CN109762792B - Porcine reproductive and respiratory syndrome virus chimeric strain and application thereof - Google Patents

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus chimeric strain and application thereof, and the porcine reproductive and respiratory syndrome virus chimeric strain is constructed by using reverse genetic technology on the basis of full-length infectious cDNA clone of a highly pathogenic PRRSV TJM-F92 vaccine attenuated strain and a NADC30 subtype-like strain FJ 1402. The chimeric strain is verified by tests that the virus growth speed and the virus titer on Marc-145 cells are lower than that of the original strain TJM-F92, the toxicity is low, the chimeric strain has better immune protection effect on HP-PRRSV strain BB0907 and the like NADC30 strain FJ1402, the effective cross immune protection can be provided for the infection of highly pathogenic strains and the like NADC30 strains, and an important basis is laid for the development of novel broad-spectrum vaccines of the disease.

Description

Porcine reproductive and respiratory syndrome virus chimeric strain and application thereof

Technical Field

The invention relates to a novel chimeric strain of porcine reproductive and respiratory syndrome virus, a construction method of the chimeric strain and application of the chimeric strain in preparation of a candidate vaccine for preventing the porcine reproductive and respiratory syndrome epidemic strain.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral epidemic disease seriously harming the swine industry caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and has complex and various clinical symptoms, and is characterized by sow reproductive failure or respiratory diseases of all-day-old pig groups. In 1996, the virus was first isolated in China and gradually spread throughout the country. The highly pathogenic PRRSV (HP-PRRSV) is more outbreak in summer and autumn in 2006, and the main clinical features are high fever, high morbidity and mortality, which causes huge economic loss to the pig industry. HP-PRRSV strains cause more severe clinical symptoms in piglets than classical PRRSV, leading to higher morbidity and mortality. The PRRSV genome is easy to mutate and recombine, and brings more difficulty to the prevention and control of the disease.

In recent years, a virus strain with high homology with the American PRRSV NADC30 strain is continuously separated in China and is called a NADC 30-like strain. Molecular epidemiological investigation research shows that the strain like NADC30 presents an epidemic situation in China. The American strain NADC30 has weak pathogenicity to piglets, but the pathogenicity of the like NADC30 strain after genetic recombination with HP-PRRSV is obviously enhanced. PRRSV is a single-stranded positive-strand RNA virus belonging to the family arteriviridae, the genus arterivirus. The viral genome is about 15kb in length and comprises at least 11 Open Reading Frames (ORFs), wherein ORF1a and ORF1b encode nonstructural proteins, and 8 ORFs at the 3' end of the genome mainly encode structural proteins of the virus. Wherein, the oligomers formed by GP2, GP3, GP4 and M protein have certain effect on inducing neutralizing antibodies. These proteins have T cell epitopes and can induce T cell immunity. GP5 is encoded by ORF5, is a viral envelope glycoprotein, and is also a main host protective antigen of PRRSV, and contains a neutralizing epitope closely related to an induced neutralizing antibody, and variation of the GP5 amino acid sequence will directly reduce the cross-protection rate of PRRS vaccine strains. The M protein is a non-glycosylated membrane protein encoded by ORF6 and is capable of mediating viral adsorption and inducing cellular immunity. The GP5 and M proteins can exist as heterodimers, and such heterodimers can facilitate the transport of GP5 protein from the endoplasmic reticulum to the golgi apparatus, thereby further enhancing the immune response induced by the GP5 protein. The N protein is a highly conserved nucleocapsid protein and has extremely strong immunogenicity, but the induced antibody has no virus neutralizing capacity. Due to the mutation, recombination and the like of viral genome RNA, strains show the trend of variation diversity and genetic diversity, and a serious challenge is brought to the PRRS prevention and control. Currently, PRRSV commercial attenuated vaccines have a good immune protection effect on homologous strain genes, but have a low cross protection effect on heterotypic strains. At present, commercial PRRS vaccines mainly comprise attenuated live vaccines and inactivated vaccines, and the attenuated vaccines have better immune efficacy than the inactivated vaccines, but have poorer cross protection on different subtype strains. For example, the attenuated vaccines such as TJM-F92, HuN4-F112, JXA1-R and R98 can effectively relieve clinical symptoms caused by HP-PRRSV and reduce the death rate of piglets, but have limited immune protection effect on the strain like NADC30, so that the research of a novel broad-spectrum vaccine is necessary.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a porcine reproductive and respiratory syndrome virus chimeric strain. The novel chimeric strain has low toxicity and good immunogenicity, can provide effective cross immune protection for infection of highly pathogenic PRRSV strains and PRRSV type NADC30 strains, and lays an important foundation for development of novel vaccines for the disease.

The PRRSV type NADC30 strain FJ1402 (called NADC30 strain FJ1402 for short) and the highly pathogenic PRRSV strain BB0907 (called HP-PRRSV BB0907 for short) are obtained by separation, and the pathogenicity of the NADC 30-like strain FJ1402 and the pathogenicity of the HP-PRRSV BB0907 are similar.

The PRRSV chimeric strain provided by the invention is obtained by deleting a gene segment expressing 323-433 th amino acid (aa) on an Nsp2 gene of a highly pathogenic PRRSV vaccine strain and simultaneously replacing ORF5-6 (open reading frames 5 and 6) of the PRRSV chimeric strain with ORF5-6 of a PRRSV type NADC30 strain.

The PRRSV vaccine strain selected by the invention is a highly pathogenic PRRSV TJM-F92 vaccine attenuated strain, and the PRRSV type NADC30 strain selected by the invention is a type NADC30 strain FJ 1402.

When constructing PRRSV chimeric strains, on the basis of full-length infectious cDNA clone of highly pathogenic PRRSV TJM-F92 vaccine attenuated strain and the like NADC30 strain FJ1402, utilizing reverse genetic technology to delete Nsp2 gene of TJM-F92 to express a gene fragment of 323-shivish 433 aa (total deletion of 111aa), and respectively replacing structural protein gene ORF2-7(ORF2, ORF3, ORF4, ORF5, ORF6 and 7) and ORF5-6(ORF5 and ORF6) regions of the highly pathogenic PRRSV TJM-F92 strain after the deletion of the 323-shivish 433 aa of Nsp2 gene to coding regions (ORF2-7 and ORF5-6) corresponding to the like NADC30 subtype strain FJ1402, constructing three infectious clones, transfecting plasmids containing the three infectious clones to obtain Mare-145 recombinant cells, rescuing the Marsp 54 strain Td 111-Td 2 strain (Td-Td 46111 strain), rTd111/F2-7 (replacement of rTd111 strain ORF2-7 with FJ1402 strain ORF2-7) and rTd111/F5-6 (replacement of rTd111 strain ORF5-6 with FJ1402 strain ORF 5-6).

The construction idea of the PRRSV chimeric strain is as follows:

(1) constructing infectious cDNA clone of a highly pathogenic PRRSV TJM-F92 vaccine attenuated strain, and naming the clone as pCMV-TJM;

(2) deleting a fragment expressing aa (111aa in total) at position 323-433 in the Nsp2 gene of the pCMV-TJM to construct an infectious cDNA clone which is named pTd 111;

(3) pTd111 is used as a framework, ORF2-7 and ORF5-6 of the porcine reproductive and respiratory syndrome NADC30 strain are respectively replaced by ORF2-7 and ORF5-6 of FJ1402, and infectious cDNA clones are constructed and named as pTd111/F2-7 and pTd 111/F5-6;

(4) pTd111, pTd111/F2-7 and pTd111/F5-6 were subjected to virus rescue, respectively, to obtain three chimeric strains designated as rTd111, rTd111/F2-7 and rTd 111/F5-6.

