CN116162600A - Porcine sapelo virus cell strain, and culture method and application thereof - Google Patents
Porcine sapelo virus cell strain, and culture method and application thereof Download PDFInfo
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Abstract
A strain of porcine sapelo virus, its culturing method and application, named PSV2019CM, is preserved in China center for type culture Collection (CCTCCNO. V202175) at 1/11/2021, has a full length of 7,567bp, has an ORF with a length of 6,996bp, encodes a polyprotein precursor of 2,331 amino acids, and has a 5'-UTR of 465bp and a 3' -UTR of 106bp on both sides of genome. The porcine sapelo virus provided by the invention is used for PSV virus vaccine, has better cross protection performance, better antigenicity and immunogenicity for PSV strains popular in China in recent years, has normal clinical characterization of infected pigs, stable weight growth, no toxin expelling phenomenon, can generate high-level PSV antibodies in vivo, the OD450 value of serum IgG antibodies can be up to 1.2, and the OD450 level of 60d antibodies after immunization is still maintained at 1.0.
Description
Technical Field
The invention belongs to the field of biological products for animals, and particularly relates to a porcine sapelo virus cell strain, a culture method and application thereof.
Background
Porcine sapelovirus (porcine sapelovirus, PSV), belonging to the genus Cosapelovirus, is a single-stranded positive-strand RNA virus without envelope coating, and the virus particles are microspheres with the diameter of about 30 nm. PSV is widely popular worldwide and is mainly transmitted through a feces-mouth way, so that multisystem syndromes such as diarrhea, pneumonia, cerebrospinal ash, reproductive system disorder and the like of a pig farm can be caused, meanwhile, asymptomatic infection can occur, and mixed infection can occur clinically with porcine parvovirus (porcine parvovirus, PPV), swine fever virus (classical swine fever virus, CSFV), porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory disorder syndrome virus, PRRSV), porcine enterovirus (porcine enterovirus, PEV) and the like, so that the morbidity of the pig farm becomes more complex, the mortality rate of the pig farm is increased, and serious harm is brought to the pig industry.
Porcine sapelo virus was originally isolated from the intestinal tract of diarrhea pigs and has therefore been previously classified as porcine enterovirus and designated porcine enterovirus type 8 (PEV-8). In 2002, krumbholz et al pointed out that porcine enterovirus type 8 has a unique L protein and highly structural specific 2A gene and 5' -terminal non-coding region (5 ' -untranslated region,5' -UTR) compared to other enteroviruses, and thus it is classified into a new genus of viruses. In 2014, the international committee on classification of viruses (ICTV) formally designated porcine sapelo virus (porcine sapelovirus, PSV) classified as a picornaviridae genus sapelo virus. In addition to porcine sapelo virus, the genus also includes avian sapelo virus (avian sapelovirus) and simian sapelo virus (simian sapelovirus). So far, PSV reported in countries around the world belongs to the same serotype, and a scholars point out that PSV has two gene subtypes, but the conclusion is not yet obtained internationally, and the PSV still needs to be confirmed.
PSV is highly resistant to the environment and can survive for several months at room temperature. Is insensitive to organic solvents such as acetic acid, chloroform and the like, and can resist pancreatin. The heat resistance is strong, the heat cannot be inactivated when the heat is acted for 10min at 56 ℃, and the lowest heat inactivation condition is that the heat is acted for 5min at 65 ℃ or 10min at 60 ℃. PSV is unable to agglutinate and adsorb various animal erythrocytes. Sodium hypochlorite, 70% ethanol and ultraviolet light can inactivate it, but the ultraviolet light does not affect its antigenicity.
PSV is usually isolated in porcine cell lines, such as LLC-PK or PK-15, but later scientists found (Li Y, du L, jin T, cheng Y, zhang X, jiao S, et al Characial and epidemiological survey of porcine sapelovirus in China; bai H, liu J, fang L, kataoka M, takeda N, wakita T, et al Characial of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells), it could also be grown in human cell lines other than porcine cell lines, milk hamster cell lines, green monkey cell lines, etc.
According to the current study, PLC/PRF/5, hepG2/C3a, 293T, vero E6, BHK21, PGMKC, ST, IPEC-J2, LLC-PK and PK-15 cells were all susceptible to PSV, whereas cells A549, MDCK, DF-1, huh7, WI38, hela, hep2C, HCT15 and PCMKC were more resistant to PSV. The sensitivity differences in PSV are of great significance for the study of PSV receptors, but their infection with different cell lines raises concerns about their potential host range beyond pigs.
