Double fluorescent labeled recombinant pseudorabies virus strain and its construction method and application

Abstract

The present invention discloses a recombinant pseudorabies virus strain with dual fluorescence labeling, a construction method and an application thereof. The present invention utilizes the PRV TJ strain Fosmid library to insert the complete sequence of the ANCHOR3 system into the non-coding region between the US9 and US2 genes of the viral genome, and simultaneously inserts the mCherry gene sequence before the stop codon of the UL10 gene, thereby obtaining a recombinant pseudorabies virus strain in which the genome and the envelope protein are labeled with green fluorescence and red fluorescence, respectively. According to in vivo and in vitro evaluations, the insertion of exogenous genes does not affect the growth characteristics, viral morphology, genetic stability and safety of the recombinant virus. The recombinant virus was used to successfully trace the complete PRV replication cycle and evaluate the effect of antiviral drugs in inhibiting PRV replication, and a PRV in vitro temperature-controlled visualized latent infection model was established, which can be used for PRV latent infection models and antiviral drug screening, neural circuit tracing, and studies on the functions of viral and host proteins.

Description

Technical Field

[0001] The invention relates to a fluorescently labeled recombinant pseudorabies virus strain, in particular to a recombinant pseudorabies virus strain with double fluorescent labels for genome and envelope protein and a construction method and application thereof, belonging to the field of fluorescently labeled recombinant pseudorabies virus strains and applications thereof. Background Art

[0002] Pseudorabies virus (PRV) is a neurotropic alpha herpes virus that can infect a variety of animals such as pigs, cattle, and sheep. PRV infects adult pigs and can form latent infection in their trigeminal ganglia, while infecting other host animals and young pigs, lytic infection is the main cause, leading to systemic neurological symptoms in the host and causing pseudorabies. The PRV genome is a double-stranded linear DNA, about 143kb long, and the genome consists of 70 open reading frames. 35 viral structural proteins have been identified, including capsid proteins, matrix proteins, and envelope proteins (Pomeranz LE, Reynolds AE, Hengartner CJ. 2005. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 69: 462-500.).

[0003] The life cycle of PRV includes invasion of host cells, genome replication, nucleocapsid assembly, and release of progeny virus particles. Infection of neurons also includes conduction along axons, conduction along axons, and conduction across synapses (Ekstrand MI, Enquist LW, Pomeranz LE. 2008. The alpha-herpesviruses: molecular pathfinders in nervous system circuits. Trends Mol Med 14(3): 134-140.). Fluorescently labeled recombinant PRV plays an important role in the study of virus tracing and neural circuits. According to different research purposes, a variety of methods for labeling PRV structural proteins and viral genomes have been reported. Among them, the tracing of virus invasion and release and along axonal conduction stages often uses fluorescent protein genes fused with gM, gE and US9 proteins (Masse MJ, A,Dijkstra JM,Mettenleiter TC,Flamand A.1999.Glycoproteins gM and gN ofpseudorabies viruses are dispensable for viral penetration and propagation in the nervous systems of adult mice.J Virol 73(12):10503-10517;Kratchmarov R,Kramer T,Greco TM,Taylor MP,Ch'ng TH,Cristea IM,Enquist LW.2013.GlycoproteinsgE and gI are required for efficient KIF1A-dependent anterograde axonaltransport of alphaherpesvirus particles in neurons.J Virol87(17):9431-9440;Kratchmarov R,Enquist LW,Taylor MP.2015.Us9-independent axonal sorting and transport of the pseudorabies virus glycoprotein gM.J Virol 89(12):6511-6524.). The tracing of viral nucleocapsid assembly, release and axonal movement is mainly based on fusion markers of capsid protein VP26 and matrix protein UL36 (Hogue IB, Bosse JB, Engel EA, Scherer J, Hu JR, Del Rio T, Enquist LW. 2015. Fluorescent protein approaches in alpha herpesvirus research. viruses 7(11):5933-5961), while the tracing technology for viral genome proliferation and movement is relatively late.In recent years, the Cas9 / gRNA complex combined with quantum dot technology has been applied to the genome marking of PRV, which can observe the invasion process of PRV, but cannot observe the maturation and release of the virus (Yang YB, Tang YD, Hu Y, Yu F, Xiong JY, SunMX, Lyu C, Peng JM, Tian ZJ, Cai XH, An TQ. 2020. Single virus tracking with quantumdots packaged into enveloped viruses using CRISPR. Nano Lett 20(2): 1417-1427).

[0004] The ANCHOR system is a new type of nucleic acid labeling technology. The system consists of a fluorescently labeled fusion protein (OR-FP) and a DNA target sequence (ANCH). The OR-FP protein can specifically bind to a short non-repetitive DNA target sequence (ANCH), aggregate and diffuse to adjacent sequences, and finally accumulate on the labeled viral genome to form specific fluorescent aggregation points. At present, the ANCHOR system has been successfully used to label the genome of human cytomegalovirus (HCMV). Cells infected with the recombinant virus ANCHOR-HCMV can observe the complete viral cycle until the cells are broken and die (Mariame B, Kappler-Gratias S, Kappler M, Balor S, Gallardo F, Bystricky K. 2018. Real-time visualization and quantification of human cytomegalovirus replication in living cells using the ANCHOR DNA labeling technology. J Virol 92 (18): e00571-18.).

