WO2007046643A1

WO2007046643A1 - Composition comprising an extract of pine needle for preventing and treating animal disease caused by viruses and the use thereof

Info

Publication number: WO2007046643A1
Application number: PCT/KR2006/004259
Authority: WO
Inventors: Chang Seon Song; Chi Ung Moon
Original assignee: Chang Seon Song; Chi Ung Moon
Priority date: 2005-10-19
Filing date: 2006-10-19
Publication date: 2007-04-26

Abstract

The present invention relates to a composition comprising an extract of Pine needle for preventing and treating animal disease caused by viruses and the use thereof. The extractof pine needle of the present invention showed anti- viral effect of influenza A viruses, Newcastle disease virus, infectious bronchitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus, Coxsakie virus, reticuloendotheliosis virus, Aujeszky s disease virus, adeno virus and coxsakie virus in vivo and in vitro. Therefore, it can be used as the veterinary composition, feed additive and anti-viral disinfectant composition for treating and preventing the disease caused by the infectionof virus in animal.

Description

COMPOSITION COMPRISING AN EXTRACT OF PINE

NEEDLE FOR PREVENTING AND TREATING ANIMAL

DISEASE CAUSED BY VIRUSES AND THE USE THEREOF

Technical Field

[1] The present invention relates to a composition comprising an extract of Pine needle for preventing and treating animal disease caused by viruses and the use thereof.

[2]

Background Art

[3] Influenza has been a major cause of morbidity in human and of mortality in the elderly and infant. Influenza viruses are members of the family Orthomyxoviridae, which is composed of four genera, i.e., influenza virus A, B, C and Thogotovirus.

[4] There are 144 kinds of viruses according to their serotypes including 16HA proteins and 9 NA proteins. HA makes virus adhere to a somatic cell and NA makes virus penetrate to intra-cell (Alexander DJ. Vet. Microbiol, 74(1-2), pp3-13, 2000).

[5] Avian flu classified into influenza A is caused by avian viral infection mediated by poultries such as chick, duck, turkey, wild avian and the like on farms and in live markets. Among them, highly pathogenic avian influenza has been designated as the 1^st group of legal communicable disease in Korea and List A infections disease in OIE (Office International des Epizooties). The pathogen, i.e., avian influenza virus, a sort of zoonotic virus is classified into highly pathogen, weakly pathogenic and nonpathogenic virus according to their pathogenecity.

[6] Among those serotypes, virulent avian virus transmitted to human such as H5N1 being called as Hong Kong flu, H7N7 and H9N2 subtype has been reported recently (Suarez DL et al., J. Virol, 72(8), pp 6678-6688, 1998).

[7] Avian influenza virus is frequently transmitted to human through intermediate host such as swine, chicken, etc, modified by genetic mutation in the host and the mutated virus is transmitted to human through respiratory tract and other pathway resulting in pathogenic avian flu.

[8] However, there have not yet developed effective agents to prevent or treat avian flu till now since avian influenza virus has lots of serotypes and shows frequent mutation.

[9] As a one of prevention methods, the viral vaccine of influenza has been recommended and it has been known to show 70-80% preventing activity (Influenza, Plenum Medical Book Company, P291, 1987). However, since the vaccine endows short duration of immunity and if provided with injection, it has several problems such as difficulty in administration into children and in initial prevention of influenza. [10] Newcastle disease virus (NDV) is a typical paramyxovirus which causes a severe respiratory infection in poultry. This disease is of great economic important, requiring control by vaccination or quarantine with slaughter of all birds in confirmed outbreaks. The genome of NDV is a single strand of RNA of negative polarity. On the outer surface of the viral envelope are the two viral glycoproteins, the haemagglutinin-neu- raminidase HN involved in the binding of the virus to host cell and the fusion protein F involved in fusion with and penetration through the membranes.

[11] According to the OIE, NDV is a disease of poultry caused by a virus of avian paramyxovirus serotype 1 (APMV-I) which has an intracerebral pathogenicity index (ICPI) in day-old chicks of 0.7 or greater. The poultry is reared or kept in captivity for breeding, the production of meat or eggs for consumption, or for restocking supplies of game. It is generally assumed the first outbreaks of NDV occurred in 1926 in Java, Indonesia, and in Newcastle-upon-Tyne, England. It later became clear that other less severe disease were caused by viruses indistinguishable from NDV.

[12] Infectious bronchitis virus (IBV), the prototype of the family Coronaviridae, is the etiological agent of infectious bronchitis (IB), and acute, highly contagious disease of the respiratory and urogenital tracts of chickens (King, DJ. et al., Disease of poultry, 9 ed. Iowa State University Press, Ames, Iowa, pp 471-484, 1991). The poultry is severely affected by epizootics of this disease. Infectious bronchitis still causes a large mortality, especially with young poultry. Besides the mortality and more or less strong respiratory symptoms, lesions to the oviducts occur and as a result thereof, egg production drops caused by an IB infection occur.

[13] Moreover, infections with IB virus may stimulate latent virus or bacterial infections and may give rise in this way to severe economical losses, especially in the broiler field. In spite of the use of vaccines for the control of IB, the disease continues to be a problem in commercial poultry because some serotypes do not cross protect against antigenically unrelated serotypes including variant strains of the virus (GeIb J. Jr. et al., Avian Dis., 35, pp 82-87, 1991; King D. G.., Avian Dis., 32, pp 362-364, 1988).

[14] IBV serotype, such as the Massachusetts type, 793B or 4/91, has been spread to the world. However, despite Massachusetts type vaccine or 793/B type vaccine has been used since 1990, there still remains a great need for IB vaccines with adequate immunizing properties. It will be appreciated that the desired improvement of these vaccines is still severely hampered due to the immunogenic change and other properties of the presently available IB viruses after a large number of passages in em- bryonated chicken eggs, the appearance of new serotype and the lack of sufficiently applicable serological and immunological test procedures, respectively.

[15] Corona virus, an aetiological virus of SARS has known to be transferred from animal to human as a mutant type and is a principle virus to give rise to the diarrhea of pigs and to cause to lose the appetite of pig, which result in inhibiting the growth of pig. Furthermore, it could not be treated with the conventional antibiotics and the basic treatment therapy has not been developed yet till now.

[16] Avian pneumovirus (APV), a member of the Paramyxoviridae family of viruses, has known to be the etiological agent of turkey rhinotracheitis (TRT), which cause an acute upper respiratory tract infection characterized by coughing, nasal discharge, tracheal rales, foamy conjunctivitis and sinusitis in young poultries. In laying birds, there is a transient drop in egg production along with mild respiratory tract illness (Cook JKA, The Veterinary Journal, 160, pp 118-125, 2000).

[17] APV has been firstly detected in South Africa in 1978, and detected in the UK,

France, Spain, Germany, Italy, Netherlands, Israel, and Asian countries later on. More recent studies have shown that sentinel Mallard ducks living in a pond adjacent to a turkey farm with an APV outbreak acquired the virus and developed anti-APV antibodies.

[18] Porcine reproductive and respiratory syndrome (PRRS) causes an important pathology in the porcine livestock, characterized by reproductive disorders in the female pigs, and an increase of the perinatal mortality as well as dyspnea and piglet pneumonia (Terpstra et al., Vet Quarterly, 13, pp 131-136, 1991). PRRSV is an enveloped RNA virus, the viral genome of which comprises a single-stranded RNA of positive polarity. In 1987 it was first detected in North America a swine disease. A very similar syndrome was first detected in Central Europe in 1990, and spread later to other European countries. European type and North American type viruses are clearly different from each other in respect to not only their serological reactivity but also the homology degree of nucleotide sequences of significant RNA fragments.

[19] The pathology is not restricted to abortion and respiratory disease. Other symptoms associated with the disease are: off feed, anorexia, bluish discolorations of the extremities, especially the ears. At the present time, PRRS is one of the most important diseases affecting economic loss on the swine sector through directly and indirectly, which are caused by secondary agents favored by PRRS virus infection.

[20] A relatively smaller sized RNA virus called as picona virus belongs to the family piconaviridae. The enterovirus belongs to the family piconaviridae. At present, 71 serotypes among the viruses have known to infect humans. The enterovirus genome is a single- stranded RNA molecule that encodes one large protein, which undergoes auto- catalytic proteolysis into a set of smaller structural and non- structural proteins. Enteroviruses are second to rhinoviruses as the most common viral infection in humans. Enteroviruses enter the body through the alimentary tract and possibly the respiratory route. Their normal site of replication is the gastrointestinal tract, where they typically cause mild GI disorder accompanied by non-specific febrile illness. Typical en- terovirus diseases are meningitis, paralysis, myocarditis, generalized infections in newborns, hand, foot and mouth-disease, herpangina, pleurodynia, hepatitis, rash, exanthemas and respiratory disease including pneumonia. Accordingly, there has been needed in the art to improve the ability to diagnose rapidly and accurately enterovirus infection.