Since PSV was first reported in 1958 in the united kingdom, many countries, regions, and so on, have detected this virus, such as canada, japan, australia, brazil, spanish, korea, china, and so on. At present, research on PSV at home and abroad has been focused on molecular epidemiology, and it has been partially studied that PSV has a detection rate of 36.6% in the czech republic, 60% in korea, 75.5% in prandia, 7.1% in india, 71% in hungary, 72.8% in italy, 9% in spanish, 31.8% in the united states, 17.2% in the eastern, south of china, four-channel, lake south, and Ningxia regions of china, 18.21%,20.4%,42.21%, and 61.25%, respectively. The data show that PSV is widely distributed, the infection rate is high, and the infection rate is concentrated between 7.1% and 75.5%.
PSV was susceptible to both domestic and wild pigs, and Prod ě lalov J et al (prodelalov J. The survey of porcine picorviruses and adenoviruses in fecal samples in Spain) investigated fecal samples collected from the czech republic, found that the infection rates of PSV to domestic and wild pigs were 33.8% and 27.8%, respectively. PSV is infectious in pigs of all ages, with 4 to 12 weeks of age being the most sensitive to the herd, chelli E et al (Chelli E, de Sabato L, vaccari G, ostanello F, di Bartolo I.detection and Characterization of Porcine Sapelovirus in Italian Pig Farms), reporting a positive rate of PSV of 72.8% in Italy with an infection rate of 98.4% between 4 and 12 weeks of age and 14.3% above 1 year old.
The results of the investigation by Harima H et al (Harima H, kajihara M, simulu E, bwalya E, qia Y, isono M, et al Gene and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia) showed that the infection rate of PSV was 36.2% for pigs of 0 to 3 weeks of age, 94% for pigs of 4 to 12 weeks of age, 46.39% for pigs of Yang T et al (Yang T, yu X, yan M, luo B, li R, qu T, et al molecular characterization of Porcine sapelovirus in Hunan, china) and 41.87% for pigs of 4 or more weeks of age. Clinically, PSV was commonly co-infected with other viruses, and epidemiological investigation results of 100 pig diarrhea stool samples from Korea by Son KY et al (Son KY, kim DS, matthijnssens J, kwon HJ, park JG, hosmillo M, et al molecular epidemiology of Korean porcine sapeloviruses) showed a PSV positive rate of 34%, with 5.9% of PSV alone and 94.1% of PSV in combination with other viruses such as PEV, PTV, porcine kokumi virus (porcine kobuvirus, PKV), porcine sapovirus (porcine sapovirus, PSaV), and the like.
PSV infection occurred in both diarrhea and non-diarrhea pigs, but no significant difference was observed in diarrhea and non-diarrhea pigs as indicated by the findings of Bak GY et al (Bak GY, kang MI, son KY, park JG, kim DS, seo JY, et al Occurrence and molecular characterization of Sapelovirus A in diarrhea and non-diarrhea feces of different age group pigs in one Korean pig farm [ J ]. J Vet Med Sci 2017,78:1911-1914 ]), whereas Zhang B et al (Zhang B, tang C, yue H, ren Y, song Z.Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China [ J ]. J Gen Virol2014, 95:1603-1611.) showed infection rates of PSV in diarrhea and healthy pigs of 48.1% and 17.2%, respectively. Similarly, 185 pig manure samples from China 2016-2017 were tested by Li Y et al (Li Y, du L, jin T, cheng Y, zhang X, jiao S, et al Characterisation and epidemiological survey of porcine sapelovirus in China [ J ]. Vet Microbiol 2019, 232:13-21.) and showed a PSV positive rate of 40.54%, with a PSV infection rate of 44.93% in diarrhea samples and 27.66% in asymptomatic samples. Lan D et al (Lan D, ji W, yang S, cui L, yang Z, yuan C, et al, isolation and characterization of the first Chinese porcine sapelovirus strain [ J ]. Arch Virol2011, 156:1567-1574.) confirmed by animal regression experiments that PSV can cause diarrhea, respiratory distress, and neurological disease.
