Identification and application of cell models supporting bocavirus infection

Abstract

The present invention relates to the identification of cell models that support bocavirus infection and its application. The present invention selected 29 human cell lines derived from kidney, lung, brain, lymphocytes, intestine, muscle, thyroid and mammary gland, and 7 animal cell lines derived from mice, monkeys, dogs, pigs and cattle for evaluation, and found that compared with Caco-2 cells, only HT-29 and MA104 cells had higher levels of intracellular viral RNA transcription; further detection of intracellular viral DNA levels through infection system found that compared with Caco-2 cells, only MA104 supported higher levels of viral replication. Finally, through experimental tests such as virus binding and internalization, virus replication level, virus particle morphology and virus particle reinfection, it was confirmed that human bocavirus type I has a complete life cycle in MA104 cells, including virus entry, replication, assembly and release.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the identification of cell models supporting bocavirus infection and applications thereof. Background Art

[0002] Human bocavirus (HBoV) is a common respiratory pathogen that causes respiratory symptoms such as fever and cough, as well as acute otitis media and pneumonia. Currently, there are no specific drugs or vaccines to treat or prevent it. HBoV belongs to the genus Bocavirus of the family Parvoviridae. It is a non-enveloped icosahedral virus with a size of approximately 26 nm and contains a linear single-stranded DNA of approximately 5.5 kb. [1] Parvoviruses typically rely on receptor-mediated endocytosis to enter cells, a process that involves viral particles binding to specific receptors on the cell surface and then internalizing into host cells. [2] In 2012, Qiu et al. successfully constructed an HBoV1 infectious clone through reverse genetics and demonstrated through a transfection system that HEK293T cells support bocavirus replication. However, HEK293T cells do not support viral infection into host cells, possibly due to the lack of specific receptor expression. [3] ; Literature reports that although Caco-2 cells allow HBoV1 infection, they cannot produce infectious progeny virus particles, and have limitations as a cell model for HBoV1 infection. [4] In addition, although HBoV1 can be isolated from well-differentiated or polarized human airway epithelial (HAE) cells by air-liquid interface (ALI), [5,6,7] However, due to the difficulty in obtaining culture materials and the time-consuming and expensive culture methods, it has not been widely used. Consequently, the current lack of suitable viral cell culture models has limited HBoV research to the analysis and detection of clinical samples, significantly hindering the development of HBoV antiviral drugs and vaccines. Summary of the Invention

[0003] By screening the sensitivity of 36 cell lines to HBoV1, the present invention found an African green monkey epithelial cell line, MA104, that supports the complete life cycle of HBoV1. This provides a cell model for anti-HBoV1 drug screening, and through screening and verification, it was found that Cidofovir, Ribavirin, and Sofosbuvir can inhibit HBoV1 infection, demonstrating the feasibility of applying this cell model to antiviral drug screening.

[0004] The present invention provides an African green monkey epithelial cell MA104 model that supports the complete life cycle of HBoV1 and its application in anti-bocavirus drug screening, which includes the steps of:

[0005] 1. Purified human bocavirus was used to infect 29 human and 7 non-human cell lines to detect the sensitivity of different cell lines to HBoV1 infection.

[0006] 2. A detailed evaluation of HBoV1 viral characteristics, including invasion, replication, and assembly, was performed in MA104 cells, confirming that MA104 supports the complete life cycle of HBoV1.

[0007] 3. Select broad-spectrum antiviral drugs and drugs that have been reported to have inhibitory effects on virus B19 in the same family, and evaluate the antiviral effects of small molecule drugs in MA104 cells.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In one aspect, the present invention provides the use of MA104 cells in constructing a cell model supporting bocavirus infection.

[0010] In some embodiments, the cell model supports the complete life cycle of bocavirus, including viral entry, replication, assembly, and release.

[0011] In some embodiments, the bocavirus infects MA104 cells, and spherical virus particles with a size of about 26 nm can be detected.

[0012] On the other hand, the present invention provides the use of MA104 cells in the study of bocavirus entry, replication, assembly, and release.

[0013] In another aspect, the present invention provides the use of MA104 cells in anti-bocavirus drug screening and vaccine evaluation.

[0014] Another invention provides a method for screening anti-bocavirus drugs, which includes the steps of culturing MA104 cells and infecting them with bocavirus for drug screening.

