Abstract:
The present invention provides a muliple DNA vaccine and/or a multivalent DNA vaccine for use in aquiring embroyonic immunity in fowl eggs. The multiple DNA vaccine contains two or more DNA constructs, each containing a DNA molecule encoding an avian viral protein or a fragment thereof capable of inducing a protective immune response against the avian viral disease in fowl. The multivalent DNA vaccine contains one DNA construct which contains two or more DNA molecules, each representing an avian viral gene or a fragment thereof. The multivalent DNA vaccine is capable of expressing two or more viral antigens and inducing protective immune responses against the avian viral diseases in fowl. Both the multiple DNA vaccine and the multivalent DNA vaccine are preferred to be injected into the amniotic fluid of the fowl egg after being fertilized for about 18 days.

Description:
RELATED APPLICATION  
       [0001]    This application claims the priority of U.S. Provisional Application Serial No. 60/362,547, filed on Mar. 8, 2002, which is herein incorporated by reference. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to either a muliple DNA vaccine or a multivalent DNA vaccine for use in aquiring embroyonic immunity in fowl eggs and methods for preparing and using the same. The multiple DNA vaccine contains two or more DNA constructs, each containing a DNA molecule encoding an avian viral protein or a fragment thereof capable of inducing a protective immune response against an avian viral disease in fowl. The multivalent DNA vaccine contains a DNA construct which contains two or more DNA molecules. Each of the DNA molecules represents an avian viral gene or a fragment thereof. The multivalent DNA vaccine is capable of expressing two or more viral antigens and inducing protective immune responses against two or more of the avian viral diseases in fowl. Both the multiple DNA vaccine and the multivalent DNA vaccine are preferred to be injected into the amniotic fluid of the fowl egg after being fertilized for about 18 days.  
         BACKGROUND OF THE INVENTION  
         [0003]    In ovo vaccination of virus-containing vaccines was extensively described by Sharma et al. (U.S. Pat. No. 4,458,630). In particular, it teaches that live Marek&#39;s disease virus can be injected into amniotic fluid within the egg, whereafter the embryo is infected and the vaccine virus replicates to a high titer which induces the formation of protective antibodies in the treated embryo. (See Sharma (1985),  Avian Diseases  29, 1155, 1167-68).  
           [0004]    It is well-known in the worldwide poultry business that certain viral diseases, such as Marek&#39;s disease virus (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), chick anemia virus (CAV), Fowlpox virus (FPV), avian influenza virus (AIV), reovirus, avian leukosis virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus and hemorrhagic enteritis virus (HEV), may cause major outbreak and result in significant economic losses in the commercial poultry industry. Among them, MDV, IBDV, NDV and IBV, are particularly important due to their virulent nature.  
           [0005]    Marek&#39;s Disease (MD) is a malignant, lymphoproliferative disorder disease that occurs naturally in chickens. The disease is caused by a herpesvirus: Marek&#39;s Disease Virus (MDV). MD is ubiquitous, occurring in poultry-producing countries throughout the world. Chickens raised under intensive production systems will inevitably suffer losses from MD. The symptoms of MD appear widely in the nerves, genital organs, internal organs, eyes and skin of the infected birds, causing motor trouble (due to paralysis when the nerves have been affected), functional trouble of the internal organs (due to tumors), and chronic undernourishment (if the internal organs are attacked by the virus). MD affects chickens from about 6 weeks of age, occurring most frequently between ages of 12 and 24 weeks.  
           [0006]    At of this time, there are no methods of treating MD. The control of the disease is based primarily on management methods such as insolating growing chickens from sources of infection, the use of genetically resistant stock, and vaccination. However, management procedures are normally not cost-effective and the progress has been disappointing with respect to the selection of poultry stock with increased genetically controlled resistance. Nowadays, control of MD is almost entirely based on vaccination.  
           [0007]    Infectious bursal disease virus (IBDV) is responsible for a highly contagious immunosuppressive disease in young chickens which causes significant losses to the poultry industry worldwide (See Kibenge (1988),  J. Gen. Virol.,  69:1757-1775). Infection of susceptible chickens with virulent IBDV strains can lead to a highly contagious immunosuppressive condition known as infectious bursal disease (IBD). Damage caused to the lymphoid follicles of the bursa of Fabricius and spleen can exacerbate infections caused by other agents and reduce a chicken&#39;s ability to respond to vaccination as well (See Cosgrove (1962),  Avian Dis.,  6:385-3894).  
           [0008]    IBDV is a member of the Birnaviridae family and its genome consists of two segments of double-stranded RNA (See Dobos et al (1979),  J. Virol.,  32:593-605). The smaller segment B (about 2800 bp) encodes VP 1, the dsRNA polymerase. The larger genomic segment A (about 3000 bp) encodes a 110 kDa precursor polypeptide in a single open reading frame (ORF) that is processed into mature VP2, VP3 and VP4 (See Azad et al (1985),  Virology,  143:35-44). From a small ORF partly overlapping with the polypeptide ORF, segment A can also encode VP5, a 17-kDa protein of unknown function (See Kibenge et al (1991),  J. Gen. Virol.  71:569-577).  
           [0009]    While VP2 and VP3 are the major structural proteins of the virion, VP2 is the major host-protective immunogen and causes induction of neutralizing antibodies (See Becht et al. (1988),  J. Gen. Virol.,  69:631-640; Fahey et al. (1989),  J. Gen. Virol.,  70:1473-1481). VP3 is considered to be a group-specific antigen because it is recognized by monoclonal antibodies (Mabs) directed against VP3 from strains of both serotype 1 and 2 (See Becht et al (1988),  J. Gen. Virol.,  69:631-640). VP4 is a virus-coded protease and is involved in the processing of the precursor protein (See Jagadish et al. (1988),  J. Virol.,  62:1084-1087).  
           [0010]    In the past, control of IBDV infection in young chickens has been achieved by live vaccination with avirulent strains, or principally by the transfer of maternal antibody induced by the administration of live and killed IBDV vaccines to breeder hens. Unfortunately, in recent years, virulent variant strains of IBDV have been isolated from vaccinated flocks in the United States (See e.g., Snyder et al. (1988),  Avian Dis.,  32:535-539; Van der Marel et al. (1990),  Dtsch. Tierarztl. Wschr.,  97:81-83), which drastically undermine the effectiveness of using live vaccination for IBDV.  
           [0011]    Efforts to develop a recombinant vaccine for IBDV have also been made, and the genome of IBDV has been cloned (See Azad et al (1985) “Virology”, 143:35-44). The VP2 gene of IBDV has been cloned and expressed in yeast (See Macreadie et al. (1990),  Vaccine,  8:549-552), as well as in recombinant fowlpox virus (See Bayliss et al (1991),  Arch. Virol.,  120:193-205). When chickens were immunized with the VP2 antigen expressed from yeast, antisera afforded passive protection in chickens against IBDV infection. When used in active immunization studies, the fowlpox virus-vectored VP2 antigen afforded protection against mortality, but not against damage to the bursa of Fabricius.  
           [0012]    Newcastle disease virus (NDV) is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome. Typically, virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (e.g., Paramyxoviridae and Rhabdoviridae) or those having segmented genomes (e.g., Orthomyxoviridae, Bunyaviridae and Arenaviridae). NDV, together with parainfluenza virus, Sendai virus, simian virus 5, and mumps virus, belongs to the Paramyxoviridae family.  
           [0013]    The structural elements of the NDV include the virus envelope which is a lipid bilayer derived from the cell plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) protrude from the envelope allowing the virus to contain both hemagglutinin and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides. The active F protein is involved in penetration of NDV into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane. The matrix protein (M), is involved with viral assembly, and interacts with both the viral membrane as well as the nucleocapsid proteins.  
           [0014]    The main protein subunit of the NDV nucleocapsid is the nucleocapsid protein (NP) which confers helical symmetry on the capsid. In association with the nucleocapsid are the P and L proteins. The phosphoprotein (P), which is subject to phosphorylation, is thought to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation and polyadenylation. The L gene, which encodes an RNA-dependent RNA polymerase, is required for viral RNA synthesis together with the P protein. The L protein, which takes up nearly half of the coding capacity of the viral genome is the largest of the viral proteins, and plays an important role in both transcription and replication.  
           [0015]    The replication of all negative-strand RNA viruses, including NDV, is complicated by the absence of cellular machinery required to replicate RNA. Additionally, the negative-strand genome can not be translated directly into protein, but must first be transcribed into a positive-strand (mRNA) copy. Therefore, upon entry into a host cell, the virus can not synthesize the required RNA-dependent RNA polymerase. The L, P and NP proteins must enter the cell along with the genome on infection. Both the NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) are encapsidated by nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. The cytoplasm is the site of NDV viral RNA replication, just as it is the site for transcription. Assembly of the viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.  
           [0016]    In U.S. Pat. No. 5,427,791, Ahmad et al. describe the embryonal vaccination against NDV, which requires the modification of the viruses through the use of ethyl methane sulfonate (EMS). However, EMS is a mutagen so that the vaccine prepared by the use of EMS is suspected to act as a mutagen as well, which is undesirable for regular administration of the vaccine. Nevertheless, without the modification with EMS, the NDV vaccine cannot be applied for in ovo vaccination as almost all of the embryos will die upon injection of the eggs with the unmodified virus.  
           [0017]    Infectious bronchitis virus (IBV), the prototype of the family Coronaviridae, is the etiological agent of infectious bronchitis (IB). The virus has a single-stranded RNA genome, approximately 20 kb in length, of positive polarity, and is usually about 80-100 nm in size, being round with projecting 20 nm spikes. IBV is the causative agent of an acute, highly contagious disease in chickens of all ages, affecting the respiratory, reproductive and renal systems.  
           [0018]    IBV contains three structural proteins: the spike (S) glycoprotein, the membrane glycoprotein, and the nucleocapsid protein. The spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus. The spike protein is believed likely to be responsible for immunogenicity of the virus, partly by analogy with the spike proteins of other corona-viruses and partly by in vitro neutralisation experiments (See, e.g., D. Cavanagh et al. (1984),  Avian Pathology,  13, 573-583). There are two spike glycoproteins, which are S1 (90,000 daltons) and S2 (84,000 daltons). The polypeptide components of the glycopolypeptides S1 and S2 have been estimated after enzymatic removal of oligosaccharides to have a combined molecular weight of approximately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the S2 polypeptide.  
           [0019]    IBV has been wide-spread in countries where an intensive poultry industry has been developed. Young chickens up to 4 weeks of age are most susceptible to IBV, infection leading to high rates of morbidity and to mortality resulting from secondary bacterial infection. Infection also results in a drop in egg production, or failure to lay at full potential, together with an increase in the number of down-graded eggs with thin, misshapen, rough and soft-shells produced, which can have a serious economic effect.  
           [0020]    Administering live vaccines to a developing chick in the egg (in-ovo) has proven to be a fast (40,000 eggs per hour), effective (100% of the eggs receive the vaccine), and labor saving ($100,000 per year per hatchery) method to vaccinate baby chicks against certain diseases before they hatch.  
           [0021]    The first in-ovo vaccination machine for use on chicken hatching eggs was developed by Embrex, Inc., of Raleigh, N.C. in the late 1980s. (See U.S. Pat. Nos. 5,056,464 and 5,699,751). This in-ovo machine is currently used in about 80% of the U.S. broiler hatcheries, primarily for administering MD vaccines. The popularity of this machine, which has proven to be safe and effective in vaccination of chicks against MD, is also being used increasingly to administer IBD vaccines and ND vaccines.  
           [0022]    In the invention to be presented in the following sections, a DNA-mediated immunization (collectively “DNA vaccines”) will be introduced. There are two kinds of DNA vaccines, i.e., a multiple DNA vaccine and a multivalent DNA vaccine. The multiple DNA vaccine of the present invention contains a combination of two or more DNA construct, each containing a single DNA molecule which is a viral gene or a fragment thereof. The multivalent DNA vaccine of the present invention contains two or more viral genes or fragments thereof linking together in one DNA construct. The viral genes or fragments used in preparation of either the multiple DNA vaccine or the multivalent DNA vaccine are those that encode viral peptides which are antigenic to and can induce both the humoral and the cellular immune system in a host. The DNA vaccines are preferably applied to the egg by needles. The injection of the DNA vaccines in ovo leads to surprisingly strong immune responses which include not only antibody induction and T-cell activation with cytokine secretion, but also the production of cytotoxic T lymphocytes (CTL).  
         SUMMARY OF THE INVENTION  
         [0023]    The present invention provides a multiple DNA vaccine for in ovo injection. The multiple DNA vaccine contains two or more DNA constructs, each DNA construct expressing an antigenic protein of an avian virus causing avian viral disease in fowl. The antigenic protein of the avian virus is capable of inducing a protective immune response against an avian viral disease. The multiple DNA vaccine is preferred to inject into the egg, particularly the amniotic fluid of the egg, of the fowl. The egg is preferred to be fertilized for about 18 days. The preferred fowl includes chicken, turkey, duck, and goose.  
           [0024]    The DNA construct contains a DNA molecule and a vector. The vector can be a plasmid or a viral carrier. The preferred vector is a plasmid. Examples of the plasmid include, but are not limited to, pcDNA3, pVAX1, pSectag, pTracer, pDisplay, pUC system plasmid (such as pUC7, pUC8, pUC18), and pGEM system plasmid. Alternatively, any plasmid which contains a promoter such as CMV promoter, SV40 promoter, RSV promoter, and β-actin promoter, can also be used for preparing the DNA construct. The most favorable plasmid is pcDNA3. The preferred viral carrier is one selected from the group consisting of a baculovirus, a herpes virus, and a pox virus.  