So far, the PSV reported in each place belongs to the same serotype. Pigs are the main host of PSV, and the infection sources mainly include sick pigs, recovery pigs and recessive infection pigs, water sources polluted by excrement, food and the like, wherein the recovery pigs and the recessive infection pigs are not easy to be found clinically, and are important infection sources. PSV is mainly transmitted through the digestive tract, and is also transmitted through the respiratory tract and other ways, and the feces, secretions of the respiratory tract and pollutants thereof can carry viruses, so that pigs are more easily infected. After the virus enters the body, it colonizes mainly the intestinal mucosal epithelium and exits the body with faeces, resulting in a large presence of the virus in the environment. Pregnant sows infected with PSV can spread the infected fetus via blood sources, resulting in embryonic death, stillbirth, mummy embryo, and fetal malformation, among others. Clinically, PSV often accompanies mixed infection, so that the disease condition of a pig farm becomes more complex, the diagnosis, prevention and control are more difficult to hold, and finally the devastating strike of the pig industry is caused.
Isolation culture is a gold standard for virus diagnosis, but virus isolation is a relatively complex process and has a relatively long isolation period, so that virus isolation is rarely used as a diagnosis standard in simple clinical detection. The molecular biological detection technology has the advantages of rapidness, specificity and sensitivity, and is the most common means in clinical detection. Currently, in the relevant reports of PSV, reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR), fluorescent quantitative PCR (quantitative real-time PCR, qPCR) and loop-mediated isothermal amplification (loop mediated isothermal amplification, LAMP) are mostly used for detection.
The fundamental measures for preventing and treating PSV are prevention, no vaccine aiming at PSV exists in the market at present, so that the prevention and control of PSV by strictly controlling the feeding environment is particularly important. When commercial pigs are introduced, strict isolation and quarantine should be performed, and PSV infection is determined to be absent before the commercial pigs are introduced into the herd. In the feeding process, reasonable cleaning and sterilizing plans are formulated, the pig house is cleaned every day, the pig house environment is sterilized regularly, temperature, humidity and ventilation work in the pig house are performed, adverse factors such as stress are eliminated, and meanwhile, a proper amount of vitamins can be added into feed or drinking water to improve the immunity of pigs. At ordinary times, observation should be carried out, and the sick pigs and suspicious pigs are found to be immediately cleared, and the disinfection of healthy pigs is timely enhanced, so that the invasion of pathogens is prevented.
In addition, the strain identification and genetic characteristic research of PSV in China are enhanced, and basic research and vaccine research and development are enlarged, so that scientific basis is provided for the follow-up prevention and control of PSV, and the method has important significance for the prevention and control of PSV.
Disclosure of Invention
The invention aims to provide a porcine sapelo virus cell strain, a culture method and application thereof, wherein the porcine sapelo virus cell strain is preserved in China center for type culture collection (China) for type 11, the name is PSV2019CM, the preservation number is CCTCC No. V202175, the cell strain belongs to PSV-1 type, is used for researching and developing PSV virus inactivated vaccines, has better antigenicity and immunogenicity for PSV strains popular in China in recent years, has normal clinical characterization of infected pigs, has stable weight growth, does not generate toxin expelling phenomenon in vivo, can generate high-level PSV antibodies in vivo, has an OD450 value of serum IgG antibodies of up to 1.2, and has an OD450 level of 60d antibodies maintained at 1.0 after immunization.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a salpeter virus cell strain of pig is named as PSV2019CM and has a preservation number of CCTCCNO. V202175.
The full length of the porcine sapelovirus cell strain PSV2019CM is 7,567bp, and Genbank accession number: MN685785, having an ORF of length 6,996bp encoding a 2,331 amino acid multimeric protein precursor flanked by a 465bp 5'-UTR and a 106bp 3' -UTR.
Phylogenetic analysis based on ORF fragment shows that the porcine sapelovirus cell strain PSV2019CM has highest homology with YC2011, the nucleotide similarity is 90.8%, the amino acid similarity is 98%, and the porcine sapelovirus cell strain PSV2019CM and the YC2011 form a single branch, are aggregated on a cluster of Chinese PSV strains and have far relativity with other sapeloviruses. The isolate may be a recombinant strain of HeB04 and ISU-SHIC, with a potential recombination breakpoint upstream of 3D. Analysis of VP1 protein amino acid mutation site shows that VP1 region has amino acid mutation phenomenon.
A method for culturing a salpeter virus cell line, comprising the steps of:
inoculating 0.5-1MOI porcine sapelovirus PSV2019CM strain to PK15 cells, adsorbing for 1-2h, discarding cell supernatant, adding DMEM culture solution containing TPCK-pancreatin, culturing for 60-72h, and collecting cell culture solution to obtain the porcine sapelovirus cell strain.
Further, the collected cell culture fluid is stored at-40℃or lower for use.
The invention provides an application of the porcine sapelo virus PSV2019CM in preparation of porcine diarrhea inactivated vaccines.
An inactivated vaccine for porcine diarrhea, comprising the porcine sapelo virus strain PSV2019CM.