[0005] PRV is a typical representative of neurotropic alphaherpesviruses. It has the characteristics of latent infection and is an ideal model virus for studying alphaherpesvirus invasion, latent infection and nerve conduction. There are few studies on viral genome proliferation, viral particle assembly and release. In addition, the lack of a suitable latent infection model limits the study of latent infection and reactivation mechanisms. Fluorescently labeled recombinant PRV plays an important role in virus tracing, viral gene function research, neural circuit research, cross-blood-brain barrier drug targeted delivery, antiviral drug screening and vaccine evaluation. Summary of the invention

[0006] One of the purposes of the present invention is to provide a recombinant pseudorabies virus strain with a fluorescently labeled genome;

[0007] The second object of the present invention is to provide a recombinant pseudorabies virus strain whose genome and envelope protein are respectively labeled with green fluorescence and red fluorescence;

[0008] The third purpose of the present invention is to apply the fluorescently labeled recombinant pseudorabies virus strain to pseudorabies virus tracing, pseudorabies virus gene function research, neural circuit research, cross-blood-brain barrier drug targeted delivery, anti-pseudorabies virus drug screening and vaccine evaluation.

[0009] The above object of the present invention is achieved through the following technical solutions:

[0010] One aspect of the present invention is to provide a recombinant pseudorabies virus strain with a fluorescent genome marker, the construction method of which includes using the pseudorabies virus (PRV) TJ strain as a skeleton, inserting the complete gene sequence of the ANCHOR3 system into the non-coding region between the US9 gene and the US2 gene of its genome to rescue the recombinant pseudorabies virus strain. Wherein, the nucleotide sequence of the complete gene of the ANCHOR3 system is shown in SEQ ID No.1; the recombinant pseudorabies virus strain is transcribed to produce an OR protein (OR-EGFP) fused with a green fluorescent protein EGFP, and OR-EGFP can specifically recognize and bind to the ANCH3 DNA sequence in the PRV genome, thereby realizing the green fluorescent marking of the viral genome, breaking through the limitation that only PRV structural proteins can be fluorescently marked in the past.

[0011] The Anchor3 system inserted between the US9 gene and the US2 gene of the present invention includes a 3979bp gene sequence, and the growth kinetic curves of the labeled recombinant strain and the PRV TJ strain are not significantly different. After 20 generations of virus passage, the PCR identification and sequencing of the exogenous gene were correct, and the results showed that the non-coding region between the US9 gene and the US2 gene can be compatible with longer exogenous genes mentioned in previous studies.

[0012] The second aspect of the present invention is to provide a recombinant pseudorabies virus strain whose genome and envelope protein are respectively marked with green fluorescence and red fluorescence, and the construction method thereof comprises: inserting the complete sequence of the ANCHOR3 system into the non-coding region between the US9 and US2 genes of the genome of the PRV TJ strain, and inserting the red fluorescent protein mCherry gene sequence before the stop codon of the UL10 gene, wherein the nucleotide sequence of the red fluorescent protein mCherry gene is shown in SEQ ID No. 2, and obtaining a recombinant pseudorabies virus strain whose genome and envelope protein are respectively marked with green fluorescence and red fluorescence, and naming it rPRV TJ-Anchor3-mCherry.

[0013] As a preferred specific embodiment of the present invention, the present invention utilizes the PRV TJ strain Fosmid library, inserts the complete sequence of the ANCHOR3 system into the non-coding region between the US9 and US2 genes of the pseudorabies virus genome, and simultaneously inserts the red fluorescent protein mCherry gene sequence before the stop codon of the UL10 gene, thereby rescuing a recombinant pseudorabies virus strain whose genome and envelope protein are respectively marked with green fluorescence and red fluorescence.

[0014] The present invention will rescue the recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry whose genome and envelope protein are respectively marked with green fluorescence and red fluorescence, and submit it to a patent-recognized institution for preservation. Its microbial preservation number is CGMCC No.45203, and its classification name is: recombinant pseudorabies virus strain with dual fluorescence marking of genome and envelope protein. The preservation time is: May 18, 2022. The preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] The present invention does not affect the replication of pseudorabies virus by fusing mCherry to the C-terminus of gM protein, indicating that mCherry protein does not affect the localization of gM and gN dimers in virus particles and the normal function of their glycosylation sites during virus infection; the viral load and LD of various tissues of mice infected with rPRV TJ-Anchor3-mCherry and PRV TJ strain 50 There was no significant difference, indicating that the fusion of mCherry and gM protein did not change the tissue tropism of the virus, and gM protein was confirmed not to be an essential protein for PRV infection of the mouse nervous system. The results of this part showed that the insertion of mCherry and Anchor3 genes would not change the pathogenicity of PRV to mice.

[0016] The recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry constructed by the present invention, whose genome and envelope protein are respectively labeled with green fluorescence and red fluorescence, has the same growth characteristics as the parent virus and good genetic stability, indicating that the insertion sites of US9 and UL10 (gM) are compatible with foreign genes such as Anchor3 and mCherry, and the insertion of foreign genes does not affect viral replication. In addition, the LD of mice infected with rPRV TJ-Anchor3-mCherry and the parent virus is 50 Both are 10 2.5 TCID 50 There was no significant difference in clinical symptoms and viral loads in various tissues, indicating that the fluorescent marker does not affect the pathogenicity of the virus to the host.

[0017] The red fluorescent protein mCherry and the green fluorescent protein EGFP are two luminescent groups of rPRV TJ-Anchor3-mCherry virus particles. Fluorescence can be observed in living cells, dead cells and infected tissues, and this fluorescence can be well compatible with cell nucleus dyes (Hoechst), cell membrane red fluorescent probes (DiI), etc., proving that rPRV TJ-Anchor3-mCherry has good compatibility. Conventional recombinant PRV expressing fluorescent protein independent reading frames requires infected cells to observe the fluorescence diffused throughout the cell (non-specific); viruses fused with structural proteins and fluorescent proteins are independent of cells, but the viral genome replication, nuclear entry and nuclear exit processes cannot be observed in real time by fluorescence; in contrast, the rPRV TJ-Anchor3-mCherry constructed by the present invention not only overcomes the above shortcomings, but also has the following advantages, namely, the complete virus particles only show specific red fluorescence (mCherry), and the virus particles or viral genomes without the envelope only show specific green fluorescence (EGFP). Therefore, the dual fluorescently labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry constructed by the present invention can be used to observe the entire virus replication cycle and cell localization, and has a broader application prospect than the previous fluorescently labeled viruses. The recombinant virus was used to successfully trace the complete PRV replication cycle, and the effect of the antiviral drug acyclovir on inhibiting PRV replication was evaluated. At the same time, a PRV in vitro temperature-controlled visualized latent infection model was established, which can be used for PRV latent infection models and antiviral drug screening, neural circuit tracing, virus and host protein function research, etc., and is a powerful tool for basic and applied research on PRV.