[21] Coxsackievirus belongs to a group of the genus Enterovirus of the family Pi- cornavidae. It is a spherical (regular icosahedron), non-enveloped virus with a diameter of about 25 to 30nm. From the antigenicity viewpoint, it is classified into two types (A and B) and each further includes a number of serotypes.

[22] Reticuloendotheliosis viruses (REVs) are the group of serologically related retroviruses antigenically distinct from avian leucosis retroviruses (ALVs). REV isolates have been obtained from turkeys, pheasants, chickens and ducks. Representative REVs include strain T (subtype 1), chick syncytial (CS; subtype 3), spleen necrosis (SN; subtype 2), and duck infectious anemia (DIA; subtype 2) viruses. A single replication-defective, acutely transforming REV isolate is known as REV-T, which carries the rel oncogene and which requires a non-defective helper virus (such as strain REV-A) for replication. REV-T causes an acute reticulum cell neoplasia in inoculated chickens. The virus has a large DNA molecule with numerous non-essential regions that allow the insertion of several immunogenic genes into the same virus for the purpose of developing multivalent vaccines. These multivalent vaccines may induce cell-mediated as well as antibody-mediated immune response in a vaccinated host. No vaccine against REV is currently available. REV consists of 1 stranded RNA however it might be 2-stranded DNA during proliferation sue to RNA dependent DNA polymer enzyme, which further inserted into chromosomal DNA and may be presented as provirus (Fritsch E. et al., Virology, 83, pp313-321, 1977). Because of their characteristics of gene types in host genome during such REV infection, the development and use of live vaccine has been reported to impossible and the preparation of vaccine is also difficult since the glycoprotein 85 (gp 85) reproducing the neutralization antibody against the virus is very unstable.

[23] Aujeszky's disease virus (ADV) is a highly neurotropic herpesvirus that contaminates domestic and wild animals (Thowley DG et al., J. AM. Vet. Med. Assoc, 176 , pplOOl-1003, 1980). Pigs are relatively resistant against PRV therefore they are considered as the natural host of the virus. The virus is able to replicate by itself in the cell of the nasal and pharyngeal mucosa, and after the infection of peripheral nerves, it is transported to the central nervous system resulting in severe encephalitis which is often fatal to young pigs. Older pigs usually survive the infection, but may develop fever and pneumonia. Infection in sensory ganglia generally results in the establishment of latency. Vaccination against Aujeszky s disease is carried out to reduce the economic damage caused by mortality and growth retardation in infected animals (Lee JB et al., Korean J. Vet. Res., 28(1), pp99-103, 1988).

[24] Human adenovirus (Ads) are non-enveloped, icosahedral DNA viruses causing predominantly respiratory disease, conjunctivitis, and intestinal infections in their natural host. The genome consists of a linear, double- stranded DNA molecule of about 36 Kb carrying inverted terminal repetitions (ITR). Infection of human cells by Ads results in a lytic and productive infection. During the lytic cycle, the entire genetic program of the virus is expressed, and this eventually leads to the production of progeny virus and the death of the host cell.

[25] Pine is belonged to the Pinaceae family including Pinus genus. There are 80 to 90 species in the world, and the various types of terpene isolated from P. palustris Miller, P. pinaster Aiton, P. sylvestris L., P. laricid Poiret, P. longifolia Rocvurgh, P. densiflora Sieb. et Zucc. or P. thunberii Palatore has been mainly used on industry.

[26] There have been reported that the main components of pine needle are terpentine oil, cineole, salinigrin, coniferin, P-cymene, densipimaric acid, retene, chlorophyll, protein, fat, phosphorus, iron, enzyme, mineral, fat soluble vitamin A, vitamin C etc., of which composition and ratio are varied according the species and season. Refined oil isolated from the needle of the Pinus densiflora Sieb. et Zucc. comprises α-pinene, β-pinene, camphene, phellandrene, borneol, bornylacetate, caryophyllene, cadinene diterpene, sesquiterpene, sesquiterpene alcohol, cerylalcohol, juniperic acid, sabinic acid, hexadecane-diol, triacontan-1-ol, matsusterin, phytosterin, chinic acid, shikimic acid, quercetin, kaempferol and so on and it shows potent treating effect on various diseases including myocarditis, angina, arrhythmia, diabetes, senile dementia, sudden deafness and hypertension.

[27] However, there has been not reported or disclosed on the effect for viral disease of pine needle extract in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[28] Accordingly, the present inventors have discovered that pine needle extract shows potent inhibiting activity of various viruses.

[29] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter.

[30]

Disclosure of Invention Technical Problem

[31] The present invention provides a veterinary composition, feed additive and disinfectant comprising an extract of pine needle as an active ingredient in an effective amount to treat and prevent the diseases caused by the infection of virus in animal. [32] The present invention also provides a use of an extract of pine needle showing antiviral activity.

[33] The present invention also provides a method for treating or preventing the diseases caused by the infection of virus in animal comprising administering to said animal an effective amount of an extract of pine needle.

[34]

Technical Solution

[35] Accordingly, it is an object of the present invention to provide the veterinary composition comprising an extract of pine needleas an active ingredient for the treatment and prevention the disease caused by the infection of virus in animal.

[36] In particular, the term "extract" disclosed herein comprises the crude extract and non-polar solvent extract of pine needle extract.

[37] The term "crude extract" disclosed herein comprises the extract prepared by extracting plant material with water, C -C lower alcohols such as methanol, ethanol,

1 4 preferably methanol and the like, or the mixtures thereof, preferably ethanol.

[38] The term "non-polar solvent soluble extract" disclosed herein can be prepared by extracting the above described crude extract with non-polar solvent, for example, hexane, ethyl acetate, dichloromethane, or chloroform, preferably dichloromethane.

[39] The term "pine needle" disclosed herein comprises the needle of Pinus densiflora

Sieb. et Zucc, Pinus thunbergii parlatore or Pinus koraiensis Sieb. et Zucc,preferably, Pinus densiflora Sieb. et Zucc.

[40] The term "virus" disclosed herein comprises RNA type virus or DNA type virus.

[41] The term "RNA type virus" disclosed herein comprises influenza virus, Newcastle disease virus, infectious bronchitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus, coxsakie virus or reticuloendotheliosis virus.

[42] The term "influenza virus" disclosed herein comprises HlNl, H9N2 or H6N5 subtype influenza virus.

[43] The term "DNA type virus" disclosed herein comprises Aujeszky's disease virus or adeno virus.

[44] The term "the disease caused by the infection of virus" disclosed herein means the viral disease caused by the internal viral infection wherein said virus comprises or- thomyxocirus family including influenza virus, paramyxovirus family including newcastle disease virus, coronavirus family including infectious bronchitis virus, picorna virus family including Coxsakie virus, retrovirus family including reticuloendotheliosis virus, herpesvirus family including Aujeszky's disease virus or adenovirus family including adeno virus.

[45] The term "viral disease caused by the internal infection of influenza virus family" disclosed herein comprises avian flu or variant infectious avian flu.

[46] The term "viral disease caused by the internal infection of paramyxo virus family" disclosed herein comprises Avian paramyxoo virus, Sendai virus, Canine distemper, Rinderpest or Turkey rhinotracheitis.

[47] The term "viral disease caused by the internal infection of coronavirus family" disclosed herein comprises Turkey corona virus, transmissible gastroenteritis virus, porcine respiratory corona virus, canine corona virus, feline enteritis corona virus, feline infectious peritonitis, rabbit corona virus, bovine corona virus or mouse hepatitis virus.

[48] The term "viral disease caused by the internal infection of picornavirus family" disclosed herein comprises Foot-and-mouth disease, Avian encephalomyelitis virus, Bovine enterovirus or Pocine enterovirus.

[49] The term "viral disease caused by the internal infection of retrovirus family" disclosed herein comprises Avian leucosis virus, roussarcoma virus, feline leukemia virus, bovine leukemia virus, equine infectious anemia virus or feline immunodeficiency virus.

[50] The term "viral disease caused by the internal infection of herpesvirus family" disclosed herein comprises Chicken infectious laryngotrachitis virus or Marek's disease.

[51] The term "viral disease caused by the internal infection of adeno virus family" disclosed herein comprises respiratory syndrome, gastroenteritis, Egg drop syndrome in animal.

[52] In accordance with another aspect of the present invention, there is also provided a use of an extract of pine needlefor manufacture of veterinarymedicines employed for treating or preventing the disease caused by the infection of virus in animal.

[53] In accordance with another aspect of the present invention, there is also provided a method for treating or preventing the disease caused by the infection of virus in animal wherein the method comprises administering a therapeutically effective amount of an extract of pine needleinto the animal suffering with the disease caused by the infection of virus in animal.

[54] An inventive extract isolated from of pine needle may be prepared in accordance with the following preferred embodiment.