           [0025]    Examples of the avian virus include, but are not limited to Marek&#39;s disease virus (MDV), infectious vursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), avian leukosis virus (ALV), fowlpox virus (FPV), avian paramyxovirus (APV), duck hepatitis virus (DHV), and hemorrhagic enteritis virus (HEV).  
           [0026]    The DNA molecules that are particularly suitable for inducing a protective immune response against the avian viral diseases as shown above include, but are not limited to, the entire of gB gene of Merk&#39;s Disease virus (MDV) having the DNA sequence of SEQ ID NO:1 or a fragment thereof; the entire VP2 gene of infectious bursal disease virus (IBDV) having the DNA sequence of SEQ ID NO:2 or a fragment thereof; the entire HN gene of Newcastle disease virus (NDV) having the DNA sequence (which is from bases 6321 to 8319) of SEQ ID NO:3 or a fragment thereof (i.e., SEQ ID NO:3 is the entire genome of the NDV); the entire S1 gene of infectious bronchitis virus (IBV) having the DNA sequence of SEQ ID NO:4 or a fragment thereof; the entire glycoprotein G gene of infectious laryngotracheitis virus (ILTV) having the DNA sequence of SEQ ID NO:5 or a fragment thereof; the entire VP1, VP0, or VP3 gene of avian encephalomyelitis virus (AEV) or a fragment thereof (the VP1 gene has the DNA sequence of SEQ ID NO:6; the VPO gene has the DNA sequence of SEQ ID NO:7; and the VP3 gene has the DNA sequence of SEQ ID NO:8); the entire paraglycoprotein G gene of avian parainfluenza virus (APV) having the DNA sequence of SEQ ID NO:9 or a fragment thereof the entire type A penton base gene of hemorrhagic enteritis virus (HEV) having the DNA sequence of SEQ ID NO:10 or a fragment thereof; and the entire envelope antigen gene of fowlpox virus (FPV) having the DNA sequence of SEQ ID NO:11 or a fragment thereof.  
           [0027]    One preferred example of the DNA vaccine contains two DNA constructs, each containing a DNA molecule capable of expressing a gene or a fragment thereof which is from Marek&#39;s disease virus (MDV), infectious vursal disease virus (IBDV), Newcastle disease virus (NDV), or infectious bronchitis virus (IBV).  
           [0028]    Another preferred example of the multiple DNA vaccine contains three or more DNA constructs, each containing a DNA molecule capable of expressing a gene or a fragment thereof which is from Marek&#39;s disease virus (MDV), infectious vursal disease virus (IBDV), Newcastle disease virus (NDV), or infectious bronchitis virus (IBV).  
           [0029]    The present invention also provides a method for vaccinating fowl egg and a method for preparing the multiple DNA vaccine. The method for vaccinating fowl egg includes injecting into the fowl egg the multiple DNA vaccine as shown above. The method for preparing the multiple DNA vaccine includes ligating a DNA molecule to a plasmid or virus carrier to form a DNA construct; and then mixing two or more said DNA constructs to form the multiple DNA vaccine. The insertion of the DNA molecule into the vector can be achieved by conventional method, i.e., by ligation the DNA molecule with an enzyme such as T4 DNA ligase when both the genes and the desired vector have been cut with the same restriction enzyme(s) as complementary DNA termini are thereby produced. For pcDNA3, the preferred restriction enzymes are BamH1 and EcoR1.  
           [0030]    In another embedment, there is provided a multivalent DNA vaccine for in ovo injection. The multivalent DNA vaccine comprises a DNA construct containing two or more DNA molecules linked together with a vector. Each of the DNA molecules expresses an antigenic protein of an avian virus, which is capable of inducing a protective immune response against that avian viral disease in fowl. The multivalent DNA vaccine is preferred to be injected into a fowl egg. Each of the DNA molecules of the multivalent DNA vaccine is a gene or a fragment thereof from Marek&#39;s disease virus (MDV), infectious vursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), avian leukosis virus (ALV), fowlpox virus (FPV), avian parainfluenza virus (APV), duck hepatitis virus (DHV), and hemorrhagic enteritis virus (HEV). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0031]    Traditional avian vaccines comprise chemically inactivated virus vaccines or modified live-virus vaccines. Inactivated vaccines require additional immunizations which are not only expensive to produce but also laborious to administer. Further, some infectious virus particles may survive the inactivation process and may cause disease after administration to the animal.  
         [0032]    In general, attenuated live virus vaccines are preferred over inactivated vaccines because they evoke an immune response often based on both humoral and cellular reactions. Such vaccines are normally based on serial passage of virulent strains in tissue culture. However, the attenuation process induces mutations of the viral genome, resulting in a population of virus particles heterogeneous with regard to virulence and immunizing properties. In addition, it is well known that the traditional attenuated live virus vaccines can revert to virulence resulting in disease outbreaks in inoculated animals and the possible spread of the pathogen to other animals.  
         [0033]    Thus, it is advantageous for the industry to employ vaccines based on recombinant DNA technology. The resulting DNA vaccines only contain and express the necessary and relevant immunogenic material that is capable of eliciting a protective immune response against the pathogens and would not display above mentioned disadvantages of the live or inactivated vaccines.  
         [0034]    For the purpose of preparing multiple DNA vaccines or multivalent recombinant DNA vaccines, the DNA sequence of the gene (also used interchangeably as “DNA molecule”) need not contain the full length of DNA encoding the polypeptides. In most cases, a fragment of the gene which encodes an epitope region should be sufficient enough for immunization. The DNA sequence of an epitope region can be found by sequencing the corresponding part of other viral strains and comparing them. The major antigenic determinants are likely to be those showing the greatest heterology. Also, these regions are likely to lie accessibly in the conformational structure of the proteins. One or more such fragments of genes encoding the antigenic determinants can be prepared by chemical synthesis or by recombinant DNA technology. These fragments of genes, if desired, can be linked together or linked to other DNA molecules.  
         [0035]    Also, the viral genes need not be in DNA. In fact, some of the frequently found avian viral diseases are caused by double- or single-stranded RNA viruses. For example, Marek&#39;s diesease virus is a double-stranded RNA virus, while infectious bursal disease virus (IBDV), Newcastle disease virus (NDV) and infectious bronchitis virus (IB) are single-stranded RNA viruses. The RNA viral sequences, however, can be reverse-transcribed into DNA using RT-Polymerase chain reaction (RT-PCR) technology and then incorporated into a vector by the conventional recombinant DNA technology  
         [0036]    In addition, because of the degeneracy of the genetic code it is possible to have numerous RNA and DNA sequences that encode a specified amino acid sequence. Thus, all RNA and DNA sequences which result in the expression of a polypeptide having the antibody binding characteristics are encompassed by this invention.  
         [0037]    To construct a recombinant DNA vaccine, either univalent or multivalent, the DNA sequence of the viral gene can be ligated to other DNA molecules with which it is not associated or linked in nature. Optionally, the DNA sequence of a viral gene can be ligated to another DNA molecule, i.e., a vector, which contains portions of its DNA encoding fusion protein sequences such as β-galactosidase, resulting in a so-called recombinant nucleic acid molecule or DNA construct, which can be used for transformation of a suitable host. Such vector is preferably derived from, e.g., plasmids, or nucleic acid sequences present in bacteriophages, cosmids or viruses.  
         [0038]    Specific vectors which can be used to clone nucleic acid sequences according to the invention are known in the art and include either a plasmid or a virus carrier. Examples of the plasmid include, but are not limited to, pBR322, pcDNA3, pVAX1, pSectag, pTracer, pDisplay, pUC system plasmids (e.g., pUC7, pUC8, pUC18), pGEM system plasmids, Bluescript plasmids or any other plasmids where CMV promoter, SV40 promoter, RSV promoter, or β-actin promoter is included. The preferred plasmid is pcDNA3. Examples of the virus carrier include, but are not limited to, bacteriophages (e.g., λ and the M13-derived phages), SV40, adenovirus, polyoma, baculoviruses, herpes viruses (HVT) or pox viruses (e.g., fowl pox virus).  
         [0039]    The methods to be used for the construction of a recombinant nucleic acid molecule are known to those of ordinary skill in the art. For example, the insertion of the nucleic acid sequence into a cloning vector can easily be achieved by ligation with an enzyme such as T4 DNA ligase when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme(s) so that complementary DNA termini are thereby produced.  
         [0040]    Alternatively, it may be necessary to modify the restriction sites so as to produce blunt ends either by digesting the single-stranded DNA or by filling in the recessive termini with an appropriate DNA polymerase. Subsequently, blunt end ligation with an enzyme such as T4 DNA ligase may be carried out. If desired, any restriction site may be produced by ligating linkers onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site sequences. The restriction enzyme cleaved vector and nucleic acid sequence may also be modified by homopolymeric tailing.  
         [0041]    The present invention provides two kinds of DNA vaccines. The first kind is a multiple DNA vaccine, which includes two or more of univalent DNA vaccines, each containing a DNA sequence encoding at least one polypeptide affording protection against one viral disease such as Marek&#39;s dosease voris (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), Fowlpox virus (FPV), avian influenza virus (AIV), avian leukosis virus (ALV), duck hepatitis virus B genome, and hemorrhagic enteritis virus (HEV), inserted into a commercially available plasmid.  
         [0042]    The second kind is a multivalent recombinant DNA vaccine, which contains two or more genes or gene fragments from different viruses. These genes or gene fragments are carried by a useful vector, which can be either a plasmid or a virus carrier. The multivalent recombinant DNA vaccine encodes two or more antigenic polypeptides which afford protection against at least two viral diseases including, but not limited to, MD, IBD, ND or IB. The viral genes or gene fragments are operatively attached to the vector in reading frame so that they can be expressed in a host. The different structural DNA sequences carried by the vector may be separated by termination and start sequences so that the proteins can be expressed separately or they may be part of a single reading frame and therefore be produced as a fusion protein by methods known in the art.  
         [0043]    The preferred DNA sequences include, but are not limited to, the entire of gB gene of Merk&#39;s Disease virus (MDV) having the DNA sequence of SEQ ID NO:1 or a fragment thereof; the entire VP2 gene of infectious bursal disease virus (IBDV) having the DNA sequence of SEQ ID NO:2 or a fragment thereof; the entire HN gene of Newcastle disease virus (NDV) having the DNA sequence of SEQ ID NO:3 or a fragment thereof; the entire S1 gene of infectious bronchitis virus (IBV) having the DNA sequence of SEQ ID NO:4 or a fragment thereof.  
         [0044]    The DNA sequence encoding the gB polypeptide of MDV has the nucleic acid sequence as SEQ ID NO:1. The DNA sequence contains 3650 bp of linear DNA.  
         [0045]    The DNA sequence encoding the VP2 polypeptide of IBDV has the nucleic acid sequence as SEQ ID NO:2. The DNA sequence contains 3004 bp of linear DNA molecule which is reversely transcribed from IBDV&#39;s RNA template.  
         [0046]    The DNA sequence of the entire genome of NDV contains 15186 bps of DNA, wherein (1) base No. 56 to 1792 encodes NP polypeptide, which is nucleocapsid protein; (2) base No. 1804-3244 encodes P polypeptide, which is a phosphoprotein; (3) base No. 3256-4487 encodes M polypeptide, which is a matrix protein; (4) base No. 4498-6279 encodes F polypeptide, which is a fusion protein; (5) base 6321-8319 encodes HN polypeptide, which is a hemagglutinin-neuraminidase; (6) base No. 8370-15073 encodes L polypeptide, which is a large polymerase protein. The NDV genome has the DNA sequence as SEQ ID NO:3.  
         [0047]    The DNA sequence of the Si polypeptide contains 1611 bp of linear DNA sequence as shown in SEQ ID NO:4, which is reversely transcribed from IBV&#39;s RNA templates.  
         [0048]    The following experimental designs are illustrative, but not limiting the scope of the present invention. Reasonable variations, such as those occur to reasonable artisan, can be made herein without departing from the scope of the present invention.  
         [0049]    I. Materials and Methods  
         [0050]    (A) Virus and Vaccines  
         [0051]    Avian infectious bronchitis virus (IBV), infectious bursal disease (IBD) and Newcastle disease (ND) vaccines were purchased from Intervet Inc.  
         [0052]    (B) Viral RNA Isolation and RT-PCR  
         [0053]    Two hundred microliter recovered attenuated vaccines (Intervet Inc.) were resolved in iced cold GTC buffer (4 M guanidium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1 M-mercaptoethanol) and sodium acetate (pH 4). An equal volume of phenol-chloroform (1:1) was added and placed on ice for 15 minutes after vortexing. The aqueous phase was collected after centrifuge and the RNA was precipitated with an equal volume of isopropanol. RNA was pelleted by centrifugation at 12000 rpm for 20 min at 4° C. and then suspended in diethylpyrocarbonate (DEPC) treated deionized distill water and stored at −70° C.  
         [0054]    (C) Oligonucleotides  
         [0055]    Oligonucleotide primers for RT-PCR amplification were purchased from Promega, and were designed according to the genome of the Avian infectious bronchitis virus (Beaudette CK strain), Newcastle disease virus (Lasota strain) and Infectious bursa disease virus respectively. The sequences of the primers used for PCR were:  
                                                                                                                                                                                                                                   IBS1F′                (SEQ ID NO:12)                    5′ CGGGATCCGCCGCCGCCATGTTGGTAACACCTCTT 3′;                           IBS1R′            (SEQ ID NO:13)                    5′ CGGAATTCTTAACGTCTAAAACGACGTGT 3′;                           NDF F′            (SEQ ID NO:14)                    5′ CGGGATCCGCCGCCGCCATGGGCTCCAGACCTTCTACC 3′;                           NDF R′            (SEQ ID NO:15)                    5′ CCGCTCGAGTTACATTTTTGTAGTGGCTCTCATT 3′;                           NDHN F′            (SEQ ID NO:16)                    5′ CGGGATCCGCCGCCGCCATGGACCGCGCCGTTAGGCAAG 3′;                           NDHN R′            (SEQ ID NO:17)                    5′ GCTCTAGATTACTCAACTAGCCAGACCTG 3′;                           IBDVP2F′            (SEQ ID NO:18)                    5′ CGGGATCCGCCGCCGCCATGACAAACCTGCAAGAT 3′;                           IBDVP2R′            (SEQ ID NO:19)                    5′ CGGAATTCTTACCTTATGGCCCGGATTAT 3′.           
 