[0018] The dual fluorescent labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry screening antiviral drug link of the present invention can provide a reference for the development of anti-alpha herpes virus drugs or small molecule compounds, and can accurately determine the specific link where the antiviral effect occurs in the virus cycle. In addition, the latent infection model can be used to identify host and viral factors related to latent infection and reactivation, and can provide new target molecules for the drug and vaccine research and development of PRV and other herpes viruses. In addition, the use of dual fluorescent labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry will greatly accelerate the research process of viral gene function, and the positioning and quantification of viral particles does not require additional technology and reagents, which greatly reduces the cost and work intensity. Finally, the dual fluorescent labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry constructed by the present invention can also be used for in vivo neural circuit research, cross-blood-brain barrier drug targeted delivery, alternative (simplified) immunohistochemical process, efficient evaluation of vaccine immune effect, and even for in vivo tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the construction of the dual fluorescent labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry; A. PRV TJ strain genome and PRV TJ strain Fosmid library; B. Schematic diagram of the construction of Fosmid-o-mCherry cosmid; C. Schematic diagram of the construction of Fosmid-s-Anchor3 cosmid; D. Composition of Fosmids used to rescue rPRV TJ-Anchor3-mCherry recombinant virus.

[0020] Figure 2 Identification of the dual-fluorescence-labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry; A: Live cell imaging effect of N2a cells infected with rPRVTJ-Anchor3-mCherry and PRV TJ strain; B. PCR identification results of rPRV TJ-Anchor3-mCherry exogenous gene insertion after 20 consecutive generations; C. Electron microscopy observation results of rPRV TJ-Anchor3-mCherry and PRV TJ strain virus particles.

[0021] Figure 3 Double fluorescently labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry genome-specific fluorescent labeling identification.

[0022] Figure 4 Growth characteristics of the dual fluorescently labeled recombinant pseudorabies virus strain rPRVTJ-Anchor3-mCherry;

[0023] A: Plaque morphology of PK-15 cells infected with rPRV TJ-Anchor3-mCherry and PRV TJ strain; B. One-step growth curve of PK-15 cells infected with rPRV TJ-Anchor3-mCherry and PRV TJ strain.

[0024] Figure 5 Viral load in various tissues of mice infected with dual fluorescently labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry.

[0025] Figure 6 Viral cycle tracking of N2a cells infected with dual fluorescently labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry.

[0026] Figure 7Live cell imaging and quantitative analysis of the dual-fluorescence-labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry in PK-15; A~C: Quantitative analysis results of fluorescence intensity at 0, 2, and 24 h after rPRV TJ-Anchor3-mCherry infection of PK-15 cells; D. Fitting analysis results of genome fluorescence value (EGFP) and corresponding genome copy number at different time points in PK-15 infected with rPRV TJ-Anchor3-mCherry; E. Quantitative analysis results of mature virus particle fluorescence value (mCherry) at different time points in PK-15 cells infected with rPRV TJ-Anchor3-mCherry.

[0027] Figure 8 Live cell imaging and quantitative analysis of dual fluorescent labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry in Vero cells; A: Replication of rPRV TJ-Anchor3-mCherry in Vero cells under the action of acyclovir (72hpi); B: Replication of rPRV TJ-Anchor3-mCherry in Vero cells under the action of oseltamivir (72hpi); C: Acyclovir inhibits high-dose virus (10 7 TCID 50 ) effect; D: Analysis results of cell infection rate under the action of acyclovir (10 7 TCID 50 ); E: Quantitative analysis results of viral DNA at different time points under the action of acyclovir; F: Trend of viral maturation ratio at different time points under the action of acyclovir.

[0028] Fig. 9 N2a cell latent infection model of dual fluorescently labeled recombinant pseudorabies virus strain rPRV TJ-Anchor3-mCherry; A: rPRV TJ-Anchor3-mCherry fluorescence images under different infection conditions; B: Confocal microscopy images of the latent state and reactivated state; D: Quantitative detection results of viral gene transcription levels during the latent infection period; C: Analysis of viral load differences at different time points during latent infection and lytic infection. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0030] Experimental Example 1 Construction, Identification, Performance Analysis and Application of Recombinant Pseudorabies Virus Strain rPRV TJ-Anchor3-mCherry with Double Fluorescence Labeling of Genome and Envelope Protein

[0031] 1 Materials and Methods

[0032] 1.1 Main Reagents

[0033] PRV TJ strain and its Fosmid library, mouse brain neuroma cells (N2a cells), pig kidney cells (PK-15 cells) and African green monkey kidney cells (Vero cells) were maintained by our laboratory; N2a, PK-15 and Vero cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; pGEM7-Anchor3 plasmid and Escherichia coli DH10B competent bacteria were maintained by our laboratory; pmCherry-C1 and pMD18-T SimpleVector plasmids were purchased from TaKaRa.