PSV is mainly colonized in the intestinal tract and the lung, and there is also a small distribution in the brain and intestinal lymph, but no viral nucleic acid is detected in the heart, liver and kidney. Animals infected with PSV can expel toxin through feces, and the virus load in feces is higher 3-6 days after toxin expelling. The proliferation of the porcine sapelo virus PSV2019CM strain on PK15 cells depends on pancreatin, belongs to cell adaptation strains, and mainly occurs in intestinal tracts and lungs of piglets, and is characterized in that: thin intestinal canal, thin and rotten intestinal contents, intestinal villus atrophy and rupture, and intestinal submucosa edema; swelling of the lungs, local bleeding, thickening of alveolar spaces and exudation of serous fluid, inflammatory cell infiltration. Thus, the isolated cell strain PSV2019CM provides a scientific material for subsequent vaccine development.
At present, the research on PSV at home and abroad is limited to epidemiological investigation, and related vaccine research on PSV at home and abroad is not yet available at present due to lack of importance. But the selective breeding of PSV vaccine strains is important, so that the prevention and control of the diarrhea of pigs can be developed from a new angle, and biological materials can be reserved in advance for outbreak of the enteroviruses of new pigs.
Compared with the prior art, the invention has the beneficial effects that:
the isolated cytotoxic PSV2019CM of the invention proliferates on PK15 cells and depends on pancreatin, belongs to cell adaptation strains, has pathogenicity to piglets, is used for preparing related vaccines of PSV, has better antigenicity and immunogenicity to PSV strains popular in China in recent years, has normal clinical characterization of infected pigs, stable weight growth, does not have toxin expelling phenomenon, can generate high-level PSV antibodies in vivo, has an OD450 value of serum IgG antibodies of up to 1.2, and maintains the OD450 level of 60d antibodies at 1.0 after immunization.
Drawings
FIG. 1 shows the results of cell subculture testing of PSV isolates in examples of the present invention, wherein M: DL2,000; G1-G6: PSV2019CM G1-G6.
FIG. 2 shows the results of virus isolation by inoculating PK-15 cells with PSV-positive samples, A being the results of non-inoculated cells, B being the results of about 30 hours of inoculation, and C being the results of about 70 hours of inoculation, in examples of the present invention.
FIG. 3 is a view of the result of electron microscopy for imaging and observing suspected PSV virus particles by using a transmission electron microscope in the embodiment of the invention.
FIG. 4 shows the results of analysis of the evolution relationship of PSV2019CM isolate and protein coding regions of other representative strains at home and abroad in the examples of the present invention.
FIGS. 5-8 are analysis results of recombination events identified according to potential recombinant strain polyprotein genes in an embodiment of the invention, wherein FIG. 5 is a similarity analysis result of PSV recombinant strain polyprotein genes by SimPlot software; FIG. 6 shows the PSV gene structure and the recombination breakpoint identification of the RDP software to the recombinant strain; FIG. 7 shows the analysis of the evolution tree of SHCM2019 and its potential parent strain protein coding region; FIG. 8 shows the P values of nine algorithms in RDP software to detect recombination events.
FIG. 9 is a comparison of viral loads in various tissues in an example of the present invention, where Log (RNA copies/ml) represents the Log of viral copy number per ml of tissue sample.
Detailed Description
The invention is further illustrated below with reference to specific examples.
DL2000 DNA marker, exTaq DNA polymerase, trizol, reverse Transcriptase M-MLV (RNase H-), 5X Reverse Transcriptase M-MLV Buffer, recominant Rnase Inhibitor, dNTP mix (2.5 mM each) were all purchased from TaKaRa; PSV monoclonal antibodies were purchased from Shandong Green both Co., ltd; other conventional reagents are all domestic analytical grade products.
Example acquisition of PSV epidemic PSV2019CM
In the embodiment, PSV detection is carried out on diarrhea samples collected in Shanghai, 1 PSV epidemic strain is successfully separated, 100 generation PSV epidemic strain PSV2019CM is obtained through subculture and screening, antigenicity and immunogenicity are good, and scientific research materials are provided for subsequent vaccine development.
1. Sample source
55 piglet diarrhea stool samples are collected from a pig farm in Shanghai, and 5 piglet diarrhea intestinal tissue samples are collected from a pig farm in Shanghai Chongming.
2. Primer design
A pair of PCR detection primers was designed and synthesized for the PSV 5' UTR gene, see Table 1 for specific sequences.
