CN116162600A - A kind of porcine sapero virus cell strain, its culture method and application - Google Patents

Abstract

A porcine Sapero virus strain, its culture method and application, named PSV2019CM, was deposited in the China Center for Type Culture Collection on November 1, 2021, with the preservation number CCTCCNO.V202175, the full length is 7,567bp, and has a length of It is a 6,996bp ORF, which encodes a polyprotein precursor of 2,331 amino acids, with a 465bp 5′-UTR and a 106bp 3′-UTR on both sides of the genome. The porcine sapero virus of the present invention is used for PSV virus vaccine, has good cross-protection to PSV strains popular in China in recent years, good antigenicity and immunogenicity, and the clinical signs of infected pigs are normal, and the body weight increases Stable, no detoxification phenomenon, high level of PSV antibody can be produced in the body, the OD450 value of serum IgG antibody can reach up to 1.2, and the OD450 level of antibody remains at 1.0 60 days after immunization.

Description

A porcine Sapelo virus cell line, its culture method and application

Technical Field

The present invention belongs to the field of veterinary biological products, and in particular relates to a porcine Sapelo virus cell line, a culture method and an application thereof.

Background Art

Porcine sapelovirus (PSV) belongs to the genus Sapelovirus of the family Picornaviridae. It is a type of non-enveloped single-stranded positive-strand RNA virus, and its virus particles are microspheres with a diameter of about 30 nm. PSV is widely prevalent worldwide and is mainly transmitted through the fecal-oral route. It can cause multi-system syndromes such as diarrhea, pneumonia, encephalitis, and reproductive system disorders in pigs. It may also cause asymptomatic infection. Clinically, it can also cause mixed infection with porcine parvovirus (PPV), classical swine fever virus (CSFV), porcine reproductive and respiratory disorder syndrome virus (PRRSV), porcine enterovirus (PEV), etc., making the morbidity of pigs more complicated, leading to an increase in the mortality rate of pigs, and causing serious harm to the pig industry.

Porcine sapelovirus was originally isolated from the intestines of diarrheal pigs, so it was previously classified as a porcine enterovirus and named porcine enterovirus type 8 (PEV-8). In 2002, Krumbholz et al. pointed out that compared with other enteroviruses, porcine enterovirus type 8 has a unique L protein and a highly specific 2A gene and 5′-untranslated region (5′-UTR) in structure, so it was classified as a new virus genus. In 2014, the International Committee on Taxonomy of Viruses (ICTV) officially named it porcine sapelovirus (PSV) and classified it as the genus sapelovirus of the picornaviridae family. In addition to porcine sapelovirus, this genus also includes avian sapelovirus and simian sapelovirus. So far, all PSV reported in countries around the world belong to the same serotype. Some scholars have pointed out that PSV has two genetic subtypes, but this conclusion has not yet been reached internationally and still needs to be confirmed.

PSV is highly resistant to the environment and can survive for several months at room temperature. It is insensitive to organic solvents such as acetic acid and chloroform and can resist pancreatic enzymes. It is highly heat-resistant and cannot be inactivated by exposure to 56°C for 10 minutes. The minimum condition for heat inactivation is 65°C for 5 minutes or 60°C for 10 minutes. PSV cannot agglutinate or adsorb various animal red blood cells. Sodium hypochlorite, 70% ethanol and ultraviolet light can inactivate it, but ultraviolet light does not affect its antigenicity.

PSV is usually isolated and cultured in porcine cell lines, such as LLC-PK or PK-15, but later scholars discovered (LiY, Du L, Jin T, Cheng Y, Zhang X, Jiao S, et al. Characterization and epidemiological survey of porcine sapelovirus in China; Bai H, Liu J, Fang L, Kataoka M, Takeda N, Wakita T, et al. Characterization of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells) that it can also grow in human cell lines other than porcine cell lines, such as baby hamster cell lines and green monkey cell lines.

According to current research, PLC/PRF/5, HepG2/C3a, 293T, Vero E6, BHK21, PGMKC, ST, IPEC-J2, LLC-PK and PK-15 cells are susceptible to PSV, while A549, MDCK, DF-1, Huh7, WI38, Hela, Hep2c, HCT15 and PCMKC cells are highly resistant to PSV. The difference in sensitivity to PSV is of great significance for the study of PSV receptors, but its infection of different cell lines has raised concerns that its potential host range exceeds pigs.

Since the first report of PSV in the UK in 1958, the virus has been detected in many countries and regions, such as Canada, Japan, Australia, Brazil, Spain, South Korea, China, etc. At present, the research on PSV at home and abroad is mostly focused on molecular epidemiology. According to some studies, the detection rate of PSV in the Czech Republic is 36.6%, in South Korea is 60%, in Zambia is 75.5%, in India is 7.1%, in Hungary is 71%, in Italy is 72.8%, in Spain is 9%, in the United States is 31.8%, and in East China, South China, Sichuan, Hunan, Ningxia in China are 17.2%, 18.21%, 20.4%, 42.21%, 61.25% respectively. The above data show that PSV is widely distributed and has a high infection rate, which is mostly concentrated between 7.1% and 75.5%.