The gene inheritance is stable when the rTd111, the rTd111/F2-7 and the rTd111/F5-6 are respectively passaged for 5 times. Further experimental validation of the rTd111, rTd111/F2-7 and rTd111/F5-6 chimeric strains revealed that: the growth rate and virus titer of the rTd111/F2-7 and rTd111/F5-6 chimeric strains on Marc-145 cells were slightly lower than those of the original TJM-F92 strain, but the growth rate and virus titer on PAM cells were similar to those of the original TJM-F92 strain. Upon infection of PAMs cells, rTd111/F5-6 induced levels of inflammatory cytokines similar to Td111, slightly higher than TJM-F92, but rTd111/F2-7 induced levels of inflammatory cytokines significantly higher than Td111, rTd111/F5-6 and TJM-F92. The rTd111, the rTd111/F2-7, the rTd111/F5-6, the TJM-F92 and the FJ1402 are respectively inoculated to piglets with the age of 30-35 days, and the results of clinical symptoms, antibody detection, autopsy lesion and pathological observation on pigs show that the rTd111/F5-6 has virulence similar to that of TJM-F92 and is obviously lower than that of FJ1402, while the rTd111/F2-7 has virulence higher than that of the rTd111/F5-6, thereby indicating that the rTd111/F5-6 has the potential as a vaccine candidate strain. Healthy piglets of 30-35 days are selected, piglet immune protection efficacy of the chimeric vaccine candidate strain rTd111/F5-6 is further researched, after immunization, 28d, the HP-PRRSV strain BB0907 and the like NADC30 strain FJ1402 are respectively utilized to carry out intramuscular injection challenge, and animal test results show that the infection levels of the BB0907 and the FJ1402 strains in the rTd111/F5-6 immune group are obviously reduced, and clinical symptoms and pathological changes are obviously alleviated. The chimeric strain is shown to have good immunogenicity, can provide effective cross immune protection for infection of highly pathogenic strains and similar NADC30 strains, and lays an important foundation for development of novel vaccines for the disease.

Based on the above experimental results, the invention selects rTd111/F5-6 as the PRRSV chimeric strain to be protected. The invention further cultures and preserves the fifth generation of the rTd111/F5-6 chimeric strain, the preservation number is CGMCC No.14762, the preservation name is porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus) rTd111/F5-6F5, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation unit address is North Chen West Lu No.1 institute No.3 of the sunward area in Beijing, and the preservation date is 2018, 5 months and 25 days.

Furthermore, in the rTd111/F5-6F5 CGMCC No.14762 strain of the porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus), the nucleotides of ORF5 and ORF6 are the same as ORF5-6 of the similar NADC30 strain FJ1402, and other open reading frames are the same as the attenuated strain of the highly pathogenic PRRSV TJM-F92 vaccine, but a fragment of 111aa (position 323-433) is deleted and expressed on the Nsp2 gene.

Furthermore, the nucleotide sequence of the rTd111/F5-6F5 CGMCC No.14762 strain of the porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus) is shown as SEQ ID No.1, the nucleotide sequence of ORF5 is shown as SEQ ID No.2, and the nucleotide sequence of ORF6 is shown as SEQ ID No. 3.

Furthermore, the chimeric strain constructed by the invention has better immune protection effect on the HP-PRRSV strain BB0907 and the like NADC30 strain FJ1402, so the application of the chimeric strain as a candidate vaccine of the porcine reproductive and respiratory syndrome epidemic strain is also within the protection range of the invention, and preferably, the PRRSV chimeric strain is rTd111/F5-6F5 CGMCC No.14762 or rTd 111/F5-6.

Furthermore, the invention also provides the application of the porcine reproductive and respiratory syndrome virus chimeric strain in preparing a porcine reproductive and respiratory syndrome vaccine, preferably, the porcine reproductive and respiratory syndrome virus chimeric strain is rTd111/F5-6F5 CGMCC No.14762 or rTd 111/F5-6.

Furthermore, the invention also provides a vaccine, the effective component of which comprises an immunizing dose of porcine reproductive and respiratory syndrome virus chimeric strain, and the chimeric strain is rTd111/F5-6F5 CGMCC No.14762 or rTd 111/F5-6. Preferably, the immunizing amount of the chimeric strain in the vaccine is: 10^5.0TCID₅₀Viral fluid/ml.

Furthermore, the invention also provides application of the vaccine in preparing a medicament for preventing porcine reproductive and respiratory syndrome. Further, the porcine reproductive and respiratory syndrome is caused by infection of a classical porcine reproductive and respiratory syndrome strain, or a highly pathogenic PRRSV strain, or a PRRSV type NADC30 strain, or cross infection of the three strains.

The invention constructs a chimeric strain of porcine reproductive and respiratory syndrome virus by using reverse genetic technology on the basis of full-length infectious cDNA clone of a highly pathogenic PRRSV TJM-F92 vaccine attenuated strain (namely the highly pathogenic porcine reproductive and respiratory syndrome virus strain TJM-F92 vaccine attenuated strain) and a similar NADC30 subtype strain FJ 1402. The chimeric strain is verified by tests that the virus growth speed and the virus titer on Marc-145 cells are lower than that of the original strain TJM-F92, the toxicity is low, the chimeric strain has better immune protection effect on HP-PRRSV strain BB0907 and the like NADC30 strain FJ1402, the effective cross immune protection can be provided for the infection of highly pathogenic strains and the like NADC30 strains, and an important basis is laid for the development of novel broad-spectrum vaccines of the disease.

Preservation information

The porcine reproductive and respiratory syndrome virus rTd111/F5-6F5 is preserved in China general microbiological culture Collection center (CGMCC) in 25.5.2018, wherein the preservation unit address is No.3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 14762.

Drawings

FIG. 1 shows the construction of infectious clone of PRRSV chimeric strain, (A) using pCMV-TJM as the skeleton, deleting 111aa fragments expressed by Nsp2 to obtain pTd111, wherein the gene of the diagram is marked with the position of the corresponding 323-433 amino acids; (B) chimeric cDNA clones pTd111/F2-7 and pTd111/F5-6 were constructed with pTd111 as a backbone, respectively.

FIG. 2 plaque morphology (A) and growth curves (B) of chimeric PRRSV strains on Marc-145 cells.

FIG. 3 lesions (A) and growth curves (B) of chimeric PRRSV strains on PAMs cells.

FIG. 4 fluorescent quantitative PCR assay results of inflammatory cytokines of virus-infected PAMs cells.

FIG. 5 temperature (A) and average daily gain (B) of piglets measured after virus inoculation.

FIG. 6 shows the results of determination of viremia (A) in piglets and viral load (B) in lung tissues after inoculation of PRRSV chimeric strains.

FIG. 7 Lung histopathological observations of piglets 14 days after inoculation (200X).

FIG. 8 shows the result of detecting PRRSV specific antibodies after immunization of piglets.

FIG. 9 shows the body temperature change (A) and survival curve (B) of piglets after challenge with HP-PRRSV BB0907 strain.

FIG. 10 measurement of viral load (B) in lung and lymph node tissues and porcine viral hemotopathy (A) after challenge with HP-PRRSV BB0907 strain.

FIG. 11 macroscopic and histopathological changes of the lungs of piglets after challenge with HP-PRRSV BB0907 strain.

FIG. 12 temperature change (A) and survival curve (B) of piglets after PRRSV-like NADC30 strain FJ1402 challenge.

FIG. 13 determination of viral load in lung and lymph node tissues (B) and porcine viremia after challenge with PRRSV-like strain NADC30 strain FJ 1402.

Figure 14 macroscopic pathological changes and histopathological changes of the lungs of piglets after virus challenge of PRRSV type NADC30 strain FJ 1402.

Detailed Description

The invention adopts a highly pathogenic porcine reproductive and respiratory syndrome strain (highly pathogenic PRRSV or HP-PRRSV for short) TJM-F92 vaccine attenuated strain infectious cDNA clone pCMV-TJM, according to the characteristics of the porcine reproductive and respiratory syndrome like NADC30 strain (like NADC30 strain for short) Nsp2 gene, the segment expressing aa (111aa in total) at position 323- rTd111 strain ORF5-6 was replaced by FJ1402 strain ORF5-6), and the genes were stably inherited after 5 passages. The growth rate and virus titer of the rTd111/F2-7 and rTd111/F5-6 chimeric strains on Marc-145 cells are lower than those of the original TJM-F92 strain, the expression level of the innate immune cytokines induced by the rTd111/F5-6 infected with the PAMs cells is similar to that of the original vaccine strain TJM-F92, and the expression level of the innate immune cytokines induced by the rTd111/F2-7 is obviously higher than that of the original vaccine strain TJM-F92. The rTd111, the rTd111/F2-7, the rTd111/F5-6, the TJM-F92 and the FJ1402 are respectively inoculated to piglets with the age of 30-35 days, and the results of clinical symptoms, antibody detection, autopsy lesion and pathological observation on pigs show that the rTd111/F5-6 has similar virulence to the TJM-F92 and is obviously lower than the FJ1402, and the rTd111/F2-7 has higher virulence than the rTd 111/F5-6. In order to further research the piglet immunoprotection efficacy of the chimeric vaccine candidate strain rTd111/F5-6, healthy piglets of 30-35 days old are selected, and are challenged by intramuscular injection at 28d after immunization by utilizing HP-PRRSV strain BB0907 and NADC 30-like strain FJ1402 respectively. The results of animal experiments show that the infection level of BB0907 and FJ1402 strains in the rTd111/F5-6 experimental group is obviously reduced. The results show that the chimeric strain has good immunogenicity, can provide effective cross immune protection for infection of highly pathogenic strains and similar NADC30 strains, and lays an important foundation for development of novel vaccines for the diseases.