[55] Hereinafter, the present invention is described in detail.

[56] For the present invention, for example, the dried whole plant of pine needleiscut into small pieces and the piece was mixed with 1 to 30-fold, preferably, 1 to 15-fold volume of polar solvent, for example, water, C 1 -C 4 lower alcohol such as methanol, ethanol, butanol, or the mixtures thereof, preferably ethanol; and is heated at the temperature ranging from 10 to 100⁰C, preferably from 20 to 5O⁰C, for the period ranging 1 to 24 hours, preferably 5 to 20 hours, by reflux extraction with hot water, cold water extraction, ultra- sonication or conventional extraction, preferably by reflux extraction or hot water; the residue was filtered and then the filtrate is dried by vacuum freeze-drying to obtain the crude extract of the present invention.

[57] In the above crude extract prepared by above step, is suspended in water, and then is mixed with non polar solvent such as hexane, chloroform, ethyl acetate or dichloromethane, preferably ethyl acetate, extracted with the non-polar solvent to obtain non-polar solvent soluble extract of the present invention.

[58] Remaining polar solvent soluble layer is collected by removing the non-polar solvent soluble extract to obtain polar solvent soluble extract of the present invention which may be soluble in water, lower alcohols such as butanol, or the mixtures thereof.

[59] In accordance with another aspect of the present invention, there is provided a veterinary composition comprising a crude extract or non-polar solvent soluble extract of pine needle prepared by the above-described preparation method for the treatment and prevention of the disease caused by viral infection as active ingredients

[60] In accordance with another aspect of the present invention, there is also provided a use of a crude extract or non-polar solvent soluble extract of pine needle prepared by the above described preparation method for manufacture of veterinary medicines employed for treating or preventing the disease caused by viral infection.

[61] In accordance with another aspect of the present invention, there is also provided a method for treating or preventing the disease caused by viral infection, wherein said method comprises administering a therapeutically effective amount of a crude extract or non-polar solvent soluble extract of pine needle prepared by the above-described preparation method into the animal suffering with virus disease.

[62] The inventive composition for treating and virus disease may comprise the above described extract as 0.1 ~ 50% by weight based on the total weight of the composition.

[63] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[64] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

[65] The composition according to the present invention can be provided as a veterinary composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti- agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[66] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[67] Veterinary formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[68] The composition of the present invention in veterinary dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other veterinary active ingredients.

[69] The desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.001 to 100mg/kg, preferably, 0.1 to 100mg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day.

[70] It is still another object of the present invention to provide a feed additive and feed comprising an extract of pine needleas an active ingredient for preventing and improving the diseases caused by viral infection.

[71] The above-described extract of pine needlecan be added to animal feed with the concentration ranging from 20 to 90% (w/w %) as high-concentrated fluid type, powder type or granule type.

[72] To develop for feed additive and feed for animal, at least one of the other ingredients selected from organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid and the like; phosphate salt such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate and the like; and natural anti-oxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further be added to the active ingredient of the present invention.

[73] For example, the inventive veterinary composition of the present invention could be prepared by the procedure consisting of the steps of: mixing inventive extract of present invention with the combination of various aid component such as amino acid, inorganic salt, vitamin, antibiotic, anti-bacterial agent, anti-oxidant, anti-fungal agent, live microbial preparation and the like; and grain such as macerated or pulverized wheat, oat, barley, corn or rice; vegetable protein feed such as bean or sunflower seed; animal protein feed such as blood powder, meat powder, bone powder or fish powder; sugar powder or milk product such as various powdered milk or powdered whey and the like together: heating to obtain remaining fluid component, for example, lipid component such as fluidized animal fat, vegetable lipid and the like; mixing the lipid component as a main component with other component such as nutrient supplement, digestive improving agent, growth stimulator, disease preventing agent and the like to prepare purposed inventive composition.

[74] The above-describe animal feed composition can be administrated into animal as a sole or the combinations with other feed additive.

[75] The inventive extract of the present invention can be administrated into animals through top dressing method, mixing method directly with animal feed or other administration route such as injection, subcutaneous injection or other route with an interval of once a day or several times a day well-known in the art.

[76] Providing that the inventive extract of the composition is administrated into the animal independently on other animal feed, the extract may be combined with pharmaceutically acceptable non-toxic edible carrier to make imprompt releasing or sustained releasing preparation. Solid type or fluid type edible carrier, for example, corn starch, lactose, sucrose, bean flake, bean oil, olive oil, sesame oil and propylene glycol may be used as an edible carrier of the present invention. In case of solid carrier, the dosage form of inventive composition can be tablet, capsule, powder, troche, sugar-containing tablet or micro-dispersion type top-dressing. In case of fluid carrier, the dosage form of the inventive composition can be soft gelatin capsule, syrup, suspension, emulsion or solution and the like. The dosage form may contain the other aid component such as preservative, stabilizer, humidifier, emulsifier, solubilizer or other substance useful in improving or preventing viral disease.

[77] The animal feed additive of the present invention can be added to animal feed for use as an appetizer, for example, conventionally available optional protein-comprising organic grain such as corn powder, bean powder or the mix therewith.

[78] Inventive composition of the present invention has no toxicity and adverse effect therefore they can be used with safe.

[79] The above-described composition therein can be added to animal feed wherein the amount of the above-described extract in feed may generally range from about 0.01 to 80w/w%, preferably 0.01 to 15w/w% of total weight of feed for the feed composition and 0.02 to 5g, preferably 0.3 to Ig on the ratio of 100ml of the feed composition.

[80] The feed and feed additive of the present invention can be prepared by any mixing means known to one skilled in the art such as, for example, mechanical blending, extrusion, palletizing, and spray drying. The order of adding individual components for mixing generally dose not alter the physical characteristics or feeding efficiency of the composition.

[81] The animal feed or feed additive of the present invention can be applied to various animals including mammals, poultry and fish, preferably, commercially important mammals, for example, pig, cow, sheep, goat, experimental rodents such as rat, mouse, hamster, or gerbil mouse, fur animal such as mink or fox, animal in zoological gardens such as monkey, livestock such as cat or dog, poultry such as chicken, turkey, duck, goose, quail and the like and raising fish such as trout and the like.

[82] The animal feed additive of the present invention can be mixed with animal feed in the amount ranging about 1 to lOOg per lkg of animal feed.

[83] For example, the inventive feed composition of the present invention could be prepared by the procedure consisting of the steps of: mixing inventive extract of present invention with other feed component to obtain cohesive granule type to be used directly or other type to purpose further processing and packaging steps, for example, adding water to said feed to perform further conventionally necessary procedure such as pellet, expansion or compression etc.

[84] In accordance with still another aspect of the present invention, there are provided an anti- viral disinfectant composition comprising an extract of pine needleas an active ingredient for preventing and improving diseases caused by the infection of virus.

[85] The disinfectant composition of the present invention can further comprise additional additive component well-known in the art to make more potent disinfectant composition having anti- viral activity.

[86] Providing that the inventive extract of the composition is used as an anti-septic composition, the composition may be prepared by fluid type or tablet type. In case of fluid type composition, the composition may be provided as a spraying antiseptic solution which may be directly sprayed to animal facility or animal body or disinfectant and etc. In case of solid type composition, the composition may be provided as a sterilizer or disinfectant as a form of tablet to disinfect hand or utensil in animal facility or the service trade facility requiring sterilization. In case of fluid type composition, the composition may add other additive known in the art, for example, ethanol or isopropanol which has sterilizing activity by itself and safety to human in the amount ranging 1.0 to 40.0 w/w (%), preferably, 5.0 to 30.0 w/w (%) of the weight of total composition. [87] The fluid type composition of the present invention may be diluted with distilled water in order to improve solubility or dispersion activity of the composition. [88] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. [89]

Advantageous Effects

[90] The present invention provides a veterinary composition, feed additive and disinfectant comprising an extract of pine needle as an active ingredient in an effective amount to treat and prevent the diseases caused by the infection of virus in animal. [91]

Brief Description of the Drawings [92] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; [93] Fig. 1 shows the anti viral activity of the extract of pine needle on MDCK cell causing A/PR/8/34(HlNl) influenza, [94] Fig. 2 shows the anti viral activity of the extract of pine needle on MARK- 145 causing cell porcine reproductive and respiratory syndrome, [95] Fig. 3 presents the anti viral activity of the non-polar solvent soluble extract of pine needle on MDCK cell causing A/PR/8/34(HlNl) influenza. [96]

Best Mode for Carrying Out the Invention [97] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. [98] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner. [99]

Mode for the Invention [100] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner. [101] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[102] Reference Example 1. Preparation of RNA virus

[103] 1-1. Preparation of Influenza A virus

[104] A/PR/8/34(HlNl) strain was obtained from Hokkaido University, A/HS/K5/01 strain was obtained from college of veterinary medicine, Konkuk University, A/ HS/MS96/96 isolation strain was obtained from National Veterinary Research and Quarantine Service and A/CSM/D2/05(H6N5) strain isolated from the migratory bird were obtained from college of veterinary medicine, Konkuk University. And the influenza A viruses were proliferated in the 10-day-old SPF egg for 48 hours for in vitro and in vivo experiment, and left at -8O⁰C.

