HN protein mutated gene VII type Newcastle disease virus recombinant vaccine strain
阅读说明：本技术 一种hn蛋白突变的基因vii型新城疫病毒重组疫苗株 (HN protein mutated gene VII type Newcastle disease virus recombinant vaccine strain ) 是由 于晓慧 王静静 刘华雷 李峥 舒波 李阳 蒋文明 于 2021-10-08 设计创作，主要内容包括：本发明提供一种HN蛋白突变的基因VII型新城疫病毒重组疫苗株,其中用于构建基因VII型新城疫病毒重组疫苗株的过程中使用的基因片段包含了毒性致弱的基因VII.2亚型新城疫病毒的囊膜糖蛋白F蛋白基因和HN蛋白基因。本发明提供的基因VII型新城疫病毒重组疫苗株在鸡胚中具有高生长滴度和低致病力的生物学特性,遗传稳定,对新城病毒具有良好的免疫保护效果,并能有效抑制排毒,能够用于防控目前流行的基因VII型新城疫病毒,具有广阔的应用前景。(The invention provides a gene VII type Newcastle disease virus recombinant vaccine strain with HN protein mutation, wherein a gene fragment used in the process of constructing the gene VII type Newcastle disease virus recombinant vaccine strain comprises a envelope glycoprotein F protein gene and an HN protein gene of attenuated gene VII.2 subtype Newcastle disease virus. The gene VII type newcastle disease virus recombinant vaccine strain provided by the invention has the biological characteristics of high growth titer and low pathogenicity in chick embryos, is stable in heredity, has a good immune protection effect on newcastle disease virus, can effectively inhibit toxin expelling, can be used for preventing and controlling the currently popular gene VII type newcastle disease virus, and has a wide application prospect.)
1. A gene segment for constructing gene VII type Newcastle disease virus recombinant vaccine strain is characterized in that the gene segment replaces the corresponding part of LaSota strain genome with envelope glycoprotein F protein gene segment and HN protein gene segment which contain attenuated gene VII.2 subtype NDV.
2. The gene fragment for constructing the gene type VII Newcastle disease virus recombinant vaccine strain according to claim 1, wherein the envelope glycoprotein F protein gene of the attenuated gene VII.2 subtype NDV is 112R-R-R-K-R-F117 mutated into 112G-R-Q-G-R-L117 at the site of the F gene with the amino acid sequence of SEQ ID NO. 1.
3. The gene fragment for constructing the gene VII type Newcastle disease virus recombinant vaccine strain according to claim 1, wherein the amino acid sequence of the HN protein gene is SEQ ID NO. 2.
4. The gene fragment for constructing the gene VII type Newcastle disease virus recombinant vaccine strain according to claim 1, wherein the amino acid sequence of the HN protein gene is SEQ ID NO. 3.
5. The gene fragment for constructing the gene VII type Newcastle disease virus recombinant vaccine strain according to claim 1, wherein the nucleotide sequence of the gene fragment is SEQ ID NO. 4.
6. Use of the gene fragment for constructing a genetic type VII Newcastle disease virus recombinant vaccine strain according to any one of claims 1-5 in constructing a genetic type VII Newcastle disease virus recombinant vaccine strain.
7. A gene VII type Newcastle disease virus recombinant vaccine strain, which is characterized in that the vaccine strain is genetically rescued after a cell is cotransfected with a helper plasmid by using the gene segment for constructing the gene VII type Newcastle disease virus recombinant vaccine strain as claimed in any one of claims 1-5.
8. The genotype VII recombinant vaccine strain of Newcastle disease virus according to claim 7, wherein said cells are BSR-T7/5 cells.
9. Use of the genetic type VII newcastle disease virus recombinant vaccine strain according to claim 7 or 8 in the preparation of a vaccine.
10. An inactivated vaccine, wherein the antigen comprises the inactivated recombinant vaccine strain of the genotype VII Newcastle disease virus according to claim 7 or 8.
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a gene VII type Newcastle disease virus recombinant vaccine strain with HN protein mutation and a construction method and application thereof.
Background
Newcastle Disease (ND) is an acute, highly contagious disease caused by infection of birds with virulent strains of Newcastle Disease Virus (NDV). The NDV virulent strain can cause the infected poultry to have the typical characteristics of dyspnea, diarrhea, nervous dysfunction, mucosal hemorrhage, serosal hemorrhage, necrosis and the like, the disease death rate can reach 100 percent, and serious economic loss is caused to the poultry industry. The world animal health Organization (OIE) ranks Newcastle disease as a legal animal epidemic disease, ranks Newcastle disease as a type of animal epidemic disease by the rural agricultural department of China, and ranks it as a major animal epidemic disease for preferential prevention and treatment in the national middle and long term animal epidemic disease prevention and treatment plan (2012-2020).
NDV is a single-stranded, negative-strand, non-segmented RNA virus with a wide host range, of which chickens are the most susceptible. NDV has genetic diversity. NDV can be divided into two types, class I (class I) and class II (class II), by genetic evolutionary analysis of the viral F gene, where class I has only 1 genotype and class II has at least 21 genotypes (I-XXI). In 1984, the gene VII type NDV is discovered for the first time in Taiwan of China. Subsequently, the genotype strain is widely prevalent in domestic poultry, mainly as the gene VII.1.1 subtype. In 2011, gene VII.2 subtype NDV is first isolated from Guangxi wild birds in China. National newcastle disease reference laboratory monitoring data show that vii.2 subtype strains are also isolated in poultry in succession in our country from 2012 to 2018 and may become dominant genotypes.
The vaccine immunity is an important means for preventing newcastle disease, the currently widely used newcastle disease vaccines mainly comprise II vaccine B1, IV vaccine LaSota, clone30 and V4 live vaccines, A-VII oil emulsion inactivated vaccines, LaSota oil emulsion inactivated vaccines and the like, the vaccines can provide certain immune protection, but can not completely prevent the spread and the transmission of newcastle disease viruses, and particularly, the genetic VII newcastle disease viruses cause huge loss to the breeding industry in the epidemic of China. The current gene VII type Newcastle disease vaccine immunized by a farm is developed aiming at a gene VII.1.1 subtype, and the vaccine has the risk of immune failure on the current gene VII.2 subtype strain. In view of this, in order to promote the healthy development of poultry industry in China and finally achieve the goal of purifying Newcastle disease, the development of the gene VII.2 subtype NDV vaccine has important significance.
Disclosure of Invention
The invention aims to provide a gene VII type Newcastle disease virus recombinant vaccine strain with HN protein mutation, a construction method and application thereof, the constructed vaccine strain has high toxicity weakened, high reproductive capacity on chicken embryos and good immunogenicity, and can be used for preventing and controlling currently epidemic gene VII.2 subtype NDV.