In the following, methods for obtaining the chimeric strains of the present invention are specifically exemplified so that those skilled in the art will understand the present invention in more detail. In the methods described below, unless otherwise indicated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Also, techniques for infectious cDNA cloning, cell culture, cell transformation, viral transfection, viral rescue, immunological experiments, recombinant plasmids, recombinant viruses, etc., as used herein are all techniques that have been reported in the art, and the steps can be performed using conventional procedures disclosed in the prior art.

Examples

1 materials and methods

1.1 viruses, cells and plasmids

The PRRSV highly pathogenic strain BB0907 and the like NADC30 strain FJ1402 are separated, identified and stored in the laboratory, the PRRSV vaccine (TJM-F92 strain) is Wuhua Heifeng from Huaweite (Thailand) bioengineering Co., Ltd, and the infectious cDNA clone plasmid pCMV-TJM of the TJM-F92 vaccine strain is constructed and stored in the laboratory. Porcine Primary Alveolar Macrophages (PAMs) were prepared according to literature methods; marc-145 cells were cryopreserved by liquid nitrogen in this laboratory and purchased from ATCC in the United states.

1.2 other reagents and test animals

RNA extraction kit

Total RNA Kit I was purchased from OMEGA; SuperScript^TMIII Reverse Transcriptase from Invitrogen; restriction enzymes AscI and SpeI were purchased from NEB; DMEM/F12 medium was purchased from Tremella; low melting agar (agar) was purchased from SIGMA; IDEXX HerdChek PRRS X3ELISA kit was purchased from IDEXX. The 30-35-day-old PRRSV, PCV2, PRV and CSFV antibodies and antigen-negative piglets are purchased from farmers in Nantong city, Jiangsu province.

1.3 construction of infectious cDNA clones of chimeric strains

1.3.1 primer design

Primers were designed based on the gene sequences of the TJM-F92 strain and the FJ1402 strain, and the sequences of the primers are shown in Table 1. The primers were synthesized by Nanjing Kinshire Biotechnology, Inc., and the purification method was RPC.

TABLE 1 infectious cDNA clone construction primers

In the above table, the sequence in italics and underlined represents the cleavage site, and the sequence in bold is the sequence of strain FJ 1402.

In the above table, each primer functions as follows:

T-A-F and Td111aa-R are primers for amplifying the front segment of the 111aa deletion site of the A fragment of the TJM strain;

td111aa-F and T-A-R are primers for amplifying the rear section of the 111aa deletion site of the A fragment of the TJM strain;

T-A-F and T-A-R are primers for carrying out fusion PCR on the front section and the rear section of the 111aa deletion site of the A fragment of the TJM strain;

T-D-F and T-1b-R are primers for amplifying the front section of ORF2 of the D fragment of the TJM strain;

FJ-2-F and FJ-7-R are primers for amplifying FJ1402 strain ORF2-ORF 7;

t-3' UTR-F, T-D-SwaI and T-D-SpeI are primers for amplifying the rear segment of ORF7 of the D fragment of the TJM strain;

T-D-F and T-4-R are primers for amplifying the front section of ORF5 of the D fragment of the TJM strain;

FJ-5-F and FJ-6-R are primers for amplifying FJ1402 strain ORF5-ORF 6;

T-7-F, T-D-SwaI and T-D-SpeI are primers for amplifying the rear segment of ORF6 of the D fragment of the TJM strain.

1.3.2 construction of infectious cDNA cloning plasmid of chimeric Strain

The specific steps for constructing the PRRSV TJM-F92 vaccine strain infectious cDNA clone pCMV-TJM laboratory are disclosed in "construction and identification of recombinant porcine reproductive and respiratory syndrome virus expressing porcine circovirus type 2 ORF2 gene" (Zhangjie et al, Prov. Virol. 2015 No. 1).

The infectious cDNA clone pTd111 (figure 1-A) is constructed by taking PRRSV TJM-F92 vaccine strain infectious cDNA clone pCMV-TJM constructed in a laboratory as a framework and deleting a gene fragment of 111aa in the 323-433 th site on the Nsp2 gene of the pCMV-TJM according to the gene characteristics of the PRRSV type NADC30 strain Nsp 2. The specific experimental steps are as follows:

the front and rear sections of the 111aa deletion site of the A fragment of the TJM strain are amplified by using the pCMV-TJM as a template and T-A-F, Td111aa-R and Td111aa-F, T-A-R respectively. Subsequently, the first and second sections were fused by SOE PCR using the amplified fragment as a template and T-A-F and T-A-R as primers to obtain 111 aa-deleted A (d 111). And carrying out double digestion on the fragment A (d111) and pCMV-TJM by PacI and XhoI respectively, recovering, purifying and connecting the digested fragment and the vector, and finally successfully replacing the 111aa deleted A (111aa) with the pCMV-TJM to obtain pTd111 with 111aa deleted Nsp 2.

The pTd111 corresponding fragment and the FJ1402 strain corresponding fragment are fused together by SOE PCR to respectively obtain a recombinant D fragment (Td111/F2-7) of the chimeric FJ1402 strain ORF2-7 and a recombinant D fragment (Td111/F5-6) of the chimeric FJ1402 strain ORF5-6, and finally the modified D fragments are respectively replaced into pTd111 by double enzyme digestion (AscI and SpeI) to obtain infectious cDNA clones pTd111/F2-7 and pTd111/F5-6 (FIG. 1-B). The specific experimental steps are as follows:

pTd111/F2-7 construction: respectively amplifying the front section of ORF2 and the rear section of ORF7 in the D fragment by taking pTd111 as a template, amplifying the sections of ORF2-ORF7 by taking cDNA of the FJ1402 strain as a template, and fusing the three sections together by fusion PCR to obtain D (Td111/F2-7) of the ORF2-7 of the chimeric FJ1402 strain; d (Td111/F2-7) and pTd111 are subjected to double enzyme digestion by AscI and SpeI respectively, and the digested fragment is connected with a vector to obtain pTd 111/F2-7.

pTd111/F5-6 construction: respectively amplifying the front section of ORF5 and the rear section of ORF6 in the D fragment by taking pTd111 as a template, amplifying the sections of ORF5-ORF6 by taking cDNA of the FJ1402 strain as a template, and fusing the three sections together by fusion PCR to obtain D (Td111/F5-6) of the ORF5-6 of the chimeric FJ1402 strain; d (Td111/F5-6) and pTd111 are subjected to double enzyme digestion by AscI and SpeI respectively, and the digested fragment is connected with a vector to obtain pTd 111/F5-6.

1.3.3 identification and sequence analysis of infectious clone plasmids of chimeric strains

By using

The Endo-free Plasmid DNA Mini Kit I extracts infectious cDNA clone plasmids pTd111, pTd111/F2-7 and pTd111/F5-6, determines the nucleotide sequence of the chimeric part, and analyzes whether the deletion and mutation exist by the BioEdit software.

1.4 rescue of chimeric strains and analysis of biological Properties

1.4.1 rescue of chimeric strains

Infectious cDNA clone plasmids pTd111, pTd111/F2-7 and pTd111/F5-6 were transfected into Marc-145 cells with cell growth density of 70-90% according to the method of lipofectamine 3000, respectively, and placed at 37 ℃ in 5% CO₂Observing the culture in the incubator. And when the cytopathic CPE in the hole reaches about 80%, harvesting the transfected cells, repeatedly freezing and thawing, centrifuging, and taking the virus supernatant to be inoculated into the monolayer Marc-145 cells cultured for 48h for continuous subculture. RNA was extracted from the virus passaged to the 5 th generation (F5), and the ORF5 gene and Nsp2 gene were amplified using the reverse-transcribed cDNA as a template and sent to Kinry Biotechnology Ltd for sequencing verification. The correctly sequenced recombinant viruses were designated rTd111, rTd111/F2-7 and rTd111/F5-6, respectively.

1.4.2 Virus plaque assay and growth Curve determination

Collecting Marc-145 cell product with 80% CPE, repeatedly freezing and thawing for 3 times, and diluting with DMEM to 10%^-2To 10^-4And (4) dilution degree. And respectively inoculating 100 mu l/well of the mixture to a 12-well plate full of a monolayer of Marc-145 cells, and performing a plaque screening test according to a conventional method. And when the plaque appears, selecting the plaque, connecting the plaque to a new full monolayer cell hole, and purifying the virus for 3-4 times to obtain the stable and purified recombinant virus.