         [0056]    (D) Reverse Transcription Polymerase Reaction (RT-PCR)  
         [0057]    Reverse transcription of IBV, NDV and IBDV RNA were carried out at 42° C. for 30 min in 2.5× Taq buffer (200 mM NaCl, 15 mM Tris-HCl, pH7.4, 15 mM MgCl 2 , 15 mM β-mercaptoethanol, and 0.25 mM each of dATP, dCTP, dGTP, and dTTP). In addition to the Taq buffer, the reaction mixture (40 μl) also contained viral RNA, 2.4 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega), 16 U of RNasin (Promega), and 0.01 nmol reverse primer (IBDVP2R, NDF F, NDHN F or IBS1R). The final volume of the reaction mixture was 40 μl. After reverse transcription, the following reagents were added to the reverse transcription mixture: 0.02 nmol of each nucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 0.01 nmol of forward primer (IBDVP2F, NDF R, NDHN R or IBS1F) and 1.5 U of Taq DNA polymerase (Strategene). Water was then added to a final volume of 100 μl. The reaction was carried out for 32 cycles in a Thermal Cycler (Perkin Elmer-Cetus). Each PCR cycle consisted of 1 min of denaturation at 94° C., 1 min of annealing at 57° C., and 2 min of DNA chain elongation at 72° C.  
         [0058]    (E) Preparation of DNA Constructs  
         [0059]    The plasmids pCMV-VP2, pCMV-S1, pCMV-NDF and pCMV-NDHN were constructed with the VP2, S1, NDF and NDHN genes from IBD vaccine, IBV vaccine and NDV vaccine respectively, placed downstream of the commercial plasmid pcDNA3. (Invitrogen, U.S.A.). All of the genes were inserted into the pcDNA3 vector using restriction enzymes BamH1, EcoR1, XbaI and XhoI (underlined characters in the sequence of the primers). Sequences of the all genes in the pcDNA3 vector were verified by sequencing in both directions.  
         [0060]    (F) Preparation of DNA and DNA Delivery  
         [0061]    The quantity of plasmid DNA that had been purified by affinity chromatography (Qiagen. Inc.) was determined by spectrophotometric measurements at 260 and 280 nm. The DNA in aliquots to 100 μg was suspended in 100 μl of PBS (0.14M NaCl, 10 mM sodium phosphate, pH 7.4). For DNA delivery, 1 cc syringe with a 20 gauge 1 and ½ inch needle were used. For the in-ovo groups, the embryos (18-day-old fertilized and developing eggs from the setting trays) were injected with 0.1 milliliters of DNA vaccine (100 μg) into the large end of each egg through the air cell with a needle. The eggs were then transferred into the hatchery where they remained until they hatched at about 21 days of age. For the IM (Intramuscular), all of the vaccines (⅕ dose of live vaccines) were injected into the chicken&#39;s thoracic muscle at 10 days post hatchery.  
         [0062]    II. Experimental Design  
         [0063]    Specific Pathogen Free (SPF) fertilized eggs (n=60) were randomized into 12 groups. All groups (five eggs each group), all eggs were given 100 μl in volume each. 100 μg pCMV-NDF+100 μg pCMV-NDHN mixture was injected in each egg of group A, 100 μg pCMV-S1 was injected in each egg of group B, 100 μg pCMV-VP2 was injected in each egg of group C, 100 μg pCMV-NDF+100 μg pCMV-NDHN+100 μg pCMV-S1(ND+IB) was injected in each egg of group D, 100 μg pCMV-NDF+100 μg pCMV-NDHN+100 μg pCMV-VP2 (ND+IBD) was injected in each egg of group E, 100 μg pCMV-VP2+100 μg pCMV-VP2 mixture (IB+IBD) was injected in each egg of group F, 100 μg pCMV-NDF+100 μg pCMV-NDHN+100 μg pCMV-S1+100 μg pCMV-VP2 mixture (ND+IB+IBD) was injected in each egg of group G, one dose of commercialized in-ovo IBD vaccine (Embrex, Inc) was injected in each egg of group H as positive control, 100 ul PBS was injected in each egg of group I, J, K and L. All chickens in this experiment were given 100 μl in volume (⅕ dose of live vaccines), injected into the chicken&#39;s thoracic muscle each at 10 days post hatchery. Chickens in group A and I were injected with NDV vaccine, group B and J were injected with IBV vaccine, group C and K were injected with IBDV vaccine, group D were injected with the mixture of NDV+IB vaccines, group E were injected with the mixture of NDV+IBD vaccines, group F were injected with the mixture of IB+IBD vaccines and group G and L were injected with the mixture of NDV, IB and IBD vaccines.  
         [0064]    III. Serology Detection  
         [0065]    All of the serum samples were collected at 10 days (injected with low dose live vaccines at the same time), 17 days, 24 days and 31 days post hatchery. The antibody titers were detected by ELISA using IB, IBD and NDV antibody test kits which purchased from IDEXX Laboratories, Inc. All of the samples were detected duplicated. Dilute test samples five hundred fold (1:500) with sample diluents prior to being assayed. The test procedure was applied according to the kit&#39;s manual. For the assay to be valid, measure and record absorbance values at 650 nm, A (650). The relative level of antibody in the unknown was determined by calculating the sample to positive (S/P) ratio. Endpoint titers were calculated using the formula: Log 10 Titer=1.09(Log 10 S/P)+3.36  
         [0066]    Results  
         [0067]    As shown in Table 1, the results demonstrated that, for the detection of anti-IBD antibodies, the IBDV recombinant antigens VP2 could be expressed and played the role of primary stimulation. The titers increased rapidly after a low dose vaccine booster. The titers of group C, E, F and G at 17 days post hatchery (i.e. 7 days post IM injection) were significantly higher than those of group K and L. Most importantly, the expression of IBDV antigen was not interfered by other monovalent DNA vaccines (NDV and IBV). The same results were also applied to IB and NDV DNA vaccines. The titers of group B, D, F and G were higher than those of group J and L at 17 days post hatchery (Table 2) and the titers of group A, D, E and G were higher than those of group I and L at 17 days post hatchery (Table 3). The only unpredicted result was the anti-NDV titer could not be highly induced by the triple valent DNA vaccine (Table 3, group G), but anti-IBD and anti-IB did (Tables 1 and 2, group G).  
                                     TABLE 1                           Serum Samples Detected by IDEXX IBD Antibody Test Kit (Ab Titers       Correspond to the Average Titers ±SD)       Immunization and Sample Collection Schedule (days)            Animal Group   10 Days PH*   17 Days PH   24 Days PH   31 Days PH               C(IBD)    —**   4535 ± 1267   16623 ± 3105   21254 ± 3852       E(IBD + ND)   —   1685 ± 655    17339 ± 2185   19041 ± 2967       F(IBD + IB)   —   8252 ± 2205   10057 ± 1295   17561 ± 2006       G(IBD + IB + ND)   —   9111 ± 1701   13127 ± 1763   16694 ± 2134       H(IBD positive)   6553 ± 851   13025 ± 2131    18015 ± 1592   18853 ± 2614       K(PBS/IBD)   —   —   1853 ± 302   17002 ± 2965       L(PBS/IBD + IB + ND)   —   —    6923 ± 1168   18063 ± 2531                                  
 
         [0068]    [0068]                                     TABLE 2                           Serum Samples Detected by IDEXX IB Antibody test kit (Ab Titers       Correspond to the Average Titers ±SD       Immunization and Sample Collection Schedule (days)            Animal Group   10 Days PH*   17 Days PH   24 Days PH   31 Days PH               B(IB)    —**   441 ± 117   2426 ± 264   3214 ± 877       D(IB + ND)   —   586 ± 182    805 ± 221   1988 ± 501       F(IB + IBD)   —   509 ± 89     685 ± 186   1192 ± 237       G(IBD+ IB +   —   499 ± 81    688 ± 78   2551 ± 531       ND)       J(PBS/IB)   —   —   485 ± 76   1662 ± 441       L(PBS/IBD +   —   —    819 ± 202   1332 ± 488       IB + ND)                                    
         [0069]    [0069]                                     TABLE 3                           Serum Samples Detected by IDEXX ND Antibody Test Kit (Ab Titers       Correspond to the Average Titers ±SD).       Immunization and Sample Collection Schedule (days)            Animal Group   10 Days PH*   17 Days PH   24 Days PH   31 Days PH               A(ND)   —   466 ± 101   2394 ± 456   8103 ± 2198       D(ND + IB)   —   706 ± 140   1778 ± 378   6811 ± 2206       E(ND + IBD)   —   517 ± 104   3021 ± 411   5991 ± 1695       G(IBD + IB +   —   —   —   783 ± 201       ND)       I(PBS/ND)   —   —   1853 ± 324   3912 ± 304        L(PBS/IBD +   —   —   4027 ± 662   5807 ± 1996       IB + ND)                                    
         [0070]    [0070] 
     
       
       