TABLE 1
3. Experimental method
3.1 sample handling and RT-PCR detection
Diarrhea stool: the diarrhea feces are collected by cotton swabs and are filled into a 10ml centrifuge tube, 1ml of sterilized PBS is added, after the diarrhea feces are fully dissolved, the mixture is stood for 30min, and the supernatant is sucked into a new centrifuge tube and is preserved at the temperature of minus 80 ℃.
Intestinal tissue sample: scraping intestinal mucosa content into a centrifuge tube, adding equal amount of PBS, fully and uniformly mixing, standing for 30min, sucking supernatant into a new centrifuge tube, and preserving at-80 ℃.
With reference to the kit instructions, 250. Mu.l of the treated sample was taken for total RNA extraction, and finally 20. Mu. l H was used 2 O solubilizes RNA and reverse transcribes with PSV-R primers. Then, 5. Mu.l of cDNA was taken for PCR detection. The PCR reaction system is as follows: 94℃for 4min,94℃for 35s, 51℃for 35s, 72℃for 45s (30 cycles) and 72℃for 8min.
The PCR products were detected by electrophoresis on a 1.5% agarose gel.
3.2 cloning and sequencing of PCR products
The PCR product gel with correct size is recovered, connected with pMD-18T Vector and transformed, positive clone is identified by plasmid extraction method and bacterial liquid PCR method, and sent to Shanghai bioengineering Co.Ltd for sequencing.
Sequences were aligned and analyzed using lasergene7.1 software.
3.3 proliferation and identification of viruses
PK15 cells with good growth state are selected, and are paved in a 6-well plate after being digested by pancreatin. The next day, cell supernatant is discarded, PBS is used for washing for 2 times, and then DMEM virus culture solution and sample treatment solution containing pancreatin (0.1-1 mug/ml TPCK-pancreatin) with different concentrations are respectively added; removing supernatant after adsorbing for 2h, adding DMEM culture solution containing certain pancreatin concentration, culturing in a 37 ℃ incubator, observing cytopathic effect, and if no cytopathic effect exists, detoxifying after 5 d; after freeze thawing 2 times, the above procedure was repeated again, and serial subculture was performed.
And taking 250 mu l of cell culture of each generation, and carrying out RT-PCR detection to verify whether the virus passage is successful or not.
3.4 Indirect immunofluorescence detection
And inoculating PSV2019CM of the 3 rd generation to PK15 cells, adsorbing for 1h, and changing into a virus culture solution to continue to culture at 37 ℃. After 72h, the cell supernatant was discarded, PBST was washed once, and fixed on 70% ethanol ice for 15min; PBST is washed 3 times and spin-dried, and 1 is added: 1000 diluted PSV monoclonal antibody, and incubating in a wet box at 37 ℃ for 1h; washing as above, adding 1:3000 diluted FITC-labeled goat anti-mouse IgG, incubated at 37℃for 30min; after washing, the cells were observed under an inverted fluorescence microscope (Axio Observer).
3.5TCID 50 Is (are) determined by
PK15 cells were plated 24h in 96-well cell plates, 10-fold specific dilutions of cytotoxic PSV2019CM were made for different generations, then added to each cell well, 100 μl/well, and 8 wells were repeated for each dilution. After adsorption for 1h, the supernatant was discarded, 200. Mu.l/well of PSV virus broth was added, and then placed at 37℃in 5% CO 2 Culturing is continued in the incubator.
TCID was then performed using an indirect immunofluorescence assay 50 Is determined by: after 120h incubation, the supernatant was discarded, washed once with PBS, 100 μl of 75% ethanol was added to each well, and the wells were fixed at-20deg.C for 15min; the fixative was discarded, washed 3 times with PBS, then 1:1500 diluted PSV monoclonal antibody, and incubating in a 37 ℃ wet box for 1h; the supernatant was discarded, washed as above, and 1:3000 diluted FITC-labeled goat anti-mouse IgG, incubated in a 37℃wet box for 30min; the supernatant was discarded, washed as above, and 100. Mu.l of PBS was added to each well, followed by observation under a fluorescence microscope. And TCID is carried out according to the method of Reed-Muench 50 Is calculated by the computer.
3.6 recombinant Virus analysis
Reference information (Meng Li. Establishment of a method for multiplex PCR detection of porcine diarrhea pathogen and analysis of major viral evolution [ D ]: shanghai ocean university; 2017), 23 pairs of primers were designed (see Table 2), the whole genome sequence of the resulting PSV2019CM strain was determined, and genetic evolution analysis was performed, further whole genome recombination analysis was performed using RDP4.1 recombination software, and in addition, the 5 'terminal sequence was determined using the 5' RACE Kit.