The invention firstly provides a gene segment for constructing gene VII type Newcastle disease virus recombinant vaccine strain, which is a gene segment of a corresponding part of LaSota strain genome replaced by a cyst membrane glycoprotein F protein gene segment and a HN protein gene segment containing attenuated gene VII.2 subtype NDV;
the envelope glycoprotein F protein gene of the gene VII.2 subtype NDV with attenuated toxicity is characterized in that a site 112R-R-R-K-R-F117 of an F gene with an amino acid sequence of SEQ ID NO:1 (a specific coding gene with a sequence of SEQ ID NO:5) is mutated into 112G-R-Q-G-R-L117;
the HN protein gene has an amino acid sequence of SEQ ID NO. 2 (the sequence of a specific coding gene is SEQ ID NO. 6);
furthermore, the length of HN protein is extended from 571aa to 577aa, and its amino acid sequence is SEQ ID NO. 3 (the sequence of a specific coding gene is SEQ ID NO. 7);
as a concrete description of an embodiment, a concrete nucleotide sequence of a gene fragment containing envelope glycoprotein F protein gene and HN protein gene of a attenuated gene VII.2 subtype NDV and used for constructing a gene VII type Newcastle disease virus recombinant vaccine strain is SEQ ID NO. 4;
the invention also provides a gene VII type Newcastle disease virus recombinant vaccine strain which is constructed by using the gene fragment, and the construction method comprises the following steps:
1) constructing a genome full-length cDNA plasmid containing gene II type Newcastle disease virus LaSota;
2) on the LaSota strain genome full-length cDNA plasmid constructed in 1), replacing corresponding partial fragments of LaSota strain genome with envelope glycoprotein F protein gene and HN protein gene fragments of gene VII.2 subtype NDV with attenuated toxicity to obtain full-length recombinant plasmid;
3) co-transfecting the full-length recombinant plasmid constructed in the step 2) and eukaryotic expression plasmids expressing genes NP, P and L of the LaSota strain of the newcastle disease virus to express cells of T7 polymerase to obtain a recombinant newcastle disease virus attenuated vaccine strain expressing genes VII type F and HN proteins;
the cells are BSR-T7/5 cells.
The invention also aims to provide the application of the gene VII type Newcastle disease virus recombinant vaccine strain with HN protein mutation in the preparation of vaccines;
the vaccine is an inactivated vaccine, and is obtained by inactivating a low virulent strain through formaldehyde.
The HN protein mutated gene VII type Newcastle disease virus recombinant vaccine strain provided by the invention replaces envelope glycoprotein F protein gene and HN protein gene fragment of attenuated gene VII type Newcastle disease virus with corresponding partial genes of LaSota strain genome, and obtains the gene VII type Newcastle disease virus recombinant attenuated vaccine strain through rescue. The recombinant attenuated strain is genetically stable and has the biological characteristics of high growth titer and low pathogenicity. The vaccine prepared by the recombinant attenuated strain can induce to generate a high neutralizing antibody, can effectively inhibit toxin expelling, can be used for preventing and controlling the invasion of a virulent gene VII and a newly epidemic gene VII.2 subtype NDV, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the construction of pOK-rLaSota full-length plasmid, in which the inverted triangle is the molecular marker (mutated single enzyme cutting site) introduced by the sequence different from the wild strain;
FIG. 2 is a diagram showing the sequencing result of the mutation position of the full-length cDNA of the rescued virus rLaSota strain;
FIG. 3 is a schematic diagram of the construction of full-length genomic plasmids pOK-rLa-VII-YN17 and pOK-rLa-VII-571-YN 17;
FIG. 4 shows the results of sequencing comparison of gene cleavage sites of candidate strains rLa-VII-YN17 and rLa-VII-571-YN17 of rLa-VII-YN17, r10, r15 and NDV YN 1106/2017F strains.
Detailed Description
The invention designs a construction method of recombinant attenuated vaccine rLa-VII-YN17 expressing gene VII type NDV F and HN gene, which is characterized in that on the basis of establishing a reverse genetic operation platform of gene II type Newcastle disease virus LaSota strain, the capsular glycoprotein F protein gene and HN protein gene fragment of a Newcastle disease virus gene VII.2 subtype isolate YN1106/2017 which is mainly popular in China are replaced with the corresponding partial gene of LaSota strain genome, simultaneously, a virulent cracking site (112R-R-R-K-R-F117) of F protein is mutated into an F protein cracking site (112G-R-Q-G-R-L117) of attenuated strain LaSota, the length of HN protein is prolonged from 571aa to 577aa, and a full-length cDNA clone pOK-rLa-VII-YN17 expressing NDV gene VII.2 subtype F and HN gene with weakening degree is constructed, after co-transfecting BSR-T7/5 cells with helper plasmids, the recombinant NDV attenuated vaccine strain rLa-VII-YN17 is rescued.
The invention also provides a construction method of the HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain rLa-VII-YN17, which comprises the following steps:
(1) constructing genome full-length cDNA plasmid pOK-rLaSota of gene II type Newcastle disease virus LaSota strain;
(2) constructing a recombinant plasmid pOK-rLa-VII-YN17, cutting a B fragment and a C fragment by Bgl II and Hind III restriction endonucleases on the basis of pOK-rLaSota, replacing a F gene and a HN gene fragment of a gene VII.2 subtype Newcastle disease virus YN1106/2017 strain with a corresponding part gene of a LaSota strain genome by combining a homologous recombination method, mutating a virulent cleavage site (112R-R-R-K-R-F117) of an F protein into an F protein cleavage site (112G-R-Q-G-R-L117) of a low virulent strain LaSota, and prolonging the length of the HN protein from 571aa to 577aa to obtain a recombinant plasmid pOK-rLa-VII-YN 17;
(3) rescuing the recombinant attenuated strain rLa-VII-YN17, co-transfecting BSR-T7/5 cells expressing T7 polymerase with full-length plasmids pOK-rLa-VII-YN17 and three eukaryotic expression plasmids of pCI-NP, pCI-P and pCI-L expressing NDV LaSota strain NP, P and L genes, adding TPCK pancreatin after 6h of transfection, incubating for 72h, repeatedly freezing and thawing the transfected samples, and inoculating 9-11-day-old SPF chick embryos to obtain the recombinant Newcastle disease virus attenuated vaccine strain rLa-VII-YN17 expressing gene VII type F and HN proteins.
In the method, pOK-rLaSota in the step (1) is constructed by the following method: according to the characteristics of the full-length cDNA sequence of LaSota strain, the full-length genome is divided into 5 segments of a, b, c, d and e. Designing a primer according to cloning requirements to perform corresponding mutation modification on a part of fragments so as to ensure the uniqueness of enzyme cutting sites used for cloning, wherein a mutated base sequence can be used as a molecular label for rescuing viruses, introducing a T7 promoter sequence at the 5 'upstream of a fragment, introducing three continuous G bases behind a T7 promoter to enhance the transcriptional activity of the fragment, and introducing a hepatitis B ribozyme (HdvRz) sequence and a T7 terminator sequence at the 3' end of an e fragment. The individual fragments were subsequently cloned into pOK12 vectors in sequence. The full-length plasmid of the completed genome was named pOK-rLaSota in the examples.
The invention further aims to provide application of the Newcastle disease virus recombinant vaccine strain rLa-VII-YN 17.
The biological product containing the gene recombination attenuated vaccine strain rLa-VII-YN17 of the expression gene VII type Newcastle disease virus F and the prolonged HN protein belongs to the protection scope of the invention.
The genetic stability of the recombinant vaccine strain rLa-VII-YN17 is analyzed, and the result shows that the rLa-VII-YN17 strain is continuously passaged to the 15 th generation in 9-11 day-old SPF chick embryos, and the viruses of the 1 st generation (F1), the 5 th generation (F5), the 10 th generation (F10) and the 15 th generation (F15) all contain F (mutation cracking site) and HN sequences of the gene VII type Newcastle disease virus through sequence determination, contain molecular tags introduced during construction, and have good virus genetic stability.
The gene VII type Newcastle disease virus recombinant vaccine strain rLa-VII-YN17 designed by the invention mutates a virulent cleavage site 112R-R-K-R-F117 of a gene VII.2 subtype Newcastle disease virus F protein into an F protein cleavage site 112G-R-Q-G-R-L117 of a low virulent strain LaSota, and prolongs the length of HN protein from 571aa to 577aa, and the sequence after mutation can reduce the toxicity and ensure better safety of the virulent strain.