[0015] In some embodiments, the method includes the steps of collecting intracellular DNA, detecting viral copies, and calculating the inhibition rate of the drug to be screened on the virus, thereby screening anti-bocavirus drugs.

[0016] In some embodiments, the method further comprises the step of culturing the MA104 cells with culture medium containing different doses of the drug, and calculating the intracellular DNA copy number to thereby detect whether the drug inhibits the bocavirus in a dose-dependent manner.

[0017] In another aspect, the present invention provides use of a drug screened by the method described above in preparing a pharmaceutical composition against Bocavirus.

[0018] On the other hand, the present invention provides the use of Cidofovir in the preparation of anti-Bocavirus drugs.

[0019] In some embodiments, the bocavirus is a human bocavirus.

[0020] In some embodiments, the human bocavirus is selected from HBoV1, HBoV2, HBoV3, and HBoV4.

[0021] definition

[0022] Human bocavirus: Human bocavirus belongs to the Parvoviridae family and the Bocavirus genus. It is a non-enveloped, spherical virus particle approximately 26 nm in diameter. Bocavirus is a single-stranded, negative-strand DNA (ssDNA) virus with a genome length of approximately 5.5 kb. Its genome consists of three open reading frames (ORF1, ORF2, and ORF3), expressing three structural proteins (VP1, VP2, and VP3) and six nonstructural proteins (NS1, NS1-70, NS2, NS3, NS4, and NP1). The structural proteins VP1, VP2, and VP3 form the viral capsid in a 1:1:10 ratio. The viral capsid carries host determinants on its surface and is involved in numerous processes, including host tropism, cellular recognition, pathogenicity, assembly, and immune response. The nonstructural proteins NS1 and NP1 are essential for viral DNA replication and are highly conserved among different bocavirus genotypes. They are commonly used as targets for human bocavirus detection. Human bocaviruses are classified into four types: HBoV1, HBoV2, HBoV3, and HBoV4. HBoV1 is detected in 0% to 44% of patients with respiratory illness. HBoV1 infection primarily causes respiratory symptoms, while HBoV2-4 primarily cause diarrhea and other symptoms. VP2, a major component of the viral capsid, is involved in regulating multiple viral processes, including viral tropism, receptor recognition, assembly, and immune response. Although the symptoms caused by HBoV1-4 infection vary, structural analysis of the VP2 structural protein of types 1-4 reveals differences between HBoV1 and HBoV2 in the VR-III region, which plays a key role in receptor recognition and explains the differences in their tropism. However, they share the same N-terminal VP rearrangement, residue-level modifications, cysteine ​​and histidine residues, and specific surface modifications of VP2. Furthermore, the VP2 sequence is relatively conserved.

[0023] NP1: NP1 is unique to the Bocavirus genus and has a highly conserved sequence. It plays a key role in viral genome replication and RNA post-transcriptional modification and processing.

[0024] NS1: Nonstructural protein. When bocavirus infects cells, it is first transcribed and translated. The replication and expression of the viral genome require the regulation of NS1 protein.

[0025] Virus Binding and Internalization: Viruses are non-cellular organisms that parasitize and replicate within living cells. Different viruses enter cells in different ways, but most require internalization by binding to specific receptor proteins or lipid structures on the cell surface, thereby initiating the invasion process and infecting the host cell.

[0026] Infectious clones: The construction of infectious clones is a reverse genetics technique, in which the entire viral genome and essential response elements are sequentially incorporated into a vector to create infectious transcripts, or infectious clones. Once introduced into cells, infectious viral particles can be rescued. Like wild-type strains, viral particles derived from infectious clones undergo mutations and recombination during their replication cycle, and remain as infectious as their parental viruses. However, their evolution within the host may not be consistent with that of the parental virus, and the direction of mutation may differ.

[0027] Natural viral infection: The natural viral infection process includes viral entry, replication, assembly, and release. After the virus enters a susceptible cell, the viral genome begins to be transcribed and translated to produce new viral capsid proteins. These proteins further assemble and package the new viral genome to form complete viral particles, which are then released outside the host cell.