[0034] Table 1 Primers used to construct and identify the recombinant virus rPRV TJ-Anchor3-mCherry

[0035]

[0036]

[0037] The antibiotics and L-arabinose (Sigma A-3256) used were purchased from Sigma; the plasmid miniprep kit was purchased from Epigenetics (ZR BAC DNA Miniprep Kit, D4049); the plasmid midi kit was purchased from TIANGEN (TIANfilter Plasmid Midi Kit, 12243); and the X-tremeGENE HPDNA transfection reagent was purchased from Roche (Cat. No. 06366546001). All primers were synthesized by Ruibo Biotechnology Co., Ltd. (Table 1).

[0038] 1.2 Construction of recombinant UL10 homology arm mCherry intermediate plasmid

[0039] Using the pmCherryC1 plasmid as a template, the mCherry fragment was obtained by PCR amplification, the PRV TJ strain genome was used as a template, and the insertion site was before the stop codon of the gM protein encoding gene UL10. The homology arms of 50 bp on the left and right were designed, and the mCherry-UL10-arm gene fragment was obtained by PCR amplification and connected to the pMD18-T vector. According to the sequencing results, the correct clone was selected and named pMD18-mCherry-UL10-arm.

[0040] 1.3 Construction of US9 homology arm Anchor3 intermediate plasmid

[0041] Using pGEM7-Anchor3 plasmid as template, PCR amplification was performed to obtain the Anchor3 fragment, using PRV TJ genome as template, and the insertion site after the US9 gene stop codon, 200 bp homology arms were designed on the left and right, and US9-arm-left and US9-arm-right gene fragments were obtained by PCR amplification; Anchor3, US9-arm-left and US9-arm-right gene fragments were amplified by fusion PCR to obtain the Anchor3-US9-arm gene fragment and connected to the pMD18-T vector. The correct clone was selected by sequencing and named pMD18-Anchor3-US9-arm.

[0042] 1.4 Inserting the mCherry gene into the cosmid containing UL10 in the PRV TJ strain Fosmid library

[0043] Gene recombination was performed using the cosmid containing UL10 in the Fosmid library of the PRV TJ strain. The detailed construction scheme is as follows: Figure 1 B. First, Fosmid-o and Red / ET plasmids were co-transformed into DH10B competent bacteria, and 100 μL of the transformed bacteria was spread on a plate containing chloramphenicol (Cm + ) and tetracycline (Tet + ) were cultured in LB with two resistances at 30°C in the dark for 30-40h; a single clone colony was picked to prepare the electrotransformation competent bacteria DH10B-Fosmid-o-Red / ET, the rpsL-neo gene fragment containing the mCherry-UL10-arm homology arm was amplified by PCR, and the rpsL-neo gene fragment was electrotransferred to the competent bacteria DH10B-Fosmid-o-Red / ET, and 100 μL of the transformed bacteria was spread on a plate containing chloramphenicol (Cm + ), tetracycline (Tet + ) and kanamycin +) and cultured them at 30°C in the dark for 30-40h. Single clone colonies were picked for PCR identification and sequencing to confirm that rpsL-neo had successfully replaced the insertion before the termination codon of the UL10 gene to obtain the recombinant bacteria DH10B-Fosmid-o-rpsL. The recombinant DH10B-Fosmid-o-rpsL electrotransformation competent bacteria were prepared, and the pMD18-mCherry-UL10-arm plasmid was used as a template to PCR amplify the mCherry-UL10-arm fragment and electrotransform the competent bacteria DH10B-Fosmid-o-rpsL. 100 μL of the transformed bacteria were spread on a plate containing chloramphenicol (Cm + ) and streptomycin + ) on a double-resistance plate and cultured in the dark at 37°C for 18-24 hours. Single clone colonies were picked for PCR identification and sequencing to confirm that mCherry had successfully replaced the rpsL-neo gene, and the recombinant bacteria DH10B-Fosmid-o-mCherry were obtained.

[0044] 1.5 Insertion of Anchor3 gene into the US9 cosmid containing the PRV TJ strain Fosmid library

[0045] The Fosmid-s and Red / ET plasmids were co-transfected into DH10b competent bacteria by electroporation in the same manner as above, and the recombinant bacteria DH10B-Fosmid-s-Anchor3 ( Figure 1 C) 1.6 Rescue of dual fluorescently labeled rPRV TJ-Anchor3-mCherry

[0046] According to the instructions of X-tremeGENE HP DNA transfection reagent, the purified cosmids Fosmid-a, Fosmid-f, Fosmid-o-mCherry, Fosmid-q and Fosmid-s-Anchor3 were co-transfected into Vero cells in a 10 cm cell culture plate with a cell confluence of 90%. The transfection dose of each cosmid was 2 μg ( Figure 1 D) After the cells become cytopathic, the cells are repeatedly frozen and thawed, and the virus is harvested. The rescued virus is named rPRV TJ-Anchor3-mCherry.

[0047] 1.7 PCR identification of rPRV TJ-Anchor3-mCherry genome

[0048] The rescued virus was repeatedly frozen and thawed at -80℃ / 37℃, and then propagated for 20 generations on PK-15 cells. The genome of the 20th generation virus was extracted, and the mCherry and Anchor3 gene fragments were amplified with specific primers, and the amplified fragments were sequenced and analyzed.

[0049] 1.8 Electron microscopic observation of rPRV TJ-Anchor3-mCherry virus particles

[0050] The virus titer was 10 8 TCID 50 1 mL of rPRV TJ-Anchor3-mCherry and PRV TJ virus solution was centrifuged at 13000 r / min for 10 min, the supernatant was collected, and the morphology of virus particles was observed by transmission electron microscopy after negative staining with 2% phosphotungstic acid.

