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Definitions

• VACCINE ACCELERATOR FACTOR VAF FOR IMPROVEMENT OF VACCINATIONS IN POULTRY
• the present invention relates to a vaccine accelerator factor (VAF) which is an in ovo nucleotides-immuno-stimulant containing one or more DNA constructs, each having a DNA molecule and a vector.
• Each ofthe DNA molecule contains one or more genes or gene fragments, each encoding an antigenic peptide of an avian virus.
• the VAF is preferably administered to the amniotic fluid of an egg, which has been fertilized for about 17-19 days.
• the VAF can be co-administered with a viral vaccine containing one or more attenuated or inactive avian viruses or a recombinant DNA vaccine.
• the VAF can be injected into egg prior to the administration of the viral vaccine.
• the viral vaccine is preferably administered at hatch or post-hatch.
• the VAF stimulates and accelerate a protective immune response of a viral vaccine against the avian virus of which the DNA molecule ofthe VAF contains a gene or a fragment thereof.
• Poultry vaccines can be administered via different routes and by various methods. For example, a post-hatch spray vaccination method has been widely used. This method can mass-immunize day old chicks through aerosol spray. Also, live or attenuated vaccines can be administered to poultry through traditional method, e., by subcutaneous injection to chicks, rearing stock and breeders. Furthermore, poultry vaccines can be delivered via eye drops and/or intranasal routes during brooding of chicks. Finally, and most prevalently, vaccines can be administered to poultry via drinking water. This vaccination method has the advantage of low cost, but its effectiveness, particularly against some infections, is limited due to less control of vaccination.
• This in-ovo machine is currently used in about 80% ofthe U.S. broiler hatcheries, primarily for administering Marek's disease (MD) vaccines.
• MD Marek's disease
• 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 infectious bursal disease (IBD) and Newcastle disease (ND) vaccines.
• IBD infectious bursal disease
• ND Newcastle disease
• Newcastle disease virus Newcastle disease virus (NDV), infectious bronchitis virus (IB V), infectious • laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), chick anemia virus
• Marek's Disease is a malignant, lymphoproliferative disorder disease that occurs naturally in chickens. The disease is caused by a herpesvirus: Marek's Disease Virus (MDV).
• MDV Marek's Disease Virus
• MD is ubiquitous, occumng 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 sldn ofthe infected birds, causing motor trouble (due to paralysis when the nerves have been affected), functional trouble ofthe 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. At of this time, there are no methods of treating MD. The control ofthe 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.
• control of MD is almost entirely based on vaccination.
• IBDV Infectious bursal disease virus
• IBD infectious bursal disease
• IBDV is a member ofthe 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 VP1, the dsRNApolymerase.
• 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
• segment A 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).
• VP2 and VP3 are the major structural proteins ofthe virion, VP2 is the major host-protective immunogen and causes induction of neutralizing antibodies (See
• 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 ofthe precursor protein (See
• Newcastle disease virus is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome.
• 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).
• NPV together with parainfluenza virus, Sendai virus, simian virus 5, and mumps virus, belongs to the Paramyxoviridae family.
• the structural elements ofthe NPV 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 NPV into host cells by facilitating fusion ofthe 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.
• the main protein subunit ofthe NPV nucleocapsid is the nucleocapsid protein (NP) which confers helical symmetry on the capsid.
• NP nucleocapsid protein
• P phosphoprotein
• L L protein
• 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 RNApolymerase, is required for viral RNA synthesis together with the P protein.
• the L protein which takes up nearly half of the coding capacity ofthe viral genome is the largest ofthe viral proteins, and plays an important role in both transcription and replication.
• RNA viruses including NPV
• NPV negative-strand genome
• mRNA positive-strand
• the L, P and NP proteins must enter the cell along with the genome on infection.
• vRNAs negative strand genomes
• cRNAs antigenomes
• the cytoplasm is the site of NPV viral RNA replication, just as it is the site for transcription. Assembly ofthe viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.
• EMS ethyl methane sulfonate
• 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 ofthe vaccine.