[105] 1-2. Preparation of Newcastle disease virus (NDV)

[106] Kr-005/00 isolation strain and KJW strain were obtained from National Veterinary

Research and Quarantine Service, proliferated in the 10-day-old SPF egg for 48 hours for in vitro and in vivo experiment and left at -8O⁰C.

[107] 1-3. Preparation of Infectious broncheitis virus (IBV)

[108] M41 strain were obtained from National Veterinary Research and Quarantine

Service, proliferated in the 10-day-old SPF egg for 48 hours for in vitro and in vivo experiment, and left at -8O⁰C.

[109] 1-4. Preparation of Avian pneumovirus (APV)

[110] APV A type Poulvac®SHS modified live vaccine (Fort Dodge Animal Health,

UK) was used.

[I l l] 1-5. Preparation of Porcine reproductive and respiratory syndrome virus

(PRRSV)

[112] ATCC VR-2332 parental virus strain of the PRRSV Ingelvac®PRRS modified live vaccine was obtained from National Veterinary Research and Quarantine Service, proliferated in MARK- 145 cell, and left at -8O⁰C.

[113] 1-6. Preparation of Coxsakie virus(CXV)

[114] Coxsakie virus was obtained from National Veterinary Research and Quarantine

Service, proliferated in Hep-2 cell, and left at -8O⁰C

[115] 1-7, Preparation of reticuloendotheliosis virus(REV)

[116] Chicken syncytial virus(CSV) was obtained from ATCC, proliferated in chicken embryo fibroblast cell, and left at -8O⁰C.

[117] Reference Example 2. Preparation of DNA virus

[118] 2-1. Preparation of aujeszky's disease virus and Adeno virus(ADV)

[119] Aujeszky's disease virus and Adeno virus were obtained from National Veterinary

Research and Quarantine Service, proliferated in Hep-2 cell, and left at -8O⁰C.

[120] Reference Example 3. Cell culture [121] 3-1. MDCK (Madin Darby Canine Kidney) cell

[122] MDCK cell (American Type Culture Collection, Manassas, VA, USA) was cultivated with Eagle's minimum essential medium (EMEM) containing 10% fetal calf serum (Gibco, USA) at 37⁰C in a humidified atmosphere of 95% air-5% CO . Trypsin (5μg/ml), 0.22% sodium hydrogen carbonate (NaHCO ) and gentamaycin (40μg/ml) were added to the Minimum essential medium (MEM) in case of the test for screening the effect of anti- virus.

[123] 3-2. Hep-2 cell

[124] Hep-2 cell was obtained from Korea Research Institute of Chemical Technology

(KRICT, Korea) and cultivated with MEM containing 5% fetal bovine serum (Gibco, USA) at 37⁰C in a humidified atmosphere of 95% air-5% CO

[125] 3-3. Chicken embryo fibroblast cell

[126] Chicken embryo fibroblast cell was cultivated with M 199 medium containing TPB

(Tryptose phosphate broth) and 8% calf serum(Gibco, USA) at 37⁰C in a humidified atmosphere of 95% air-5% CO

[127] 3-4. MARK-145 cell

[128] MARK-145 cell was obtained from National Veterinary Research and Quarantine

Service and cultivated with MEM containing 5% fetal bovine serum(Gibco, USA) at 37⁰C in a humidified atmosphere of 95% air-5% CO

[129] Reference Example 4. Quantitative suspension test with viruses

[130] Quantitative suspension test with viruses was performed by National Veterinary

Research and Quarantine Service regulation 30 (Jan. 27, 2003). To determine the antiviral activity, sample was diluted with 100-fold in distilled water and the diluted extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. 2.5ml of influenza virus was mixed with 2.5ml of respective diluted sample at the interval of 1 min, exactly reacted for 30min with shaking at the interval of 10 mins at 4⁰C. As a negative control group, distilled water was used instead of the diluted sample in experiment.

[131] The cells were prepared by the incubation to proliferate into monolayer on 96 well plates. 25μl of the mixture was inoculated in various dilutions, i.e., un-diluted solution, 10^"1, 10^"2, 10³, 10^"4, 10^"5 and 10^~6 diluted solution on 8 well/dilution. 30min after the change of medium, the medium was cultured for 3 to 4 days at 37⁰C in 5% CO and 95% air condition in a humidified incubator. The cytopathic effects (CPE) of virus were determined using by inverted phase microscope. In case that the virus did not show the cytophatic effect in cell i.e., H9N2 influenza virus and infectious broncheitis virus, 0.2ml of the mixture was inoculated into 10-day-old SPF egg. 3 days after the inoculation, the hemagglutination test and dot immunoassay were performed. The amount of virus was calculated using by Karber method (Payment P et al., Methods and Techniques in Virology, p 33, 1993). [132] Reference Example 5. Plaque reduction assay

[133] 6xlO⁵ cell/well of MDCK cell was cultured in 6 well-tissue cultural plate. After the formation of mono layer, the medium was removed. 0.1ml of virus was inoculated in the concentration of 400pfu/ml, and adhered for 1 hour at 37⁰C in 5% CO and 95% air condition in a humidified incubator. The sample was diluted to 100, 500, 250, 125, 62.5, 31.25μg/ml in MEM medium, double-layered with 1% agarose diluted in same concentration as that of the samples on the cell wherein virus was inoculated, cultured for 2-3 days at 37⁰C in 5% CO and 95% air condition in a humidified incubator. The number of plaque was calculated.

[134] Reference Example 6. Neutral red colorimetric assay

[135] To determine the anti- viral activity, the sample was diluted with 500-fold in distilled water. 2.5ml of virus solution was mixed with 2.5ml of the diluted sample, exactly reacted for 30min with shaking for 10 min intervals at 4⁰C. As a negative control group, distilled water was used instead of the diluted sample in experiment.

[136] The cells were prepared by the incubation to proliferate into monolayer on 96 well plates. 25μl of the mixture was inoculated in various dilutions, i.e., un-diluted solution, 10^"1, 10^"2, 10³, 10^"4, 10^"5 and 10^~6 diluted solution on 8 well/dilution. 30min after the change of medium, the medium was cultured for 3 to 4 days at 37⁰C in 5% CO and 95% air condition in a humidified incubator. 4 days after, neutral red assay was performed according to the method disclosed in the literature (Donald FS et al., Antimicrobial Agents and Chemotherapy, 45(3), pp 743-748, 2001).

[137] Example 1. Preparation of polar solvent soluble extract of pine needle

[138] 1 - 1. Preparation of water soluble extract

[139] The leave of Pinus denstifora Sieb. et Zucc, Pinus thunbergii parlatore and Pinus koraiensis Sieb. et Zucc. were collected in Wha-Seong city was washed, dried and cut into small pieces.

[140] 80g of the dried powder of each pine needle was mixed with 800ml of distilled water and the mixture was heated at 90? for 4 hours by reflux extraction with hot water. Each extract was centrifuged at 10,000xg for 30min and the supernatant was dried with freezing dryer (ModulyoD, ThermoSavant, USA) to obtain 5g (yield: 6.25%) of the extract of Pinus denstifora , 6g of the extract of Pinus thunbergii, and 8g of the extract of Pinus koraiensis respectively.

[141] 1-2. Preparation of ethanol soluble extract

[142] 80g of the dried powder of the each pine needle prepared in Example 1-1 was mixed with 800ml of 30%, 60% and 100% ethanol and the mixtures were extracted at room temperature for 18 hours by reflux extraction. Each extract was centrifuged at 10,000 xg for 30min and the supernatant was dried with dry oven (Program oven, MOV-313P, Sanyo, Japan) at 8O⁰C to 12O⁰C. 200ml of distilled water was added to each sample and the mixture was dried with freezing dryer (ModulyoD, ThermoSavant, USA) to obtain 9g (30% ethanol. yield: 11.25%), 2Og (60% ethanol, yield:25%) and 2Og (100% ethanol, yield:25%) of the extract of Pinus denstifora, 9g (30% ethanol), 2 Ig (60% ethanol) and 2 Ig (100% ethanol) of the extract of Pinus thunbergii and 9g (30% ethanol), 22g (60% ethanol) and 22g (100% ethanol) of the extract of Pinus koraiensis respectively. ( See Table 1)

[143] Table 1

[144] 1-3. Preparation of methanol soluble extract [145] 80g of the dried powder of the each pine needle was mixed with 800ml of 100% methanol and all the procedure was performed according to the procedure disclosed in Example 1-2. At the result, 2Og (yield:25%) of the extract of Pinus denstifora, 21g of the extract of Pinus thunbergii and22g of the extract of Pinus koraiensis were obtained respectively.