The TJM-F92 vaccine strain and the rescued 3 chimeric strains are respectively inoculated to Marc-145 cells at the dose of MOI (maximum organization of 14) 0.1, and cell supernatants are respectively collected at 12h, 24h, 36h, 48h and 72h after infection to determine TCID (T cell identity)₅₀One-step growth curves of the viruses were plotted against the virus titer at different time points (3 parallel wells were set per time point for each virus).

1.5 Induction of Primary PAMs innate immune cytokines by chimeric strains

In order to solve the change of main cell factors generated after the porcine alveolar macrophage PAMs are infected with the chimeric strains, the infection mechanism of the chimeric strains is further clarified. Selecting common weaned piglets which are negative to PRRSV, PCV2 and PRV antigens and antibodies, separating and culturing PAMs, inoculating each virus (TJM-F92, rTd111, rTd111/F2-7 and rTd111/F5-6) with cells at a dose of MOI of 0.1 respectively (3 parallel wells are arranged at each time point for each virus), and simultaneously arranging a non-inoculated Mock control well. Cell supernatants were harvested at different time points (6h, 12h, 24h, 36h, 48h and 72h) after virus inoculation, and virus one-step growth curves were constructed by detecting mRNA levels of various cytokines and determining virus titers using Real-time PCR.

1.6 pathogenicity test of candidate strains of chimeric strain vaccine on piglets

30 PRRSV negative piglets were randomly divided into six groups (n ═ 5). Injecting rTd111, rTd111/F2-7, rTd111/F5-6, TJM-F92 and FJ1402 virus solution into the 1 st to 5 th groups respectively, wherein the injection amount is as follows: intramuscular injection of 3mL 10 per pig⁵TCID₅₀Viral fluid/mL, group 6 injected with an equal volume of PBS, and piglets were observed for temperature and clinical symptoms within 14 days. Blood was collected at 7d and 14d after immunization, and the serum was used for determination of viremia. 14d after immunization, performing killing and autopsy on all piglets, observing general pathological changes of the lung, and taking frozen lung tissues for virus load measurement; another part of lung tissue was fixed with 4% paraformaldehyde for pathological observation.

1.7 immunoprotection test of candidate strains of chimeric Strain vaccine

Dividing 30 PRRSV negative piglets into 3 groups (n is 10) at random, and isolating and feeding. One group was intramuscularly injected with 3mL of TJM-F92 (10) per pig⁵TCID₅₀/mL), another group was intramuscular injected with 3mL rTd111/F5-6 (10) per pig group⁵TCID₅₀mL), 3mL PBS was injected intramuscularly in each pig in the last group, and clinical symptoms were observed daily in pigs. The blood was collected at 7d, 14d, 21d and 28d after immunization for immunogenicity testing. At 28d after immunization, 5 heads of each group are attacked by PRRSV highly pathogenic strain BB0907, 5 heads of each group are attacked by NADC30 subtype-like strain FJ1402, and 4 mL/head. After the challenge, the state of the pig body is observed every day, the rectal temperature is measured, and viremia is measured by blood sampling at 7d, 14d and 21d after the challenge respectively. And 21d after the challenge, performing killing and autopsy on all piglets, observing general pathological changes of the lung, and taking lung tissues and tonsils for measuring viral load and histopathological observation.

2 results

2.1 biological characterization of chimeric strains

As shown in FIG. 2, the three recombinant viruses rTd111, rTd111/F2-7 and rTd111/F5-6 have similar plaque morphology and size and no significant difference from the parent strain TJM-F92. Growth curve results show that the growth characteristics of rTd111 are essentially identical compared to the parental strain, differing only at individual time points. However, the growth titer peak values of the chimeric strains rTd111/F2-7 and rTd111/F5-6 in Marc-145 cells are at 48h after infection and are lower than the virus infection titer of the parent strain TJM-F92 and the recombinant virus rTd 111.

2.2 infection of PAMs with chimeric strains

After inoculation of TJM-F92, rTd111/F2-7 and rTd111/F5-6 strains with PAMs, obvious lesions appear, which are characterized by rough cell edges and local bunching (FIG. 3-A). The results of the one-step growth curves show that the growth characteristics of the recombinant viruses rTd111, rTd111/F2-7 and rTd111/F5-6 and the parental strain TJM-F92 in PAMs cells are also substantially consistent (FIG. 3-B).

The mRNA expression levels of IL-6, TNF-alpha, IFN-beta and IFN-gamma are detected by a fluorescent quantitative Real-time PCR method at 6h, 12h and 24h after inoculation, and the results show that the level of inflammatory factors induced by the recombinant strain rTd111 is not obviously different from that of TJM-F92 strain (P is more than 0.05), the up-regulation effect of rTd111/F2-7 on the mRNA level of the inflammatory factors is most obvious (P is less than 0.05), and the level of cytokine induced by the rTd111/F5-6 is between the rTd111 and the rTd111/F2-7 (figure 4).

2.3 pathogenicity test of candidate strains of chimeric virus vaccine for piglets

2.3.1 clinical changes

3ml (10) of piglet inoculation⁵TCID₅₀Ml) rTd111, rTd111/F2-7, rTd111/F5-6, TJM-F92 and FJ1402 virus liquid, no obvious clinical clinic is found in the piglets in the test groupAbnormal symptoms, the average body temperature is maintained at about 39.5-40 ℃. Further statistics were carried out on the weight data of the immunized piglets at 0d and 14d, and the results showed that there was no significant difference in average daily gain of piglets in the chimeric virus immunized group and the PBS group (P >0.05), as shown in FIG. 5.

2.3.2 viremia and viral load assay

The PRRSV load in the serum after each virus inoculation is detected by a fluorescent quantitative PCR method, and the results are shown in figure 6-A, the PRRSV viremia levels of the rTd111/F5-6, the rTd111 and the TJM-F92 immune groups of piglets are similar (P is more than 0.05), while the rTd111/F2-7 immune group is slightly higher than the viremia levels caused by the three viruses. The results of the viral load detection in lung tissues are shown in FIG. 6-B, wherein the viral load in the lungs of piglets in each immune group of rTd111/F2-7, rTd111/F5-6, rTd111 and TJM-F92 is significantly reduced compared with that in the lungs of the FJ1402 challenge control group (P < 0.05).

2.3.3 histopathological Observation

Killing all piglets 14d after inoculation, collecting lung tissues, preparing tissue slices, observing histopathological changes, and obtaining the result: the rTd111/F5-6, rTd111 and TJM-F92 groups showed only mild lesions with complete alveolar structure and slightly thickened alveolar spaces, while the rTd111/F2-7 and FJ1402 groups showed significant interstitial pneumonia lesions with increased inflammatory cells or infiltration of erythrocytes and more pronounced thickening of alveolar walls (FIG. 7). Indicating that rTd111/F5-6 is very low pathogenic, while rTd111/F2-7 is somewhat pathogenic.

2.4 immunoprotective potency of chimeric vaccine candidate strains

2.4.1 immune antibody assay

And (3) carrying out antibody detection on the collected immune pig serum at different time points by using an IDEXX PRRS X3ELISA kit. The result shows that the chimeric strain rTd111/F5-6 has equivalent immune effect (p is more than 0.05) with the parental vaccine strain TJM-F92, and can effectively stimulate the antibody immune response of piglets. The serum PRRSV antibodies of the piglets in the PBS group were always negative (S/P < 0.4) (FIG. 8).

2.4.2 HP-PRRSV BB0907 strain challenge test result

2.4.2.1 clinical symptoms

After the HP-PRRSV BB0907 strain is attacked, the uninfected piglets have obvious hyperpyrexia symptoms (more than or equal to 40 ℃), long duration, listlessness, inappetence and death of 2 heads. After the chimeric strain rTd111/F5-6 and the parental vaccine strain TJM-F92 are immunized, the body temperature of piglets is not obviously increased and clinical symptoms do not appear, and all piglets are healthy and alive (figure 9). The evaluation results show that: the chimeric strain rTd111/F5-6 has good protection effect on the attack of the PRRSV virulent strain BB 0907.

2.4.2.2 viremia and tissue viral load

The fluorescence quantitative PCR method detects the PRRSV load after challenge, the result is shown in figure 10-A, the PRRSV virus blood level of the rTd111/F5-6 immune group and the piglet PRRSV virus blood level of the parental vaccine strain TJM-F92 immune group are similar (P is more than 0.05), and the levels are all obviously lower than those of the non-immune group (P is less than 0.05).