[0051] 1.9 Identification of the specificity of rPRV TJ-Anchor3-mCherry genomic fluorescent protein labeling

[0052] Vero cells were inoculated into confocal cell culture dishes, placed in a 37°C, 5% CO2 cell culture incubator for overnight culture, Hoechst dye was added, and staining was performed for 5 minutes. The staining solution was discarded, and the cells were washed 3 times with PBS. The OR3-mCherry eukaryotic expression plasmid was transfected into Vero cells at a dose of 1 μg / dish, and rPRV TJ-Anchor3-EGFP was infected 24 hours after transfection (MOI=1). The cells transfected with OR3-mCherry eukaryotic expression plasmid and infected only with rPRV TJ-Anchor3-EGFP in the same manner were used as controls. The cell culture dishes were placed under a confocal microscope and photographed 24 hours after infection.

[0053] 1.10 Analysis of growth characteristics of rPRV TJ-Anchor3-mCherry

[0054] The virus was inoculated into a monolayer of PK-15 cells at a dose of MOI = 5. After 1 hour of adsorption, the virus solution was discarded, the cells were washed 3 times with PBS, and fresh culture medium was replaced. The virus was harvested at 0, 6, 12, 24, 36, 48, 72, 96 and 120 hours after infection. The virus titers harvested at different time points were determined by IFA and the virus growth curve was plotted.

[0055] The virus stock solution was diluted 10 times with DMEM culture medium and the final volume was 1 mL, inoculated into PK-15 cells cultured in a 6-well cell culture plate, and placed in a 37°C, 5% CO2 cell culture incubator for 2 hours to discard the virus solution; 1×DMEM culture medium containing 2% FBS and 1% low-melting point agarose was heated and melted in an 80°C water bath, and after melting, placed in a cell culture incubator to cool to about 37°C, 2 mL of gel solution was taken to cover the monolayer cells, placed at room temperature for about 15 minutes to solidify the low-melting point agarose, and then continued to be cultured in a 37°C, 5% CO2 cell culture incubator for 72 hours; 600 μL of 4% paraformaldehyde was added to each well for fixation for 20 minutes, and the low-melting point agarose gel solid on the cell surface was gently peeled off with a 1 mL syringe needle, and then the cells were stained with crystal violet staining solution for 5 to 10 minutes, and the crystal violet staining solution was sucked out and the cell culture wells were gently rinsed with clean water, and photographed and the plaque diameter was measured.

[0056] 1.11 Evaluation of the pathogenicity of rPRV TJ-Anchor3-mCherry in mice

[0057] Forty-five 6-week-old SPF female BALB / c mice were randomly divided into 9 groups, and each group of mice was housed in separate cages. 4 , 10 3 , 10 2 or 10 1 TCID 50 The PRV TJ strain was used. Groups 5 to 8 were inoculated with the same dose of rPRV TJ-Anchor3-mCherry, and group 9 was inoculated with 100 μL DMEM as a control. The inoculation route was intramuscular injection. The weight and clinical symptoms of the mice were recorded every day for 14 days. The onset time and death time of the mice were also recorded, and the mortality rate was calculated. After that, 10 4 The heart, liver, spleen, lung, kidney, gastrocnemius muscle and brain tissues of mice in the infection group and the control group were dissected and stored at -20°C for detection of the viral load in each tissue.

[0058] 1.12Tracing of viral replication cycle in N2a cells infected with rPRV TJ-Anchor3-mCherry

[0059] N2a cells were inoculated into confocal cell culture dishes, cultured overnight in a 37°C, 5% CO2 cell culture incubator, Hoechst dye was added, stained for 5 min, the staining solution was discarded, the cells were washed 3 times with PBS, infected with rPRV TJ-Anchor3-mCherry (MOI=1), placed in a live cell imaging system for continuous culture, and photographed continuously.

[0060] 1.13 rPRV TJ-Anchor3-mCherry was used for real-time quantification of viral genome and mature virus. Vero cells were inoculated in confocal cell culture dishes and cultured overnight in a 37°C, 5% CO2 cell culture incubator. Hoechst dye was added and stained for 5 min. The staining solution was discarded, and the cells were washed 3 times with PBS. rPRV TJ-Anchor3-mCherry (MOI = 0.1) was infected and cultured continuously in a live cell imaging system, and continuous photography was performed. Samples infected for 24 hours were collected for viral genome quantification and linear analysis was performed with the fluorescence intensity at the same time. Based on the analysis results, the fluorescence value of a single viral genome or mature viral particle was determined. Data analysis and quantification in this section were completed using Image J (https: / / imagej.net / Downloads) and multidimensional image analysis software Imaris 8.

[0061] 1.14rPRV TJ-Anchor3-mCherry for antiviral drug screening

[0062] Vero cells were inoculated into 96-well cell culture dishes for high-throughput live cell imaging (Corning, USA, cat#3603) or regular 96-well cell culture dishes, and cultured overnight in a 37°C, 5% CO2 cell culture incubator. Hoechst dye was added and stained for 5 min. The staining solution was discarded and the cells were washed 3 times with PBS. The experiment was divided into two groups, A and B. Group A was first infected with rPRV TJ-Anchor3-mCherry (10 1 ~10 7 TCID 50 ) After 1 hour, the virus solution was discarded, and the highest safe concentration of acyclovir (Acyclovir, 1mM) was added for continuous culture; Group B and Group A were treated in the same way, and the highest safe concentration of oseltamivir (Oseltamivir, 0.5mM) was added for continuous culture. At 12, 24, 36, 48, 60, and 72 hours after infection, the samples were placed in a high-throughput live cell imaging system for scanning and image acquisition, and samples of the same period were collected for quantitative analysis of viral genome copy number. The above data analysis and quantification were completed using Image J (https: / / imagej.net / Downloads) and high-resolution live cell confocal microscopy analysis software (LSM800-ZEISS-2.30).