[0028] MA-104 cells are derived from green monkey embryonic kidneys and are known as African green monkey kidney cells in Chinese. Their normal cell morphology is epithelial-like and adherent. These cells are commonly used for the production of porcine reproductive and respiratory syndrome virus (PRRS) and simian rotavirus, and have been commercially validated as a cell line capable of infecting African swine fever virus. MA-104 cells typically proliferate for 48 to 72 hours and do not require any specialized culture medium.

[0029] Replication intermediates: These structures appear during viral nucleic acid replication. They are typically formed during the replication of ssDNA or ssRNA in the viral genome, forming dsDNA or dsRNA. Detection of replication intermediates indicates that the virus is capable of completing viral genome replication within the cell.

[0030] Beneficial effects

[0031] To develop a cell model that allows HBoV1 infection and completes the entire viral life cycle of entry, replication, assembly, and release, the inventors evaluated 29 human cell lines derived from kidney, lung, brain, lymphocytes, intestine, muscle, thyroid, and mammary gland, as well as seven animal cell lines derived from mouse, monkey, dog, pig, and cow. They found that only HT-29 and MA104 cells exhibited higher levels of intracellular viral RNA transcripts compared to Caco-2 cells. Furthermore, an infection system measuring intracellular viral DNA levels revealed that only MA104 cells supported higher levels of viral replication compared to Caco-2 cells. Assays for viral binding and internalization, viral replication, viral particle morphology, and virion reinfection confirmed that human bocavirus type 1 (HBoV1) carries out the complete life cycle in MA104 cells, including viral entry, replication, assembly, and release. The inventors also screened antiviral drugs in MA104 cells and found that cidofovir, ribavirin, and sofosbuvir could inhibit HBoV1 replication to some extent, demonstrating the feasibility of this cell model for antiviral drug screening.

[0032] In addition, the cell model of the present invention can support the natural infection process of bocavirus and produce progeny viruses. When used for drug screening, it can more accurately simulate the viral infection process and virulence level, thereby more correctly and effectively screening anti-bocavirus drugs.

[0033] Since HBoV1 has a very high respiratory tropism, it may be an ideal viral vector for lung gene therapy. Therefore, the present invention can also be applied to the construction of lung gene therapy viral vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 2 shows the results of a sensitivity test for HBoV1 against different cell lines. A, q-PCR analysis of viral transcriptome levels after infection with HBoV1 in 36 cell lines. B, Quantification of intracellular HBoV1 DNA levels after infection with HBoV1 in HEK293T, Caco-2, HT-29, MA104, HeLa, RD, A172, and A549 cells. HEK293T served as a negative control, and Caco-2 served as a positive control.

[0035] Figure 2Figure 1 shows the results of an analysis of the replication characteristics of HBoV1 in MA104 cells. A, Expression levels of the viral nonstructural protein NP1 in MA104 cells under transfection conditions. HEK293T cells served as a positive control. B, Immunofluorescence analysis of NS1 expression and subcellular localization in MA104 cells under transfection conditions. HEK293T cells served as a positive control. C, Southern blotting analysis of viral replication intermediates in MA104 cells under transfection conditions. HEK293T cells served as a positive control. D, Negative staining of viral particles using purified HBoV1 expressed in MA104 cells. HEK293T cells served as a positive control.

[0036] Figure 3 Figure 3 shows the results of an analysis of the infectious properties of HBoV1 in MA104 cells. A & B, Virus binding and internalization assays. HEK293T cells served as a negative control. C, MA104 cells infected with HBoV1, and intracellular HBoV1 DNA levels were measured. D, MA104 cells infected with HBoV1, cell samples and supernatant samples were collected for concentration and purification of viral particles, and viral morphology was observed using negative staining. E, Supernatant from MA104 cells following infection was collected, concentrated (100×), and then reinfected into MA104 cells. 8 days later, the viral supernatant was collected for HBoV1 DNA extraction and analysis of viral copies.

[0037] Figure 4 Figure 1 shows the results of an anti-HBoV1 drug screening application analysis in MA104 cells. A, Analysis of the effects of six small molecule compounds (Cidofovir 200μM, Nevirapine 50μM, Remdesivir 10μM, Ribavirin 50μM, Sofosbuvir 50μM, Zidovudine 50μM) on HBoV1. B, Analysis of the anti-HBoV1 efficacy of cidofovir. MA104 cells were pretreated with a gradient of drug concentrations (400μM, 2-fold dilutions, 3 gradients) for 2 hours. Cell samples were collected 72 hours after HBoV1 infection, and intracellular viral DNA levels were measured.