The detection results of the viral loads in lung tissues and lymph nodes are shown in FIG. 10-B, and the viral loads in the lungs and lymph nodes of the piglets of the rTd111/F5-6 immune group are similar to those of the parent vaccine strain TJM-F92 (P >0.05), and are both significantly lower than those of the nonimmunized control group (P <0.05), which indicates that the rTd111/F5-6 immunity can reduce the infection level of the HP-PRRSV BB0907 strain.

2.4.2.3 Observation of Lung Pathology

The lung pathological changes of the 21d killed piglets after the challenge are shown in figure 11, and compared with the non-immune challenge control group, the lungs of the piglets in the rTd111/F5-6 and TJM-F92 immune groups have no obvious visual lesions. Histological observation of the lung showed that the alveolar structures of the rTd111/F5-6 and TJM-F92 immune groups were intact and the alveolar spaces were not significantly thickened. The results show that the rTd111/F5-6 immunized piglet can effectively reduce clinical symptoms and pathological changes caused by the HP-PRRSV BB0907 strain.

2.4.3 PRRSV-like NADC30 strain FJ1402 challenge test result

2.4.3.1 clinical symptoms

After the FJ1402 strain is attacked, the body temperature of the non-immunized piglets is obviously increased (not less than 40 ℃), the piglets die for 1 head, the TJM-F92 immunized piglets are also fever (not less than 40 ℃), the death phenomenon does not exist, and the rTd111/F5-6 immunized piglets have no obvious clinical symptoms (figure 12), which shows that the rTd111/F5-6 immunization can effectively resist the attack of the similar NADC30 strain FJ 1402.

2.4.3.2 viremia and tissue viral load

The fluorescent quantitative PCR method detects the PRRSV load after challenge, the result is shown in figure 13-A, the PRRSV viremia levels of the TJM-F92 immune group and the non-immune group of piglets are similar (P is more than 0.05), and the viremia of the rTd111/F5-6 immune group is obviously lower than that of the non-immune group (P is less than 0.05).

The virus load detection results in lung tissues and lymph nodes show that the virus load of the TJM-F92 immune group piglet in the lung is not significantly different from that of the non-immune challenge control group (P is greater than 0.05), while the virus load of the rTd111/F5-6 immune group is significantly lower than that of the non-immune control group (P is less than 0.05) (figure 13-B), which indicates that the rTd111/F5-6 immune group can reduce the infection level of the FJ1402 strain, and the protection effect of the TJM-F92 vaccine strain is relatively poor.

2.4.3.3 Observation of Lung Pathology

As shown in FIG. 14, the lungs of piglets killed 21d after challenge, TJM-F92 immune group and nonimmune challenge control group showed marked enlargement, diffuse and focal consolidation, and dark red lung color. Histological examination revealed interstitial pneumonia, thickening of alveolar walls, inflammatory cell proliferation, or infiltration of erythrocytes. No obvious visual lesions of the lungs of the rTd111/F5-6 immunized piglets exist, and the histological examination shows that the piglets have mild lesions, the alveolar structures are complete, and the alveolar spaces are slightly thickened.

The results show that the rTd111/F5-6 immunized piglet can effectively reduce the attack of the PRRSV-type NADC30 strain FJ1402, while the TJM-F92 can not completely provide clinical protection for the FJ1402 strain infection.

Sequence listing

<110> Nanjing university of agriculture

<120> porcine reproductive and respiratory syndrome virus chimeric strain and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 14631

<212> DNA

<213> porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus Td111/F5-6F5 CGMCC No.14762)