Both domestic pigs and wild boars are susceptible to PSV. Prodělalová J et al. (Prodelalova J. The survey of porcine picorviruses and adenoviruses in fecal samples in Spain) investigated fecal samples collected from the Czech Republic and found that the infection rates of PSV in domestic pigs and wild boars were 33.8% and 27.8%, respectively. PSV is infectious to pigs of all ages, among which pigs aged 4 to 12 weeks are the most sensitive. 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) reported that the PSV positive rate in Italy was 72.8%, of which the infection rate was 98.4% in pigs aged 4 to 12 weeks and 14.3% in pigs over 1 year old.

The results of Harima H et al. (Harima H, Kajihara M, Simulundu E, Bwalya E, Qiu Y, Isono M, et al. Genetic and Biological Diversity of Porcine Sapeloviruses Prevailing in Zambia) in their survey on Zambia showed that the PSV infection rate in pigs aged 0 to 3 weeks was 36.2%, and that in pigs aged 4 to 12 weeks was 94%. The results 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) in their survey on China showed that the PSV infection rate was 46.39%, of which the infection rate in pigs under 4 weeks of age was 4.52%, and that in pigs over 4 weeks of age was 41.87%. Clinically, PSV often causes mixed infection with other viruses. The epidemiological survey results of Son KY et al. (Son KY, Kim DS, Matthijnssens J, Kwon HJ, Park JG, Hosmillo M, et al. Molecular epidemiology of Korean porcinesapeloviruses) on 100 pig diarrhea stool samples from South Korea showed that the PSV positivity rate was 34%, of which 5.9% were PSV infections alone, and 94.1% were PSV mixed infections with other viruses such as PEV, PTV, porcine kobuvirus (PKV), porcine sapovirus (PSaV), etc.

PSV infection can occur in both diarrheal pigs and pigs without clinical symptoms. The survey results 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.) showed that the PSV infection rate was 60%, and there was no significant difference between diarrheal pigs and non-diarrhea pigs. However, 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 Virol 2014, 95: 1603-1611.) showed that the infection rates of PSV in diarrheal pigs and healthy pigs were 48.1% and 17.2%, respectively. Similarly, Li Y et al. (Li Y, Du L, Jin T, Cheng Y, Zhang X, Jiao S, et al. Characterization and epidemiological survey of porcinesapelovirus in China [J]. Vet Microbiol 2019, 232: 13-21.) tested 185 pig feces samples from China from 2016 to 2017, and the results showed that the PSV positive rate was 40.54%, of which the PSV infection rate in diarrheal samples was 44.93%, and in asymptomatic samples it was 27.66%. 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 porcinesapelovirus strain[J]. Arch Virol 2011, 156: 1567-1574.) confirmed through animal regression experiments that PSV can cause diarrhea, respiratory distress, and neurological diseases.

So far, all PSV reported in various places belong to the same serotype. Pigs are the main hosts of PSV, and the main sources of infection are sick pigs, recovered pigs, latently infected pigs, water sources and food contaminated by feces. Among them, recovered pigs and latently infected pigs are often not easy to be found clinically and are important sources of infection. PSV is mainly transmitted through the digestive tract, and can also be transmitted through the respiratory tract and other routes. The feces, respiratory secretions and their pollutants can carry the virus, which makes the pig herd more susceptible to infection. After the virus enters the body, it mainly colonizes in the epithelial cells of the intestinal mucosa and is excreted from the body with feces, resulting in the presence of a large number of the virus in the environment. Pregnant sows infected with PSV can infect the fetus through blood transmission, resulting in embryonic death, stillbirth, mummification and fetal malformations. Clinically, PSV is often accompanied by mixed infections, making the morbidity of the pig herd more complicated, and diagnosis and prevention and control will be more difficult, which will eventually cause a devastating blow to the pig farming industry.

Isolation and culture is the gold standard for virus diagnosis, but virus isolation is a relatively complex process and the isolation cycle is long, so virus isolation is rarely used as a diagnostic standard in simple clinical testing. Molecular biological detection technology has the advantages of rapidity, specificity, and sensitivity, and is the most commonly used method in clinical testing. At present, in the relevant reports on PSV, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP) are mostly used to detect it.