• the NPV vaccine cannot be applied for in ovo vaccination as almost all ofthe embryos will die upon injection ofthe eggs with the unmodified virus.
• IBV Infectious bronchitis virus
• Coronaviridae the prototype ofthe 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.
• 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 ofthe virus.
• the spike protein is believed likely to be responsible for immunogenicity ofthe virus, partly by analogy with the spike proteins of other corona- viruses and partly by in vitro neutralization experiments (See, e.g., P. Cavanagh et al. (1984), Avian Pathology, 13, 573-583).
• the polypeptide components ofthe glycopolypeptides SI 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.
• 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.
• VNF® Human vaccinate baby chicks against certain diseases before they hatch.
• Embrex, Inc. has developed a live viral vaccine called VNF® (Viral
• the VNF contains an antibody (immunoglobulin) specific for the virus used in the vaccine. This specific antibody is mixed in an appropriate ratio with the vaccine virus to form a virus-antibody complex (immune complex) vaccine.
• the amount ofthe antibody in a complex vaccine is so small that it does not provide passive immunity or neutrialize the vaccine virus.
• the amount of antibody added to the vaccine virus is enough to delay by several days the normal course of vaccine virus replication.
• This delayed vaccine virus replication allows for the safe in ovo administration of moderately attenuated vaccine viruses in young animals.
• Moderately attenuated vaccine viruses are better at overcoming maternal immunity and at stimulating strong protective immune responses than highly attenuated vaccine viruses. Therefore, the virus-antibody complex vaccine technology allows for the safe hatchery use of vaccine viruses that are not eliminated by maternal antibody. Vaccination in the hatchery (either in ovo or at hatch) is better controlled and more uniform than field vaccination.
• virus-antibody complex vaccine technology improves vaccine efficacy as well as safety.
• immuno-stimulatory nucleotides particularly a PNA sequence, specific for avian viral vaccines (collectively “vaccine accelerator factor” and abbreviated as “VAF" will be introduced.
• the VAF can be either co-administered with or separately administered from the commercially available vaccines.
• the VAF can be either a single or multiple PNA constructs, each containing a single PNA molecule which is a viral gene or a fragment thereof. Additionally, the VAF can be a multivalent DNA construct, which contains two or more viral genes or fragments thereof linking together in one DNA construct.
• the viral genes or fragments used in preparation ofthe VAFs 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 VAFs are preferably applied in ovo in an appropriate quantity.
• the VAFs share the same advantages as the VNF,_Le., to improve vaccine efficacy as well as safety, the VAFs differ from the antibody used in Embrex
• the VAF stimulates immunological responses, such as antibody induction, T-cell activation with cytokine secretion, and the production of cytotoxic T lymphocytes, while the antibody in VNF acts to delay the normal course of vaccines much faster and enables the live vaccines to generate much higher titres of antibody than without the VAF.
• the VAF's effect on stimulating the immune response is not influenced by maternal immunity.
• the VAF in sum, improves pre-hatch immune response, which in turn improves life long poultry health and reduces vaccination costs.
• the present invention provides a vaccine accelerator factor (VAF), which is a nucleotides- or DNA-containing in ovo immuno-stimulant.
• VAF vaccine accelerator factor
• the VAF contains one or more DNA constructs. Each ofthe DNA constructs comprises a DNA molecule and a vector.
• the DNA molecule comprises at least one gene or a fragment ofthe gene encoding an antigenic peptide of an avian virus.
• the VAF stimulates and accelerates a protective immune response of a viral vaccine against the avian virus of which the DNA molecule contains a gene or a fragment thereof.
• the first kind of VAF contains one DNA construct.
• the DNA molecule ofthe DNA construct contains one gene or a fragment of this gene encoding an antigenic peptide of an avian virus.
• This kind of VAF stimulates and accelerate a viral vaccine against the same avian virus.
• the second kind of VAF also contains one DNA construct. But the DNA molecule ofthe DNA construct in the second kind of VAF contains more than one viral gene or a fragment thereof.