The gene VII type Newcastle disease virus recombinant vaccine strain rLa-VII-YN17 designed by the invention uses NP, P, M and L genes of a Newcastle disease virus gene II type classical vaccine strain LaSota strain as a framework, the characteristic of high propagation titer of the LaSota virus strain is kept, and the recombinant vaccine strain shows excellent growth characteristic on a chick embryo.
The vaccine is an inactivated vaccine, and is obtained by inactivating a low virulent strain through formaldehyde.
The immune protection experiment result shows that the inactivated vaccine prepared by the recombinant vaccine strain rLa-VII-YN17 is safe to chickens and has no side reaction; the antibody can be generated at a higher level after immunizing SPF chickens of 28 days old for 2 weeks, and the antibody level is higher after 3 weeks; after 3 weeks of immunization, the wild strain YN1106/2017 of gene VII type Newcastle disease virus virulent strain is used for virus challenge, the immunized group of chickens have no disease symptoms or death, the immune protection rate reaches 100%, and can effectively inhibit detoxification, and the mortality rate of the control group and the detoxification detection rate of the swab virus are both 100%.
The present invention will be further described with reference to the following specific examples.
The experimental procedures in the examples, if not otherwise specified, are conventional and well known to those skilled in the art, and all starting materials are commercially available. The test materials used in the examples were purchased from conventional biochemicals, unless otherwise specified.
Example 1: rescue of LaSota Strain
1. Virus purification
The purification of the virus was carried out by limiting dilution method to purify the LaSota strain in order to obtain a single virus clone, and the detailed procedure was as follows: diluting the virus solution by 10 times, and respectively taking each titer of virus solution to inoculate SPF (specific pathogen free) chick embryos (100 mu L/embryo) of 9-11 days old after dilution, and inoculating 5 virus embryos at each dilution. Chick embryo allantoic fluid was harvested after 4d for HA activity. Allantoic fluid with the highest dilution of HA activity was selected as the next generation of purified virus fluid. The virus is continuously purified for 5 generations by the same method for multiple dilution and chick embryo inoculation, and the virus liquid of the 5 th generation is subpackaged and stored as the original seed virus for the next test.
2. Viral sequence determination
The purified cDNA of the viral reverse transcription genome was used as a template, and the LaSota genome was amplified by PCR using 13 pairs of primers (Table 1) designed to amplify the full-length sequence of the LaSota strain, and the two terminal sequences of the viral genome were determined using 3 '-RACE and 5' -RACE kits. The determined sequences were spliced by DNA Star biology software. The result shows that the total genome length of the NDV LaSota strain is 15186 bp.
Table 1: primer table for LaSota strain NDV whole genome sequencing
3. Construction of LaSota full-Length cDNA cloning plasmid
According to the sequence characteristics of the LaSota genome, the full-length genome is divided into 5 segments (segments a-e) by enzyme cutting site screening. Primers are designed to amplify the 5 segments (table 2), and three enzyme cutting sites on the P gene, the M gene and the L gene are mutated so as to ensure the unicity of the enzyme cutting sites of Bgl II and Hind III, and the mutated base sequence can be used as a molecular tag for rescuing viruses. The T7 promoter sequence is introduced into the 5 'upstream of the a fragment, three continuous G bases are introduced behind the T7 promoter to enhance the transcriptional activity, and the hepatitis D ribozyme (Hdvrz) sequence and the T7 terminator sequence are introduced into the 3' end of the e fragment. The individual fragments were then ligated sequentially into the pOK12 cloning vector to construct the strategy as shown in FIG. 1. After the full-length genome cloning was completed, sequencing was performed, and the constructed and sequenced correct viral genome plasmid was named pOK-rLaSota.
Table 2: primer table for construction of LaSota strain genome full-length cDNA clone
4. Construction of helper plasmids
According to the determined LaSota strain genome sequence, primers are designed to respectively amplify the open reading frames (table 3) of the encoded nucleocapsid protein NP, phosphoprotein P and polymerase protein L, 3 ORFs are respectively cloned to the downstream of a CMA promoter of a eukaryotic expression vector pCI-neo, 3 auxiliary plasmids pCI-NP, pCI-P and pCI-L are constructed, and no gene mutation is generated through sequencing verification.
Table 3: primer table for LaSota helper plasmid construction
5. Rescue of virus
The constructed full-length plasmid (pOK-rLaSota) and 3 helper plasmids (pCI-NP, pCI-P, and pCI-L) were co-transfected into BSR-T7/5 cells. After 6h of transfection, the medium was changed to DMEM medium containing 2% serum, 1% antibiotic, 5. mu.g/mL TPCK-Trypsin. And (3) after transfection for 72h, harvesting culture solution and cells, inoculating 9-11 day-old SPF (specific pathogen free) chick embryos (200 mu L/embryo), incubating at 37 ℃, harvesting chick embryo allantoic fluid after 3-4 days, and performing Hemagglutination (HA) test. The result shows that HA is positive, which preliminarily indicates that the virus rescue is successful.
6. Identification and biological characteristic analysis of rescued virus rLaSota
And extracting HA positive virus liquid RNA, and performing whole genome sequence determination on the rescued virus. The sequence alignment result shows that the rescued virus genome contains a molecular tag (mutated restriction enzyme cutting site, figure 2) introduced during construction, and shows that the LaSota strain genome cDNA is cloned intoSuccessfully rescued and the rescued virus was named rLaSota. Continuously transmitting the SPF chick embryos to 5 generations in SPF chick embryos of 9-11 days old. The hemagglutination titer of each generation can be detected to be 210～211In the meantime. Sequence determination is carried out on F5-generation virus liquid, and the result shows that the molecular label has no back mutation, which indicates that the rescued rLaSota has better stability. Determination of the amount of infection (EID) in half of the embryos of chickens that rescued the virus from the parental virus50) Average chick embryo Mortality (MDT) and intracerebral inoculation pathogenicity index (ICPI) for 1 day old chicks, table 4 shows that the rescued viruses have the same biological properties and pathogenicity as the parental viruses.
Table 4: biological characterization of rescued virus and parental strains
In conclusion, the reverse genetic operation platform of the Newcastle disease virus LaSota strain is successfully constructed, and can be used as a good vaccine vector.
Example 2: rescue of recombinant newcastle disease vaccine candidate seed virus
1. Screening and purification of viruses
Selecting genes VII.2 subtype chicken source Newcastle disease viruses which are separated and stored in a laboratory and mainly epidemic in China at present for propagation and rejuvenation, and further purifying 5 strains by adopting a limiting dilution method, wherein the hemagglutination titer of each strain in different generations is shown in Table 5. And finally, selecting the strain YN1106/2017 purified after 5 generations, and using the strain as an original seed virus for constructing a full-length cDNA clone.
Table 5: hemagglutination titer table for different strains of gene VII type Newcastle disease virus
2. Determination and analysis of full-length genome sequence of strain YN1106/2017
The YN1106/2017 genome was PCR-amplified using 12 pairs of primers (Table 6) designed to amplify the full-length sequence of type VII NDV using the purified retrogenomic cDNA of the YN1106/2017 virus as a template, and the two terminal sequences of the viral genome were determined using 3 'RACE and 5' RACE kits. The determined sequences were spliced by DNA Star biology software. As a result, the total length of the genome of the strain YN1106/2017 was 15192 bp.
Table 6: primer table for VII type NDV whole genome sequencing
3. Construction of full-length cDNA of vaccine candidate strain genome
The method is characterized in that pOK-LaSota full-length plasmid is used as a platform to design and synthesize a new gene sequence YN17-571HN-bc from a Bgl II enzyme cutting site to a Hind III enzyme cutting site in the full-length sequence, wherein the new gene sequence YN17-571HN-bc is 6539bp in total, the new gene sequence comprises a membrane glycoprotein F protein gene and an HN protein gene fragment of a gene VII Newcastle disease virus YN1106/2017 strain and a corresponding part gene of a LaSota strain genome are replaced, meanwhile, a virulent cleavage site (112R-R-R-K-R-F117) of an F protein (SEQ ID NO:1) is mutated into an F protein cleavage site (112G-R-Q-G-R-L117) of a low virulent strain LaSota, other sequences are kept unchanged, and a recombinant virus rLa-VII-571-YN17 strain is designed.