The fundamental measure to prevent and control PSV is prevention. There is currently no vaccine for PSV on the market, so strict control of the breeding environment is particularly important for the prevention and control of PSV. When introducing commercial pigs, strict isolation and quarantine should be carried out. Only after confirming that there is no PSV infection can they be introduced into the pig herd. During the breeding process, a reasonable cleaning and disinfection plan should be formulated, the pig house should be cleaned every day and the pig house environment should be disinfected regularly. The temperature, humidity, and ventilation in the pig house should be well controlled, and adverse factors such as stress should be eliminated. At the same time, appropriate amounts of vitamins can be added to feed or drinking water to improve the immunity of the pig herd. At ordinary times, attention should be paid to observation, and sick and suspicious pigs should be removed immediately, and the disinfection of healthy pigs should be strengthened in a timely manner to prevent the invasion of pathogens.

In addition, we should strengthen the identification of PSV strains and research on their genetic characteristics in my country, and increase basic research and vaccine development, which will provide a scientific basis for the subsequent prevention and control of PSV and is of great significance to the prevention and control of PSV.

Summary of the invention

The object of the present invention is to provide a porcine Sapelo virus cell strain, a culture method and application thereof. The porcine Sapelo virus cell strain was deposited in the China Center for Type Culture Collection on November 1, 2021, with the name PSV2019CM and the preservation number CCTCC NO.V202175. The cell strain belongs to PSV-1 type, and is used for the research and development of PSV virus inactivated vaccine. It has good antigenicity and immunogenicity against the PSV strains prevalent in China in recent years. The infected pigs have normal clinical manifestations, stable weight gain, no detoxification phenomenon, and can produce high levels of PSV antibodies in the body. The OD450 value of serum IgG antibodies can reach up to 1.2, and the OD450 level of antibodies 60 days after immunization is still maintained at 1.0.

In order to achieve the above object, the present invention provides the following technical solutions:

A porcine Sapelo virus cell line, named PSV2019CM, with a deposit number of CCTCCNO.V202175.

The porcine Sapelo virus cell line PSV2019CM of the present invention has a full length of 7,567 bp, a Genbank accession number of MN685785, and has an ORF of 6,996 bp in length, which encodes a polyprotein precursor of 2,331 amino acids, and has a 465 bp 5′-UTR and a 106 bp 3′-UTR on both sides of its genome.

Phylogenetic analysis based on ORF fragments showed that the porcine Sapelo virus strain PSV2019CM had the highest homology with YC2011, with a nucleotide similarity of 90.8% and an amino acid similarity of 98%. The two formed a separate branch, clustered in the cluster of Chinese PSV strains, and were distantly related to other Sapelo viruses. The isolate may be a recombinant strain of HeB04 and ISU-SHIC, with a potential recombination breakpoint upstream of 3D. Analysis of the amino acid mutation site of the VP1 protein showed that there were amino acid mutations in the VP1 region.

A method for culturing a porcine Sapelo virus cell line comprises the following steps:

The porcine Sapelo virus PSV2019CM strain was inoculated into PK15 cells at 0.5-1 MOI. After adsorption for 1-2 hours, the cell supernatant was discarded, and DMEM culture medium containing TPCK-trypsin was added. The cells were cultured for 60-72 hours, and the cell culture medium was collected to obtain the porcine Sapelo virus cell line.

Furthermore, the collected cell culture fluid was stored below -40°C for future use.

The present invention provides the use of the porcine Sapelo virus PSV2019CM in preparing an inactivated vaccine for porcine diarrhea.

An inactivated vaccine for porcine diarrhea, comprising the porcine Sapelo virus strain PSV2019CM.

PSV is mainly colonized in the intestines and lungs, and is also distributed in small amounts in the brain and intestinal lymph, but no viral nucleic acid has been detected in the heart, liver, and kidneys. Animals infected with PSV can excrete toxins through feces, and the viral load in the feces is high 3 to 6 days after the attack. The proliferation of the porcine Sapelo virus PSV2019CM strain on PK15 cells of the present invention depends on pancreatic enzymes. It is a cell-adapted strain, and the lesions mainly occur in the intestines and lungs of piglets. The pathological manifestations are: thinning of the intestinal tract, rotten intestinal contents, atrophy and rupture of intestinal villi, edema of the intestinal submucosa; swelling of the lungs, local bleeding, thickening of the alveolar septa and serous exudation, and infiltration of inflammatory cells. Therefore, the isolated cell strain PSV2019CM provides scientific research materials for subsequent vaccine development.

At present, the research on PSV at home and abroad is limited to epidemiological investigation. Due to the lack of attention, there is no relevant vaccine research on PSV at home and abroad at this stage. However, the selection and breeding of PSV vaccine strains is very important. It can not only carry out the prevention and control of swine diarrhea from a new perspective, but also reserve biological materials in advance for the outbreak of new porcine enterovirus.