• the DNA construct is a "multivalent" DNA VAF where two or more genes or fragments thereof, each from a different avian virus, are linked together with a vector.
• the multivalent DNA VAF is capable of stimulating and accelerating two or more viral vaccines or a viral vaccine containing two or more viral antigens against the same avian viruses represented in the VAF.
• the third kind of VAF contains more than one DNA construct. Each ofthe DNA molecule ofthe DNA construct in this VAF contains only one gene or a fragment ofthe gene. In other words, this kind of VAF is a "multiple" PNA VAF. A “multiple" PNA VAF is also capable of stimulating and accelerating two or more viral vaccines or a viral vaccine containing two or more viral antigens against the same avian viruses represented in the VAF.
• the fourth kind of VAF contains more than one PNA construct.
• Each of the PNA molecule of the PNA construct either contains one gene or a fragment thereof or more than one gene or a fragment thereof.
• this kind of VAF is a "multiple multivalent" PNA VAF.
• This multiple multivalent PNA VAF is capable of stimulating and accelerating no less than three viral vaccines or a viral vaccine containing no less than three viral antigens against the same avian viruses as represented in the VAF.
• the viral vaccines used in combination with the VAF include, but are not limited to, any commercially available viral vaccines, such as vaccines containing live- virus, inactivated virus, or recombinant PNA viral vaccines.
• the viral vaccines are administered to the fowl either together with or subsequent to the in ovo injection of the VAF.
• a live vaccine at 1/5 volume ofthe full dose recommended by the manufacturer ofthe viral vaccine is injected into the fowl post hatch after the fowl eggs receive an in ovo injection of VAF.
• the vector ofthe PNA construct can be a plasmid or a viral carrier.
• the preferred vector is a plasmid.
• the plasmid include, but are not limited to, pcDNA3, pVAXl, pSectag, pTracer, pDisplay, pUC system plasmid (such as pUC7, pUC8, pUCl 8), and pGEM system plasmid.
• any plasmid which contains a promoter such as CMV promoter, SV40 promoter, RSV promoter, and /3-actin promoter, can also be used for preparing the DNA construct.
• the most favorable plasmid is pcDNA3.
• the preferred viral carrier is one ofthe following ⁇ viruses: a bacteriophage, SV40, an adenovirus, a polyoma virus, a baculovirus, a herpes virus, a vaccinia virus, or a pox virus.
• avian virus examples include, but are not limited to Marek'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).
• the genes contained in the DNA molecules that are particularly suitable for stimulating and accelerating a protective immune response against avian viral diseases include, but are not limited to, the entire of gB gene of Merl 's Pisease virus (MPV) having the PNA sequence of SEQ IP NO:l or a fragment thereof; the entire VP2 gene of infectious bursal disease virus (IBPV) having the PNA sequence of SEQ IP NO:2 or a fragment thereof; the entire HN gene of Newcastle disease virus (NPV) having the PNA sequence (which is from bases 6321 to 8319) of SEQ IP NO:3 or a fragment thereof (i.e., SEQ IP NO:3 is the entire genome ofthe NPV); the entire SI gene of infectious bronchitis virus (IBV) having the PNA sequence of SEQ IP NO:4 or a fragment thereof; the entire glycoprotein G gene of infectious laryngotracheitis virus
• ILTV having the PNA sequence of SEQ IP NO: 5 or a fragment thereof; the entire VP1, VPO, or VP3 gene of avian encephalomyelitis virus (AEV) or a fragment therof (the VP1 gene has the PNA sequence of SEQ IP NO: 6; the VPO gene has the PNA sequence of SEQ IP NO: 7; and the VP3 gene has the PNA sequence of SEQ IP NO: 8); the entire paraglycoprotein G gene of avian parainfluenza virus (APV) having the PNA sequence of SEQ IP 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.
• AEV avian encephalomyelitis virus
• AEV avian encephal
• the VAF is preferred to be injected into the egg, particularly in the amniotic fluid ofthe egg, of a fowl.
• the egg is preferred to be fertilized for about 17-19 days.