         1 
         
           
             19  
           
           
             1  
             3650  
             DNA  
             Marek′s disease virus  
           
            1 

tcgagctcgc cggggatgtt tagtcacgat agacatcggt tcgcccagcc gtcgaataca     60 

gcattatatt ttagtgttga aaatgtaggg ctgcttcctc acttaaagga ggaaatggct    120 

cgattcatgt ttcatagcag tagaaaaaca gattggaccg tcagtaagtt tagagggttt    180 

tatgacttta gcactataga taatgtaact gcggcccatc gcatggcttg gaaatatatc    240 

aaagaactga tttttgcaac agctttattt tcttctgtat ttaaatgtgg cgaattgcac    300 

atctgtcgtg ccgacagttt gcagatcaac agcaatggag actatgtatg gaaaaatgga    360 

atatatataa catatgaaac cgaatatcca cttataatga ttctggggtc agaatcaagc    420 

acttcagaaa cgcaaaatat gactgcaatt attgatacag atgttttttc gttgctttat    480 

tctattttgc agtatatggc ccccgttacg gcagatcagg tgcgagtaga acagattacc    540 

aacagccacg cccccatctg acccgtccaa tattcttgtg tccctgcatt ttatctcaca    600 

caatttatga acagcatcat taagatcatc tcactatgca ctattttagg cggaattgca    660 

tttttttcct tatagttatt ctatatggta cgaactcatc tccgagtacc caaaatgtga    720 

catcaagaga agttgtttcg agcgtccagt tgtctgagga agagtctacg ttttatcttt    780 

gtcccccacc agtgggttca accgtgatcc gtctagaacc gccgcgaaaa tgtcccgaac    840 

ctagaaaagc caccgagtgg ggtgaaggaa tcgcgatatt atttaaagag aatatcagtc    900 

catataaatt taaagtgacg ctttattata aaaatatcat tcagacgacg acatggacgg    960 

ggacgacata tagacagatc actaatcgat atacagatag gacgcccgtt tccattgaag   1020 

agatcacgga tctaatcgac ggcaaaggaa gatgctcatc taaagcaaga taccttagaa   1080 

acaatgtata tgttgaagcg tttgacaggg atgcgggaga aaaacaagta cttctaaaac   1140 

catcaaaatt caacacgccc gaatctaggg catggcacac gactaatgag acgtataccg   1200 

tgtggggatc accatggata tatcgaacgg gaacctccgt caattgtata gtagaggaaa   1260 

tggatgcccg ctctgtgttt ccgtattcat attttgcaat ggccaatggc gacatcgcga   1320 

acatatctcc attttatggt ctatccccac cagaggctgc cgcagaaccc atgggatatc   1380 

cccaggataa tttcaaacaa ctagatagct atttttcaat ggatttggac aagcgtcgaa   1440 

aagcaagcct tccagtcaag cgtaactttc tcatcacatc acacttcaca gttgggtggg   1500 

actgggctcc aaaaactact cgtgtatgtt caatgactaa gtggaaagag gtgactgaaa   1560 

tgttgcgtgc aacagttaat gggagataca gatttatggc ccgtgaactt tcggcaacgt   1620 

ttatcagtaa tacgactgag tttgatccaa atcgcatcat attaggacaa tgtattaaac   1680 

gcgaggcaga agcagcaatc gagcagatat ttaggacaaa atataatgac agtcacgtca   1740 

aggttggaca tgtacaatat ttcttggctc tcgggggatt tattgtagca tatcagcctg   1800 

ttctatccaa atccctggct catatgtacc tcagagaatt gatgagagac aacaggaccg   1860 

atgagatgct cgacctggta aacaataagc atgcaattta taagaaaaat gctacctcat   1920 

tgtcacgatt gcggcgagat attcgaaatg caccaaatag aaaaataaca ttagacgaca   1980 

ccacagctat taaatcgaca tcgtctgttc aattcgccat gctccaattt ctttatgatc   2040 

atatacaaac ccatattaat gatatgttta gtaggattgc cacagcttgg tgcgaattgc   2100 

agaatagaga acttgtttta tggcacgaag ggataaagat taatcctagc gctacagcga   2160 

gtgcaacatt aggaaggaga gtggctgcaa agatgttggg ggatgtcgct gctgtatcga   2220 

gctgcactgc tatagatgcg gaatccgtca ctttgcaaaa ttctatgcga gttatcacat   2280 

ccactaatac atgttatagc cgaccattgg ttctattttc atatggagaa aaccaaggaa   2340 

acatacaggg acaactcggt gaaaacaacg agttgcttcc aacgctagag gctgtagagc   2400 

catgctcggc taatcatcgt agatattttc tgtttggatc cggttatgct ttatttgaaa   2460 

actataattt tgttaagatg gtagacgctg ccgatataca gattgctagc acatttgtcg   2520 

agcttaatct aaccctgcta gaagatcggg aaattttgcc tttatccgtt tacacaaaag   2580 

aagagttgcg tgatgttggt gtattggatt atgcagaagt agctcgccgc aatcaactac   2640 

atgaacttaa attttatgac ataaacaaag taatagaagt ggatacaaat tacgcgttta   2700 

tgaacggttt ggccgaattg tttaacggta tgggtcaggt agggcaagct ataggcaaag   2760 

ttgtagtagg ggctgccggt gcaatcgtat ctaccatatc tggtgtctct gctttcatgt   2820 

caatcccttt ggggctttcg gcaatcggtt taatcattat agcaggactc gtggctgcat   2880 

ttttagcata tcgttatgta aacaagctta aaagcaatcc aatgaaagcc ctttatccta   2940 

tgacaacaga agtgcttaag gcacaggcaa cgcgtgagtt gcatggcgag gaatcagatg   3000 

atttggaacg aacatctatt gatgaaagaa aattagaaga agctagagaa atgataaaat   3060 

atatggcgtt agtctccgcg gaagaacgcc acgagaaaaa actgcggaga aagaggcgag   3120 

gcactaccgc cgttctatcg gaccacctgg caaaaatgag gattaaaaat agtaacccta   3180 

aatatgataa gttacctact acatattcag actcagaaga tgatgctgtg taagtgggca   3240 

ctattatatt tgaactgaat aaaacgcata gagcatgata tggtttactc atttattgcg   3300 

agatataaag catattcaat acgatatatt gcgaacgtga tgctaaaaac atagctccct   3360 

gtattattga tgcgccatca tttgattaat aaatacatcg acgccggcat cactggtgcg   3420 

gtgtatacca gctacggcgc tagcattcat ggtatcccgt gattgctcga tgctttcctt   3480 

ctgaattccg tcggaacgct cctgagagat ggtcgcagtt attggtacat ttcgaccagc   3540 

ctccggatct gaaactggca caggaatgca ccgtggaatt ggtagaagtt tttccttccg   3600 

tggaaggcat agggcgttcg actcccatgg gccatgaaac tgtgggatgt              3650 

 
           
             2  
             3004  
             DNA  
             infectious bursal disease virus (IBDV)  
           
            2 

tgatgccaac aaccggaccg gcgtccattc cggacgacac cctggagaag cacactctca     60 

ggtcagagac ctcgacctac aatttgactg tgggggacac agggtcaggg ctaattgtct    120 

ttttccctgg attccctggc tcaattgtgg gtgctcacta cacactgcag agcaatggga    180 

actacaagtt cgatcagatg ctcctgactg cccagaacct accggccagt tacaactact    240 

gcaggctagt gagtcggagt ctcacagtga ggtcaagcac acttcctggt ggcgtttatg    300 

cactaaacgg caccataaac gccgtgacct tccaaggaag cctgagtgaa ctgacagatg    360 

ttagctacaa tgggttgatg tctgcaacag ccaacatcaa cgacaaaatt gggaacgtcc    420 

tagtagggga aggggtcacc gtcctcagct tacccacatc atatgatctt gggtatgtga    480 

ggcttggtga ccccattccc gcaatagggc ttgacccaaa aatggtagcc acatgtgaca    540 

gcagtgacag gcccagagtc tacaccataa ctgcagccga tgattaccaa ttctcatcac    600 

agtaccaacc aggtggggta acaatcacac tgttctcagc caacattgat gccatcacaa    660 

gcctcagcgt tgggggagag ctcgtgtttc gaacaagcgt ccacggcctt gtactgggcg    720 

ccaccatcta cctcataggc tttgatggga caacggtaat caccagggct gtggccgcaa    780 

acactgggct gacgaccggc accgacaacc ttatgccatt caatcttgtg attccaacaa    840 

acgagataac ccagccaatc acatccatca aactggagat agtgacctcc aaaagtggtg    900 

gtcaggcagg ggatcagatg ttatggtcgg caagagggag cctagcagtg acgatccatg    960 

gtggcaacta tccaggggcc ctccgtcccg tcacgctagt ggcctacgaa agagtggcaa   1020 

caggatccgt cgttacggtc gctggggtga gcaacttcga gctgatccca aatcctgaac   1080 

tagcaaagaa cctggttaca gaatacggcc gatttgaccc aggagccatg aactacacaa   1140 

aattgatact gagtgagagg gaccgtcttg gcatcaagac cgtctggcca acaagggagt   1200 

acactgactt tcgtgaatac ttcatggagg tggccgacct caactctccc ctgaagattg   1260 

caggagcatt cggcttcaaa gacataatcc gggccataag gaggatagct gtgccggtgg   1320 

tctccacatt gttcccacct gccgctcccc tagcccatgc aattggggaa ggtgtagact   1380 

acctgctggg cgatgaggca caggctgctt caggaactgc tcgagccgcg tcaggaaaag   1440 

caagagctgc ctcaggccgc ataaggcagc tgactctcgc cgccgacaag gggtacgagg   1500 

tagtcgcgaa tctattccag gtgccccaga atcccgtagt cgacgggatt cttgcttcac   1560 

ctggggtact ccgcggtgca cacaacctcg actgcgtgtt aagagagggt gccacgctat   1620 

tccctgtggt tattacgaca gtggaagacg ccatgacacc caaagcattg aacagcaaaa   1680 

tgtttgctgt cattgaaggc gtgcgagaag acctccaacc tccatctcaa agaggatcct   1740 

tcatacgaac tctctctgga cacagagtct atggatatgc tccagatggg gtacttccac   1800 

tggagactgg gagagactac accgttgtcc caatagatga tgtctgggac gacagcatta   1860 

tgctgtccaa agatcccata cctcctattg tgggaaacag tggaaatcta gccatagctt   1920 

acatggatgt gtttcgaccc aaagtcccaa tccatgtggc tatgacggga gccctcaatg   1980 

cttgtggcga gattgagaaa gtaagcttta gaagcaccaa gctcgccact gcacaccgac   2040 

ttggccttaa gttggctggt cccggagcat tcgatgtaaa caccgggccc aactgggcaa   2100 

cgttcatcaa acgtttccct cacaatccac gcgactggga caggctcccc tacctcaacc   2160 

taccatacct tccacccaat gcaggacgcc agtaccacct tgccatggct gcatcagagt   2220 

tcaaagagac ccccgaactc gagagtgccg tcagagcaat ggaagcagca gccaacgtgg   2280 

acccactatt ccaatctgca ctcagtgtgt tcatgtggct ggaagagaat gggattgtga   2340 

ctgacatggc caacttcgca ctcagcgacc cgaacgccca tcggatgcga aattttcttg   2400 

caaacgcacc acaagcaggc agcaagtcgc aaagggccaa gtacgggaca gcaggctacg   2460 

gagtggaggc tcggggcccc acaccagagg aagcacagag ggaaaaagac acacggatct   2520 

caaagaagat ggagaccatg ggcatctact ttgcaacacc agaatgggta gcactcaatg   2580 

ggcaccgagg gccaagcccc ggccagctaa agtactggca gaacacacga gaaataccgg   2640 

acccaaacga ggactatcta gactacgtgc atgcagagaa gagccggttg gcatcagaag   2700 

aacaaatcct aagggcagct acgtcgatct acggggctcc aggacaggca gagccacccc   2760 

aagctttcat agacgaagtt gccaaagtct atgaaatcaa ccatggacgt ggcccaaacc   2820 

aagaacagat gaaagatctg ctcttgactg cgatggagat gaagcatcgc aatcccaggc   2880 

gggctctacc aaagcccaag ccaaaaccca atgctccaac acagagaccc cctggtcggc   2940 

tgggccgctg gatcaggacc gtctctgatg aggaccttga gtgaggctcc tggaagtctc   3000 

ccga                                                                3004 

 
           
             3  
             15186  
             DNA  
             Newcastle disease virus (NDV)  
           