On the basis of the above mutation, a recombinant virus rLa-VII-YN17 strain was designed by synthesizing a new gene sequence YN17-577HN-bc (SEQ ID NO:4) comprising the HN protein extended from 571aa (SEQ ID NO:2) to 577aa (SEQ ID NO:3) in a total of 6539 bp.
The pOK-rLaSota full-length plasmid was double digested with Bgl II and Hind III restriction enzymes, and the original b and c fragments were cut off. Primers were designed to amplify the synthesized target genes YN17-571HN-bc and YN17-577HN-bc fragments (Table 7), and the target fragments YN17-571HN-bc and YN17-577HN-bc fragments recovered from the gel were ligated to the digested vector, respectively, to construct the strategy shown in FIG. 3. After the cloning of the full-length genome was completed, the virus genome plasmids that were constructed and sequenced correctly were designated pOK-rLa-VII-571-YN17 and pOK-rLa-VII-YN17, respectively.
TABLE 7 primer tables for amplifying YN17-571HN-bc and YN17-577HN-bc fragments
4. Rescue of vaccine candidate strains
Two full-length recombinant plasmids (pOK-rLa-VII-571-YN17 and pOK-rLa-VII-YN17) which have been constructed were co-transfected with 3 helper plasmids (pCI-NP, pCI-P and pCI-L) of LaSota virus to BSR-T7/5 cells, respectively. After 6h of transfection, the medium was changed to DMEM medium containing 2% serum, 1% antibiotic, 5. mu.g/mL TPCK-Trypsin. And (3) harvesting culture solution and cells 72h after transfection, inoculating 9-11 day-old SPF (specific pathogen free) chick embryos (200 mu L/embryo), incubating at 37 ℃, harvesting chick embryo allantoic fluid after 3-4 days, and performing Hemagglutination (HA) test. The results are all shown to be HA positive, which preliminarily shows that the virus rescue is successful.
5. Sequence identification and genetic stability of vaccine candidate strains
And (3) selecting HA positive virus liquid to extract RNA, carrying out reverse transcription, and then carrying out whole genome sequence determination. The sequence alignment result shows that the genome of the rescued virus rLa-VII-571-YN17 takes a LaSota strain as a framework, F and HN genes of a chimeric YN1106/2017 strain are embedded, and the F gene cleavage site is mutated; the genome of the rescued virus rLa-VII-YN17 takes a LaSota strain as a framework, F and HN genes of YN1106/2017 strains are embedded, the F gene cleavage site is mutated, and the length of HN protein is prolonged from 571aa to 577aa, which indicates that both strains of viruses are successfully rescued.
Continuously carrying out continuous passage on the rescued virus in SPF chick embryos of 9-11 days old for 15 times. The hemagglutination titer of each generation of virus is 210～211In the meantime. Selecting F and F of the 1 st, 5 th, 10 th and 15 th generation recombinant virusesThe HN gene was sequenced. Sequencing results show that the F and HN genes of the 1 st, 5 th, 10 th and 15 th recombinant viruses are consistent with the inserted sequences, and the F protein cleavage sites are not subjected to back mutation (figure 4), which shows that the two recombinant viruses are stable in heredity in the process of passage.
6. Biological characterization of vaccine candidate strains
Selecting 15 th generation rLa-VII-YN17 and rLa-VII-571-YN17 vaccine candidate strains to measure EID thereof50MDT and ICPI, and comparing the virulence changes of the viruses by taking the Lasota virus strain and the YN1106/2017 virus strain as controls. The test data in Table 8 show that the rescued rLa-VII-YN17 and rLa-VII-571-YN17 vaccine candidate strains have obviously reduced toxicity, and compared with the rLa-VII-571-YN17 vaccine candidate strain, the rLa-VII-YN17 vaccine candidate strain with the HN protein length prolonged has higher growth titer in chick embryos and has the biological characteristics of lower pathogenicity to 1-day-old chicks. Therefore, the rLa-VII-YN17 vaccine candidate strain constructed by the invention can be used as a preferable vaccine strain for vaccine production.
Table 8: biological characterization of rescued virus and parental strains
Example 3 safety test and immune Effect test of rLa-VII-YN17 vaccine Strain on SPF chickens
1. Preparation of inactivated vaccine
Diluting the rLa-VII-YN17 vaccine strain by 10000 times with sterilized normal saline, inoculating SPF (specific pathogen free) chick embryos of 9-11 days old, inoculating 0.1mL of each embryo, and placing at 37 ℃ for continuous incubation. Discarding the dead embryos within 24h after inoculation, immediately placing the dead embryos for 24 h-120 h at 4 ℃, collecting the mixed sample, and determining the HA of the vaccine preparation poison to be 28Viral titer of 109.65EID500.1 mL. Introducing the newcastle disease virus solution with the determined titer into an inactivation tank, adding a formaldehyde solution with the final concentration of 0.1% to fully mix the newcastle disease virus solution, inactivating the newcastle disease virus solution at 4 ℃ for 48 hours, and shaking the newcastle disease virus solution once every 2 hours. Preparing the inactivated oil adjuvant inactivated vaccine by the inactivated virus stock solution according to a conventional method.
2. Overdose safety test for inactivated vaccine
In order to determine the safety of the rLa-VII-YN17 vaccine strain rescued in the embodiment 2 of the invention to SPF chickens, the prepared rLa-VII-YN17 inactivated vaccine is used for immunizing 16 SPF chickens with the age of 7 days, 1.0mL of vaccine is injected into each muscle, 5 controls are set at the same time, the control is raised under the same condition, the feeding, drinking and clinical conditions of the test chickens are continuously observed for 14 days, and the feeding, drinking and clinical conditions of the test chickens are recorded. 3 chickens were necropsied 3 days and 7 days after immunization, and the presence of lesions in the liver, spleen, pancreas, duodenum, glandular stomach, trachea, caecal tonsil, bursa of fabricius, heart, brain, lung, kidney, thymus and other tissue organs was observed. The result shows that the vaccine has good absorption effect after the immunization of the chicken, the diet, the drinking water and the defecation are normal, no local or systemic adverse reaction is shown, and all the organs of the chicken subjected to the autopsy have no pathological changes.
3. Evaluation of immune Effect of inactivated vaccine
To determine the immunoprotection effect of the rLa-VII-YN17 vaccine strain rescued in example 2 of the present invention on SPF chickens, 20 SPF chickens of 28 days old, which were kept in an isolator, were randomly divided into two groups of 10, one group being the rLa-VII-YN17 inactivated vaccine immunization group prepared in step 1 of example 3, and the other group being the negative control group. Each chicken in the immunized group was given intramuscular injection of 20. mu.L of vaccine, and the control group was given the same dose of PBS. Blood was collected 7 days, 14 days and 21 days after immunization, and serum was separated and antibody was measured. After the blood collection for 21 days, the virulent YN1106/2017 strain which is clinically separated and wild in gene VII type is used for counteracting the virus, and 10 strains are taken for each chicken6EID50The dose is detoxified by intramuscular injection, the chickens are raised in an isolator, observed for 14 days, and the morbidity and mortality of the chickens are recorded every day; collecting two groups of chicken oropharynx swabs, cloaca swab and cloaca swab inoculated chick embryos for virus separation 3 days, 5 days, 7 days and 10 days after virus challenge, and detecting the condition of toxin expelling.