• the preferred fowl includes chicken, turkey, duck, and goose.
• the present invention also provides a method for vaccinating fowl egg, which includes injecting into a fowl egg the VAF as shown above.
• the VAF is prepared by ligating a DNA molecule to a plasmid or virus carrier to form a DNA construct.
• the VAF which contains more than one DNA construct two or more ofthe PNA constructs are mixed together.
• the insertion ofthe PNA molecule into the vector can be achieved by conventional method, L , by ligation the PNA molecule with an enzyme such as T4 PNA ligase when both the genes and the desired vector have been cut with the same restriction enzyme(s) as complementary PNA termini are thereby produced.
• the preferred restriction enzymes are BamHl and EcoRl .
• 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.
• 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.
• the attenuation process induces mutations ofthe viral genome, resulting in a population of virus particles heterogeneous with regard to virulence and immunizing properties.
• the traditional attenuated live virus vaccines can revert to virulence resulting in disease outbreaks in inoculated animals and the possible spread ofthe pathogen to other animals.
• a comprehensive post-hatch vaccination program involves administrations of different vaccines at various times.
• the vaccination program starts from 1 day of age and lasts up to 65 weeks of age.
• Many ofthe vaccines require repeated administrations, i.e., using vaccines of different immunogenic activities or using different route of administration at various times.
• the employment ofthe VAF to stimulate and accelerate the immune response of a vaccine would be advantageous for the poultry industry.
• the VAF is injected into an egg of a fowl at 17-19 days of fertilization. Because the VAF contains at least a gene or a fragment ofthe gene capable of expressing an antigenic peptide of an avian virus in ovo, the administration ofthe VAF to an egg induces an initial immune response against the avian virus.
• the dosage ofthe vaccine used in combination with the use of VAF for achieving the same immune response is far less than that without the administration ofthe VAF (as low as 20% ofthe normal dosage).
• the time period for achieving the same immune response is much shorter when the VAF is used.
• some ofthe commercially available viral vaccines such as the vaccines for infectious bronchitis virus (IB) and Newcastle disease virus (NP), are not suitable for in ovo injection.
• the vaccine for IB is Icnown to cause embryonic damage, and the vaccine for NP can only be properly stimulated through local muscular injection.
• the introduction ofthe VAF to the immunization of these diseases will improve the overall prevention ofthe diseases.
• the PNA sequence of a gene need not contain the full length of PNA encoding the polypeptides. In most cases, a fragment of the gene which encodes an epitope region should be sufficient enough for immunization.
• the PNA 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 ofthe proteins.
• One or more such fragments of genes encoding the antigenic determinants can be prepared by chemical synthesis or by recombinant PNA technology. These fragments of genes, if desired, can be linked together or linked to other PNA molecules.
• Marek's disease virus is a double-stranded RNA virus, while infectious bursal disease virus (IBPV), Newcastle disease virus (NPV) and infectious bronchitis virus
• RNA viruses are single-stranded RNA viruses.
• the RNA viral sequences can be reverse-transcribed into PNA using RT-Polymerase chain reaction (RT-PCR) technology and then incorporated into a vector by the conventional recombinant PNA technology.
• RT-PCR RT-Polymerase chain reaction
• RNA and PNA sequences that encode a specified amino acid sequence.
• all RNA and PNA sequences which result in the expression of a polypeptide having the antibody binding characteristics are encompassed by this invention.
• the PNA sequence ofthe viral gene can be ligated to other PNA molecules with which it is not associated or linked in nature.
• the PNA sequence of a viral gene can be ligated to another PNA molecule, ie., a vector, which contains portions of its PNA encoding fusion protein sequences such as ⁇ -galactosidase, resulting in a so-called recombinant nucleic acid molecule or PNA construct, which can be used for transformation of a suitable host.
• a vector which contains portions of its PNA encoding fusion protein sequences such as ⁇ -galactosidase, resulting in a so-called recombinant nucleic acid molecule or PNA 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.
• the plasmid include, but are not limited to, pBR322, pcPNA3, pVAXl, pSectag, pTracer, pPisplay, 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 -actin promoter is included.