[0038] Note: UI in the figure stands for uninfected. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0040] Cell culture

[0041] All cells were cultured at 37°C in an atmosphere of 5% CO2. HEK293T, MA104, Caco-2, A549, HeLa, and RD cells were obtained from ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gbico) supplemented with 2 mM HEPES (Gbico), 10% fetal bovine serum (FBS) (Invitrogen), and 1% penicillin-streptomycin (P / S) (Life Technologies). A172 cells were purchased from Procell and cultured in DMEM supplemented with 10% FBS and 1% P / S. HT-29 cells were cultured in RPMI1640 (Gbico) supplemented with 10% FBS and 1% P / S.

[0042] Viral expression and purification

[0043] The infectious clone pHBoV1 (donated by Wang Hanzhong's group at the Wuhan Institute of Virology, Chinese Academy of Sciences) was transfected into HEK293T cells using linearized polyethyleneimine PEI 40000 transfection reagent. After 72 hours, the cell samples were collected and centrifuged at 4°C 100g for 10 minutes. The supernatant was discarded to remove cell debris, the cell pellet was resuspended in PBS, and the cells were lysed by three repeated freeze-thaw cycles. Then, the cells were digested with nuclease (50U / mL) for 1.5 hours. After centrifugation at 4°C 10,000g for 30 minutes. The supernatant was collected and further concentrated and purified using a 20% (wt / vol) sucrose cushion at 4°C 100,000g ultracentrifugation for 2 hours. The precipitate was resuspended in PBS and dissolved overnight. Viral DNA was extracted using the TIANamp Viral DNA / RNA Fast Kit (TIANGEN) and quantitative detection was performed by qPCR.

[0044] Viral infection

[0045] Target cells were infected with HBoV1 at 3000 genome equivalents per cell for 12 hours. Cells were washed three times with PBS and cultured in DMEM supplemented with 2% FBS. On the designated days after infection, cells and culture supernatants were harvested for viral DNA extraction and assays.

[0046] qRT-PCR

[0047] Total cellular RNA was extracted using Trizol reagent according to the manufacturer's instructions. Specific mRNA was quantified using the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme). β-actin was used for normalization. Viral RNA transcriptome was quantified using the HiScript II One Step qRT-PCR Probe Kit (Vazyme). -ΔΔCt Methods Relative expression levels were calculated. The primer sequences used for quantification are shown in Table 1.

[0048] Table 1 Primer sequences used in the present invention

[0049]

[0050] q-PCR

[0051] Supernatant and intracellular viral genomes were extracted using the TIANamp Viral DNA / RNA Fast Kit. DNA copies were quantified using the TaqPro HS Universal Probe Master Mix (Vazyme), and absolute quantification was calculated using a standard curve. The NS1 primer sequences used for quantitative PCR are shown in Table 1.

[0052] Western blotting

[0053] Yiqiao Shenzhou was commissioned to produce four antibodies against HBoV-1NP1. Western blotting showed that αNP1-3 and αNP1-4 antibodies specifically recognized the viral protein NP1. The cells were lysed in lysis buffer at 4°C for 30 minutes, then centrifuged at 12,000 rpm for 10 minutes at 4°C and quantified. The samples were separated by 12% SDS-PAGE, electrotransferred to a nitrocellulose filter (Millipore), then blocked with 5% skim milk solution for 1 hour, and then blotted with NP1 or β-Actin antibodies. Proteins were visualized using HRP-labeled secondary antibodies (Jackson Immuno Research) and WesternBright Sirius HRP substrate (advansta).

[0054] Statistical analysis

[0055] Statistical analysis was performed using GraphPad Prism software. Groups were compared using a two-tailed Student's t-test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA). Data are presented as mean ± standard deviation. Differences were considered statistically significant when *P < 0.05, **P < 0.01, or ***P < 0.001.