Table 2PSV SHCM2019 Whole genome amplification primers
3.7 animal experiments
Generation screening of 3.7.1 strain
The 6-day-old 5-day-old PSV negative piglets were randomly divided into 3 groups, and after grouping, 3d were observed to adapt the piglets to the new environment and to determine the health of the piglets. The piglets are fasted and forbidden for 2 hours before the poison attack, after the poison attack, the piglets are guaranteed to drink enough water, and fed with commercial feed, and the specific grouping and poison attack conditions are shown in table 3:
TABLE 3 grouping and treatment modes for animals
After toxin is removed, the clinical manifestations of piglets including feeding, drinking water, spirit, feces, diarrhea and the like are observed, and the anal swab of the piglets is collected every day to detect the viral load in the feces. If the piglets develop, the onset time and onset condition are recorded. After 6d of toxin attack, all piglets are dissected, tissues such as brain, heart, liver, lung, kidney, duodenum, jejunum, ileum, blind transmission, colon, rectum and intestinal lymph are collected for quantitative detection of nucleic acid, and the diseased pig intestine and lung tissues are fixed by 4% paraformaldehyde, and then immediately sent to Shanghai Weiao Biotechnology Co for slicing and HE staining.
3.7.2 fluorescent quantitative PCR
100mg of animal tissue collected in 3.2.1 was taken, 1mL of sterile PBS was added thereto, followed by grinding, centrifugation at 4,000rpm for 10min at 4℃and, thereafter, 250. Mu.L of the supernatant was aspirated into a new RNase-free EP tube. The daily collected anal swabs were resuspended in 1mL PBS, vortexed, mixed well, centrifuged and 250 μl of supernatant was removed to an rnase-free EP tube. The total viral nucleic acid in the disease supernatant was extracted by TRIzol method, and the extraction concentration was measured by a nucleic acid protein meter. According to PrimeScript TM RT instructions for genomic DNA removal followed by reverse transcription reactions followed by absolute quantitative determination of cDNA template strands using qPCR techniques. The primers used in this example to detect viral copy number were 1 pair of specific primers PSV-1/PSV-2 designed for PSV 5' -UTR by Chen J et al (see Table 4).
Table 4 PSV fluorescent quantitative PCR amplification primers
Blunt-zero plasmid containing PSV 5' -UTR fragment was subjected to 10-fold gradient dilution and its concentration was measured, using specific primers PSV-1/PSV-2 and following TB
Premix Ex Taq TM II instructions for known concentrations of recombinant plasmid and reverse transcription into cDNA disease samples for qPCR detection, each sample 3 multiple hole. The test uses an ABI 7500 fluorescent quantitative PCR instrument to carry out data determination analysis, and the calculation formulas of a standard curve and the number of copies of viruses in a sample are as follows: "ct=slope lgx + intercept). The specific calculation method comprises the steps of substituting the concentration of each dilution plasmid and the Ct value thereof into a standard curve, substituting the Ct value of each measured sample into a formula of the standard curve, and obtaining the virus copy number in the sample by using a linear extrapolation method.
Coating 100TCID50PSV virus on each hole, coating overnight at 4 ℃, removing solution in the holes, adding 200 mu l of PBST washing solution into each hole, shaking on a shaker for 3min, pouring, drying, and repeatedly washing for 5 times; 250 μl of PBST blocking solution containing 5% skimmed milk is added into each well, and the mixture is placed in a 37 ℃ incubator for blocking for 2 hours and then poured out, and washing is repeated for 5 times; diluting the serum sample with PBS, adding 100 μl of the sample into each well, incubating in a 37 ℃ incubator for 1.5 hours, pouring out, and repeating washing for 5 times; mu.l PSV monoclonal antibody (1:2000 dilution) was added to each well, the supernatant was discarded after incubation in a 37℃incubator for 1h, and washed 5 times; adding 100 μl of PBST diluted enzyme-labeled secondary antibody into each well, incubating in a 37 ℃ incubator for 1h, pouring out, and repeating washing for 5 times; a, B liquid was added per well at 1:1, 50 μl of the prepared color development liquid is developed for 10min at 37 ℃ in a dark place; mu.l of stop solution was added to each well and the results were measured within 10min.
4. Results
4.1 RT-PCR detection results of samples
The results of the previous 6-generation virus RT-PCR are shown in FIG. 1, and each of the G1 to G6 generation has a single band, and the unvaccinated cells do not amplify any band. Further sequencing results confirm that the amplified fragment is PSV and has a size of 624bp.