[0063] 1.15 Establishment of temperature-controlled latent infection model of N2a cells infected with rPRV TJ-Anchor3-mCherry. N2a cells were inoculated in confocal cell culture dishes or conventional cell culture dishes, placed in a 37°C, 5% CO2 cell culture incubator for overnight culture, and the cell culture medium was discarded. Infected with rPRV TJ-Anchor3-mCherry (MOI = 0.01) and placed in a 37°C, 5% CO2 cell culture incubator for 2 hours. The virus solution was discarded, the cells were washed 3 times with PBS, and fresh complete culture medium was replaced. The cells were placed in a 42°C, 5% CO2 cell culture incubator for overnight culture for 72 hours as the latent infection treatment group. The culture medium was changed every day during the culture period; the cells were placed in a 37°C, 5% CO2 cell culture incubator for overnight culture for 72 hours after the same virus infection as the lytic infection group. The culture temperature of the latent infection group was reduced to 37°C and continued to be cultured for 48 hours as the reactivation treatment group. The cells of the lytic infection group and the latent infection group were collected, and the genome and total RNA were extracted for quantitative detection of the genome and transcripts.

[0064] 2. Test results

[0065] 2.1 Identification of rPRV TJ-Anchor3-mCherry

[0066] Using the existing PRV TJ strain Fosmid operating system in the inventor's laboratory, this experiment successfully constructed a recombinant virus rPRV TJ-Anchor3-mCherry double-labeled with Anchor3 and mCherry. After the recombinant virus infected the cell, a single virus particle in the cell could be successfully observed. Specific green fluorescence appeared in the cell nucleus, indicating that the genome in the cell nucleus was labeled with green fluorescence. Red fluorescence appeared around the cell membrane, and there were some specific single virus particles, indicating that the progeny virus particles completed the viral envelope assembly ( Figure 2 A). PCR identification results showed that using the rPRV TJ-Anchor3-mCherry genome as a template, the PCR amplified fragments containing the Anchor3 and mCherry exogenous genes were 4078bp and 1500bp, which were consistent with expectations, while the PRVTJ strain genome control had only 400bp and 750bp ( Figure 2 B). Electron microscopic observation of rPRV TJ-Anchor3-mCherry and PRV TJ strain virus particles showed that the two had the same morphology and size. The above results indicate that the dual-fluorescence-labeled rPRVTJ-Anchor3-mCherry was successfully rescued, and the insertion of exogenous genes did not affect the morphology of virus particles.

[0067] 2.2 Validation of Anchor3-specific markers on the PRV genome

[0068] Only the Anchor3-EGFP sequence was inserted into the genome of the PRV TJ strain to obtain rPRV TJ-Anchor3-EGFP. The rPRV TJ-Anchor3-EGFP and OR3-mCherry expression plasmids can express the same OR3 protein, but they are fused with two different colors of fluorescent proteins, EGFP and mCherry. When Vero cells were first transfected with the OR3-mCherry expression plasmid and then infected with rPRVTJ-Anchor3-EGFP, Figure 3 From the results, it can be observed that OR3-mCherry and OR3-EGFP are co-localized in the cell nucleus, while in the control group transfected with only OR3-mCherry expression plasmid, OR3-mCherry is only diffusely distributed in the cytoplasm, and in the control group infected with only rPRV TJ-Anchor3-EGFP, OR3-EGFP is mainly distributed in the cell nucleus, indicating that OR3-mCherry and OR3-EGFP compete for the Anchor3 sequence on the rPRV TJ-Anchor3-EGFP viral genome. When cells are infected with rPRVTJ-Anchor3-EGFP, the OR3-EGFP protein encoded by it can bind to the viral genome and achieve green fluorescence labeling.

[0069] 2.3 Replication characteristics of rPRV TJ-Anchor3-mCherry

[0070] The same dose of rPRV TJ-Anchor3-mCherry and PRV TJ strain infected PK-15 cells, and the plaque staining results at 72 hours after infection showed that there was no significant difference in the size of single plaques formed by rPRV TJ-Anchor3-mCherry and PRV TJ infection ( Figure 4 A), the one-step growth curves of the two strains on PK-15 cells were basically the same ( Figure 4 B). The above results indicate that inserting the complete gene sequence of the Anchor3 system between the US9 gene and the US2 gene in the PRVTJ strain genome, and inserting the mCherry gene before the stop codon of the UL10 gene, do not affect viral replication.

[0071] 2.4 Analysis of pathogenicity and tissue tropism of rPRV TJ-Anchor3-mCherry in mice

[0072] 45 6-week-old SPF female BALB / c mice were randomly divided into 9 groups and inoculated with rPRV TJ-Anchor3-mCherry and PRV TJ strains in the same manner and at the same dose. Mice inoculated with DMEM served as controls. 4 TCID 50All mice in the infection group died. The average onset and death time of rPRV TJ-Anchor3-mCherry strain group was 2.2 days and 3 days, and the average onset and death time of PRV TJ strain group was 2.2 days and 2.9 days; 1 TCID 50 There was no disease or death in the mice in the infection group and the control group; 3 TCID 50 The mortality rate in the infected group was higher than 10 2 TCID 50 There was no significant difference between the two strains, and the onset time and death time were similar. The median lethal dose (LD 50 ) are both 10 2.5 TCID 50 The above results show that there is no significant difference in pathogenicity between rPRVTJ-Anchor3-mCherry and PRV TJ strains to mice. 4 TCID 50 The viral load of tissues from the dead mice in the infection group was tested, and the results showed that there was no significant difference in the genome copy numbers of the two strains in tissues such as heart, liver, spleen, lung, kidney, gastrocnemius muscle and brain ( Figure 5 ), indicating that the insertion of Anchor3 and mCherry genes into the PRV TJ genome does not affect the tissue tropism of the virus.