[0056] Example 1 Detection of the sensitivity of different cells to bocavirus infection

[0057] To obtain cell lines that support human bocavirus type 1 infection, the inventors selected 29 human cell lines derived from kidney, lung, brain, lymphocytes, intestine, muscle, thyroid, and breast, as well as 7 animal cell lines derived from mice, monkeys, dogs, pigs, and cattle, and evaluated them according to the above-mentioned bocavirus infection method. Compared with Caco-2 cells, only HT-29 and MA104 cells had higher levels of intracellular viral RNA transcripts ( Figure 1A). Then, cells with slightly higher transcriptome levels were further tested for intracellular viral DNA copies using an infection system. The results showed that compared with Caco-2 cells, only MA104 cells had a higher intracellular viral DNA copy number than Caco-2 cells, with the intracellular viral DNA copy number in MA104 cells being 7.9 times higher than that in Caco-2 cells ( Figure 1 B) These results suggest that the monkey kidney cell line MA104 cells have the potential to support HBoV-1 infection.

[0058] Example 2 Analysis of the replication characteristics of HBoV1 in MA104 cells

[0059] 1. Detection of viral protein expression and subcellular localization: In HEK293T or MA104 cells, the vector and HBoV1 infectious clone were transfected via liposomes. After 48 hours, intracellular samples were collected to detect the expression of bocavirus protein NP1 and the localization of NS1.

[0060] 2. Southern Blotting: HEK293T or MA104 cells were transfected with vector or HBoV1-WT infectious clone. The genome was extracted using the Hirt DNA extraction method 48 hours later. The replication ability of HBoV1 in MA104 cells was detected by Southern Blotting.

[0061] 3. Expression and Purification of Viral Particles: HEK293T or MA104 cells were transfected with the HBoV1 infectious clone using lipofectamine (linearized polyethyleneimine PEI 40000 transfection reagent). After 48 hours, cells were harvested and centrifuged at 100g to remove cell debris. Viral particles were released by lysing the cells by three freeze-thaw cycles. The cells were then digested with nuclease (50 U / mL) at 37°C for 1.5 hours, centrifuged at 10,000g for 30 minutes at 4°C, and the cell pellet was removed. The pellet was then ultracentrifuged at 100,000g for 2 hours at 4°C using a 20% (wt / vol) sucrose cushion. The pellet was then collected and resuspended in PBS and dissolved overnight. Finally, viral particles were visualized using negative staining techniques and transmission electron microscopy.

[0062] Results: In MA104 cells, HBoV1 viral protein NP1 was expressed normally, and NS1 was mainly localized in the nucleus, which was consistent with the literature reports ( Figure 2 A and 2B). Southern Blotting results showed that HBoV transfection in MA104 cells could produce replication intermediates (dRF and mRF) resistant to DpnⅠ restriction endonuclease digestion ( Figure 2 C). Negative staining electron microscopy results showed that spherical virus particles of approximately 26 nm in size could be detected in MA104 cells, and their morphology and size were similar to those of virus particles purified from HEK293T cells ( Figure 2 D).

[0063] The above experimental results show that HBoV1 can replicate correctly in MA104 cells and assemble spherical virus particles with a size of approximately 26 nm.

[0064] Example 3 Analysis of infection characteristics of HBoV1 in MA104 cells

[0065] 1. Virus Binding and Internalization: HEK293T or MA104 cells were infected with HBoV on ice for 11 hours, washed five times with PBS, and intracellular DNA was harvested to measure viral copies for calculation of viral binding efficiency. For viral internalization, after 1 hour of binding, cells were transferred to a 37°C incubator and incubated for 6 hours. The cells were then digested with 500 ng / mL proteinase K for 1 hour, washed three times with PBS, and intracellular DNA was harvested to measure viral copies for calculation of viral internalization efficiency.

[0066] 2. Detection of viral copies in the supernatant: MA104 cells were infected with HBoV1 for 12 hours. 96 hours after infection, the supernatant DNA was collected for genome extraction and viral particles were detected in the supernatant.

[0067] 3. Viral Particle Purification in the Infection System: MA104 cells were infected with HBoV1 for 12 hours. After 96 hours, the supernatant and cell samples were collected and the virus was purified. The supernatant was concentrated using PEG8000 precipitation; the cell samples were repeatedly freeze-thawed to release viral particles. Subsequently, the pellet was collected by ultracentrifugation at 100,000g for 2 hours at 4°C using a 20% (wt / vol) sucrose cushion and resuspended in PBS overnight. Negatively stained samples were prepared, and viral particles were examined using transmission electron microscopy.