4.2 Virus isolation
PSV positive samples were inoculated with PK-15 cells for virus isolation and CPE was first observed when blinded to G3 passage. As shown in FIG. 2, the uninoculated cells grew normally (A), and at about 30 hours of inoculation, some cells became round, shrunken, slightly sloughed off (B), and at about 70 hours of inoculation, most cells had completely escaped the cell flask (C).
4.3 electron microscope observations
To determine the morphological characteristics of the virus, suspected PSV virus particles were observed by imaging using a transmission electron microscope. Electron microscopy of the negatively stained samples as seen in fig. 3, it can be seen from fig. 3 that the virions were microspheroidal, without a capsule, approximately 30nm in diameter, which was consistent with previous reports (Li Y, du L, jin T, cheng Y, zhang X, jiao S, et al, characialization and epidemiological survey of porcine sapelovirus in China [ J ]. Vet Microbiol 2019, 232:13-21), and no other virus-like particles were observed, indicating successful virus isolation.
4.4 determination of viral titres
Collecting cell culture solutions of the isolates G3 to G6 generation respectively, diluting with 10 times ratio, inoculating PK-15 cells, and measuring TCID50, wherein the results are shown in tables 2-4, the TCID50 of the isolate increases with passage number, the toxicity is increased, and the maximum of the 5 th generation reaches 10 6.6 1TCID50/mL。
TABLE 5 isolate TCID 50 Measurement results
4.5 genome-wide sequence information
The cDNA of PSV2019CM isolate is used as a template, the whole genome of the virus is amplified by utilizing a specific primer, each sequencing fragment is verified by BLAST, after the sequencing is determined to be correct, each fragment is sheared and spliced by DNAstar, and finally the full length of the isolate is 7,567bp, the cDNA has an ORF with the length of 6,996bp, and two sides of the genome are respectively provided with a 465bp 5'-UTR and a 106bp 3' -UTR. The sequence has been uploaded to NCBI and specific information can be looked up for the sequence number of Genbank number MN 685785.
4.6 phylogenetic analysis of isolates
The evolution relationship of PSV2019CM isolate and protein coding regions of other representative strains at home and abroad is analyzed by utilizing MEGA7.0, and the result is shown in figure 4, wherein the evolution relationship of PSV2019CM and YC2011 isolate is nearest and is accumulated in a group of Chinese PSV; the Chinese PSV isolate has the closest relationship with the Korean PSV isolate and has a far relationship with the European isolate; PSV isolates from each country clustered in the same group, with distinct groupings of sapelo viruses from other species. Further, the nucleotide and amino acid similarity of the strain is analyzed by DNAstar, and the result shows that the PSV2019CM and YC2011 isolate have higher homology, the nucleotide similarity is 90.8 percent, and the amino acid similarity is 98 percent; the nucleotide similarity of the Chinese PSV strain and the Korean PSV strain is 87.2% -89.8%, and the nucleotide similarity of the Chinese PSV strain and the European PSV strain is 78.7% -86.4%. Furthermore, phylogenetic analysis of the VP1 gene of PSV revealed that VP1 tree is different from ORF tree in topology and tree branches.
4.6 recombinant Virus analysis
The separated strains are subjected to recombination analysis by using SimPlot 3.5.1 and RDP 4.10 software, the similarity analysis of PSV recombinant strain polyprotein genes is carried out by using SimPlot software according to the recombination event junctions identified by potential recombinant strain polyprotein genes and referring to figures 5-8, and the similarity comparison result of the analyzed sequence PSV2019CM and reference sequence ISU-SHIC (KX 810820)/PoSav VIRES HeBo 4C 1 (MK 378925) is shown in figure 5; the PSV gene structure and the recombination breakpoint identification condition of the RDP software on the recombinant strain are analyzed by Bootstrap, and the diagram is shown in FIG. 6; the analysis of the evolutionary tree of the SHCM2019 and the protein coding region of the potential parent strain is shown in FIG. 7, wherein the tree (Potential recombinant parent part) is respectively made according to different parts of the recombination sequence, only the confirmed recombination region is made (i.e. minor parent part), and only the confirmed non-recombination region is made (i.e. major parent part); the P values for nine algorithms in RDP software to detect recombination events are shown in figure 8,Confirmation table, which demonstrates that 9 different methods are used to detect the number of strains that have undergone the recombination event and the degree of compliance with respect to the presently detected recombination event.