[146] Example 2. Preparation of non-polar solvent soluble extract of pine needle [147] 2-1. Preparation of dichloromethane soluble fraction [148] 800g of the dried powder of the Pinus denstifora in the above Example 1, was mixed with 8L of 60% ethanol and the mixture was extracted at 42⁰C by reflux extraction, centrifuged at 10,000xg for 30min. The supernatant was concentrated by rotary evaporator to obtain the extract of Pinus denstifora. The above-described extract of Pinus denstifora was mixed with 800ml of distilled water. 800ml of dichloromethane was added to the mixture and then divided into 800ml of water soluble layer and 800ml of dichloromethane soluble layer.

[149] The dichloromethane soluble layer was concentrated by rotary evaporator and dried with freeze dryer to obtain 4.97g of dichloromethane soluble extract.

[150] 2-2. Preparation of ethyl acetate soluble fraction [151] 800ml of the water soluble layer prepared from Example 2- 1 was mixed with 800ml of ethyl acetate and then divided into 800ml of water soluble layer and 800ml of ethyl acetated soluble layer. [152] The ethyl acetate soluble layer was concentrated by rotary evaporator and dried with freeze dryer to obtain 7.534g of ethyl acetate soluble extract.

[153] 2-3. Preparation of butanol soluble fraction

[154] 800ml of the water soluble layer prepared from Example 2-2 was mixed with

800ml of n-butanol and then divided into 800ml of water soluble layer and 800ml of n- butanol soluble layer.

[155] The n-butanol soluble layer was concentrated by rotary evaporator and dried with freeze dryer to obtain 17.23g of n-butanol soluble extract and 47.1g of water soluble extract.

[156] Experimental Example 1. Anti- viral effect of the each extract of pine needle in vitro

[157] To determine the antiviral activity of the extract of pine needle on various virus, the

60% ethanol extract of pine needle prepared in Example 1-2 was diluted with 500-fold in distilled water. 2.5ml of influenza virus solution (HlNl) was mixed with 2.5ml of the diluted extract of pine needle, exactly reacted for 30min with shaking for 10 min intervals at 4⁰C. As a negative control group, distilled water was used instead of the diluted extract of pine needle in experiment. MDCK (Mardin-Darby Canine Kidney, ATCC, USA) cell line derived from dog kidney was used in the experiment.

[158] The cells were prepared by the incubation to proliferate into monolayer on 96 well plates. 25μl of the mixture was inoculated in various dilutions, i.e., un-diluted solution, 10^"1, 10^"2, 10³, 10^"4, 10^"5 and 10^~6 diluted solution on 8 well/dilution. 30min after the change of medium, the medium was cultured for 3 to 4 days at 37⁰C in 5% CO and 95% air condition in a humidified incubator. The cytopathic effects (CPE) of virus were determined using by inverted phase microscope.

[159] In case that the virus did not show the cytophatic effect in cell i.e., H9N2 influenza virus and infectious bronchitis virus, 0.2ml of the mixture was inoculated into 10-day-old SPF egg. 3 days after the inoculation, the haemagglutination test and dot immunoassay were performed. The amount of virus was calculated using by Karber method (Payment P et al., Methods and Techniques in Virology, p 33, 1993) ( See Table 2).

[160] Table 2

[161]

[162] As can be seen in Table 2, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[163] Table 3

[164]

[165] As can be seen in Table 3, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[166] Table 4

of P. koraiensis dilution(500μg/ml) dilution(500μg/ml) dilution(500μg/ml)

*Infectious bronchitis virus (10 TCID /ml) was treated thereto, and reacted for

30min at 4⁰C

[167]

[168] As can be seen in Table 4, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[169] Table 5

[170]

[171] As can be seen in Table 5, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[172] Table 6

[173] [174] As can be seen in Table 6, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[175] Table 7

[176] [177] As can be seen in Table 7, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[178] Table 8

[179] [180] As can be seen in Table 8, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the anti- viral activity of present composition.

[181] Table 9

[182] [183] As can be seen in Table 9, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[184] Table 10

[185] [186] As can be seen in Table 10, the extract of P. denstifora, P. thunbergii and P. koraiensis showed strong inhibitory effect on the virus, which can confirm the antiviral activity of present composition.

[187] Experimental Example 2. Anti- viral activity of the pine extract prepared by various preparation methods [188] To determine the anti- viral activity of pine needle extract, each 30%, 60% and 100% ethanol soluble extract of pine needle prepared in Example 1-2 was diluted to 100-fold with distilled water and the diluted extract was further diluted to 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. 2.5ml of diluted solution with influenza virus (HlNl) was mixed with 2.5ml of respective diluted extract of pine needle at the interval of 1 min, exactly reacted for 30min with shaking at the interval of 10 mins at 4⁰C. As a negative control group, distilled water was used instead of the diluted sample in experiment.

[189] The cells were prepared by the incubation to proliferate into monolayer on 96 well plates. 25μl of the mixture was inoculated in various dilutions, i.e., un-diluted solution, 10^"1, 10^"2, 10^"3, 10^"4, 10^"5 and 10^~6 diluted solution on 8 well/dilution. 30min after the change of medium, the medium was cultured for 3 to 4 days at 37⁰C in 5% CO and 95% air condition in a humidified incubator. The cytopathic effects (CPE) of virus were determined using by inverted phase microscope. In case that the virus did not show the cytophatic effect in cell i.e., H9N2 influenza virus and infectious bronchitis virus, 0.2ml of the mixture was inoculated into 10-day-old SPF egg. 3 days after the inoculation, the haemagglutination test and dot immunoassay were performed. The amount of virus was calculated using by Karber method (Payment P et al., Methods a nd Techniques in Virology, p 33, 1993).

[190] Table 11

[191]

[192] As can be seen in Table 11, the groups treated with 2000 fold diluted 100% ethanol extract (500μg), 16000-fold diluted 60% ethanol extract (62.5μg), 4000-fold diluted 30% ethanol extract (250μg), and 1000-fold diluted water extract (lmg) for 30 minutes completely reduced A/H1N1 (10 TCID /ml) virus. The groups treated with 8000-fold diluted 100% ethanol extract (125μg), 32000-fold diluted 60% ethanol extract (31.25μg), 16000-fold diluted 30% ethanol extract (62.5μg), and 2000-fold diluted water extract (500μg) for 30 minutes completely reduced A/H1N1 (10 TCID /ml) virus to 10 . It has confirmed that 60% ethanol extract of P. denstifora showed most potent inhibitory activity among them.

[193] Furthermore, the inhibitory effect of 60% ethanol extract of P. denstifora on the other viruses such as Newcastle disease virus; chic infectious bronchitis virus; pneumo virus; pig genitalis respiratory syndrome virus; Coxsakie virus; reticuloendotheliosis virus; Aujeszky's disease virus; and adeno virus etc was also determined as follows.

[194] Experimental Example 3. in vitro anti- viral effect of the extract of pine needle

[195] 3-1. RNA virus

[196] 3-1-1. Anti-viral effect of pine needle extract on A/PR/8/34(ΗlNn influenza in

MDCK cell

[197] To determine the anti- viral activity of pine needle extract on A/PR/8/34(HlNl) influenza in MDCK cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted by 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. The anti- viral activity of pine needle extract on A/PR/8/34(HlNl) influenza in MDCK cell was determined in accordance with the similar method to the procedure disclosed in Reference Example 4 and the result was shown in Fig. 1 and Table 12.

[198] Table 12

[199]

[200] As can be seen in Table 12, the group treated with 16000 fold diluted 60% ethanol extract (62.5 μg) for 30 minutes completely reduced A/PR/8/34 influenza ( 10⁵⁶ TCID /

50 ml) virus. The group treated with 32000-fold diluted 60% ethanol extract (31.25μg) for 30 minutes reduced A/PR/8/34 influenza (10⁵⁶TCID /ml) virus to 10².

50

[201] As can be seen in Fig. 1, IC of the group treated with 60% ethanol extract of P. denstifora showed 31.25μg, and it has confirmed that 60% ethanol extract of P. denstifora showed potent in vitro virucidal activity and in vivo anti-viral activity of A/

PR/8/34(HlNl) influenza virus. [202] 3-1-2. Anti- viral effect of the extract of pine needle on A/HS/MS96/96(H9N2^S) influenza virus in SPF egg

[203] To determine the anti-viral activity of the extract of pine needle on A/ HS/MS96/96(H9N2) influenza virus in SPF egg, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the above extract was diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold and following experiment was performed according to the procedure disclosed in the above Reference Example 4 and the result was shown in Table. 13.