            3 

accaaacaga gaatccgtga gttacgataa aaggcgaagg agcaattgaa gtcgcacggg     60 

tagaaggtgt gaatctcgag tgcgagcccg aagcacaaac tcgagaaagc cttctgccaa    120 

catgtcttcc gtatttgatg agtacgaaca gctcctcgcg gctcagactc gccccaatgg    180 

agctcatgga gggggagaaa aagggagtac cttaaaagta gacgtcccgg tattcactct    240 

taacagtgat gacccagaag atagatggag ctttgtggta ttctgcctcc ggattgctgt    300 

tagcgaagat gccaacaaac cactcaggca aggtgctctc atatctcttt tatgctccca    360 

ctcacaggta atgaggaacc atgttgccat tgcagggaaa cagaatgaag ccacattggc    420 

cgtgcttgag attgatggct ttgccaacgg cacgccccag ttcaacaata ggagtggagt    480 

gtctgaagag agagcacaga gatttgcgat gatagcagga tctctccctc gggcatgcag    540 

caacggaacc ccgttcgtca cagccggggc agaagatgat gcaccagaag acatcaccga    600 

taccctggag aggatcctct ctatccaggc tcaagtatgg gtcacagtag caaaagccat    660 

gactgcgtat gagactgcag atgagtcgga aacaaggcga atcaataagt atatgcagca    720 

aggcagggtc caaaagaaat acatcctcta ccccgtatgc aggagcacaa tccaactcac    780 

gatcagacag tctcttgcag tccgcatctt tttggttagc gagctcaaga gaggccgcaa    840 

cacggcaggt ggtacctcta cttattataa cctggtaggg gacgtagact catacatcag    900 

gaataccggg cttactgcat tcttcttgac actcaagtac ggaatcaaca ccaagacatc    960 

agcccttgca cttagtagcc tctcaggcga catccagaag atgaagcagc tcatgcgttt   1020 

gtatcggatg aaaggagata atgcgccgta catgacatta cttggtgata gtgaccagat   1080 

gagctttgcg cctgccgagt atgcacaact ttactccttt gccatgggta tggcatcagt   1140 

cctagataaa ggtactggga aataccaatt tgccagggac tttatgagca catcattctg   1200 

gagacttgga gtagagtacg ctcaggctca gggaagtagc attaacgagg atatggctgc   1260 

cgagctaaag ctaaccccag cagcaatgaa gggcctggca gctgctgccc aacgggtctc   1320 

cgacgatacc agcagcatat acatgcctac tcaacaagtc ggagtcctca ctgggcttag   1380 

cgaggggggg tcccaagctc tacaaggcgg atcgaataga tcgcaagggc aaccagaagc   1440 

cggggatggg gagacccaat tcctggatct gatgagagcg gtagcaaata gcatgaggga   1500 

ggcgccaaac tctgcacagg gcactcccca atcggggcct cccccaactc ctgggccatc   1560 

ccaagataac gacaccgact gggggtattg atggacaaaa cccagcctgc ttccacaaaa   1620 

acatcccaat gccctcaccc gtagtcgacc cctcgatttg cggctctata tgaccacacc   1680 

ctcaaacaaa catccccctc tttcctccct ccccctgctg tacaactccg cacgccctag   1740 

ataccacagg cacaatgcgg ctcactaaca atcaaaacag agccgaggga attagaaaaa   1800 

agtacgggta gaagagggat attcagagat cagggcaagt ctcccgagtc tctgctctct   1860 

cctctacctg atagaccagg acaaacatgg ccacctttac agatgcagag atcgacgagc   1920 

tatttgagac aagtggaact gtcattgaca acataattac agcccagggt aaaccagcag   1980 

agactgttgg aaggagtgca atcccacaag gcaagaccaa ggtgctgagc gcagcatggg   2040 

agaagcatgg gagcatccag ccaccggcca gtcaagacaa ccccgatcga caggacagat   2100 

ctgacaaaca accatccaca cccgagcaaa cgaccccgca tgacagcccg ccggccacat   2160 

ccgccgacca gccccccacc caggccacag acgaagccgt cgacacacag ttcaggaccg   2220 

gagcaagcaa ctctctgctg ttgatgcttg acaagctcag caataaatcg tccaatgcta   2280 

aaaagggccc atggtcgagc ccccaagagg ggaatcacca acgtccgact caacagcagg   2340 

ggagtcaacc cagtcgcgga aacagtcagg aaagaccgca gaaccaagtc aaggccgccc   2400 

ctggaaacca gggcacagac gtgaacacag catatcatgg acaatgggag gagtcacaac   2460 

tatcagctgg tgcaacccct catgctctcc gatcaaggca gagccaagac aatacccttg   2520 

tatctgcgga tcatgtccag ccacctgtag actttgtgca agcgatgatg tctatgatgg   2580 

aggcgatatc acagagagta agtaaggttg actatcagct agatcttgtc ttgaaacaga   2640 

catcctccat ccctatgatg cggtccgaaa tccaacagct gaaaacatct gttgcagtca   2700 

tggaagccaa cttgggaatg atgaagattc tggatcccgg ttgtgccaac atttcatctc   2760 

tgagtgatct acgggcagtt gcccgatctc acccggtttt agtttcaggc cctggagacc   2820 

cctctcccta tgtgacacaa ggaggcgaaa tggcacttaa taaactttcg caaccagtgc   2880 

cacatccatc tgaattgatt aaacccgcca ctgcatgcgg gcctgatata ggagtggaaa   2940 

aggacactgt ccgtgcattg atcatgtcac gcccaatgca cccgagttct tcagccaagc   3000 

tcctaagcaa gttagatgca gccgggtcga tcgaggaaat caggaaaatc aagcgccttg   3060 

ctctaaatgg ctaattacta ctgccacacg tagcgggtcc ctgtccactc ggcatcacac   3120 

ggaatctgca ccgagttccc ccccgcagac ccaaggtcca actctccaag cggcaatcct   3180 

ctctcgcttc ctcagcccca ctgaatggtc gcgtaaccgt aattaatcta gctacattta   3240 

agattaagaa aaaatacggg tagaattgga gtgccccaat tgtgccaaga tggactcatc   3300 

taggacaatt gggctgtact ttgattctgc ccattcttct agcaacctgt tagcatttcc   3360 

gatcgtccta caaggcacag gagatgggaa gaagcaaatc gccccgcaat ataggatcca   3420 

gcgccttgac ttgtggactg atagtaagga ggactcagta ttcatcacca cctatggatt   3480 

catctttcaa gttgggaatg aagaagccac tgtcggcatg atcgatgata aacccaagcg   3540 

cgagttactt tccgctgcga tgctctgcct aggaagcgtc ccaaataccg gagaccttat   3600 

tgagctggca agggcctgtc tcactatgat agtcacatgc aagaagagtg caactaatac   3660 

tgagagaatg gttttctcag tagtgcaggc accccaagtg ctgcaaagct gtagggttgt   3720 

ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg aaagcgccag agaagattcc   3780 