The results show that the immunization group produced higher levels of antibody after 14 days of immunization and higher levels of antibody after 21 days of immunization, and the results are shown in Table 9. After the toxin is attacked, no morbidity or mortality occurs in an immune group in an observation period of 14 days, and the immune protection rate reaches 100%; the control group of chickens died after 3-4 times of toxic attack, and the results are shown in Table 10. The detoxification results in table 11 show that only one chicken in the rLa-VII-YN17 inactivated vaccine immunization group detoxified 5 days after challenge, and the negative control group detoxified 100%.
Table 9: table for measuring antibody titer after immunization
Table 10: table of immunopotency test results
Table 11: test chicken detoxification test result table after challenge
In conclusion, the inactivated gene VII type Newcastle disease virus recombinant vaccine strain rLa-VII-YN17 prepared by the invention has better safety and protection effect on immunized chickens and has wide application prospect.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention should not be limited thereby, that is, all the simple changes, equivalents and modifications made to the above embodiment according to the claims and the description of the present invention are within the scope of the present invention.
Sequence listing
<110> China center for animal health and epidemiology
<120> gene VII type Newcastle disease virus recombinant vaccine strain with HN protein mutation
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<213> Newcastle disease virus (Newcastle disease virus)
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Thr Leu Ile Ile Leu Ile Leu Ser Phe Ile Cys Leu Ala Ser Ser Leu
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Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys
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Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Arg Ala Pro
65 70 75 80
Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly
85 90 95
Asp Ser Ile Arg Lys Ile Gln Gly Ser Ala Ser Thr Ser Gly Gly Arg
100 105 110
Arg Arg Lys Arg Phe Ile Gly Ala Ile Ile Gly Ser Val Ala Leu Gly
115 120 125
Val Ala Thr Ala Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala
130 135 140
Asn Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala
145 150 155 160
Thr Asn Glu Ala Val His Glu Val Thr Asn Gly Leu Ser Gln Leu Ser
165 170 175
Val Ala Val Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Asn
180 185 190
Thr Ala Arg Glu Leu Asp Cys Ile Lys Ile Thr Gln Gln Val Gly Val
195 200 205
Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly Pro Gln
210 215 220
Ile Thr Ser Pro Ala Leu Thr Gln Leu Thr Ile Gln Ala Leu Tyr Asn
225 230 235 240
Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Val Gly
245 250 255
Asn Asn Gln Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Tyr
260 265 270
Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Asn
275 280 285
Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu
290 295 300
Thr Leu Ser Val Ser Thr Thr Lys Gly Phe Ala Ser Ala Leu Val Pro
305 310 315 320
Lys Val Val Thr Gln Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser
325 330 335
Tyr Cys Ile Glu Ser Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr
340 345 350
Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser
355 360 365
Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Thr Thr Pro Tyr Met
370 375 380
Thr Leu Lys Gly Ser Val Ile Ala Asn Cys Lys Ile Thr Thr Cys Arg
385 390 395 400
Cys Ala Asp Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val
405 410 415
Ser Leu Ile Asp Arg Arg Ser Cys Asn Val Leu Ser Leu Asp Gly Ile
420 425 430
Thr Leu Arg Leu Ser Gly Glu Phe Asp Ala Thr Tyr Gln Lys Asn Ile
435 440 445
Ser Ile Leu Asp Ser Gln Val Ile Val Thr Gly Asn Leu Asp Ile Ser
450 455 460
Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asp Lys
465 470 475 480
Leu Ala Glu Ser Asn Ser Lys Leu Asp Lys Val Asn Val Lys Leu Thr
485 490 495
Ser Thr Ser Ala Leu Ile Thr Tyr Ile Val Leu Thr Val Ile Ser Leu
500 505 510
Val Ser Gly Ala Leu Ser Leu Ile Leu Thr Cys Tyr Leu Met Tyr Lys
515 520 525
Gln Lys Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu
530 535 540
Asp Gln Met Arg Ala Thr Thr Arg Ala
545 550
<210> 6
<211> 571
<212> PRT
<213> Newcastle disease virus (Newcastle disease virus)
<400> 6
Met Asp Arg Val Val Asn Arg Val Val Leu Glu Asn Glu Arg Arg Glu
1 5 10 15
Ala Lys Asn Thr Trp Arg Leu Val Phe Arg Ile Thr Val Leu Leu Leu
20 25 30
Leu Ala Met Thr Leu Ala Ile Ser Ala Ala Ala Leu Ala His Ser Met
35 40 45
Arg Ala Cys Thr Leu Arg Asp Leu Ala Gly Ile Ser Thr Gly Ile Ser
50 55 60
Lys Thr Glu Asp Arg Ile Thr Ser Leu Leu Ser Leu Ser Gln Asp Val
65 70 75 80
Ile Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu
85 90 95
Leu Asn Thr Glu Ser Ile Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr
100 105 110
Gln Ile Asn Gly Ala Ala Asn Ser Ser Gly Cys Gly Ala Pro Val His
115 120 125
Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp
130 135 140
Thr Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Tyr Gln Glu His Leu
145 150 155 160
Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro
165 170 175
Ser Phe Asp Met Ser Thr Thr His Tyr Cys Tyr Thr His Asn Val Ile
180 185 190
Phe Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu
195 200 205
Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Ile Phe Phe Ser Thr Leu
210 215 220
Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val
225 230 235 240
Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Val Thr Glu
245 250 255
Thr Glu Glu Glu Asp Tyr Arg Ser Val Thr Pro Thr Ser Met Val His
260 265 270
Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Thr
275 280 285
Thr Val Leu Phe Lys Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly
290 295 300
Gly Ser Phe Ile Asn Asp Arg Val Trp Phe Pro Val Tyr Gly Gly Leu
305 310 315 320
Lys Pro Asn Ser Pro Ser Asp Thr Ala Gln Glu Gly Lys Tyr Val Ile
325 330 335
Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile
340 345 350
Arg Met Ala Lys Ser Ser Tyr Lys Pro Gln Arg Phe Gly Gly Lys Arg
355 360 365
Val Gln Gln Ala Ile Leu Ser Ile Glu Val Ser Thr Ser Leu Gly Lys
370 375 380
Asp Pro Val Leu Thr Ile Pro Pro Asn Thr Ile Thr Leu Met Gly Ala
385 390 395 400
Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg
405 410 415
Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Ile Val His
420 425 430
Asn Lys Thr Ala Thr Leu His Asn Pro Tyr Thr Phe Asn Ala Phe Thr
435 440 445
Arg Pro Gly Ser Val Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser
450 455 460
Cys Ile Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe His Arg
465 470 475 480
Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Asp Gly Gln
485 490 495
Ala Arg Leu Asn Pro Thr Ser Ala Val Phe Asp Asn Ile Ser Arg Ser
500 505 510
Arg Val Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr
515 520 525
Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser
530 535 540
Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro
545 550 555 560
Leu Leu Ile Glu Ile Leu Lys Asp Asp Arg Val
565 570
<210> 7
<211> 577
<212> PRT
<213> Newcastle disease virus (Newcastle disease virus)
<400> 7
Met Asp Arg Val Val Asn Arg Val Val Leu Glu Asn Glu Arg Arg Glu
1 5 10 15
Ala Lys Asn Thr Trp Arg Leu Val Phe Arg Ile Thr Val Leu Leu Leu
20 25 30
Leu Ala Met Thr Leu Ala Ile Ser Ala Ala Ala Leu Ala His Ser Met
35 40 45
Arg Ala Cys Thr Leu Arg Asp Leu Ala Gly Ile Ser Thr Gly Ile Ser
50 55 60
Lys Thr Glu Asp Arg Ile Thr Ser Leu Leu Ser Leu Ser Gln Asp Val
65 70 75 80
Ile Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu
85 90 95
Leu Asn Thr Glu Ser Ile Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr
100 105 110
Gln Ile Asn Gly Ala Ala Asn Ser Ser Gly Cys Gly Ala Pro Val His
115 120 125
Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp
130 135 140
Thr Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Tyr Gln Glu His Leu
145 150 155 160
Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro
165 170 175
Ser Phe Asp Met Ser Thr Thr His Tyr Cys Tyr Thr His Asn Val Ile
180 185 190
Phe Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu
195 200 205
Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Ile Phe Phe Ser Thr Leu
210 215 220
Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val
225 230 235 240
Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Val Thr Glu
245 250 255
Thr Glu Glu Glu Asp Tyr Arg Ser Val Thr Pro Thr Ser Met Val His
260 265 270
Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Thr
275 280 285
Thr Val Leu Phe Lys Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly
290 295 300
Gly Ser Phe Ile Asn Asp Arg Val Trp Phe Pro Val Tyr Gly Gly Leu
305 310 315 320
Lys Pro Asn Ser Pro Ser Asp Thr Ala Gln Glu Gly Lys Tyr Val Ile
325 330 335
Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gln Asp Tyr Gln Ile
340 345 350
Arg Met Ala Lys Ser Ser Tyr Lys Pro Gln Arg Phe Gly Gly Lys Arg
355 360 365
Val Gln Gln Ala Ile Leu Ser Ile Glu Val Ser Thr Ser Leu Gly Lys
370 375 380
Asp Pro Val Leu Thr Ile Pro Pro Asn Thr Ile Thr Leu Met Gly Ala
385 390 395 400
Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg
405 410 415
Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Ile Val His
420 425 430
Asn Lys Thr Ala Thr Leu His Asn Pro Tyr Thr Phe Asn Ala Phe Thr
435 440 445
Arg Pro Gly Ser Val Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser
450 455 460
Cys Ile Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe His Arg
465 470 475 480
Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Asp Gly Gln
485 490 495
Ala Arg Leu Asn Pro Thr Ser Ala Val Phe Asp Asn Ile Ser Arg Ser
500 505 510
Arg Val Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr
515 520 525
Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser
530 535 540
Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro
545 550 555 560
Leu Leu Ile Glu Ile Leu Lys Asp Asp Arg Val Arg Glu Ala Arg Ser
565 570 575
Gly
<210> 4
<211> 6539
<212> DNA
<213> Newcastle disease virus (Newcastle disease virus)
<400> 4
agatcttgtc ttgaaacaga catcctccat ccctatgatg cggaccgaaa tccaacagct 60
gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc tggatcccgg 120
ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc acccggtttt 180
agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa tggcacttaa 240
taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca ctgcatgcgg 300
gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac gcccaatgca 360
cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga tcgaggaaat 420
caggaaaatc aagcgccttg ctctaaatgg ttaattacta ctgccacacg tagcgggtcc 480
ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac ccaaggtcca 540
actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc gcgtaaccgt 600
aattaatcta gctacattta agattaagaa aaaatacggg tagaattgga gtgccccaat 660
tgtgccaaga tggactcatc taggacaatt gggctgtact ttgattctgc ccattcttct 720
agcaacctgt tagcatttcc gatcgtccta caagacacag gagatgggaa gaagcaaatc 780
gccccgcaat ataggatcca gcgccttgac ttgtggactg atagtaagga ggactcagta 840
ttcatcacca cctatggatt catctttcaa gctgggaatg aagaagccac tgtcggcatg 900
atcgatgata aacccaagcg cgagttactt tccgctgcga tgctctgcct aggaagcgtc 960
ccaaataccg gagaccttat tgagctggca agggcctgtc tcactatgat agtcacatgc 1020
aagaagagtg caactaatac tgagagaatg gttttctcag tagtgcaggc accccaagtg 1080
ctgcaaagct gtagggttgt ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg 1140
aaagcgccag agaagattcc cgggagtgga accctagaat acaaggtgaa ctttgtctcc 1200
ttgactgtgg taccgaagaa ggatgtctac aagatcccag ctgcagtatt gaaggtttct 1260
ggctcgagtc tgtacaatct tgcgctcaat gtcactatta atgtggaggt agacccgagg 1320
agtcctttgg ttaaatctct gtctaagtct gacagcggat actatgctaa cctcttcttg 1380
catattggac ttatgaccac cgtagatagg aaggggaaga aagtgacatt tgacaagctg 1440
gaaaagaaaa taaggagcct tgatctatct gtcgggctca gtgatgtgct cgggccttcc 1500
gtgttggtaa aagcaagagg tgcacggact aagctcttgg cacctttctt ctctagcagt 1560
gggacagcct gctatcccat agcaaatgct tctcctcagg tggccaagat actctggagt 1620
caaaccgcgt gcctgcggag cgttaaaatc attatccaag caggtaccca acgcgctatc 1680