In SimPlot FIG. 5, PSV2019CM was subjected to standard similarity node analysis as an independent recombination event to find the presence or absence of recombination event, and as a result, PSV2019CM isolates were highly similar to HeB04 nucleotide sequences in VP1, VP2 and VP3 regions, and ISU-SHIC in the 3D region, as shown in FIGS. 6-7. Subsequently, in RDP 4.10, the recombinant strain was analyzed using nine algorithms RDP, chimaera, bootscan, 3Seq, maxchi, phylpro, GENECONV, LARD and SISCAN to find the potential breakpoint of the recombinant strain, bootscan analysis PSV2019CM isolate had recombination breakpoint in the 3D region, and the evolutionary tree of the recombinant strain was constructed using the orthographic method, the recombination region of the PSV2019CM isolate was located at 5,817-6,352bp, which further validated the recombination region of PSV2019CM. In addition, when the recombinant strain was analyzed using nine algorithms, six of which detected a recombinant signal at a P value <0.05, as shown in fig. 8.
4.7 piglet challenge test of PSV strain
After the toxin is removed, the piglets have poor appetite and listlessness, and then have the symptoms of slight abdominal distension and attached feces around anus. One piglet in the experimental group 1 (low dose group) showed watery diarrhea at 5d after inoculation, while one piglet in the experimental group 2 (high dose group) showed watery diarrhea at 2d after inoculation. All piglets are killed by human in 6d after the toxicity is removed, then, the intestinal canal and the lung of the sick pigs are obviously lesions of the intestinal canal of the piglets inoculated with PSV are blown up, thinned, the intestinal canal content is thin and soft, the lung has local bleeding points, the lung is relatively swollen, and the lesions of the high-dose group are slightly obvious compared with those of the low-dose group. The control group piglets have no obvious clinical symptoms, clean anus, normal intestinal tracts and lungs.
4.8 tissue viral load detection
After the dissection, piglet tissues (heart, liver, intestinal lymph, lung, kidney, brain, duodenum, jejunum, ileum, cecum, colon, rectum) were collected and the viral load in each tissue was detected by qPCR. The results show that the content of the virus in the intestinal tract and the lung is higher, and the content in the cecum, the colon and the rectum is slightly higher than that in the duodenum, the jejunum and the ileum; the virus content in brain and lymph was low, while no virus nucleic acid was detected in heart, liver, kidney (fig. 9).
The PSV2019CM isolate was pathogenic to piglets:
and inoculating 9-day-old piglets with PSV2019CM with different dosages, observing the morbidity of the piglets, collecting anal swabs every day, detecting the viral load in feces by qPCR, and if the piglets are ill, performing pathological observation and nucleic acid quantitative detection on the tissues of the ill piglets. The results show that the water-based diarrhea appears in the 1 st piglet of the experiment 1 group after the inoculation, the water-based diarrhea appears in the 1 st piglet of the experiment 2 group after the inoculation, the water-based diarrhea appears in the 2 nd piglet after the inoculation, all piglets are examined by the 6 th section, and the pathological changes are mainly found in the intestinal tracts and the lungs of the piglets, and the pathological changes are shown as follows: thin intestinal canal, thin and rotten intestinal contents, intestinal villus atrophy and rupture, and intestinal submucosa edema; swelling of the lungs, local bleeding, thickening of alveolar spaces and exudation of serous fluid, inflammatory cell infiltration. PSV is mainly colonized in the intestinal tract and the lung, and there is also a small distribution in the brain and intestinal lymph, but no viral nucleic acid is detected in the heart, liver and kidney. Animals infected with PSV can expel toxin through feces, and the virus load in feces is higher 3-6 days after toxin expelling.
Claims (5)
1. A salpeter virus strain of pig is named PSV2019CM and has a preservation number of CCTCC
NO.V202175。
2. A method for culturing a salpeter virus cell line, comprising the steps of:
inoculating 0.5-1MOI (Multiplicity of infection) porcine sapelovirus PSV2019CM strain to PK15 cells, adsorbing for 1-2h, discarding cell supernatant, adding DMEM culture solution containing TPCK-pancreatin, culturing for 60-72h, and collecting cell culture solution to obtain the porcine sapelovirus cell strain.
3. The method for culturing a porcine sapelo virus cell strain according to claim 2, wherein the collected cell culture fluid is stored below-40 ℃ for later use.
4. Use of porcine sapelo virus PSV2019CM according to claim 1 for the preparation of an inactivated vaccine against porcine diarrhea.
5. A porcine diarrhea inactivated vaccine comprising the porcine sapelo virus strain PSV2019CM of claim 1.
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