cgggagtgga accctagaat acaaggtgaa ctttgtctcc ttgactgtgg taccgaagaa   3840 

ggatgtctac aagatcccag ctgcagtatt gaaggtttct ggctcgagtc tgtacaatct   3900 

tgcgctcaat gtcactatta atgtggaggt agacccgagg agtcctttgg ttaaatcttt   3960 

gtctaagtct gacagcggat actatgctaa cctcttcttg catattggac ttatgaccac   4020 

cgtagatagg aaggggaaga aagtgacatt tgacaagctg gaaaagaaaa taaggagcct   4080 

tgatctatct gtcgggctca gtgatgtgct cgggccttcc gtgttggtaa aagcaagagg   4140 

tgcacggact aagcttttgg cacctttctt ctctagcagt gggacagcct gctatcccat   4200 

agcaaatgct tctcctcagg tggccaagat actctggagt caaaccgcgt gcctgcggag   4260 

cgttaaaatc attatccaag caggtaccca acgcgctgtc gcagtgaccg ccgaccacga   4320 

ggttacctct actaagctgg agaaggggca cacccttgcc aaatacaatc cttttaagaa   4380 

ataagctgcg tctctgagat tgcgctccgc ccactcaccc agatcatcat gacacaaaaa   4440 

actaatctgt cttgattatt tacagttagt ttacctgtct atcaagttag aaaaaacacg   4500 

ggtagaagat tctggatccc ggttggcgcc ctccaggtgc aagatgggct ccagaccttc   4560 

taccaagaac ccagcaccta tgatgctgac tatccgggtt gcgctggtac tgagttgcat   4620 

ctgtccggca aactccattg atggcaggcc tcttgcagct gcaggaattg tggttacagg   4680 

agacaaagcc gtcaacatat acacctcatc ccagacagga tcaatcatag ttaagctcct   4740 

cccgaatctg cccaaggata aggaggcatg tgcgaaagcc cccttggatg catacaacag   4800 

gacattgacc actttgctca ccccccttgg tgactctatc cgtaggatac aagagtctgt   4860 

gactacatct ggagggggga gacaggggcg ccttataggc gccattattg gcggtgtggc   4920 

tcttggggtt gcaactgccg cacaaataac agcggccgca gctctgatac aagccaaaca   4980 

aaatgctgcc aacatcctcc gacttaaaga gagcattgcc gcaaccaatg aggctgtgca   5040 

tgaggtcact gacggattat cgcaactagc agtggcagtt gggaagatgc agcagtttgt   5100 

taatgaccaa tttaataaaa cagctcagga attagactgc atcaaaattg cacagcaagt   5160 

tggtgtagag ctcaacctgt acctaaccga attgactaca gtattcggac cacaaatcac   5220 

ttcacctgct ttaaacaagc tgactattca ggcactttac aatctagctg gtggaaatat   5280 

ggattactta ttgactaagt taggtgtagg gaacaatcaa ctcagctcat taatcggtag   5340 

cggcttaatc accggtaacc ctattctata cgactcacag actcaactct tgggtataca   5400 

ggtaactcta ccttcagtcg ggaacctaaa taatatgcgt gccacctact tggaaacctt   5460 

atccgtaagc acaaccaggg gatttgcctc ggcacttgtc cccaaagtgg tgacacaggt   5520 

cggttctgtg atagaagaac ttgacacctc atactgtata gaaactgact tagatttata   5580 

ttgtacaaga atagtaacgt tccctatgtc ccctggtatt tattcctgct tgagcggcaa   5640 

tacgtcggcc tgtatgtact caaagaccga aggcgcactt actacaccat acatgactat   5700 

caaaggttca gtcatcgcca actgcaagat gacaacatgt agatgtgtaa accccccggg   5760 

tatcatatcg caaaactatg gagaagccgt gtctctaata gataaacaat catgcaatgt   5820 

tttatcctta ggcgggataa ctttaaggct cagtggggaa ttcgatgtaa cttatcagaa   5880 

gaatatctca atacaagatt ctcaagtaat aataacaggc aatcttgata tctcaactga   5940 

gcttgggaat gtcaacaact cgatcagtaa tgctttgaat aagttagagg aaagcaacag   6000 

aaaactagac aaagtcaatg tcaaactgac tagcacatct gctctcatta cctatatcgt   6060 

tttgactatc atatctcttg tttttggtat acttagcctg attctagcat gctacctaat   6120 

gtacaagcaa aaggcgcaac aaaagacctt attatggctt gggaataata ctctagatca   6180 

gatgagagcc actacaaaaa tgtgaacaca gatgaggaac gaaggtttcc ctaatagtaa   6240 

tttgtgtgaa agttctggta gtctgtcagt tcagagagtt aagaaaaaac taccggttgt   6300 

agatgaccaa aggacgatat acgggtagaa cggtaagaga ggccgcccct caattgcgag   6360 

ccaggcttca caacctccgt tctaccgctt caccgacaac agtcctcaat catggaccgc   6420 

gccgttagcc aagttgcgtt agagaatgat gaaagagagg caaaaaatac atggcgcttg   6480 

atattccgga ttgcaatctt attcttaaca gtagtgacct tggctatatc tgtagcctcc   6540 

cttttatata gcatgggggc tagcacacct agcgatcttg taggcatacc gactaggatt   6600 

tccagggcag aagaaaagat tacatctaca cttggttcca atcaagatgt agtagatagg   6660 

atatataagc aagtggccct tgagtctccg ttggcattgt taaatactga gaccacaatt   6720 

atgaacgcaa taacatctct ctcttatcag attaatggag ctgcaaacaa cagtgggtgg   6780 

ggggcaccta tccatgaccc agattatata ggggggatag gcaaagaact cattgtagat   6840 

gatgctagtg atgtcacatc attctatccc tctgcatttc aagaacatct gaattttatc   6900 

ccggcgccta ctacaggatc aggttgcact cgaataccct catttgacat gagtgctacc   6960 

cattactgct acacccataa tgtaatattg tctggatgca gagatcactc acattcatat   7020 

cagtatttag cacttggtgt gctccggaca tctgcaacag ggagggtatt cttttctact   7080 

ctgcgttcca tcaacctgga cgacacccaa aatcggaagt cttgcagtgt gagtgcaact   7140 

cccctgggtt gtgatatgct gtgctcgaaa gtcacggaga cagaggaaga agattataac   7200 

tcagctgtcc ctacgcggat ggtacatggg aggttagggt tcgacggcca gtaccacgaa   7260 

aaggacctag atgtcacaac attattcggg gactgggtgg ccaactaccc aggagtaggg   7320 

ggtggatctt ttattgacag ccgcgtatgg ttctcagtct acggagggtt aaaacccaat   7380 

tcacccagtg acactgtaca ggaagggaaa tatgtgatat acaagcgata caatgacaca   7440 

tgcccagatg agcaagacta ccagattcga atggccaagt cttcgtataa gcctggacgg   7500 

tttggtggga aacgcataca gcaggctatc ttatctatca aggtgtcaac atccttaggc   7560 

gaagacccgg tactgactgt accgcccaac acagtcacac tcatgggggc cgaaggcaga   7620 

attctcacag tagggacatc tcatttcttg tatcaacgag ggtcatcata cttctctccc   7680 

gcgttattat atcctatgac agtcagcaac aaaacagcca ctcttcatag tccttataca   7740 

ttcaatgcct tcactcggcc aggtagtatc ccttgccagg cttcagcaag atgccccaac   7800 

tcgtgtgtta ctggagtcta tacagatcca tatcccctaa tcttctatag aaaccacacc   7860 

ttgcgagggg tattcgggac aatgcttgat ggtgtacaag caagacttaa ccctgcgtct   7920 

gcagtattcg atagcacatc ccgcagtcgc attactcgag tgagttcaag cagtaccaaa   7980 

gcagcataca caacatcaac ttgttttaaa gtggtcaaga ctaataagac ctattgtctc   8040 

agcattgctg aaatatctaa tactctcttc ggagaattca gaatcgtccc gttactagtt   8100 

gagatcctca aagatgacgg ggttagagaa gccaggtctg gctagttgag tcaattataa   8160 

aggagttgga aagatggcat tgtatcacct atcttctgcg acatcaagaa tcaaaccgaa   8220 

tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac aatcagccag tgctcatgcg   8280 

atcagattaa gccttgtcat taatctcttg attaagaaaa aatgtaagtg gcaatgagat   8340 

acaaggcaaa acagctcatg gtaaataata cgggtaggac atggcgagct ccggtcctga   8400 

aagggcagag catcagatta tcctaccaga gccacacctg tcttcaccat tggtcaagca   8460 

caaactactc tattactgga aattaactgg gctaccgctt cctgatgaat gtgacttcga   8520 

ccacctcatt ctcagccgac aatggaaaaa aatacttgaa tcggcctctc ctgatactga   8580 

gagaatgata aaactcggaa gggcagtaca ccaaactctt aaccacaatt ccagaataac   8640 

cggagtgctc caccccaggt gtttagaaca actggctaat attgaggtcc cagattcaac   8700 

caacaaattt cggaagattg agaagaagat ccaaattcac aacacgagat atggagaact   8760 

gttcacaagg ctgtgtacgc atatagagaa gaaactgctg gggtcatctt ggtctaacaa   8820 

tgtcccccgg tcagaggagt tcagcagcat tcgtacggat ccggcattct ggtttcactc   8880 

aaaatggtcc acagccaagt ttgcatggct ccatataaaa cagatccaga ggcatctgat   8940 

ggtggcagct aagacaaggt ctgcggccaa caaattggtg atgctaaccc ataaggtagg   9000 

ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat acgaatgaga acaagttcac   9060 