<400> 1

atgacgtata ggtgttggct ctatgccacg gcatttgtat tgtcaggagc tgtgaccata 60

ggcacagccc aaaacttgct gcacgggaac accctcctgt gacagccctc ttcaggggga 120

ttaggggtct gtccctaaca ccttgcttcc ggagttgcac tgctttacgg tctctccacc 180

cctttaacca tgtctgggat acttgatcgg tgcacgtgta cccccaatgc cagggtgttt 240

gtggcggagg gccaggtcta ctgcacacga tgtctcagtg cacggtctct ccttcctctg 300

aatctccaag ttcctgagct tggggtgctg ggtctattct ataggcccga agagccactc 360

cggtggacgt tgccacgtgc attccccact gtcgagtgct cccccgccgg ggcctgctgg 420

ctttctgcga tctttccgat tgcacgaatg actagtggaa acctgaactt tcaacaaaga 480

atggtgcggg tcgcagctga aatctacaga gccggccaac tcacccctac agttctaaag 540

actctacaag tttatgaacg gggttgtcgc tggtacccca ttgtcgggcc cgtccctggg 600

gtgggcgttt acgccaactc cctgcatgtg agtgacaaac ctttcccggg agcaactcat 660

gtgttaacca acttgccgct cccgcagagg cccaaacctg aggacttttg cccttttgag 720

tgtgctatgg ctgacgtcta tgacattggt cgtggcgccg tcatgtatgt ggccggagga 780

aaggtctctt gggcccctcg tggtgggaat gaagtgaaat ttgaacctgt ccccaaggag 840

ttgaagttgg ttgcgaaccg actccacacc tccttcccgc cccatcacgt agtggacatg 900

tccaggttta ccttcatgac ccctgggagt ggtgtctcca tgcgggttga gtaccaatac 960

ggctgcctcc ccgctgacac tgtccctgaa ggaaactgct ggtggcgctt gtttgactcg 1020

ctcccaccgg aagttcagta caaagaaatt cgccacgcta accaatttgg ctatcaaacc 1080

aagcatggtg tccctggcaa gtacctacag cggaggctgc aagttaatgg tcttcggaca 1140

gtgaccgaca cacatggacc tatcgtcata cagtacttct ctgttaagga gagttggatc 1200

cgccacctga agttggtgga agaacccagc ctccccgggt ttgaggatct cctcagaatc 1260

agggttgagc ccaatacgtc accactggct ggaaaggatg agaagatttt ccggtttggc 1320

agtcataagt ggtacggtgc cggaaagaga gcaaggaaaa cacgctctgg tgcgactact 1380

atggtcgctc atcacgcttc gtccgctcat aaaatccggc aggccacgaa gcacgagggt 1440

gccggcgcta acaaggctga gcatctcaag cgctactctc cgcctgccga agggaactgt 1500

ggttggcact gcatttccgc catcgccaac cggatggtga attccaactt tgagaccacc 1560

cttcctgaaa gagtaaggcc ttcagatgac tgggccactg acgaggatct tgtgaatacc 1620

atccaaatcc tcaggctccc tgcggccttg gacaggaacg gcgcttgcgg tagcgccaag 1680

tacgtgctta aactggaggg tgagcattgg actgtctctg tgatccctgg gatgtcccct 1740

actttgctcc cccttgaatg tgttcagggt tgttgtgaac ataagggcgg tcttgtttcc 1800

ccggatgcgg tcgaaatttc cggatttgat cctgcctgcc ttgaccgact ggctaaggta 1860

atgcacttgc ctagcagtac catcccagcc gctctggccg aattgtccga cgactccaac 1920

cgtccggttt ccccggccgc tactacgtgg actgtttcgc aattctatgc tcgtcataga 1980

ggaggagatc atcatgacca ggtgtgctta gggaaaatca tcagcctttg tcaagttatt 2040

gaggactgct gctgccatca gaataaaacc aaccgggcta ctccggaaga ggtcgcggca 2100

aagattgatc agtacctccg tgacgcaaca agtcttgagg aatgcttggc caaacttgag 2160

agagtttccc cgccgagcgc agcggacacc tcctttgatt ggaatgttgt gcttcctggg 2220

gttgaggcga cgaatcagac aaccgaacaa cctcacgtca actcatgctg caccccggtc 2280

cctcccgtga ctcaagagcc tttgccaaga gttcaacctc gaagaacaaa gtctgtcaaa 2340

agtttgccag aggacaagcc tgtccctgct ccgcgcagga aggtcagatc cgattgcggc 2400

agcccggttt tgatgggcga caatgtccct aacggttcgg aagaaactgt cggtggtctc 2460

ctcaattttc cgacaccatc cgagccgatg acacctatga gtgagcccgt acttgtgccc 2520

gcgtcgcgac gtgtccccaa gctgatgaca cctttgagtg ggtcggcacc agttcctgca 2580

ccgcgtagaa ctgtgacaac aacgctgacg caccaggatg agcctctgga tttgtctgcg 2640

tcctcacaaa cggaatatga ggcttccccc ctaacaccat cgcagaacat gggcatcctg 2700

gaggcggggg ggcaagaagc tgagggagtc ctgagtgaaa tctcggatat actaaatgac 2760

accaaccctg cacctgtgtc atcaagcagc tccctgggtt cagtggccac cgaggatgtt 2820

ccacgcatcc tcgggaaaat aggagacact gacgagctgc ttgaccgagg tccctcggca 2880

ccctccaagg gagaaccggt ctgtgaccaa cctgccaaag atccccggat gtcgccgcgg 2940

gagtctgacg agagcataat agttccgccc gcagatacag gtggtgtcgg ctcattcact 3000

ggtttgccgt cttcagatgg tgtggatgtg gacggggggg ggccgttaag aacggtaaaa 3060

acaaaagcag aaaggctctt agatcaactg agctgccagg tttttagcct cgtttcccat 3120

ctccctattt tcttctcaca cctcttcaaa tctgacagtg gttattctcc gggtgattgg 3180

ggttttgcag cttttactct attttgcctc tttttatgtt acagttaccc attcttcggt 3240

tttgctcccc tcttgggtgt attttctggg tcttctcggc gtgtgcgaat gggggttttt 3300

ggctgctggt tggcttttgc tgttggtctg ttcaagcctg tgtccgaccc agtcggcact 3360

gcttgtgagt ttgactcgcc agagtgtagg aacgtccttc attcttttga gcttctcaaa 3420

ccttgggacc ctgtccgcag ccttgttgtg ggccccgtcg gtctcggcct tgccattctt 3480

ggcaggttac tgggcggggc acgctacatc tggcactttt tgcttaggct tggcattgtt 3540

gcagactgtg tcttggctgg agcttatgtg ctttctcaag gtaggtgtaa aaagtgctgg 3600

ggatcttgtg taagaactgc tcctaatgag atcgccttca acgtgttccc ttttacacgt 3660

gcgaccaggt cgtcactcat cgacctgtgc gatcggtttt gcgcaccaaa aggcatggac 3720

cccatttttc tcgccactgg gtggcgtggg tgctggaccg gccggagtcc cattgagcaa 3780

ccttctgaaa aacccatcgc gttcgcccag ctggatgaga agaggattac ggctagaact 3840

gtggtcgctc agccttatga tcccaaccag gccgtaaagt gcttgcgggt attacaggcg 3900

ggtggggcga tggtggccga ggcagtccca aaagtggtca aagtttccgc tattccattc 3960

cgagctcctt tctttcccgc tggagtgaaa gttgatcctg agtgcagaat cgtggttgat 4020

cccgatactt ttactacagc cctccggtct ggctattcca ccgcgaacct cgtccttggt 4080

acgggggact ttgcccagct gaatggacta aagatcaggc aaatttccaa gccttcaggg 4140

ggaggcccac acctcattgc tgccttgcat gttgcctgct cgatggcgtt acacatgctt 4200

gctggtgttt atgtaactgc agtggggtcc tgcggtgccg gtaccaacga tccgtggtgc 4260

actaacccgt ttgctgtccc tggctatgga cctggctctc tttgcacgtc tagattgtgc 4320

atctcccaac acggcctcac cttgcccttg acagcacttg tggcgggatt cggccttcaa 4380

gagattgcct tggtcgtttt gatctttgtc tccatcggag gcatggctca taggttgagt 4440

tgtaaggctg acatgttgtg catcttactc gcaatcgcta gttatgtttg ggtacctctt 4500

acctggttgc tttgtgtgtt tccttgttgg ttgcgctggt tctctttgca ccccctcacc 4560

atcctgtggt tggtgttttt cttgatttct gtaaatatac cctcgggaat cttggccgtg 4620

gtgttattgg tttctctctg gcttttaggt cgttatacta acattgctgg tctcgtcacc 4680

ccttatgaca ttcatcatta caccagtggc ccccgcggtg tcgccgcctt ggccaccgcg 4740

ccagatggaa cctacttggc tgccgtccgc cgtgctgcgc tgactggtcg taccatgctg 4800

ttcaccccgt ctcagcttgg gtccctcctt gagggcgctt tcagaactca aaaaccctca 4860

ctgaacaccg tcaatgtggt cgggtcctcc atgggctctg gcggagtgtt cactattgac 4920

gggaaaatca agtgcgtgac tgccgcacat gtccttacgg gtaactcagc tagggtttcc 4980

ggggtcggct tcaatcaaat gcttgacttt gatgtaaaag gggacttcgc catagctgat 5040

tgcccgaatt ggcaaggggt tgctcccaag gcccagttct gcgaggatgg gtggactggt 5100

cgcgcctatt ggctgacatc ctctggcgtt gaacccggtg ttattgggaa tgggttcgcc 5160

ttctgcttca ccgcgtgtgg cgattctgga tccccagtga ttaccgaagc cggtgagctt 5220

gtcggcgttc acacaggatc aaacaaacaa ggaggaggca ttgtcacgcg cccctcaggc 5280

cagttttgta atgtgaagcc catcaagctg agcgagttga gtgaattctt cgctggacct 5340

aaggtcccgc tcggtgatgt gaaaattggc agtcacataa ttaatgacac atgcgaggtg 5400

ccttcagatc tttgtgccct gcttgctgcc aaacccgaac tggaaggagg cctttccaca 5460

gttcaacttc tgtgtgtgtt tttcctcctg tggagaatga tggggcatgc ctggacgccc 5520

ttggttgctg tggggttttt catcctgaat gagattctcc cagctgttct ggtccggagt 5580

gttttctcct ttgggatgtt tgtgctatct tggctcacac catggtctgc gcaagtcctg 5640

atgatcaggc ttctgacagc agcccttaac agaaacagat ggtctcttgg tttttacagc 5700

cttggtgcaa taaccagttt tgtcgcagat cttgcggtaa ctcaagggca tccgttacag 5760