[204] Table 13

[205] [206] As can be seen in Table 13, the group treated with 16000-fold diluted 60% ethanol extract (62.5μg) for 30 minutes completely reduced A/HS/MS96/96(H9N2) (10 5 6 , EID /ml) virus. The group treated with 32000-fold diluted 60% ethanol extract (31.25μg) for 30 minutes reduced A/HS/MS96/96(H9N2) (10 ,5 6 , EID /ml) virus to 10 . It has

50 confirmed that 60% ethanol extract of P. denstifora showed potent inhibitory activity of A/HS/MS96/96(H9N2) (10⁵⁶EID /ml) virus.

[207] 3-1-3. Anti- viral effect of the extract of pine needle on Newcastle disease virus in chicken embryo fibroblast cell [208] To determine the anti-viral activity of the extract of pine needle on Newcastle disease virus in chicken embryo fibroblast cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro virucidal activity using KJW strain, a sort of NDV was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 14).

[209] Table 14

[210] [211] As can be seen in Table 14, the group treated with 4000-fold diluted 60% ethanol extract (250μg) for 30 minutes completely reduced Newcastle disease virus KJW strain(10 -.5 6 TCID /ml). The group treated with 8000-fold diluted 60% ethanol extract

50

(125μg) for 30 minutes reduced Newcastle disease virus KJW strain(10 ,5 6 r TCID /ml)

50 virus to 10 . It has confirmed that 60% ethanol extract of P. denstifora showed potent inhibitory activity of Newcastle disease virus KJW strain(10 TCID /ml).

[212] 3-1-4. Anti- viral effect of the extract of pine needle on infectious bronchitis in SPF

[213] To determine the anti viral activity of the extract of pine needle on infectious bronchitis in SPF egg, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 15).

[214] Table 15

[215] [216] As can be seen in Table 14, the group treated with 4000-fold diluted 60% ethanol extract (250μg) for 30 minutes completely reduced infectious bronchitis virus M41 strain (10 TCID /ml). It has confirmed that 60% ethanol extract of P. denstifora

50

5 6 _r showed potent inhibitory activity of infectious bronchitis virus M41 strain (10 TCID /ml).

50

[217] 3-1-5. Anti- viral effect of the extract of pine needle on avian pneumovirus in chicken embryo fibroblast cell [218] To determine the anti viral activity of the extract of pine needle on avian pneumovirus in chicken embryo fibroblast cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 16).

[219] Table 16

In vitro virucidal activity

The virus was completely reduced The virus was reduced by 10 concentration 500-fold dilution (2mg) 2000-fold dilution (500μg)

* Avian pneumo virus (10 TCID /ml) was treated thereto for 30min at 4⁰C.

[220] [221] As can be seen in Table 16, the group treated with 500-fold diluted 60% ethanol

5 6 _r extract (2mg) for 30 minutes completely reduced Avian pneumo virus (10 TCID / ml). The group treated with 2000-fold diluted 60% ethanol extract (500μg) for 30 minutes reduced Avian pneumo virus (10 5 6 r TCID /ml) to 10 . It has confirmed that

50

60% ethanol extract of P. denstifora showed potent inhibitory activity of Avian pneumo virus (10 TCID /ml).

[222] 3-1-6. Anti- viral effect of the extract of pine needle on porcine reproductive and respiratory syndrome virus (PRRSV) in MARK- 145 cell [223] To determine the anti viral activity of the extract of pine needle on porcine reproductive and respiratory syndrome virus in MARK- 145 cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro inhibitory activity of viral proliferation using by NR analysis was determined according to the procedure disclosed in the above Reference Example 6 and the result was shown in Fig. 2.

[224] As can be seen in Fig. 2, EC of 60% extract of P. denstifora on PRRSV was 121.25μg, which can confirm that 60% extract of P. denstifora showed potent antiviral activity of PRRSV.

[225] 3-1-7. Anti- viral effect of the extract of pine needle on Coxakie virus (CXV) in Hep-2 cell [226] To determine the anti- viral activity of the extract of pine needle on Coxakie virus in Hep-2 cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See. Table 17).

[227] Table 17

[228] As can be seen in Table 17, the group treated with 500-fold diluted 60% ethanol extract (2mg/ml) for 30 minutes completely reduced Coxsakie virus (105 6 r TCID /ml).

50

The group treated with 2000-fold diluted 60% ethanol extract (500μg) for 30 minutes reduced Coxsakie virus (10 _Λ5 6 TCID /ml) to 10 . It has confirmed that 60% ethanol

50 extract of P. denstifora showed potent inhibitory activity of Coxsakie virus (10 TCID /ml).

50

[229] 3-1-8. Anti- viral effect of the extract of pine needle on reticuloendotheliosis virus(REV) in chicken embryo fibroblast cell [230] To determine the anti- viral activity of the extract of pine needle on reticuloendotheliosis virus in chicken embryo fibroblast cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 18).

[231] Table 18

[232] As can be seen in Table 18, the group treated with 4000-fold diluted 60% ethanol extract (250μg/ml) for 30 minutes completely reduced reticuloendotheliosis virus (10 TCID /ml). The group treated with 8000-fold diluted 60% ethanol extract (125μg/ml)

50

5 6 _r for 30 minutes reduced reticuloendotheliosis virus (10 TCID /ml). It has confirmed

50 that 60% ethanol extract of P. denstifora showed potent inhibitory activity of reticu-

5 6 _r loendotheliosis virus (10 TCID /ml).

[233] 3-2. DNA virus [234] 3-2-1. Anti- viral effect of the extract of pine needle on Aujeszky s disease virus (ADV) in MDCK cell

[235] To determine the anti- viral activity of the extract of pine needle on Aujeszky's disease virus (ADV) in MDCK cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 19).

[236] Table 19

In vitro virucidal activity

The virus was completely reduced The virus was reduced by 10 concentration 4000-fold dilution(250μg/ml) 8000-fold dilution(125μg/ml)

* Aujeszky's disease virus (10 TCID /ml) was treated thereto for 30min at 4⁰C.

[237] [238] As can be seen in Table 19, the group treated with 4000-fold diluted 60% ethanol extract (250μg/ml) for 30 minutes completely reduced Aujeszky's disease virus (10 TCID /ml). The group treated with 8000-fold diluted 60% ethanol extract (125μg/ml)

50 for 30 minutes reduced Aujeszky's disease virus (10 5 6 r TCID /ml). It has confirmed

50 that 60% ethanol extract of P. denstifora showed potent inhibitory activity of

Aujeszky's disease virus (10 TCID /ml).

[239] 3-2-2. Anti- viral effect of the extract of pine needle on adenovirus (ADV) in Hep-2 cell [240] To determine the anti- viral activity of the extract of pine needle on adeno virus in Hep-2 cell, the 60% ethanol extract of P. denstifora was diluted with 100-fold in dis tilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table 20).

[241] Table 20

[242] [243] As can be seen in Table 20, the group treated with 100-fold diluted 60% ethanol extract (10mg/ml) for 30 minutes completely reduced Adeno virus (10 TCID /ml). It has confirmed that 60% ethanol extract of P. denstifora showed potent inhibitory activity of Adeno virus (10 TCID /ml).

[244] Experimental Example 4. Anti- viral effect of the non-polar solvent soluble extract of pine needle in vitro [245] 4-1. Anti- viral effect of the extract of pine needle on A/PR/8/34(ΗlNl^s) influenza virus in MDCK cell [246] To determine the anti viral activity of the non-polar solvent soluble extract of pine needle on A/PR/8/34(HlNl) influenza virus in MDCK cell, the non-polar solvent soluble extract of P. denstifora prepared from Example 2 was diluted with 100-fold in distilled water and the extract was diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See Table

21).

[247] Table 21

[248] [249] As can be seen in Table 21, dichloromethane soluble extract (62.5μg/ml), ethyl acetate soluble extract (1 mg/ml), n-butanol soluble extract (125 μg/ml) and water soluble extract (31.25μg/ml)completely reduced Influenza virus HlNl(IO TCID / ml).

[250] Also, to determine the anti A/PR/8/34(HlNl) influenza viral activity of the non- polar solvent soluble extract of pine needle in MDCK cell, plaque reduction assay was performed by following procedure disclosed in the above Reference Example 5.

[251] As can be seen in Fig. 3, each non-polar solvent soluble extract showed potent antiviral effect (%), i.e., 47.5% (dichloromethane soluble extract), 13% (ethyl acetate soluble extract), 28.4% (n-butanol soluble extract) and 86.5% (water soluble extract) in the concentration of 50μg/ml. Among them, dichloromethane soluble extract and water soluble extract of P. denstifora showed strong virucidal activity and antiviral activity of A/PR/8/34(HlNl) influenza virus.

[252] 4-2. in vitro anti- viral effect of the extract of pine needle on Newcastle disease virus in chicken embryo fibroblast cell [253] To determine the anti- viral effect of the non-polar solvent soluble extract of pine needle on Newcastle disease virus in chicken embryo fibroblast cell, the non-polar solvent soluble extract of P. denstifora prepared from Example 2 was diluted with 100-fold in distilled water and the extract was further diluted with 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold and 32000-fold serially. In vitro virucidal activity was determined according to the procedure disclosed in the above Reference Example 4 ( See. Table 22).