atgtcttacc caggaacttg tattgatgta tgcagatatg atggagggca gagatatggt   9120 

caacataata tcaaccacgg cggtgcatct cagaagctta tcagagaaaa ttgatgacat   9180 

tttgcggtta atagacgctc tggcaaaaga cttgggtaat caagtctacg atgttgtatc   9240 

actaatggag ggatttgcat acggagctgt ccagctactc gagccgtcag gtacatttgc   9300 

aggagatttc ttcgcattca acctgcagga gcttaaagac attctaattg gcctcctccc   9360 

caatgatata gcagaatccg tgactcatgc aatcgctact gtattctctg gtttagaaca   9420 

gaatcaagca gctgagatgt tgtgtctgtt gcgtctgtgg ggtcacccac tgcttgagtc   9480 

ccgtattgca gcaaaggcag tcaggagcca aatgtgcgca ccgaaaatgg tagactttga   9540 

tatgatcctt caggtactgt ctttcttcaa gggaacaatc atcaacgggt acagaaagaa   9600 

gaatgcaggt gtgtggccgc gagtcaaagt ggatacaata tatgggaagg tcattgggca   9660 

actacatgca gattcagcag agatttcaca cgatatcatg ttgagagagt ataagagttt   9720 

atctgcactt gaatttgagc catgtataga atatgaccct gtcaccaacc tgagcatgtt   9780 

cctaaaagac aaggcaatcg cacaccccaa cgataattgg cttgcctcgt ttaggcggaa   9840 

ccttctctcc gaagaccaga agaaacatgt aaaagaagca acttcgacta atcgcctctt   9900 

gatagagttt ttagagtcaa atgattttga tccatataaa gagatggaat atctgacgac   9960 

ccttgagtac cttagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt  10020 

gaaagttaat ggacggatct tcgctaagct gacaaagaag ttaaggaact gtcaggtgat  10080 

ggcggaaggg atcctagccg atcagattgc acctttcttt cagggaaatg gagtcattca  10140 

ggatagcata tccttgacca agagtatgct agcgatgagt caactgtctt ttaacagcaa  10200 

taagaaacgt atcactgact gtaaagaaag agtatcttca aaccgcaatc atgatccgaa  10260 

aagcaagaac cgtcggagag ttgcaacctt cataacaact gacctgcaaa agtactgtct  10320 

taattggaga tatcagacaa tcaaattgtt cgctcatgcc atcaatcagt tgatgggcct  10380 

acctcacttc ttcgaatgga ttcacctaag actgatggac actacgatgt tcgtaggaga  10440 

ccctttcaat cctccaagtg accctactga ctgtgacctc tcaagagtcc ctaatgatga  10500 

catatatatt gtcagtgcca gagggggtat cgaaggatta tgccagaagc tatggacaat  10560 

gatctcaatt gctgcaatcc aacttgctgc agctagatcg cattgtcgtg ttgcctgtat  10620 

ggtacagggt gataatcaag taatagcagt aacgagagag gtaagatcag acgactctcc  10680 

ggagatggtg ttgacacagt tgcatcaagc cagtgataat ttcttcaagg aattaattca  10740 

tgtcaatcat ttgattggcc ataatttgaa ggatcgtgaa accatcaggt cagacacatt  10800 

cttcatatac agcaaacgaa tcttcaaaga tggagcaatc ctcagtcaag tcctcaaaaa  10860 

ttcatctaaa ttagtgctag tgtcaggtga tctcagtgaa aacaccgtaa tgtcctgtgc  10920 

caacattgcc tctactgtag cacggctatg cgagaacggg cttcccaaag acttctgtta  10980 

ctatttaaac tatataatga gttgtgtgca gacatacttt gactctgagt tctccatcac  11040 

caacaattcg caccccgatc ttaatcagtc gtggattgag gacatctctt ttgtgcactc  11100 

atatgttctg actcctgccc aattaggggg actgagtaac cttcaatact caaggctcta  11160 

cactagaaat atcggtgacc cggggactac tgcttttgca gagatcaagc gactagaagc  11220 

agtgggatta ctgagtccta acattatgac taatatctta actaggccgc ctgggaatgg  11280 

agattgggcc agtctgtgca acgacccata ctctttcaat tttgagactg ttgcaagccc  11340 

aaatattgtt cttaagaaac atacgcaaag agtcctattt gaaacttgtt caaatccctt  11400 

attgtctgga gtgcacacag aggataatga ggcagaagag aaggcattgg ctgaattctt  11460 

gcttaatcaa gaggtgattc atccccgcgt tgcgcatgcc atcatggagg caagctctgt  11520 

aggtaggaga aagcaaattc aagggcttgt tgacacaaca aacaccgtaa ttaagattgc  11580 

gcttactagg aggccattag gcatcaagag gctgatgcgg atagtcaatt attctagcat  11640 

gcatgcaatg ctgtttagag acgatgtttt ttcctccagt agatccaacc accccttagt  11700 

ctcttctaat atgtgttctc tgacactggc agactatgca cggaatagaa gctggtcacc  11760 

tttgacggga ggcaggaaaa tactgggtgt atctaatcct gatacgatag aactcgtaga  11820 

gggtgagatt cttagtgtaa gcggagggtg tacaagatgt gacagcggag atgaacaatt  11880 

tacttggttc catcttccaa gcaatataga attgaccgat gacaccagca agaatcctcc  11940 

gatgagggta ccatatctcg ggtcaaagac acaggagagg agagctgcct cacttgcaaa  12000 

aatagctcat atgtcgccac atgtaaaggc tgccctaagg gcatcatccg tgttgatctg  12060 

ggcttatggg gataatgaag taaattggac tgctgctctt acgattgcaa aatctcggtg  12120 

taatgtaaac ttagagtatc ttcggttact gtccccttta cccacggctg ggaatcttca  12180 

acatagacta gatgatggta taactcagat gacattcacc cctgcatctc tctacaggtg  12240 

tcaccttaca ttcacatatc caatgattct caaaggctgt tcactgaaga aggagtcaaa  12300 

gaggggaatg tggtttacca acagagtcat gctcttgggt ttatctctaa tcgaatcgat  12360 

ctttccaatg acaacaacca ggacatatga tgagatcaca ctgcacctac atagtaaatt  12420 

tagttgctgt atcagagaag cacctgttgc ggttcctttc gagctacttg gggtggtacc  12480 

ggaactgagg acagtgacct caaataagtt tatgtatgat cctagccctg tatcggaggg  12540 

agactttgcg agacttgact tagctatctt caagagttat gagcttaatc tggagtcata  12600 

tcccacgata gagctaatga acattctttc aatatccagc gggaagttga ttggccagtc  12660 

tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa  12720 

tacccgaaat tggatcagtg aagctcagaa ttcagatgtg gtccgcctat ttgaatatgc  12780 

agcacttgaa gtgctcctcg actgttctta ccaactctat tacctgagag taagaggcct  12840 

agacaatatt gtcttatata tgggtgattt atacaagaat atgccaggaa ttctactttc  12900 

caacattgca gctacaatat ctcatcccgt cattcattca aggttacatg cagtgggcct  12960 

ggtcaaccat gacggatcac accaacttgc agatacggat tttatcgaaa tgtctgcaaa  13020 

actattagta tcttgcaccc gacgtgtgat ctccggctta tattcaggaa ataagtatga  13080 

tctgctgttc ccatctgtct tagatgataa cctgaatgag aagatgcttc agctgatatc  13140 

ccggttatgc tgtctgtaca cggtactctt tgctacaaca agagaaatcc cgaaaataag  13200 

aggcttaact gcagaagaga aatgttcaat actcactgag tatttactgt cggatgctgt  13260 

gaaaccatta cttagccccg atcaagtgag ctctatcatg tctcctaaca taattacatt  13320 

cccagctaat ctgtactaca tgtctcggaa gagcctcaat ttgatcaggg aaagggagga  13380 

cagggatact atcctggcgt tgttgttccc ccaagagcca ttattagagt tcccttctgt  13440 

gcaagatatt ggtgctcgag tgaaagatcc attcacccga caacctgcgg catttttgca  13500 

agagttagat ttgagtgctc cagcaaggta tgacgcattc acacttagtc agattcatcc  13560 

tgaactcaca tctccaaatc cggaggaaga ctacttagta cgatacttgt tcagagggat  13620 

agggactgca tcttcctctt ggtataaggc atctcatctc ctttctgtac ccgaggtaag  13680 

atgtgcaaga cacgggaact ccttatactt agctgaaggg agcggagcca tcatgagtct  13740 

tctcgaactg catgtaccac atgaaactat ctattacaat acgctctttt caaatgagat  13800 

gaaccccccg caacgacatt tcgggccgac cccaactcag tttttgaatt cggttgttta  13860 

taggaatcta caggcggagg taacatgcaa agatggattt gtccaagagt tccgtccatt  13920 

atggagagaa aatacagagg aaagtgacct gacctcagat aaagcagtgg ggtatattac  13980 

atctgcagtg ccctacagat ctgtatcatt gctgcattgt gacattgaaa ttcctccagg  14040 

gtccaatcaa agcttactag atcaactagc tatcaattta tctctgattg ccatgcattc  14100 

tgtaagggag ggcggggtag taatcatcaa agtgttgtat gcaatgggat actactttca  14160 

tctactcatg aacttgtttg ctccgtgttc cacaaaagga tatattctct ctaatggtta  14220 

tgcatgtcga ggagatatgg agtgttacct ggtatttgtc atgggttacc tgggcgggcc  14280 

tacatttgta catgaggtgg tgaggatggc aaaaactctg gtgcagcggc acggtacgct  14340 

cttgtctaaa tcagatgaga tcacactgac caggttattc acctcacagc ggcagcgtgt  14400 

gacagacatc ctatccagtc ctttaccaag attaataaag tacttgagga agaatattga  14460 

cactgcgctg attgaagccg ggggacagcc cgtccgtcca ttctgtgcgg agagtctggt  14520 

gagcacgcta gcgaacataa ctcagataac ccagattatc gctagtcaca ttgacacagt  14580 

tatccggtct gtgatatata tggaagctga gggtgatctc gctgacacag tatttctatt  14640 

taccccttac aatctctcta ctgacgggaa aaagaggaca tcacttatac agtgcacgag  14700 

acagatccta gaggttacaa tactaggtct tagagtcgaa aatctcaata aaataggcga  14760 

tataatcagc ctagtgctta aaggcatgat ctccatggag gaccttatcc cactaaggac  14820 

atacttgaag catagtacct gccctaaata tttgaaggct gtcctaggta ttaccaaact  14880 

caaagaaatg tttacagaca cttctgtatt gtacttgact cgtgctcaac aaaaattcta  14940 

catgaaaact ataggcaatg cagtcaaagg atattacagt aactgtgact cttaacgaaa  15000 

atcacatatt aataggctcc ttttttggcc aattgtattc ttgttgattt aatcatatta  15060 

tgttagaaaa aagttgaacc ctgactcctt aggactcgaa ttcgaactca aataaatgtc  15120 

ttaaaaaaag gttgcgcaca attattcttg agtgtagtct cgtcattcac caaatctttg  15180 

tttggt                                                             15186 

 
           
             4  
             1611  
             DNA  
             infectious bronchitis virus (IBV)  
           
            4 

atgttggtaa cacctctttt actagtgact cttttgtgtg cactatgtag tgctgctttg     60 

tatgacagta gttcttacgt gtactactac caaagtgcct tcagaccacc tgatggttgg    120 

catttacatg ggggtgcgta tgcggttgtt aatatttcta gtgaatctaa taatgcaggc    180 

tcttcatctg ggtgtactgt tggtattatt catggtggtc gtgttgttaa tgcttcttct    240 

atagctatga cggcaccgtc atcaggtatg gcttggtcta gcagtcagtt ttgtactgca    300 

tactgtaact tttcagatac tacagtgttt gttacacatt gttacaaaca tgttgggtgt    360 

cctataactg gcatgcttca acagcattct atacgtgttt ctgctatgaa aaatggccag    420 

cttttttata atttaacagt tagtgtagct aagtacccta cttttaaatc atttcagtgt    480 

gttaataatt taacatccgt atatttaaat ggtgatcttg tttacacctc taatgagacc    540 

acagatgtta catctgcagg tgtttatttt aaagctggtg gacctataac ttataaagtt    600 

atgagagaag ttagagccct ggcttatttt gttaatggta ctgcacaaga tgttattttg    660 

tgtgatgggt cacctagagg cttgttagca tgccagtata atactggcaa tttttcagat    720 

ggcttttatc cttttactaa tagtagttta gttaagcaga agtttattgt ctatcgtgaa    780 

aatagtgtta atactacttt tacgttacac aatttcactt ttcataatga gactggcgcc    840 

aacccaaatc ctagtggtgt ccagaatatt caaacttacc aaacacaaac agctcagagt    900 

ggttattata attttaattt ttcctttctg agtagttttg tttataagga gtctaatttt    960 

atgtatggat cttatcaccc aagttgtaat tttagactag aaactattaa taatggtttg   1020 

tggtttaatt cactttcagt ttcaattgct tacggtcctc ttcaaggtgg ttgcaagcaa   1080 

tctgtcttta gtggtagagc aacctgttgt tatgcttact catatggagg tcctttgctg   1140 

tgtaaaggtg tttattcagg tgagttagat cataattttg aatgtggact gttagtttat   1200 

gttactaaga gcggtggctc tcgtatacaa acagccactg aaccgccagt tataactcaa   1260 

cacaattata ataatattac tttaaatact tgtgttgatt ataatatata tggcagaact   1320 

ggccaaggtt ttattactaa tgtaaccgac tcagctgtta gttataatta tctagcagac   1380 

gcaggtttgg ctattttaga tacatctggt tccatagaca tctttgtcgt acaaagtgaa   1440 

tatggtctta attattataa ggttaaccct tgcgaagatg tcaaccagca gtttgtagtt   1500 

tctggtggta aattagtagg tattcttact tcacgtaatg agactggttc ccagcttctt   1560 

gagaatcagt tttacatcaa aatcactaat ggaacacgtc gttttagacg t            1611 

 
           
             5  
             960  
             DNA  
             infectious laryngotracheitis virus (ILTV)  
           
            5 

ggaggggaga gagacaactt cagctcgaag tctgaagaga catcatgagc ggcttcagta     60 

acataggatc gattgccacc gtttccctag tatgctcgct tttgtgcgca tctgtattag    120 

gggcgccggt actggacggg ctcgagtcga gccctttccc gttcgggggc aaaattatag    180 

cccaggcgtg caaccgcacc acgattgagg tgacggtccc gtggagcgac tactctggtc    240 

gcaccgaagg agtgtcagtc gaggtgaaat ggttctacgg gaatagtaat cccgaaagct    300 

tcgtgttcgg ggtggatagc gaaacgggca gtggacacga ggacctgtct acgtgctggg    360 

ctctaatcca taatctgaac gcgtctgtgt gcagggcgtc tgacgccggg atacctgatt    420 

tcgacaagca gtgcgaaaaa gtgcagagaa gactgcgctc cggggtggaa cttggtagtt    480 

acgtgtctgg caatggatcc ctggtgctgt acccagggat gtacgatgcc ggcatctacg    540 

cctaccagct ctcagtgggt gggaagggat ataccgggtc tgtttatcta gacgtcggac    600 

caaaccccgg atgccacgac cagtatgggt acacctatta cagcctggcc gacgaggcgt    660 

cagacttatc atcttatgac gtagcctcgc ccgaactcga cggtcctatg gaggaagatt    720 

attccaattg tctagacatg cccccgctac gcccatggac aaccgtttgt tcgcatgacg    780 

tcgaggagca ggaaaacgcc acggacgagc tttacctatg ggacgaggaa tgcgccggtc    840 

cgctggacga gtacgtcgac gaaaggtcag agacgatgcc caggatggtt gtcttttcac    900 

cgccctctac gctccagcag tagccacccg agagtgtttt ttgtgagcgc ccacgcaaca    960 

 
           
             6  
             810  
             DNA  
             avian encephalomyelitis virus (AEV)  
           
            6 

gggaaagagg atgaaggagg atttttcagt gtgcctgaag tggagcaaca tgttgttgag     60 

gataaggaac cacagggacc tttgcacgtg acaccttttg gcgctgttaa agctatggag    120 

gacccccaat tggccaggaa aacacctggc acattccctg aattagctcc tggtaaacct    180 

cgacatacag tggaccacat ggatctgtat aagttcatgg ggcgtgccca ttacttgtgg    240 

ggacatgaat tcaccaaaac tgacatgcag tacacattcc agataccatt aagtcccatt    300 

aaagagggtt ttgtgacggg tacacttagg tggtttttaa gtcttttcca actgtatcgt    360 

ggttctctcg acattaccat gacatttgca ggaaaaacta atgtggatgg cattgtgtac    420 

tttgtgcctg agggtgttgc gatagagact gagagggagg agcagacccc tttgctcaca    480 

ttgaactata aaacatcggt aggtgccatt aggtttaata ctggacaaac tacgaatgtc    540 

cagtttagga tccctttcta cacgccactg gaacacatcg caacccattc taaaaatgcg    600 

atggattcag tcttgggggc aatcacaacc cagatcacta actatagtgc tcaggatgag    660 

tatttgcagg ttacctacta catcagtttc aatgaagatt cacagttttc tgttcccaga    720 

gcggtgccag tggtcagctc attcactgac acatctagca aaacagtgat gaatacatat    780 

tggcttgatg atgacgagtt ggtagaagag                                     810 

 
           
             7  
             726  
             DNA  
             avian encephalomyelitis virus (AEV)  
           
            7 

atgagcaaac tattttctac tgtaggcagg actgttgatg aggttttgtc tgtgctcaat     60 

gatgaggata ctgaatctta tgctggccct gatcgcactg cagtagttgg cggaggattt    120 

ctgacaacgg tagaccagag ttcagttagc acggctacaa tgggaagttt acaagatgta    180 

cagtacagga ctgcagtcga tattcctggt tctagagtga cacaaggtga gaggttcttc    240 

cttatcgatc agcgtgagtg gaactcaaca cagagtgaat ggcagttatt gggcaagatt    300 

gacatagtaa aagagctgct tgatcagtcg tatgctgttg atggcctttt gaagtaccat    360 

tcttatgcaa ggtttggctt ggatgtcatt gttcagatta atccaacatc attccaggca    420 

gggggcctca tagcagctct cgtaccttat gaccaggttg acattgaatc aattgttgcc    480 

atgaccactt attgccatgg caaggttaat tgcaacataa actacgttgt aaggatgaag    540 

gtgccatata tatacagtcg aggttgttac aaccttagga actcagcata ctccatttgg    600 

atgcttgtga taagagtgtg gtcacggctg cagttgggat ctggcacttc aacacagatt    660 

actatcacca ccttggctag gtttgtggat ttggaactgc atggacttag ccctttggtc    720 

gcacag                                                               726 

 
           
             8  
             735  
             DNA  
             avian encephalomyelitis virus (AEV)  
           
            8 

atgatgcgca acgaatttcg actgtcgtca tctagcaaca ttgtcaattt ggctaattat     60 

gacgatgcaa gagccaaagt gtctctagcg ctgggacaag aagagttttc cagagactcg    120 

tcaagtaccg ggggggaatt ggtgcatcat ttttcacagt ggacgtccat tccgtgcctt    180 

gccttcactt ttacattccc cggcacggta gggccaggca ctcacatctg gtcaaccacg    240 

gtggaccctt tttcctgtaa cttgagggcg tctagcactg tgcaccccac taacttgagc    300 

tcgattgcgg gtatgttctg tttttggaga ggtgacattg tatttgagtt tcaagtcttt    360 

tgcaccaagt atcattccgg caggttgatg tttgtgtatg tgcctggcga tgaaaacaca    420 

aaaatcagca ccttaactgc aaaacaagca tctactggtc ttactgctgt ttttgatatc    480 

aatggtgtaa attcaacact ggtgtttaga tgccctttca tctctgacac accttacagg    540 

gtgaatccaa cgactcataa gtccctctgg ccttatgcaa ctggcaagct tgtgtgctat    600 

gtctacaata tactgaacgc acctgccagt gtatcaccaa ccctgcccat taatgtgtac    660 

aaaagtgctg cggatctgga gttgtatgca cctgtttatg gggtttctcc caccaacacc    720 

tcaatttttg ttcaa                                                     735 

 
           
             9  
             1500  
             DNA  
             avian parainfluenza virus (APV)  
           