gtggtaatga acttaagcac ctatgccttc ctgccccgga tgatggttgt gacctcgcca 5820

gtcccagtga tcgcgtgtgg tgttgtgcac ctccttgcca taattttgta cttgtttaag 5880

taccgctgcc ttcacaatgt ccttgttggc gatggggtgt tctcttcggc tttcttcttg 5940

cgatactttg ccgagggaaa gttgagggaa ggggtgtcgc aatcctgtgg gatgagtcat 6000

gagtcgctga ctggtgccct cgccatgaga ctcactgacg aggacttgga tttccttacg 6060

aaatggactg attttaagtg ctttgtttct gcgtccaaca tgaggaatgc agcgggccaa 6120

tttatcgagg ctgcttatgc aaaagcacta agaattgaac ttgctcagtt ggtgcaggtt 6180

gataaggtcc gaggcaccat ggccaaactc gaggctttcg ccgataccgt ggcaccccaa 6240

ctctcgcccg gtgacattgt tgttgccctt ggccacacgc ctgttggcag catcttcgac 6300

ctaaaggttg gtagcaccaa gcatactctc caagctattg agactagagt ccttgccggg 6360

tccaaaatga ctgtggcgcg tgtcgttgac ccaacccccg cacccccgcc cgtacctgtg 6420

cccatccctc tcccaccgaa agttctggag aacggtccca atgcctgggg ggatgaggac 6480

cgtttgaaca agaagaagag gcgcaggatg gaagccgtcg gcatttttgt catggacggg 6540

aaaaagtacc agaaattttg ggacaagaat tccggtgatg tgttttatga ggaggtccat 6600

attagcacag acgagtggga gtgccttaga actggcgacc ctgtcgactt tgatcctgag 6660

acagggattc agtgtgggca tatcaccatt gaagataagg tttacaatgt cttcacctcc 6720

ccatctggta ggagattctt ggtccccgcc aaccccgaga atagaagagc tcagtgggaa 6780

gccgccaagc tttccgtgga gcaagccctt ggtatgatga acgtcgacgg cgaactgact 6840

gccaaagaac tggagaaact gaaaagaata attgacaaac tccagggcct gactaaggag 6900

cagtgtttaa actgctagcc gccagcggcc tgacccgctg tggtcgcggc ggcttagttg 6960

ttactgagac agcggtaaaa atagtcaaat ttcacaaccg gaccttcacc ctaggacctg 7020

tgaacttaaa agtggccagt gaggttgagc taaaagacgc ggttgagcac aaccaacatc 7080

cggttgccag accggttgat ggtggtgtcg tgctcctgcg ctctgcagtt ccttcgctta 7140

tagatgtctt gatctccggc gctgatacat ctcctaagtt actcgcccgc cacgggccgg 7200

gaaacactgg gattgatggc acgctttggg attttgaggc cgaggctact aaagaggaag 7260

ttgcactcag tgcgcaaata atacaggctt gtgatattag gcgcggcgac gcacctgaaa 7320

ttggtctccc ttataagttg taccctgtta ggggcaaccc tgagcgggta aaaggagttt 7380

tacagaacac aaggtttgga gacatacctt acaaaacccc cagtgacact ggaagcccgg 7440

tgcacgcggc tgcctgcctc acgcctaatg ctactccggt gactgatggg cgctccgtct 7500

tggctacaac catgccctct ggctttgagt tgtatgtgcc gaccattcca gcgtccgtcc 7560

ttgattatct tgattctagg cctgactgcc ctaaacagtt aacagagcac ggttgtgagg 7620

atgctgcatt aagagacctc tccaagtatg atttgtccac ccaaggcttt gttttgcctg 7680

gagttcttcg ccttgtgcgg aagtacctgt tcgcccacgt gggtaagtgc ccgcccgttc 7740

atcggccttc cacttaccct gctaagaatt ctatggctgg aataaatggg aacaggtttc 7800

caaccaagga cattcagagc gtccctgaaa tcgacgttct gtgcgcacag gctgtgcgag 7860

aaaactggca aactgttacc ccttgtaccc tcaagaaaca gtactgtggg aagaagaaga 7920

ctaggacaat acttggcacc aataacttca ttgcgttggc ccatcgggca gcgttgagtg 7980

gtgttaccca gggcttcatg aaaaaagcgt tcaactcgcc catcgccctc gggaaaaaca 8040

aatttaagga gctacaagcc ccggtcctag gcaggtgcct tgaagctgat cttgcgtcct 8100

gcgatcgatc cacacctgca attgtccgct ggtttgccgc caatcttctt tatgaactcg 8160

cctgtgctga ggagcatcta ccgtcgtacg tgctgaactg ctgccacgac ttactggtca 8220

cgcagtccgg cgcggtgact aagagaggtg gcctgtcgtc tggcgacccg attacctctg 8280

tgtcaaacac catttacagc ttagtgatat atgcacagca catggtgctc agttacttca 8340

aaagtgctca ccctcatggc cttctgtttc tgcaagacca gctgaagttt gaggacatgc 8400

tcaaggttca acccctgacc gtctattcgg acgaccttgt gctgtatgcc gagtctccct 8460

ccatgccaaa ctaccactgg tgggttgaac atctgaacct tatgctgggt ttccagacgg 8520

acccaaagaa gacaaccatc acagactcac catcattcct aggttgcagg ataataaatg 8580

ggcgccagct ggtccctaac cgtgacagga tcctcgcggc cctcgcctac cacatgaagg 8640

cgagcaatgt ttctgaatac tacgcctcgg cggctgcaat actcatggac agctgtgctt 8700

gtttggagta tgatcctgaa tggtttgaag agctcgtggt tgggatagcg cagtgcgccc 8760

gcaaggacgg ctacagcttt cctggcccac cgttcttctt gtccatgtgg gaaaaactca 8820

ggtccaatca tgaggggaag aagtccagaa tgtgcgggta ctgcggggcc ccggctccgt 8880

acgccactgc ctgtggtctc gatgtctgtg tttaccacac ccacttccac cagcattgtc 8940

ctgttataat ctggtgtggc cacccggcgg gttctggttc ttgtagtgag tgcgaacccc 9000

ccctaggaaa aggcacaagc cctctagatg aggtgttaga acaagttccg tacaagcctc 9060

cgcggactgt gatcatgcat gtggagcagg gtctcacccc tcttgaccca ggtagatacc 9120

agactcgccg cggattggtc tccgttaggc gtggcatcag gggaaatgaa gtcgacctac 9180

cagacggtga ttacgccagt accgccttgc tccctacttg taaagagatc aacatggtcg 9240

ctgtcgcctc taacgtgttg cgcagcaggt ttatcatcgg cccacccggt gctgggaaaa 9300

cacactggct tcttcaacaa gtccaggatg gtgatgtcat ttacacgcca actcaccaga 9360

ccatgctcga catgattagg gctttgggga cgtgccggtt caacgttcca gcaggtacaa 9420

cgctgcaatt ccctgccccc tcccgtaccg gcccatgggt tcgcatcttg gccggcggtt 9480

ggtgtcctgg caagaactcc ttcctggatg aagcggcgta ttgcaatcac cttgacgtct 9540

tgaggcttct cagtaaaaca actctcactt gcctagggga cttcaaacaa ctccaccctg 9600

tgggttttga ctcccattgc tatgtatttg acatcatgcc tcagacccaa ttaaagacca 9660

tctggaggtt cgggcagaat atctgtgatg ccattcaacc agattacagg gacaaactta 9720

tggccatggt caacacgacc cgtgtgacct acgtggaaaa acctgtcagg tacgggcaag 9780

tcctcacccc ctaccacagg gaccgagagg acggcgccat tactatcgac tccagtcaag 9840

gcgccacatt tgatgtggtt acactgcatt tgcccactaa agattcactc aacaggcaaa 9900

gagctcttgt tgctatcacc agggcaaggc atgctatctt cgtgtatgac ccacacaggc 9960

aattgcagag catgtttgat cttcccgcga gaggcacacc cgtcaacctc gcagtgcacc 10020

gtgacgaaca gctgatcgta ttagacagaa acaacagaga aatcacggtt gctcaggctc 10080

taggcaatgg agataaattc agggccacag ataagcgcgt tgtagattct ctccgcgcta 10140

tttgcgcaga cctggaaggg tcgagctccc cgctccccaa ggtcgcgcat aacttgggat 10200

tccatttctc acctgatttg actcagtttg ctaaactccc ggcagaactt gcaccccact 10260

ggcccgtggt gacaacccag aacaatgaaa ggtggccaga tcggctggta gccagcctcc 10320

gccctatcca taaatatagc cgcgcgtgca ttggtgccgg ctatatggtg ggcccctcgg 10380

tgtttttagg cacccctggg gttgtgtcat actatctcac aaaatttgtt agaggcgagg 10440

ctcaagtgct tccggagaca gtcttcagca ccggccgaat tgaggtagat tgtcgagagt 10500

atcttgatga tcgggagcga gaagttgctg agtccctccc acatgccttc atcggcgatg 10560

tcaaaggtac caccgttggg ggatgtcatc acgttacctc caaatacctt ccgcgcttcc 10620

ttcccaagga atcagttgcg gtggtcgggg tttcgagccc cgggaaagcc gcgaaagcag 10680

tttgcacatt gacggatgtg tacctcccag accttgaagc gtacctccac ccagagaccc 10740

agtccaggtg ctggaaagtg atgttggact ttaaggaggt tcgactgatg gtatggaaag 10800

acaagacggc ctattttcaa cttgaaggcc gccattttac ctggtatcaa cttgcaagct 10860

acgcctcata catccgagtt cctgttaatt ctactgtgta cttggacccc tgcatgggcc 10920

ctgctctttg caacaggagg gttgtcgggt ccacccattg gggagctgac ctcgcagtca 10980

ccccttatga ttacggtgcc aaaattattc tgtctagtgc ataccatggt gaaatgcctc 11040

caggttacaa aattctggcg tgcgcggagt tctcgcttga tgatccagta aggtacaaac 11100

acacctgggg atttgaatcg gatacagcgt atctgtacga gtttactgga aatggtgagg 11160

actgggagga ttacaatgat gcgtttcggg cgcgccagaa agggaaaatt tataaagcta 11220

atgccaccag catgaggttt cattttcccc cgggccctgt cattgaacca actttaggcc 11280

tgaattgaaa tgaaatgggg tctatgcaaa gcctctttga caaaattggc caactttttg 11340

tggatgcttt cacggaattt ctggtgtcca ttgttgatat catcatattt ttggccattt 11400

tgtttggctt cacaatcgcc ggttggctgg tggtcttatg catcagactg gtttgctccg 11460

cggtactccg tgcgcgctct accgttcacc ctgagcaatt acagaagatc ttatgaggcc 11520