[0073] Table 2 Determination of median lethal dose of mice infected with rPRV TJ-Anchor3-mCherry and PRV TJ strain

[0074]

[0075]

[0076] 2.5Tracing of viral replication cycle in N2a cells infected with rPRV TJ-Anchor3-mCherry

[0077] N2a cells were inoculated into confocal cell culture dishes, infected with rPRV TJ-Anchor3-mCherry at a dose of MOI = 1, and placed in a live cell imaging system for continuous photography. Figure 6It can be observed from the results that the virus particles marked with red fluorescence approach the cell membrane, adsorb on the cell membrane at 2min 30s, fuse with the virus envelope and cell membrane after 10min, the red fluorescence remains on the cell membrane, and the virus that has shed its envelope emits green fluorescence. Another virus particle is attached to the cell membrane, enters the cell by endocytosis after 25min, and the complete virus particle shows red fluorescence. At 50min, the virus particle approaches the cell nucleus, at which time the virus sheds its envelope and the virus particle shows green fluorescence. After a period of replication, the number of specific fluorescence points of a single virus genome that enters the cell nucleus increases rapidly; while the virus genome proliferates, some envelope proteins begin to be expressed and assemble into mature virus particles (red light) near the cell nucleus, followed by a large number of virus particles accumulated near the cell nucleus reaching the cell membrane and being released.

[0078] The above results indicate that rPRV TJ-Anchor3-mCherry invades N2a cells by membrane fusion and endocytosis (this experiment is the first to discover that PRV invades nerve cells through two pathways on nerve cells). While the viral genome is replicating, the envelope protein gM is expressed, and the viral genome is packaged to form mature progeny virus particles that are released from the cell membrane. rPRVTJ-Anchor3-mCherry can be used to trace the complete cycle of PRV from infection to the release of progeny virus particles.

[0079] 2.6 Real-time quantification of rPRV TJ-Anchor3-mCherry in infected cells

[0080] PK-15 cells were inoculated in confocal cell culture dishes, infected with rPRV TJ-Anchor3-mCherry at a dose of MOI = 1, and placed in a live cell imaging system for continuous photography. Starting from the start of infection, the fluorescence values ​​at the following time points were analyzed: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours. The fluorescence values ​​of the viral genome (EGFP) and complete viral particles (mCherry) were statistically analyzed, expressed in fluorescence units (FU), and the viral genome copy number of samples with the same treatment method was quantitatively detected at the same time. The results showed that the fluorescence value corresponding to a single viral genome was 28FU, and the fluorescence value of a mature single viral particle was 12FU ( Figure 7 A, 7B, 7C). According to the fluorescence value and the number of viral genome copies, the fitting analysis of the two showed that the viral fluorescence intensity value and the number of viral genome copies had the same growth trend ( Figure 7 D). That is, dual-fluorescence labeled rPRV TJ-Anchor3-mCherry can be used for real-time quantitative analysis of viral genome and mature virus particles ( Figure 7 E).

[0081] 2.7 Results of the sensitivity evaluation of rPRV TJ-Anchor3-mCherry to acyclovir

[0082] Vero cells were inoculated into 96-well cell culture dishes for high-throughput live cell imaging and infected with different doses of rPRVTJ-Anchor3-mCherry. The high-throughput live cell imaging system was used to scan and collect images every 12 hours, and the cell infection rate, viral genome, and mature virus were quantitatively analyzed. The results showed that acyclovir could significantly inhibit the proliferation of PRV genome and the formation of mature virus particles, while oseltamivir had almost no effect on the proliferation of PRV ( Figure 8 A, 8B). The number of fluorescent spots in the entire culture well did not increase significantly over time, indicating that acyclovir can inhibit viral replication and significantly reduce the cell infection rate ( Figure 8 C, 8D).

[0083] Through quantitative analysis of the fluorescence values ​​at each time point, it was found that acyclovir can effectively reduce viral genome replication and delay the maturation of viral particles ( Figure 8 E, 8F). The above results indicate that rPRV TJ-Anchor3-mCherry can be used for the screening of anti-PRV drugs (compounds), which has the advantages of being intuitive, efficient and simple to operate.

[0084] 2.8 Characteristics of the temperature-controlled latent infection model of rPRV TJ-Anchor3-mCherry in N2a cells