[0068] 4. Reinfection of supernatant virus particles: MA104 cells were infected with HBoV1 for 12 hours. The supernatant was collected 96 hours after infection, concentrated 100×, and reinfected into MA104 cells for 24 hours. The cells were then washed four times with PBS and the supernatant virus copies were detected 8 days after infection.

[0069] Results: The results of virus binding and internalization experiments showed that MA104 cells supported HBoV1 entry, but HEK293T cells did not support HBoV1 entry ( Figure 3 A and 3B). When MA104 cells were infected with HBoV1, viral copies could be detected in the supernatant. In addition, negative staining of purified intracellular and supernatant samples from infected MA104 cells revealed spherical viral particles approximately 26 nm in size, indicating that viral particles were successfully assembled and released in MA104 cells ( Figure 3C and 3D). 100-fold concentrated HBoV1-infected MA104 cell supernatant was used to reinfect MA104 target cells, and viral copies were successfully detected in the supernatant, indicating that the viral particles produced by HBoV1-infected MA104 cells are infectious ( Figure 3 E).

[0070] The above experimental results show that HBoV1 has a complete life cycle in MA104 cells, including virus entry, replication, assembly, and release.

[0071] Example 4 Analysis of Anti-HBoV1 Drug Screening Application in MA104 Cells

[0072] 1. Antiviral Drug Screening Application: In MA104 cells, six candidate small molecule drugs (Cidofovir 200μM, Nevirapine 50μM, Remdesivir 10μM, Ribavirin 50μM, Sofosbuvir 50μM, Zidovudine 50μM) were selected and pretreated for 6 hours. Among them, Cidofovir has been reported to inhibit the replication of Parvoviridae B19V. The other five are long-standing antiviral drugs used in clinical practice, mainly targeting viral replication inhibitors of RNA-dependent RNA polymerase (RdRp) and RNA / DNA-dependent DNA polymerase. Subsequently, HBoV1 was infected for 12 hours, washed three times with PBS, and replaced with fresh medium containing the drugs. After 72 hours, intracellular DNA was collected and viral copies were detected.

[0073] 2. Antiviral efficacy testing of the best candidate drugs: Select the small molecule drugs with the most obvious antiviral efficacy and perform gradient dilution to test their antiviral efficacy in MA014 cells.

[0074] Conclusion: Cidofovir 200μM, Ribavirin 50μM and Sofosbuvir 50μM have a certain degree of inhibitory effect on HBoV1 replication; among them, Cidofovir has an inhibitory effect on HBoV1, and compared with other small molecule drugs, Cidofovir has a more obvious inhibitory effect ( Figure 4 A). In MA104 cells, Cidofovir can inhibit HBoV1 intracellular DNA copies in a dose-dependent manner ( Figure 4 B).

[0075] The above experimental results show that MA104 cells can be used in the evaluation of anti-HBoV1 drugs and as a cell model for evaluating anti-HBoV1 drug screening.

[0076] References

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[0084] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of MA104 cells in constructing a bocavirus-infected cell model.

2. The use according to claim 1, characterized in that The cell model supports the complete life cycle of bocavirus, including viral entry, replication, assembly, and release.

3. Application of MA104 cells in anti-bocavirus drug screening and vaccine evaluation.

4. A method for screening anti-Bocavirus drugs, characterized in that: The method comprises the steps of culturing MA104 cells and infecting the cells with bocavirus for drug screening.

5. The method according to claim 4, characterized in that The method includes the steps of collecting intracellular DNA, detecting viral copies, and calculating the inhibition rate of the drug to be screened on the virus, thereby screening anti-bocavirus drugs.

6. The method according to claim 4 or 5, characterized in that The method comprises the steps of contacting different doses of a candidate drug with MA104, calculating the intracellular DNA copy number, and thereby detecting whether the drug inhibits the bocavirus in a dose-dependent manner.

7. The use according to any one of claims 1 to 3 or the method according to claim 6, characterized in that The bocavirus is human bocavirus.

8. The use or method according to claim 7, characterized in that: The human bocavirus is selected from HBoV1, HBoV2, HBoV3 and HBoV4.

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