tttctttctc agtgtcaggt agacattccc acctggggcg tcaaacaccc tttgggggtg 11580

ctttggcacc ataaggtgtc aaccctgatt gatgaaatgg tgtcgcgtcg aatgtaccgc 11640

gtcatggaaa aagcagggca ggctgcctgg aaacaggtgg tgagcgaggc tacattgtct 11700

cgcattagtg gtttggatgt ggtggctcat tttcaacatc ttgccgctat tgaagccgag 11760

acttgtaaat atttggcttc ccggctaccc atgctgcaca acctgcgctt gacagggtca 11820

aatgtaacca tagtgtataa tagtactttg gatcaggtgt ttgccatttt cccaacccct 11880

ggttcccggc caaagcttca tgattttcag caatggctaa tagctgtaca ttcctccata 11940

ttttcctccg ttgcagcttc ttgtactctt tttgttgtgc tgtggttgcg aattccaatg 12000

ctacgttctg tttttggttt ccgctggtta ggggcaactt ttcttttgaa ctcatggtga 12060

attacacggt atgcccgctt tgcccaaccc ggcaggcagc cgccgagatc cttgaacccg 12120

gcaagtcttt ttggtgcagg atagggcatg accgatgtag tgagaacgat catgacgaac 12180

tagggttcat ggttccgcct ggcctctcca gcgaaggcca cttgaccagt gtttacgcct 12240

ggttggcgtt cctgtccttc agctacacgg cccagttcca tcccgagata tttgggatag 12300

ggaatgtgag tcaagtttat gttgacatca agcaccaact catctgcgct gttcatgacg 12360

gggataacgc caccttgcct cgccatgaca atatttcagc cgtatttcag acctactacc 12420

aacaccaggt cgacggcggc aattggtttc acctggaatg gctacgccct ttcttttcct 12480

cttggttggt tttaaatgtt tcgtggtttc tcaggcgttc gcctgcaagc catgtttcag 12540

ttcgagtctt tcggacatca aaaccaacac caccgcagca tcaggcttcg ttgtcctcca 12600

ggacatcagc tgccttaggc atggcgactc gtcctctccg acgattcgca aaagttctca 12660

gtgccgcacg gcgataggga cgcccgtgta catcaccatc actgccaatg tcacagatga 12720

aaattatcta cattcttctg atctcctcat gctttcttct tgccttttct atgcttccga 12780

gatgagtgaa aagggattca aagtggtgtt tggcaatgtg tcaggcatcg tggctgtgtg 12840

cgtcaacttt accagctacg tccaacacgt caaggagttt acccaacgct ccttagtggt 12900

cgatcatgtg cgactgcttc atttcatgac acctgagacc atgaggtggg caaccgtttt 12960

agcctgtctt tttgccatcc tactggcaat ttgaatgttc aagtatgttg gggaaatgct 13020

tgaccgcggg ttactactcg caattgcctt ttttgtggta tatcgtgcca ttctgttttg 13080

ctgcgctcgt caacgccagc agcaacagca gctcccattt acagttgatt tataacctga 13140

cgatatgtga gctgaatggc acagattggc tgagcacaaa atttgactgg gcagtggaga 13200

ctttcgttat ctttcctgtg ttgactcata ttgtctctta cggcgccctt accactagcc 13260

attttcttga cacggtcggc ctgatcactg tgtccaccgc cggttattat cacaagcggt 13320

atgtattgag tagcatctac gctgtctgtg ccctggctgc gttggtttgc ttcgccatta 13380

ggttggcgaa aaattgcatg tcctggcgct actcatgcac cagatatacc aattttcttc 13440

tggatactaa gggcaaactc taccgctggc ggtcacccgt catcattgag aaggggggta 13500

aagttgatgt tgaaggtcat ttaatcgacc tcaagacagt tgtgcttgat ggttccgcgg 13560

caacccctgt aaccaagatt tcagcggaac aatggggtcg tccatagacg acttctgcaa 13620

tgacagcacg gctgtacaaa aggtgctctt ggcgttttct atcacctaca cgccaataat 13680

gatatatgcc ttaaaagtaa gtcgtggtcg actgctgggg cttttacacc tcttgatttt 13740

cctgaactgt gctttcactt ttgggtatat gacatttgtc cattttcaga gtacaaacaa 13800

ggtcgcactt accctggggg cagttgtcgc tctcctctgg ggggtgtatt cagccctgga 13860

aacctggaga ttcatcacct ccaggtgccg gttgtgcttg ctaggccgca agtacattct 13920

ggcccctgcc caccacgtag aaagtgccgc aggctttcat ccgataacgg caagtgataa 13980

ccacgcattt gtcgtccggc gtcccggctc cactacggtc aacggcacac tggtgcccgg 14040

gttgaaaagc ctcgtgttgg gtggcagaag agctgtcaaa cgaggagtgg tgaaccttgt 14100

taaatatgcc aaataacaac ggcaagcagc aaaagaaaaa gaaggggaat ggccagccag 14160

tcaatcagct gtgccaaatg ctgggtaaga tcatcgccca acaaaaccag tccagaggca 14220

agggaccggg gaagaaaaat aggaagaaaa acccggggaa gccccatttc cctctagcga 14280

ctgaagatga cgtcaggcat cactttaccc ctagtgagcg gcaattgtgt ctgtcgtcga 14340

tccagactgc cttcaatcag ggtgctggaa cttgtgccct gtcagattca gggaggataa 14400

gttacactgt ggagtttagt ttgccgacgc aacatactgt gcgtctgatc cgcgccacag 14460

catcaccctc agcatgatgg gctggcattc tttggcacct cagtgttaga attgggagaa 14520

tgtgtggtga atggcactga ttgacactgt gcctctaagt cacctattca attagggcga 14580

ccgtgtgggg gtaaagttta attggcgaga accatgcggc cgtaattaaa a 14631

<210> 2

<211> 603

<212> DNA

<213> porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus Td111/F5-6F5 CGMCC No.14762)

<400> 2

atgttgggga aatgcttgac cgcgggttac tactcgcaat tgcctttttt gtggtatatc 60

gtgccattct gttttgctgc gctcgtcaac gccagcagca acagcagctc ccatttacag 120

ttgatttata acctgacgat atgtgagctg aatggcacag attggctgag cacaaaattt 180

gactgggcag tggagacttt cgttatcttt cctgtgttga ctcatattgt ctcttacggc 240

gcccttacca ctagccattt tcttgacacg gtcggcctga tcactgtgtc caccgccggt 300

tattatcaca agcggtatgt attgagtagc atctacgctg tctgtgccct ggctgcgttg 360

gtttgcttcg ccattaggtt ggcgaaaaat tgcatgtcct ggcgctactc atgcaccaga 420

tataccaatt ttcttctgga tactaagggc aaactctacc gctggcggtc acccgtcatc 480

attgagaagg ggggtaaagt tgatgttgaa ggtcatttaa tcgacctcaa gacagttgtg 540

cttgatggtt ccgcggcaac ccctgtaacc aagatttcag cggaacaatg gggtcgtcca 600

tag 603

<210> 3

<211> 525

<212> DNA

<213> porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus Td111/F5-6F5 CGMCC No.14762)

<400> 3

atggggtcgt ccatagacga cttctgcaat gacagcacgg ctgtacaaaa ggtgctcttg 60

gcgttttcta tcacctacac gccaataatg atatatgcct taaaagtaag tcgtggtcga 120

ctgctggggc ttttacacct cttgattttc ctgaactgtg ctttcacttt tgggtatatg 180

acatttgtcc attttcagag tacaaacaag gtcgcactta ccctgggggc agttgtcgct 240

ctcctctggg gggtgtattc agccctggaa acctggagat tcatcacctc caggtgccgg 300

ttgtgcttgc taggccgcaa gtacattctg gcccctgccc accacgtaga aagtgccgca 360

ggctttcatc cgataacggc aagtgataac cacgcatttg tcgtccggcg tcccggctcc 420

actacggtca acggcacact ggtgcccggg ttgaaaagcc tcgtgttggg tggcagaaga 480

gctgtcaaac gaggagtggt gaaccttgtt aaatatgcca aataa 525

Claims (7)

1. A porcine reproductive and respiratory syndrome virus chimeric strain is characterized in that: the name is porcine reproductive and respiratory syndrome virus rTd111/F5-6F5, and the preservation number is CGMCC No. 14762.

2. The chimeric strain of porcine reproductive and respiratory syndrome virus of claim 1, which is characterized by: the nucleotide sequence is shown in SEQ ID NO. 1.

3. Use of the chimeric strain of porcine reproductive and respiratory syndrome virus of claim 1 or 2 for the preparation of a vaccine candidate for an epidemic strain of porcine reproductive and respiratory syndrome.

4. Use of the chimeric strain of porcine reproductive and respiratory syndrome virus of claim 1 or 2 in the preparation of a porcine reproductive and respiratory syndrome vaccine.

5. A vaccine, characterized by: the effective component comprises an immunizing dose of the porcine reproductive and respiratory syndrome virus chimeric strain as claimed in claim 1 or 2.

6. Use of the vaccine of claim 5 in the manufacture of a medicament for the prevention of porcine reproductive and respiratory syndrome.

7. Use according to claim 6, characterized in that: the porcine reproductive and respiratory syndrome is caused by infection of a highly pathogenic porcine reproductive and respiratory syndrome strain, or infection of a porcine reproductive and respiratory syndrome type NADC30 strain, or cross infection of the two strains.

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