            9 

ggggggtgtg catggtaggg tggggaaggt agccaattcc tgcccattgg gccgaccgta     60 

ccaagagaag tcaacagaag tatagatgca gggcgacatg gagggtagcc gtgataacct    120 

cacagtagat gatgaattaa agacaacatg gaggttagct tatagagttg tatccctcct    180 

attgatggtg agtgccttga taatctctat agtaatcctg acgagagata acagccaaag    240 

cataatcacg gcgatcaacc agtcgtatga cgcagactca aagtggcaaa cagggataga    300 

agggaaaatc acctcaatca tgactgatac gctcgatacc aggaatgcag ctcttctcca    360 

cattccactc cagctcaata cacttgaggc aaacctgttg tccgccctcg gaggttacac    420 

gggaattggc cccggagatc tagagcactg tcgttatccg gttcatgact ccgcttacct    480 

gcatggagtc aatcgattac tcatcaatca aacagctgac tacacagcag aaggccccct    540 

ggatcatgtg aacttcattc cggcaccagt tacgactact ggatgcacaa ggatcccatc    600 

cttttctgta tcatcatcca tttggtgcta tacacacaat gtgattgaaa caggttgcaa    660 

tgaccactca ggtagtaatc aatatatcag tatgggggtg attaagaggg ctggcaacgg    720 

cttaccttac ttctcaacag tcgtgagtaa gtatctgacc gatgggttga atagaaaaag    780 

ctgttccgta gctgcgggat ccgggcattg ttacctcctt tgtagcctag tgtcagagcc    840 

cgaacctgat gactatgtgt caccagatcc cacaccgatg aggttagggg tgctaacaag    900 

ggatgggtct tacactgaac aggtggtacc cgaaagaata tttaagaaca tatggagcgc    960 

aaactaccct ggggtagggt caggtgctat agcaggaaat aaggtgttat tcccatttta   1020 

cggcggagtg aagaatggat caacccctga ggtgatgaat aggggaagat attactacat   1080 

ccaggatcca aatgactatt gccctgaccc gctgcaagat cagatcttaa gggcagaaca   1140 

atcgtattat cctactcgat ttggtaggag gatggtaatg cagggagtcc taacatgtcc   1200 

agtatccaac aattcaacaa tagccagcca atgccaatct tactatttca acaactcatt   1260 

aggattcatc ggggcggaat ctaggatcta ttacctcaat ggtaacattt acctttatca   1320 

aagaagctcg agctggtggc ctcaccccca aatttaccta cttgattcca ggattgcaag   1380 

tccgggtacg cagaacattg actcaggcgt taacctcaag atgttaaatg ttactgtcat   1440 

tacacgacca tcatctggct tttgtaatag tcagtcaaga tgccctaatg actgcttatt   1500 

 
           
             10  
             1440  
             DNA  
             hemorrhagic enteritis virus (HEV)  
           
            10 

gaaatgttaa tgttagacca tactgaccaa ttcctggttc attttagatg gaatcttcga     60 

acactgccac tagaattttt gctccaacgg aagggagaaa cagtataatt tacagcaact    120 

tgcctcctgt tcaagataca accaaaatat tttatataga taacaaggcc attgatatag    180 

agtcatataa tcaagagaaa gatcattcta attattatac taatataatt caaacacaga    240 

acatttcaac tattgattca agtatacagc aaattcagtt agatgaaagg tctagatggg    300 

gaggagaact acatacaagc ttagtaacat ctgttatgaa ttgtactaaa cattttaatt    360 

cagataggtg tttagtgaaa attcagacta ttaagagtcc acctacattt gaatggaaag    420 

aattgaaaat acctgaggga aactatgttt taaatgagtt tattgattta ttaaatgaag    480 

gtattacttc tttatacctt cagtatggca ggcaacaggg tgtacttgaa gaagacatag    540 

gaataaaatt tgatactcgc aattttgaaa ttggtaaaga tccaactact aatcttgtta    600 

ctcctggtaa atacttgttt aagggttatc atgctgatat aatacttctt cctggttggg    660 

ctattgattt ttctttttct agattgggta acattttagg tattagaaaa cgtgagactt    720 

ataaagctgg ctttttgatt gaatatgatg acttgacaaa tggtaatatt ccaccactgt    780 

tggatgttgc taactataag tctacaagtc aagctaaacc attattacag gatccatctg    840 

gcagatctta ccacgttatg gatagtgatt ctaacagacc tgtgactgca tataggtctt    900 

ttgttttgtc atataacaat gaaggtgctg caaaattaaa gtttttgatg tgtatgagtg    960 

atataacggg gggtctcaat cagctgtatt ggtgtttgcc tgattcttat aaaccgccag   1020 

tatcttttaa gcaagaaacg caagtagata aactgcctgt tgttggtatg caactttttt   1080 

tcctttttgt ttgtaaatct gtgtattctg gtgctgctgt ttacacacag ttaattgaac   1140 

agcagactaa tttgacacaa atttttaaca gatttcatga taatgaaatt ttaaaacaag   1200 

ctccatatgt gaatcaagtt ttattggctg aaaatgtgcc cataaatgtt aatcagggaa   1260 

caataccaat attttcaact cttccaggag tacagagagt ggttgtggaa gacgatagga   1320 

gaagaactgt accctacgtt accaagtcac ttgctacagt atatccgaag gttttgtcta   1380 

gcaaaacttt gcaataatgc attctgttgt ttattctcca ggggacagta gaggatgggg   1440 

 
           
             11  
             2995  
             DNA  
             fowlpox virus (FPV)  
           
            11 

ctcttaattc gtttcaaaaa tgggaaatat ttttaagcct attccaaagg ccgattatca     60 

gattgtggaa acagtaccac aaagcttaac agctattaat tctactaatc tttctactta    120 

tgaatgtttt aaacgtttaa tagatctagc aaaaaaagag atctacatag ctacgttttg    180 

ttgtaaccta agtactaatc ctgagggtac tgacatacta aacagattaa tcgatgtttc    240 

gagtaaagtt tctgtatata ttttagtaga tgagagcagt cctcataaag attatgaaaa    300 

gattaagtct tcccatatta gttatattaa agtagatata ggtgtgctta ataatgaatc    360 

agtaggaaac ttgttaggta atttctgggt agtggataag cttcactttt atataggtag    420 

tgcgtctctt atgggaaatg cgctaacaac tattaaaaat atgggcatat attccgaaaa    480 

taattcttta gcaatggatt tatatttcag atcgttggac tataaaatta taagcaagaa    540 

aaaatgttta ttctttacca gaatggccac aaagtaccat ttcttcaaaa accataacgg    600 

tatattcttt tcagattctc cagaacatat ggtaggtaga aaaagaactt ttgacttgga    660 

ttgtgttatt cattatatag acgcggcgaa gtctactata gatctagcga tagtatctct    720 

tcttcctaca aagagaacaa aagattctat cgtctattgg cctataataa aagatgcatt    780 

aatacgggcc gtattagaac gaggtgtcaa actacgagtg ctattaggat tttggaaaaa    840 

aacggatgtt atatcaaaag catctataaa aagccttaac gaactaggag ttgaccatat    900 

agatatctct actaaagtat ttaggtttcc cgttaattct aaagtagatg atattaataa    960 

ttctaaaatg atgattatag atggaaggta tgctcatgtt atgactgcta acctagacgg   1020 

gtctcatttt aatcaccatg cttttgttag ctttaactgt atggatcaac aatttacaaa   1080 

gaaaatagct gaagtgtttg aaagggactg gatatctcct tacgcaaaag aaatagatat   1140 

gtctcaaata tagtatatat gataaaaaga tcctaataaa taaatatagc atggcactaa   1200 

tagaacagtt acaatcttct gaacaatcaa tactttcacc gtttagatat tatggtttta   1260 

aagattttca taatgtaatt tttaccacaa tagatgacga aacattaata gtaattacag   1320 

tcaacaatgt accattagta actaggttaa taacgtttga aaaaataaca ttttttagat   1380 

cgtttaatag tacttgtatt ataacttcca acaataattc ggatattgat acagatactt   1440 

attttatacc aaattcgtta tcactactag atattttgaa gaaaagagca tatgatgtag   1500 

aactaagaga tctatcattt gctataatgt cggaaatgaa taacgatgaa ttgagaaata   1560 

gtgatattgt atctctaaac aaatggctac ataagcataa tttactagac tacaaattag   1620 

tactaataag tgatatcgat agaagatata aattatacaa taaaaaaaat acaataattg   1680 

atgttatatc cgtaaatggt agaaattata atatatgggt taaagatgtt atagaatatt   1740 

attcaccgga atacttaaga tggtctatag atattaaaag agccacagaa agtaataact   1800 

ggttaccgta tagccagtct ataaaccctt tgaatgaaaa tatatacgct tttgaattta   1860 

tagctacttt agaaagatcc aatgagcgct taaatatcgg agcgatattc ctgtatccgg   1920 

atataataat tacaggtaga aacaacgaag atataataga aaagttttta gatcagttag   1980 

aagaagtaat atataaaaaa aattctgata gtattgtttt aacaggttat catctaacat   2040 

ttttagagaa tactatttta gagagatata tcagtaagta taaagactgg atttttacat   2100 

gtaatcgtct agtacattgt aaaaccggca ctgaagtatt cttatttgat gccgctatat   2160 

tttttccatc ctctaataag aaaggatatg taaaacattg gacaggtaaa aaattaaatt   2220 

ttaaaaactt tttccaaaaa gatagtcagc tagaaaaata cataaataat aacagtgtag   2280 

cagaacgtat atattattta cagtcttctt tacacaagca tatatcctgt ctaatagaaa   2340 

ttttcgagtt aaatggattt gattttaatt tttctgggtt gttagatata cttattttca   2400 

gtattcgtgt taagaataat aatggtaatt actattaccc taaacattct tcagctgtga   2460 

atttgatgtt gtcatctatt tacacggact attatgctat tgatgatata gataaagata   2520 

gtaagaaact tgtttttaac tctatttttc ctttaataat ggaaggatat taccctgaag   2580 

gaaaacctta ttatacgaaa acacccaaag aagggtattt gtcaatatgt ttatgtgatg   2640 

tagaaatatc taatgatata aagaatccta tattgtattg taaagaaaac aagtcagcta   2700 

ggaagtttac aggagtattc acatctgtag atatagatac cgctgtaaaa ctaagaggat   2760 

ataaaattaa aatattagaa tgtattgaat ggcctaataa aataaaatta ttcgacaata   2820 

tatgttatct gaataaatta tttatagaac atcaggatta cacacacgat gaaaaatctt   2880 

tacaaggcta tcttttttct tatttactta aaggcaacgt taccgaagat gttttagcta   2940 

tgaaaagttg tagaaataat ctttctataa tatcatttat aataagttac tgcag        2995 

 
           
             12  
             35  
             DNA  
             artificial sequence  
             
               PCR primer, IBS1F′ 
             
           
            12 

cgggatccgc cgccgccatg ttggtaacac ctctt                                35 

 
           
             13  
             29  
             DNA  
             artificial sequence  
             
               PCR primer, IBS1R′ 
             
           
            13 

cggaattctt aacgtctaaa acgacgtgt                                       29 

 
           
             14  
             38  
             DNA  
             artificial sequence  
             
               PCR primer, NDF F′ 
             
           
            14 

cgggatccgc cgccgccatg ggctccagac cttctacc                             38 

 
           
             15  
             34  
             DNA  
             artificial sequence  
             
               PCR primer, NDF R′ 
             
           
            15 

ccgctcgagt tacatttttg tagtggctct catt                                 34 

 
           
             16  
             39  
             DNA  
             artificial sequence  
             
               PCR primer, NDHN F′ 
             
           
            16 

cgggatccgc cgccgccatg gaccgcgccg ttaggcaag                            39 

 
           
             17  
             29  
             DNA  
             artificial sequence  
             
               PCR primer, NDHN R′ 
             
           
            17 

gctctagatt actcaactag ccagacctg                                       29 

 
           
             18  
             35  
             DNA  
             artificial sequence  
             
               PCR primer, IBDVP2F′ 
             
           
            18 

cgggatccgc cgccgccatg acaaacctgc aagat                                35 

 
           
             19  
             29  
             DNA  
             artificial sequence  
             
               PCR primer, IBDVP2R′ 
             
           
            19 

cggaattctt accttatggc ccggattat                                       29