[0085] The recombinant virus rPRV TJ-Anchor3-mCherry was infected with N2a cells at MOI = 0.01. After continuous culture at 42°C for 72 hours, there was no obvious change in green fluorescence, and no red fluorescence was observed. However, the same virus was infected with N2a cells at the same dose and cultured at 37°C for 48 hours, and a large amount of red and green fluorescence was observed, indicating that the recombinant virus rPRV TJ-Anchor3-mCherry did not proliferate under 42°C culture conditions ( Fig. 9 A, 9B). Only LAT and low-level IE180 transcripts were detected from latently infected cell samples, while the transcripts of the early gene EP0 and the late gene gB were not detected ( Fig. 9 C), quantitative detection of viral genome in the supernatant after continuous culture at 42°C for 24h and 48h showed no significant changes ( Fig. 9 D). All of these are consistent with the characteristics of PRV latent infection, indicating that this experiment has successfully established PRV latent infection in N2a cells cultured at 42°C. The establishment of the temperature-controlled latent infection model has laid a good foundation for subsequent studies on the mechanism of PRV latent infection reactivation. SEQUENCE LISTING <110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin Branch of China Animal Health and Epidemiology Center) <120> Double fluorescent labeled recombinant pseudorabies virus strain and its construction method and application <130> HLJ-2001-220504A <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 3677 <212> DNA <213> Artificial sequence <400> 1 acgcgttaag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 60 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 120 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg 180 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcagttat 240 ctagatccgg tggatcccgg gcccgcggta ccgcttctat acgctcccgc ttgcctgtgc 300 agcgttctgt gccagcactt cccttaccaa ggcctcgaga cggggtgcaa gcagatgctg 360 ctgctgctcg gtcaggttct tgagcttgag ttcggtgcgg ccatcctgga agcccttcag 420 ctcgcccagt tcgattccct cttcccccgc gaactgaacg cgcgttctgt aatgcgcctt 480 ggttcggcgc agaggctcgc ctccttccgc ccgccggttc atctcctgca gctgtttaag 540 accttgttct caagcaaacg ggtccaagag gatttgctcg gcgagttctt cgccttttcg 600 cttataaatc agtttgatgt tgtaggccgt ggccaagccg acgccttctt cgttttcagc 660 catgctcctc agcagatggc tgggaaggac cgtcaactcg ccggctaaga ccctttctgc 720 tttgctcaag tcaagggtag cggccaatgc atcctggtca ggattccgtc aagctgagcg 780 tcttggagac ctggcgattg catcgtcgaa gacggtttga gcggttcgat ctttgttgat 840 gcggtttgcg accaagtaaa gatcagcgtc agagagctcc gggaagattt ccccgtcgat 900 aaactcgtcg ccaaggctct tctttgcgcg ggagcggtac tccccgtcga tgatgacgta 960 cttgccaggg aatcgatggt tcttcgtgac cttgatcgct tcgtactggc catcccgttt 1020 gagcttcact gcaagggcgt cgatcgcctc tgggttgtaa aactctcgcg ggttgaaagg 1080 gttggagacg caatctgcca gacgaatctg ctggcgtagc ttcacggaag acgtgttccc 1140 gcgcccgcca tagtgcagcg cctcgttggc gcttgcactt gaggaagtgc cctcttcaga 1200 aaccttgctg ttggcattca ggcgtgcatc gacctcccct ctttcggtcg caagcccctt 1260 tcgcaaagcg ctacccagtc gactgcggtt tggatttgaa gatttatcac tcatttgcgg 1320 cctcctagat ctgagtccgg acttgtacag ctcgtccatg ccgagagtga tcccggcggc 1380 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc 1440 ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt 1500 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt 1560 gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt 1620 gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt 1680 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta 1740 gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga 1800 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta 1860 ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa 1920 cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt 1980 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc 2040 ctcgcccttg ctcaccatgg tggcgaccgg tagctttttg caaaagccta ggcctccaaa 2100 aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata 2160 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg 2220 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg 2280 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag 2340 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg 2400 acacacattc cacagctagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2460 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2520 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2580 atgaccatga ttacgaattc cttgaacttc ccggcatcgc gtcctgttgt cacggaatta 2640 agcttgccaa gctcggcatg atcacctcag ttaatgtgtg taattctggg caggcgctat 2700 cccgaatccc tgtttttccc tggtttgcgg cgactggatg gggttagtgg ccgctgtgac 2760 gggcttctgt tggccgagtc cagttaatgt gtgtaattct gaccattgcc tacagaggag 2820 tctcgccgtc atggatagat tgaagcagta acaagtcgag ggctgccacc aaggccttgg 2880 ctgtgaacgg cagttaatgt gtgtaattct gcttcgacag ccgacggtgc ccccaggggg 2940 aaaatcgact gagggcggat gacgccaagc tagcgggcac ttaaaacagc gtcagttaat 3000 gtgtgtaatt ctggcagaag accgaggggc tgggtcgcta cagaggcaga atcactggtg 3060 agccccacgc cccactgggg cccagttaat gtgtgtaatt ctggctgcga aagcacgatc 3120 acgctcaagc aattgcacgt tcgtgcaaca tctctgaaag agacttttcg tcgtcggtta 3180 atgtgtgtaa ttctggcaac cggaaccaac gcctcaactc gccaatctca cctgccgcga 3240 gcctgcctac gaaacaaatg gctcaatagc cagcgtggac ccttgccagt taatgtgtgt 3300 aattctatcg acggcatatc gttcttgtag cagatatgag atgaacgcag caccacattc 3360 cacacttcat ccattcggcc gctccttcct gacacggcca gttaatgtgt gtaattctgg 3420 cccctgatg gacgcacgtg gccagtgccc cgtcccggta gagatggact tttgtaaggc 3480 ttaacgcaag agagccacaa gccactcggc tcaatgcttt cagttaatgt gtgtaattct 3540 gctggcgcag cgttgcagtc acaaacaccc accgcgctga tgcttacgcc ccacagttaa 3600 tgtgtgtaat tctgtggaag atcctgccaa agtgacggat caattccgt gactcctcgt 3660 taccggggtc ctgcagg 3677 <210> 2 <211> 711 <212> DNA <213> Artificial sequence <400> 2 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711

Claims

1. A double-fluorescence-labeled recombinant pseudorabies virus strain in which the genome and envelope protein are labeled with green fluorescence and red fluorescence respectively, characterized in that: Its microbial preservation number is CGMCC No.45203.

2. Use of the dual fluorescently labeled recombinant pseudorabies virus strain according to claim 1 in the preparation of a reagent for tracing pseudorabies virus in vivo or tracing the complete pseudorabies virus replication cycle.

3. Use of the dual fluorescently labeled recombinant pseudorabies virus strain according to claim 1 in constructing an in vitro temperature-controlled visualized latent infection model of pseudorabies virus.

4. Use of the double fluorescently labeled recombinant pseudorabies virus strain according to claim 1 in screening anti-pseudorabies virus drugs.

5. Use of the dual fluorescently labeled recombinant pseudorabies virus strain according to claim 1 in screening drugs for preventing or treating pseudorabies virus.

6. Use of the double fluorescently labeled recombinant pseudorabies virus strain according to claim 1 in preparing a real-time quantitative analysis reagent for pseudorabies virus genome and mature virus particles.

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