• the preferred plasmid is pcPNA3.
• virus carrier examples include, but are not limited to, bacteriophages (e.g., ⁇ and the M13-derived phages), SV40, adenovirus, polyoma, baculoviruses, herpes viruses (HVT), vaccinia virus, or pox viruses (e.g., fowl pox virus).
• bacteriophages e.g., ⁇ and the M13-derived phages
• SV40 e.g., a and the M13-derived phages
• adenovirus e.g., polyoma
• baculoviruses baculoviruses
• HVT herpes viruses
• vaccinia virus vaccinia virus
• pox viruses e.g., fowl pox virus
• the insertion of the nucleic acid sequence into a cloning vector can easily be achieved by ligation with an enzyme such as T4 PNAligase when both the genes and the desired cloning vehicle have been cut with the same restriction enzyme(s) so that complementary PNA termini are thereby produced.
• an enzyme such as T4 PNAligase
• any restriction site may be produced by ligating linkers onto the PNA termini.
• 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.
• the present invention provides four ldnds of VAFs.
• the first kind is a VAF containing one PNA construct (i.e., univalent VAF), which comprises a PNA molecule and a vector.
• the PNA molecule contains one PNA sequence (which can be a gene or a fragment of a gene) encoding one antigenic peptide which provide immuno-protection against one avian virus.
• the second kind of VAF comprises two or more ofthe kind ofthe univalent VAF, each carrying a different PNA sequence against a different avaian virus.
• the third kind of VAF contains one PNA construct in which the PNA molecule contains two or more genes or gene fragments linked together, each from a different avian virus (i.e., a multivalent VAF or multivalent recombinant VAF). These genes or gene fragments are carried by a useful vector, which can be either a plasmid or a virus carrier.
• the multivalent recombinant VAF encodes two or more antigenic polypeptides which afford protection against at least two viral diseases including, but not limited to, MP, IBP, NP 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.
• PNA 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.
• the fourth kind of VAF is a mixture ofthe univalent and multivalent PNA VAF.
• the viral genes or gene fragments are preferably derived from Marek's disease virus (MPV), infectious bursal disease virus (IBPV), 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
• IBPV infectious bursal disease virus
• Newcastle disease virus having the DNA sequence of SEQ ID NO: 3 or a fragment thereof; the entire SI gene of infectious bronchitis virus (IBV) having the DNA sequence of SEQ ID NO:4 or a fragment thereof.
• the DNA sequence encoding the gB polypeptide of MDV has the nucleic acid sequence as SEQ ID NO:l.
• the DNA sequence contains 3650 bp of linear PNA.
• the PNA sequence encoding the VP2 polypeptide of IBPV has the nucleic acid sequence as SEQ IP NO:2.
• the PNA sequence contains 3004 bp of linear PNA molecule which is reversely transcribed from IBD V's RNA template.
• the DNA sequence ofthe 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.
• the NDV genome has the DNA sequence as SEQ ID NO:3.
• the DNA sequence ofthe SI polypeptide contains 1611 bp of linear DNA sequence as shown in SEQ ID NO:4, which is reversely transcribed from IBV's RNA templates.
• Newcastle disease (ND) vaccines were purchased from Intervet Inc. (B) Viral RNA isolation and RT-PCR
• DEPC diethylpyrocarbonate
• Oligonucleotide primers for RT-PCR amplification were purchased from Promega, and were designed according to the genome ofthe Avian infectious bronchitis virus (Beaudette CK strain), Newcastle disease virus (Lasota strain) and Infectious bursa disease virus respectively.
• the sequences ofthe primers used for PCR were :
• reaction mixture (40 ⁇ l) also contained viral RNA, 2.4 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega), 16 U of RNasin
• the plasmids pCMV-VP2, pCMV-Sl, pCMV-NPF and pCMV-NPHN were constructed with the VP2, S 1 , NPF and NPHN genes from IBP vaccine, IBV vaccine and NPV vaccine respectively, placed downstream ofthe commercial plasmid pcPNA3.