[254] Table 22

[255] [256] As can be seen in Table 22, dichloromethane soluble extract (2mg/ml) and water soluble extract (500μg/ml)showed strong virucidal activity and antiviral activity of Newcastle disease virus KJW strain.

[257] Experimental Example 5. Anti- viral effect of the extract of pine needle in vivo [258] 5-1. .Anti-viral effect of the extract of pine needle in mouse [259] 5-1-1. The inhibitory activities of pine needle extract against influenza virus by drinking water route

[260] To determine the inhibitory activity of pine needle extract in mouse infected with influenza virus, 60% ethanol extract of P. denstifora prepared from Example 2 was diluted with 5000-fold in distilled water to the extent to the final concentration of 0.2mg/ml. The groups were classified into three groups; i.e., test group treated with pine needle extract after inoculating the virus, positive control group not treated with pine needle extract after inoculating the virus, and negative control group which was not inoculated with the virus.

[261] 6- weeks-old SPF ICR mouse (Orient Co., Korea) was used and influenza virus A/ PR/8/34(HlNl) which causes high death rate and significant clinical signs in mouse was used in the experiment. 50μl/mouse of virus solution (Titer: 10 TCID /ml) was inoculated to mouse which was anesthetized. After observing the clinical signs and the number of dead mouse for 2- weeks, pathogenic index (PI) was calculated. The test was repeated in case the mouse in negative control group did not alive more than 80% among the mice. The therapeutic effect and the amount of daily feed consumption and drink consumption were shown in Table 23 and Table 24 respectively.

[262] Table 23

[263] [264] Table 24

[265] [266] As can be seen in Table 23 and Table 24, the group treated with 5000-fold diluted 60% ethanol extract of pine needleshowed significant change in the amount of feed and water from the 2° day to 10 day after the virus treatment comparing with positive control group together with reduced clinical signs, death rate and pathogenic index comparing with positive control group.

[267] 5-1-2. The inhibitory activities of pine needle extract against influenza virus through gastric tube administration using by zonde [268] To determine the inhibitory activity of pine needle extract in mouse which was infected by influenza virus, 60% ethanol extract of P. denstifora prepared from Example 2 was diluted with distilled water. The groups were classified into two groups, i.e., test group which was treated with the pine needle extract after inoculating the virus and negative control group which was not treated with the pine needle extract after inoculating the virus.

[269] Female SPF BALB/c mouse (Orient, Korea) weighing from 18 to 2 Ig was used and influenza virus A/PR/8/34(HlNl) which causes high death rate and significant clinical signs in mouse was used in the experiment. 50μl/mouse of virus solution (Titer: 10 TCID /ml) was nasally inoculated to mouse which was anesthetized. 10mg/kg/day of diluted pine needle extract were administrated before treating the virus through gavage administration for five days twice a day. Equivalent amount of physiological solution was administrated instead of pine needle extract twicw a day as a negative control group. After the observing the clinical signs and the number of dead mouse for 2-weeks, pathogenic index (PI) was calculated. Also, the lung of 5 mice per group was enucleated at 3 day and 5 day after inoculating virus and other clinical signs on lung such as lung consolidation rate, lung weight and the amount of virus were measured (Sidwell RW et al., Antiviral Research, 51, pp 179-187, 2001), The therapeutic result was shown in Table 25 and Table 26.

[270] Table 25

[271] Table 26

[272] [273] As can be seen in Table 25 and Table 26, the group treated with the extract of pine needleshowed significantly (P<0.05) strong reducing activity of death rate, and moreover reduced the viral concentration in lung, lung consolidation the 6 days after the treatment and lung weight the 3^r days after the treatment comparing with negative control group.

[274] 5-2. .Anti-viral effect of the extract of pine needle in SPF chicken [275] 5-2-1. The inhibitory activities of pine needle extract against avian influenza virus [276] To determine the inhibitory activity of pine needle extract in SPF chicken infected with avian influenza virus, 60% ethanol extract of P. denstifora prepared from Example 2 was mixed with chicken feed to be the concentration of the extract of 1%, 0.4% and 0.1% (w/w). The groups were classified into two groups; test group treated with the pine needle extract after inoculating the virus and negative control group which was not treated with the pine needle extract after inoculating the virus.

[277] 6- weeks-old SPF chicken (Nam-dug senitech Co., Korea) was used and low pathogenic avian influenza virus (LPAI) A/HS/K5/01(H9N2) which causes high death rate of 1-30% and reduced egg production in chicken was used in the experiment. lOOμl of virus solution (10 TCID /ml) was nasally inoculated and various concentrations of the feed mixed with the extract of pine needle had been supplied for 5-days 4 hours before the inoculation. After 5 days, the air duct and caecum tonsil of the chicken in test group and negative group were delivered and the virus was re- isolated again with the method of inoculating the suspension to 10-days-old chicken embryo. The re-isolation rate and the titer of virus was calculated and the result was shown in Table 27.

[278] Table 27

[279] [280] As can be seen in Table 27, the group treated with 0.1% and 0.4% extract of pine needle showed significantly (P<0.05) strong reducing effect on virus re-isolation rate and virus titer in air duct and caecum tonsil compaing with negative control group.

[281] 5-2-2. The inhibitory activities of pine needle extract against Newcastle disease virus [282] To determine the inhibitory activity of pine needle extract in SPF chicken infected with Newcastle disease virus, 60% ethanol extract of P. denstifora prepared from Example 2 was diluted to the concentration of 500μg/ml, 250 μg/ml and 125 μg/ml respectively. The group was classified into two groups; i.e., test group treated with the pine needle extract after inoculating the virus and negative control group which was not treated with the pine needle extract after inoculating the virus.

[283] 0.2ml of dead oil vaccine of Newcastle disease virus was intramuscularly inoculated into 13- weeks-old SPF chicken. After 7-days, the blood was collected and Kr-005/00 strain (10 EID /30ul) was inoculated after determining its titer.

[284] The distilled water containing various concentrations of the extract of pine needle had been supplied for 5-days from 4 hours before the inoculation. After 6-days, the amount of released virus collected by scratching the surface of chic mouth and cloaca by cotton rod was measured in chicken embryo fibroblast cell and the result was shown in Table 28.

[285] Table 28

[286] [287] As can be seen in Table 28, all the groups treated with 500μg/ml, 250 μg/ml, and 125μg/ml of pine needle extract reduced about more than 10²³⁴TCID /ml of Newcastle disease virus. In particular, the groups treated with 500μg/ml and 250μg/ml of pine needle extract released 10²³⁴TCID /ml and 10²⁹⁸ TCID /ml of virus respectively,

50 50 ^{r J} which result more potent reducing effect comparing with negative control group (10 TCID /ml) significantly(P<0.01).

[288] 5-3. .Anti-viral effect of the extract of pine needle in duck [289] To determine the inhibitory activity of pine needle extract in duck which was infected by influenza virus, 60% ethanol extract of P. denstifora prepared from Example 2 was mixed with duck feed as 1%, 0.4% and 0.1% (w/w). The group was classified as experimental group which was treated by the pine needle extract after inoculating of the virus and negative control group which was not inoculated the virus.

[290] 3-weeks-old duck (Yang seong farm, Korea) and low pathogenic avian influenza virus (LPAI) A/CSM/D2/05(H6N5) isolated from wild duck in Korea were used in the experiment. 50μl of virus solution (10 TCID /ml) was inoculated into the duck through air duct and nasal cavity respectively.

[291] Various concentrations of the feed mixed with the extract of pine needle had been supplied for 5-days from 4 hours before the inoculation. The air duct and caecum tonsil of the duck in test group and negative group were delivered at 3^r day and 6 day and the virus was re-isolated again with the method of inoculating the suspension to 10-days-old chicken embryo. The re-isolation rate and the titer of virus were calculated and the result was shown in Table 29. [292] Table 29

[293] [294] As can be seen in Table 29, the group treated with 0.1% extract of pine needle s howed significantly (P<0.05) more reduced virus re-separation rate at 3^r day after the inoculation comparing with negative control group. The group treated with 0.1% and 0.4% extract of pine needleshowed significantly (P<0.05) more reduced virus titer in air duck at 3^r day after the inoculation comparing with negative control group. Also, all the group treated with 0.1%, 0.4% and 1% extract of pine needleshowed sig- nificantly(P<0.05) more reduced virus re-separation rate and virus titer in caecum tonsil at 6 day after the inoculation comparing with negative control group.

[295]

[296] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[297]

[298] Preparation of feed composition

[299] The 60% ethanol extract of pine needle 240mg

[300] Adjuvant 76Og

[301] Feed compostion preparation was prepared by mixing above components.