gcagtgaccg ccgaccacga ggttacctct actaagctgg agaaggggca cacccttgcc 1740
aaatacaatc cttttaagaa ataagctgcg tctctgagat tgcgctccgc ccactcaccc 1800
agatcatcat gacacaaaaa actaatctgt cttgattatt tacagttagt ttacctgtct 1860
atcaagttag aaaaaacacg ggtagaagat tctggatccc ggttggcgcc ctccaggtgc 1920
aagatgggtt ccaactcttc taccaggacc ccaacacccc cgatgctggt cactctgatc 1980
atactgatat taagcttcat ctgcctggca agctctctcg atggcaggcc tcttgcggct 2040
gcagggattg tggtaacagg agacaaggca gtcaatatct acacctcgtc tcaaacaggg 2100
tcaatcatag tcaagttgct cccgaatatg cccaaagata aagaggcgtg tgcaagagcc 2160
ccattagaag catataatag aacattgact acattgctca ctccccttgg cgattccatc 2220
cgtaagatcc aagggtccgc gtccacgtca ggaggaggga gacaggggcg ccttataggt 2280
gccattattg gcagtgtcgc tcttggagtt gcaacagcgg cacagataac tgcagctgcg 2340
gccctaatac aagccaacca gaatgccgcc aacatcctcc ggcttaagga gagcattgct 2400
gcgaccaatg aagctgtgca tgaagtcacc aacggattat cacaactatc agtggcagtt 2460
ggaaagatgc agcagtttgt caatgaccag tttaataata cggcacgaga attggattgt 2520
ataaaaatta cacaacaggt tggtgtagaa ctcaacctat acctgactga attgactaca 2580
gtattcgggc cacagatcac ttcccctgcc ttaactcagc tgactatcca ggcactttat 2640
aatttagctg ggggcaatat ggattacttg ttaactaagt taggtgtagg gaacaatcaa 2700
ctcagctcat taattggtag cggcttgatt accggatacc ccatactgta tgactcacag 2760
actcaactct tgggtataca agtgaatttg ccctcagtcg gaaacctaaa taatatgcga 2820
gccacctact tggagacctt atctgtgagt acaacaaagg ggtttgcctc agcgcttgtc 2880
cctaaagtag tgacacaagt tggttctgtg atagaagagc ttgacacctc atactgtata 2940
gagtctgatc tggatttata ttgcacaaga atagtgacat tccccatgtc cccaggcatt 3000
tattcttgtt tgagcggcaa cacatcggcc tgcatgtatt caaagactga aggagcactc 3060
actacgccat atatgaccct taaaggctca gttattgcca attgtaagat aacaacatgt 3120
agatgtgcag atcctcctgg tatcatatcg caaaattacg gagaagctgt gtctctgata 3180
gacagacgtt cgtgcaatgt cctatcatta gacgggataa ctctgaggct cagtggggag 3240
tttgatgcaa cttatcaaaa gaatatctca atactagatt ctcaagttat cgtgacaggc 3300
aatctcgata tatcaactga gcttggaaat gtcaacaatt caatcagtaa tgccctagat 3360
aagctggcgg aaagcaacag caagctagat aaagtcaatg tcaaactaac cagcacatca 3420
gctctcatta cttatatcgt tttaacagtc atttctcttg tttctggtgc acttagtttg 3480
attttaacat gttacttgat gtacaagcag aaggcacaac agaagacctt actatggctt 3540
gggaataata ctcttgatca gatgagagcc accacaagag catgaacaca gatgaggaac 3600
gaaggtttcc ctaatagtaa tttatgtgaa agttctggta gtctgtcagt tcagagagtt 3660
aagaaaaaac taccggttgt agatgaccaa aggacgatat acgggtagaa cggtaagaga 3720
ggccgcccct caattgcgag ccaggcttca caacctccgt tctaccgctt caccgacaac 3780
agtcctcaat catggaccgt gtggtcaata gggtcgtgct ggagaatgag agaagagaag 3840
caaagaatac atggcgcttg gtcttccgta tcacagtctt acttttattg gcaatgactc 3900
tagccatctc tgcagccgcc ctggcgcata gcatgagggc ctgtacgctg cgtgacctcg 3960
caggcatatc gacagggatc tccaagacag aagatagaat tacgtcttta ctcagtttaa 4020
gtcaagatgt gatagatagg atatataagc aggtggctct tgaatctccg ctggcgttac 4080
taaatactga atctataatt atgaatgcaa taacctctct ttcctatcaa atcaacggag 4140
ctgcgaatag tagcggatgt ggggcgcctg ttcatgaccc agattatatc ggggggatag 4200
gcaaagaact catagtagac gatactagtg acgtcacttc gttttatcct tctgcgtatc 4260
aagaacactt gaatttcatt ccagcaccca cgacaggatc cggttgcact cggataccct 4320
catttgacat gagcaccact cattattgtt atactcacaa tgtgatattt tctggttgca 4380
gagaccactc acactcacac caatacctag cacttggcgt gctccggaca tctgcaacag 4440
ggaggatatt cttctctact ctgcgttcca tcaatttaga tgacacccaa aatcggaagt 4500
cctgcagtgt gagtgcaact cctttaggtt gtgatatgct atgctctaaa gtcacagaga 4560
ctgaggaaga agattacagg tcagttaccc ccacatcaat ggtgcacgga aggctagggt 4620
ttgatggtca ataccatgag aaggacttag ataccacagt cttatttaaa gattgggtgg 4680
caaattaccc tggggtggga ggtgggtctt ttattaacga ccgtgtatgg ttcccagttt 4740
atggagggct caaacccaat tcacctagtg acaccgcaca agaagggaaa tatgtaatat 4800
acaagcgcta taatgacaca tgccccgatg aacaagatta ccaaatccgg atggctaaat 4860
cttcatataa gccccagcga tttggtggga agcgcgtaca gcaagccatc ttatctattg 4920
aggtgtcaac atctttgggt aaggatccgg tgctgactat tccacctaat acaattacac 4980
tcatgggggc cgaaggcaga attctcacgg tagggacatc tcacttcctg tatcaacgag 5040
gatcttcata tttctccccc gctttattat accccatgat tgtgcataac aaaacggcta 5100
ctctccataa tccttataca tttaatgcct tcactcggcc gggtagcgtc ccttgccagg 5160
catcagcaag atgccccaac tcatgcatca ccggggttta tactgatcca tacccattaa 5220
tcttccatag gaatcatact ttacgagggg tcttcgggac gatgcttgat gatggacaag 5280
caagacttaa tcccacatct gcagtatttg acaacatatc ccgcagtcgt gtaactcggg 5340
tgagttcaag cagcaccaag gcagcataca caacatcaac atgttttaaa gttgtcaaga 5400
ccaataaaac ttattgtctt agtattgcag aaatatccaa caccctattc ggggaattta 5460
ggattgttcc cttactgatt gagatcctca aggatgatag agtcagagaa gccaggtctg 5520
gctagttgag tcaattataa aggagttgga aagatggcat tgtatcacct atcctctgcg 5580
acatcaagaa tcaaaccgaa tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac 5640
aatcagccag tgctcatgcg atcagattaa gccttgtcaa tagtctcttg atttagaaaa 5700
aatgtaagtg gcaatgagat acaaggcaaa acagctcatg gtaaataata cgggtaggac 5760
atggcgagct ccggtcctga aagggcagag catcagatta tcctaccaga gtcacacctg 5820
tcttcaccat tggtcaagca caaactactc tattactgga aattaactgg gctaccgctt 5880
cctgatgaat gtgacttcga ccacctcatt ctcagccgac aatggaaaaa aatacttgaa 5940
tcggcctctc ctgatactga gagaatgata aaactcggaa gggcagtaca ccaaactctt 6000
aaccacaatt ccagaataac cggagtgctc caccccaggt gtttagaaga actggctaat 6060
attgaggtcc cagattcaac caacaaattt cggaagattg agaagaagat ccaaattcac 6120
aacacgagat atggagaact gttcacaagg ctgtgtacgc atatagagaa gaaactgctg 6180
gggtcatctt ggtctaacaa tgtcccccgg tcagaggagt tcagcagcat tcgtacggat 6240
ccggcattct ggtttcactc aaaatggtcc acagccaagt ttgcatggct ccatataaaa 6300
cagatccaga ggcatctgat ggtggcagct aggacaaggt ctgcggccaa caaattggtg 6360
atgctaaccc ataaggtagg ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat 6420
acgaatgaga acaagttcac atgtcttacc caggaacttg tattgatgta tgcagatatg 6480
atggagggca gagatatggt caacataata tcaaccacgg cggtgcatct cagaagctt 6539
<210> 1
<211> 1662
<212> DNA
<213> Newcastle disease virus (Newcastle disease virus)
<400> 1
atgggttcca actcttctac caggacccca acacccccga tgctggtcac tctgatcata 60
ctgatattaa gcttcatctg cctggcaagc tctctcgatg gcaggcctct tgcggctgca 120