• All ofthe genes were inserted into the pcPNA3 vector using restriction enzymes BamHl , EcoRl, Xbal sn ⁇ Xhol (underlined characters in the sequence ofthe primers). Sequences ofthe all genes in the pcPNA3 vector were verified by sequencing in both directions.
• the quantity of plasmid PNA that had been purified by affinity chromatography was determined by spectrophotometric measurements at 260 and 280 nm.
• the PNA in aliquots to 100 ⁇ g was suspended in 100 ⁇ l of PBS (0.14M NaCl, 1 OmM sodium phosphate, pH 7.4).
• PBS 0.14M NaCl, 1 OmM sodium phosphate, pH 7.4
• SPF Specific Pathogen Free fertilized eggs
• All groups (five eggs each group ), all eggs were given 100 ⁇ l in volume each.
• 100 ⁇ g pCMV-NPF+ 100 ⁇ g pCMV-NPHN mixture was inj ected in each egg of group A
• 100 ⁇ g pCMV-Sl was injected in each egg of group B
• 100 ⁇ g pCMV-VP2 was injected in each egg of group C
• 100 ⁇ g pCMV-NPF + 100 ⁇ g pCMV-NPHN+100 ⁇ g ⁇ CMV-Sl(NP+IB) was injected in each egg of group P
• 100 ⁇ g pCMV-NPF+ 100 ⁇ g pCMV-NPHN+100 ⁇ g pCMV-VP2 (NP+IBP) was injected in each egg of group E
• lOO ⁇ g pCMV-VP2+100 ⁇ g pCMV-VP2 mixture IB
• All chickens in this experiment were given 100 ⁇ l in volume ( 1/5 dose of live vaccines), injected into the chicken's thoracic muscle each at 10 days post hatchery.
• Chickens in group A and I were injected with NPV vaccine
• group B and J were injected with IBV vaccine
• group C and K were injected with IBPV vaccine
• group P were injected with the mixture of NPV+IB vaccines
• group E were injected with the mixture of NPV+IBP vaccines
• group F were injected with the mixture of IB+IBP vaccines
• group G and L were injected with the mixture of NPV, IB and IBP vaccines.
• the in ovo injection could either be performed by automatic in ovo vaccination system (such as Embrex Inovoject® system) or by manual procedure.
• a manual in ovo procedure was described as follows: 1. The eggs were candled to remove infertile, contaminated, and/or upside down eggs. Also, dirty eggs were moved to avoid bacterial contamination during the injection.
• the top ofthe air cell of each egg was sanitized by a diluted bleach solution (prepared by mixing a 10% solution of household bleach in distilled water to reach a final concentration of 0.5% hypochlorite).
• a diluted bleach solution prepared by mixing a 10% solution of household bleach in distilled water to reach a final concentration of 0.5% hypochlorite.
• the diluted bleach solution was prepared fresh and mixed just before use.
• a cotton swab soaked in the diluted bleach was used to wipe the surface on top (air cell) ofthe egg.
• iodine could be used to replace the diluted bleach, although the diluted bleach was more effective. If clean eggs were used, there was no need to clean the eggs with the diluted bleach solution.
• the eggs were sprayed with 70% isopropyl alcohol. After the 70% isopropyl alcohol was dried, a hole was punched in the air cell end on top ofthe egg by inserting an 18 gauge 1 and 1/2 inch needle through a rubber stopper so that 1/4 to 1/2 inch ofthe needle tip emerges from the stopper. This hole would be used to insert the needle when injecting the vaccine.
• vaccine or VAF was inserted into the egg, the needle and syringe must be straight up and down (not at an angle). The embryo was not hurt by this way so that the fowl could hatch normally. 4.
• the VAF was preferably inj ected into the egg by a 1 cc syringe with a 20 gauge 1 inch needle.
• the PNA constructs were mixed under sterile conditions.
• the sterility ofthe VAF could be confirmed by sampling it before and after the injection by putting 1/2 mL of agar to determine the presence of bacteria.
• 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.

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