[302]

[303] Preparation of disinfectant composition

[304] The 60% ethanol extract of pine needle 3.0g

[305] Hydrogen peroxide 7g

[306] Thymol 1.Og

[307] Ethyl paraben 0.4g

[308] Glutaraldehyde 0.8g

[309] Eugenol 0.5g

[310] disinfectant composition preparation was prepared by mixing the above components together, and then controlling the pH to pH 4.0 by adding H SO thereto and adjusting the components to 10OD. The above describe composition has passed prEN 1276 test (0.03% albumin/light water) according to European Committee for Standardization (CEN).

[311]

[312] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[313]

Industrial Applicability

[314] As described in the present invention, the extractof pine needleshowed anti- viral effect of influenza A viruses, newcastle disease virus, infectious broncheitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus, coxsakie virus, reticuloendotheliosis virus, aujeszky's disease virus, adeno virus and coxsakie virus in vivo and in vitro. Therefore, it can be used as veterinary composition, feed additive and disinfection composition for treating and preventing the diseases caused by viral infectionin animal.

Claims

[I] A veterinary composition comprising a extract of pine needle as an active ingredient for the treatment and prevention diseases caused by the infection of virus.

[2] The veterinary composition of according to claim 1, wherein said extract is crude extract. [3] The veterinary composition of according to claim 1, wherein said crude extract is extracted with the polar solvent selected from the group consisting of water, C -

C 4 alcohols, or the mixtures thereof.

[4] The veterinary composition of according to claim 1, wherein said extract of non- polar solvent soluble extract. [5] The veterinary composition of according to claim 4, wherein said non-polar solvent soluble extract is extracted with the non-polar solvent selected from dichloromethane or ethyl acetate. [6] The veterinary composition of according to claim 1, wherein said pine needle is the needle of Pinus densiflora Sieb.et Zucc, Pinus thunbergii parlatore or Pinus koraiensis Sieb. et Zucc. [7] The veterinary composition of according to claim 1, wherein said virus is RNA type virus or DNA type virus. [8] The veterinary composition of according to claim 7, wherein said RNA type virus is influenza virus, Newcastle disease virus, infectious bronchitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus,

Coxsakie virus or reticuloendotheliosis virus. [9] The veterinary composition of according to claim 8, wherein said influenza virus is HlNl, H9N2 or H6N5 subtype influenza virus. [10] The veterinary composition of according to claim 7, wherein said DNA type virus is Aujeszky's disease virus or adeno virus.

[I I] The veterinary composition of according to claim 1, wherein said disease caused by the infection of virus is viral disease caused by the internal infection of or- thomyxocirus family including influenza virus, paramyxovirus family including newcastle disease virus, coronavirus family including infectious broncheitis virus, picorna virus family including coxsakie virus, retrovirus family including reticuloendotheliosis virus, herpesvirus family including aujeszky's disease virus or adenovirus family including adeno virus.

[12] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of influenza virus family is avian flu or variant infectious avian flu. [13] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of paramyxo virus family is Avian paramyxoo virus, Sendai virus, Canine distemper, Rinderpest or Turkey rhinotracheitis.

[14] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of corona virus family is Turkey corona virus, transmissible gastroenteritis virus, porcine respiratory corona virus, canine corona virus, feline enteritis corona virus, feline infectious peritonitis, rabbit corona virus, bovine corona virus or mouse hepatitis virus

[15] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of picornavirus family is Foot-and-mouth disease, Avian encephalomyelitis virus, Bovine enterovirus or Pocine enterovirus.

[16] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of retrovirus family is Avian leucosis virus, roussarcoma virus, feline leukemia virus, bovine leukemia virus, equine infectious anemia virus or feline immunodeficiency virus.

[17] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of herpesvirus family is Chicken infectious laryngotrachitis virus or Marek's disease.

[18] The veterinary composition of according to claim 11, wherein said viral disease caused by the internal infection of adeno virus family is respiratory syndrome, gastroenteritis, Egg drop syndrome in animal.

[19] The veterinary composition of according to claim 1, wherein said composition comprises 0.1 ~ 50% the extract by weight based on the total weight of the composition.

[20] A method for treating or preventing the diseases caused by the infection of virus in animal comprising administering to said animal an effective amount of an extract of pine needle together with a pharmaceutically acceptable carrier thereof.

[21] A use of an extract of pine needle for manufacture of medicines employed for treating or preventing the diseases caused by the infection of virus in animal.

[22] A feed additive comprising an extract of pine needleas an active ingredient for preventing and improving the diseases caused by viral infection.

[23] The feed additive according to claim 22, wherein said extract is a crude extract.

[24] The feed additive according to claim 23, wherein said crude extract is extracted with the polar solvent selected from the group consisting of water, C -C alcohols, or the mixtures thereof.

[25] The feed additive according to claim 22, wherein said extract of non-polar solvent soluble extract.

[26] The feed additive according to claim 25, wherein said non-polar solvent soluble extract is extracted with the non-polar solvent selected from dichloromethane or ethyl acetate.

[27] The feed additive according to claim 22, wherein said pine needle is the needle of Pinus densiflora Sieb.et Zucc, Pinus thunbergii parlatore or Pinus koraiensis Sieb. et Zucc.

[28] The feed additive according to claim 22, wherein said virus is RNA type virus or

DNA type virus.

[29] The feed additive according to claim 28, wherein said RNA type virus is influenza virus, newcastle disease virus, infectious broncheitis virus, avian pneumo virus, porcine reproductive and respiratory syndrome virus, coxsakie virus or reticuloendotheliosis virus.

[30] The feed additive according to claim 29, wherein said influenza virus is HlNl,

H9N2 or H6N5 subtype influenza virus.

[31] The feed additive according to claim 28, wherein said DNA type virus is aujeszky's disease virus or adeno virus.

[32] The feed additive according to claim 22, wherein said disease caused by the infection of virus is the viral disease caused by the internal infection of or- thomyxocirus family including influenza virus, paramyxovirus family including newcastle disease virus, coronavirus family including infectious broncheitis virus, picorna virus family including coxsakie virus, retrovirus family including reticuloendotheliosis virus, herpesvirus family including aujeszky's disease virus or adenovirus family including adeno virus.

[33] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of influenza virus family is avian flu or variant infectious avian flu.

[34] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of paramyxo virus family is Avian paramyxoo virus, Sendai virus, Canine distemper, Rinderpest or Turkey rhinotracheitis.

[35] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of coronavirus family is Turkey corona virus, transmissible gastroenteritis virus, porcine respiratory corona virus, canine corona virus, feline enteritis corona virus, feline infectious peritonitis, rabbit corona virus, bovine corona virus or mouse hepatitis virus

[36] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of picornavirus family is Foot-and-mouth disease, Avian encephalomyelitis virus, Bovine enterovirus or Pocine enterovirus. [37] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of retrovirus family is Avian leucosis virus, roussarcoma virus, feline leukemia virus, bovine leukemia virus, equine infectious anemia virus or feline immunodeficiency virus. [38] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of herpesvirus family is Chicken infectious laryngotrachitis virus or Marek's disease. [39] The feed additive according to claim 32, wherein said viral disease caused by the internal infection of adeno virus family is respiratory syndrome, gastroenteritis,

Egg drop syndrome in animal.

[40] A feed comprising the feed additive as set forth in claim 22.

[41] An anti- viral disinfectant composition comprising an extract of pine needle as an active ingredient for the treatment and prevention diseases caused by the infection of virus [42] The anti-viral disinfectant composition of according to claim 41, wherein said extract is crude extract. [43] The anti-viral disinfectant composition of according to claim 42, wherein said crude extract is extracted with the polar solvent selected from the group consisting of water, C -C alcohols, or the mixtures thereof. [44] The anti-viral disinfectant composition of according to claim 41, wherein said extract of non-polar solvent soluble extract. [45] The anti-viral disinfectant composition of according to claim 44, wherein said non-polar solvent soluble extract is extracted with the non-polar solvent selected from dichloromethane or ethyl acetate. [46] The anti-viral disinfectant composition of according to claim 41, wherein said pine needle is the needle of Pinus densiflora Sieb.et Zucc, Pinus thunbergii parlatore or Pinus koraiensis Sieb. et Zucc. [47] The anti-viral disinfectant composition of according to claim 41, wherein said virus is RNA type virus or DNA type virus. [48] The anti-viral disinfectant composition of according to claim 47, wherein said

RNA type virus is influenza virus, newcastle disease virus, infectious broncheitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus, coxsakie virus or reticuloendotheliosis virus. [49] The anti-viral disinfectant composition of according to claim 48, wherein said influenza virus is HlNl, H9N2 or H6N5 subtype influenza virus. [50] The anti-viral disinfectant composition of according to claim 47, wherein said

DNA type virus is aujeszky's disease virus or adeno virus.