer culture is used for infection. The freeze-dried virus is reconstituted in sterile distilled water and used for infection at a multiplicity of infection of 0.01 TCIDso- After adsorption ^no"separate adsorption step for roller culture) for one hour in the incubator at 37°C with intermittent shaking, cells are washed twice and added with maintenance medium. The cells are daily examined and medium is changed after 2 days. The cells are daily examined for appearance of CPE which is characterized by rounding and clumping, ballooning and increased refractility of cells with degeneration of the monolayer. Medium is changed once more on day 4th completely or partially with 50% medium replenished depending on the extent of CPE. The flask is finally frozen when CPE is.about 80-95% and subjected to two cycles of freezing and thawing. Virus is lyophilized after blending with equal volume of (sterile) stabilizer containing 5% lactalbumin hydrolysate plus 10% sucrose.
4.4. Vaccine quality control
4.4.1. Identity and sterility
For testing the identity of the vaccine, virus neutralization is used using hyperimmune serum raised against the goatpox virus "Sambalpur isolate" available in our laboratory. PCR assay (Heine et al., 2000) is also used for detecting viral nucleic acid.
The vaccine is tested for bacterial and fungal contamination by employing standard tests as described in the OIE Manual of Standards for diagnostic tests and vaccines.
4.5. Advantages of this technology:
1. The vaccine is homologous developed using indigenous strain for control of
goatpox in caprines.
2. The vaccine is safe, potent, and efficacious providing immunity for at least 26
months (tested so far) and immunity is possibly life long following single
immunization. Long-term immunity trial is on.

3. Since Vero cell line is a heterologous host system as compared to goat or sheep -
derived primary cultures, the attenuation is considered to be more effective. Stable attenuation of the virus has been demonstrated by reversion studies.
4. Vaccine is derived from Vero cell producing consistently high liters facilitating cost-
effective production in roller bottle cullure, hence economically viable under commercial condilions. Produclion in Vero cell line also precludes Ihe presence of adventitious pathogens mat may be possibly associated with vaccine derived from primary cultures.
5. The vaccine also gives partial cross protection in sheep under experimental conditions.

5. CLAIMS We claim:
1. The development of a live homologous vaccine for goatpox for use in caprines.
The vaccine is safe when used in non-pregnant, healthy animals aged more than 3
months. It provides protective immunity for at least 3 years of protection on single
vaccination.
2. The vaccine is safe when used in combination with other live viral vaccines such
as PPR vaccine used in small ruminants in the endemic regions