gggattgtgg taacaggaga caaggcagtc aatatctaca cctcgtctca aacagggtca 180
atcatagtca agttgctccc gaatatgccc aaagataaag aggcgtgtgc aagagcccca 240
ttagaagcat ataatagaac attgactaca ttgctcactc cccttggcga ttccatccgt 300
aagatccaag ggtccgcgtc cacgtcagga ggaaggagac ggaaacgctt tataggtgcc 360
attattggca gtgtcgctct tggagttgca acagcggcac agataactgc agctgcggcc 420
ctaatacaag ccaaccagaa tgccgccaac atcctccggc ttaaggagag cattgctgcg 480
accaatgaag ctgtgcatga agtcaccaac ggattatcac aactatcagt ggcagttgga 540
aagatgcagc agtttgtcaa tgaccagttt aataatacgg cacgagaatt ggattgtata 600
aaaattacac aacaggttgg tgtagaactc aacctatacc tgactgaatt gactacagta 660
ttcgggccac agatcacttc ccctgcctta actcagctga ctatccaggc actttataat 720
ttagctgggg gcaatatgga ttacttgtta actaagttag gtgtagggaa caatcaactc 780
agctcattaa ttggtagcgg cttgattacc ggatacccca tactgtatga ctcacagact 840
caactcttgg gtatacaagt gaatttgccc tcagtcggaa acctaaataa tatgcgagcc 900
acctacttgg agaccttatc tgtgagtaca acaaaggggt ttgcctcagc gcttgtccct 960
aaagtagtga cacaagttgg ttctgtgata gaagagcttg acacctcata ctgtatagag 1020
tctgatctgg atttatattg cacaagaata gtgacattcc ccatgtcccc aggcatttat 1080
tcttgtttga gcggcaacac atcggcctgc atgtattcaa agactgaagg agcactcact 1140
acgccatata tgacccttaa aggctcagtt attgccaatt gtaagataac aacatgtaga 1200
tgtgcagatc ctcctggtat catatcgcaa aattacggag aagctgtgtc tctgatagac 1260
agacgttcgt gcaatgtcct atcattagac gggataactc tgaggctcag tggggagttt 1320
gatgcaactt atcaaaagaa tatctcaata ctagattctc aagttatcgt gacaggcaat 1380
ctcgatatat caactgagct tggaaatgtc aacaattcaa tcagtaatgc cctagataag 1440
ctggcggaaa gcaacagcaa gctagataaa gtcaatgtca aactaaccag cacatcagct 1500
ctcattactt atatcgtttt aacagtcatt tctcttgttt ctggtgcact tagtttgatt 1560
ttaacatgtt acttgatgta caagcagaag gcacaacaga agaccttact atggcttggg 1620
aataatactc ttgatcagat gagagccacc acaagagcat ga 1662
<210> 2
<211> 1716
<212> DNA
<213> Newcastle disease virus (Newcastle disease virus)
<400> 2
atggaccgtg tggtcaatag ggtcgtgctg gagaatgaga gaagagaagc aaagaataca 60
tggcgcttgg tcttccgtat cacagtctta cttttattgg caatgactct agccatctct 120
gcagccgccc tggcgcatag catgagggcc tgtacgctgc gtgacctcgc aggcatatcg 180
acagggatct ccaagacaga agatagaatt acgtctttac tcagtttaag tcaagatgtg 240
atagatagga tatataagca ggtggctctt gaatctccgc tggcgttact aaatactgaa 300
tctataatta tgaatgcaat aacctctctt tcctatcaaa tcaacggagc tgcgaatagt 360
agcggatgtg gggcgcctgt tcatgaccca gattatatcg gggggatagg caaagaactc 420
atagtagacg atactagtga cgtcacttcg ttttatcctt ctgcgtatca agaacacttg 480
aatttcattc cagcacccac gacaggatcc ggttgcactc ggataccctc atttgacatg 540
agcaccactc attattgtta tactcacaat gtgatatttt ctggttgcag agaccactca 600
cactcacacc aatacctagc acttggcgtg ctccggacat ctgcaacagg gaggatattc 660
ttctctactc tgcgttccat caatttagat gacacccaaa atcggaagtc ctgcagtgtg 720
agtgcaactc ctttaggttg tgatatgcta tgctctaaag tcacagagac tgaggaagaa 780
gattacaggt cagttacccc cacatcaatg gtgcacggaa ggctagggtt tgatggtcaa 840
taccatgaga aggacttaga taccacagtc ttatttaaag attgggtggc aaattaccct 900
ggggtgggag gtgggtcttt tattaacgac cgtgtatggt tcccagttta tggagggctc 960
aaacccaatt cacctagtga caccgcacaa gaagggaaat atgtaatata caagcgctat 1020
aatgacacat gccccgatga acaagattac caaatccgga tggctaaatc ttcatataag 1080
ccccagcgat ttggtgggaa gcgcgtacag caagccatct tatctattga ggtgtcaaca 1140
tctttgggta aggatccggt gctgactatt ccacctaata caattacact catgggggcc 1200
gaaggcagaa ttctcacggt agggacatct cacttcctgt atcaacgagg atcttcatat 1260
ttctcccccg ctttattata ccccatgatt gtgcataaca aaacggctac tctccataat 1320
ccttatacat ttaatgcctt cactcggccg ggtagcgtcc cttgccaggc atcagcaaga 1380
tgccccaact catgcatcac cggggtttat actgatccat acccattaat cttccatagg 1440
aatcatactt tacgaggggt cttcgggacg atgcttgatg atggacaagc aagacttaat 1500
cccacatctg cagtatttga caacatatcc cgcagtcgtg taactcgggt gagttcaagc 1560
agcaccaagg cagcatacac aacatcaaca tgttttaaag ttgtcaagac caataaaact 1620
tattgtctta gtattgcaga aatatccaac accctattcg gggaatttag gattgttccc 1680
ttactgattg agatcctcaa ggatgataga gtctga 1716
<210> 3
<211> 1734
<212> DNA
<213> Newcastle disease virus (Newcastle disease virus)
<400> 3
atggaccgtg tggtcaatag ggtcgtgctg gagaatgaga gaagagaagc aaagaataca 60
tggcgcttgg tcttccgtat cacagtctta cttttattgg caatgactct agccatctct 120
gcagccgccc tggcgcatag catgagggcc tgtacgctgc gtgacctcgc aggcatatcg 180
acagggatct ccaagacaga agatagaatt acgtctttac tcagtttaag tcaagatgtg 240
atagatagga tatataagca ggtggctctt gaatctccgc tggcgttact aaatactgaa 300
tctataatta tgaatgcaat aacctctctt tcctatcaaa tcaacggagc tgcgaatagt 360
agcggatgtg gggcgcctgt tcatgaccca gattatatcg gggggatagg caaagaactc 420
atagtagacg atactagtga cgtcacttcg ttttatcctt ctgcgtatca agaacacttg 480
aatttcattc cagcacccac gacaggatcc ggttgcactc ggataccctc atttgacatg 540
agcaccactc attattgtta tactcacaat gtgatatttt ctggttgcag agaccactca 600
cactcacacc aatacctagc acttggcgtg ctccggacat ctgcaacagg gaggatattc 660
ttctctactc tgcgttccat caatttagat gacacccaaa atcggaagtc ctgcagtgtg 720
agtgcaactc ctttaggttg tgatatgcta tgctctaaag tcacagagac tgaggaagaa 780
gattacaggt cagttacccc cacatcaatg gtgcacggaa ggctagggtt tgatggtcaa 840
taccatgaga aggacttaga taccacagtc ttatttaaag attgggtggc aaattaccct 900
ggggtgggag gtgggtcttt tattaacgac cgtgtatggt tcccagttta tggagggctc 960
aaacccaatt cacctagtga caccgcacaa gaagggaaat atgtaatata caagcgctat 1020
aatgacacat gccccgatga acaagattac caaatccgga tggctaaatc ttcatataag 1080
ccccagcgat ttggtgggaa gcgcgtacag caagccatct tatctattga ggtgtcaaca 1140
tctttgggta aggatccggt gctgactatt ccacctaata caattacact catgggggcc 1200
gaaggcagaa ttctcacggt agggacatct cacttcctgt atcaacgagg atcttcatat 1260
ttctcccccg ctttattata ccccatgatt gtgcataaca aaacggctac tctccataat 1320
ccttatacat ttaatgcctt cactcggccg ggtagcgtcc cttgccaggc atcagcaaga 1380
tgccccaact catgcatcac cggggtttat actgatccat acccattaat cttccatagg 1440
aatcatactt tacgaggggt cttcgggacg atgcttgatg atggacaagc aagacttaat 1500
cccacatctg cagtatttga caacatatcc cgcagtcgtg taactcgggt gagttcaagc 1560
agcaccaagg cagcatacac aacatcaaca tgttttaaag ttgtcaagac caataaaact 1620
tattgtctta gtattgcaga aatatccaac accctattcg gggaatttag gattgttccc 1680
ttactgattg agatcctcaa ggatgataga gtcagagaag ccaggtctgg ctag 1734