Compared with the prior art, the present invention has the following beneficial effects:

The isolated cytotoxic PSV2019CM of the present invention depends on pancreatic enzymes for proliferation on PK15 cells, belongs to a cell-adapted strain, is pathogenic to piglets, is used for the preparation of PSV-related vaccines, has good antigenicity and immunogenicity to the PSV strains prevalent in China in recent years, has normal clinical manifestations in infected pigs, has stable weight gain, does not detoxify, can produce high levels of PSV antibodies in the body, the OD450 value of serum IgG antibodies can reach up to 1.2, and the OD450 level of antibodies 60 days after immunization is still maintained at 1.0.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a cell subculture test result of the PSV isolate in an embodiment of the present invention, wherein M: DL 2,000; G1-G6: PSV2019CM G1-G6.

Figure 2 shows the results of virus isolation by inoculating PK-15 cells with PSV-positive samples in an embodiment of the present invention, where A is the result of cells not inoculated, B is the result at about 30 hours of inoculation, and C is the result at about 70 hours of inoculation.

FIG. 3 is an electron microscopic observation result of imaging suspected PSV virus particles using a transmission electron microscope in an embodiment of the present invention.

Figure 4 is the analysis result of the evolutionary relationship between the protein coding regions of the PSV2019CM isolate and other representative strains at home and abroad in the embodiments of the present invention.

Figures 5-8 are the results of recombination event analysis based on the identification of the polyprotein gene of potential recombinant strains in the embodiments of the present invention, wherein Figure 5 is the similarity analysis result of the polyprotein gene of the PSV recombinant strain using SimPlot software; Figure 6 is the PSV gene structure and the identification of the recombination breakpoints of the recombinant strain by RDP software; Figure 7 is the evolutionary tree analysis of the protein coding region of SHCM2019 and its potential parent strains; Figure 8 is the P value of the nine algorithms in the RDP software for detecting recombination events.

FIG9 is a comparison of viral loads in various tissues in an embodiment of the present invention, wherein Log (RNA copies/ml) represents the logarithm of the number of viral copies per milliliter of a tissue sample.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific embodiments.

DL2000 DNA marker, ExTaq DNA polymerase, Trizol, Reverse Transcriptase M-MLV (RNase H-), 5×Reverse Transcriptase M-MLV Buffer, Recominant RNase Inhibitor, and dNTP Mixture (2.5 mM each) were all purchased from TaKaRa; PSV monoclonal antibody was purchased from Shandong Ludu Co., Ltd.; other conventional reagents were all domestic analytical grade products.

Example: Acquisition of PSV epidemic strain PSV2019CM

In this example, PSV detection was performed on diarrhea samples collected in Shanghai, and one PSV epidemic strain was successfully isolated. The 100th generation PSV epidemic strain PSV2019CM was obtained by subculture and screening, which had good antigenicity and immunogenicity, providing scientific research materials for subsequent vaccine development.

1. Sample source

55 fecal samples of piglets with diarrhea were collected from a pig farm in Shanghai, and 5 intestinal tissue samples of piglets with diarrhea were collected from a pig farm in Chongming, Shanghai.

2. Primer Design

A pair of PCR detection primers were designed and synthesized for the PSV 5’UTR gene. The specific sequences are shown in Table 1.

Table 1

3. Experimental Methods

3.1 Sample processing and RT-PCR detection

Diarrhea stool: Collect diarrhea stool with a cotton swab and place it in a 10ml centrifuge tube. Add 1ml of sterile PBS, let it stand for 30 minutes after it is fully dissolved, aspirate the supernatant into a new centrifuge tube, and store it at -80℃.

Intestinal tissue samples: scrape the intestinal mucosal contents into a centrifuge tube, add an equal amount of PBS, mix thoroughly, let stand for 30 minutes, aspirate the supernatant into a new centrifuge tube, and store at -80℃.

According to the kit instructions, 250 μl of the treated sample was taken for total RNA extraction, and finally 20 μl of H ₂ O was used to dissolve the RNA, and reverse transcription was performed using PSV-R primers. Then, 5 μl of cDNA was taken for PCR detection. The PCR reaction system was: 94°C for 4 min, 94°C for 35 s, 51°C for 35 s, 72°C for 45 s (30 cycles), and 72°C for 8 min.

The PCR products were detected by 1.5% agarose gel electrophoresis.

3.2 Cloning and sequencing of PCR products

After the PCR product of the correct size was recovered from the gel, it was connected to the pMD-18T Vector and transformed. The positive clones were double-identified by the plasmid extraction method and bacterial liquid PCR method, and sent to Shanghai Bioengineering Co., Ltd. for sequencing.

Lasergene7.1 software was used to compare and analyze the sequences.

3.3 Virus proliferation and identification

PK15 cells with good growth status were selected, digested with trypsin and plated in 6-well plates. On the second day, the cell supernatant was discarded, washed twice with PBS, and then DMEM virus culture medium and sample treatment solution containing different concentrations of trypsin (0.1-1μg/ml TPCK-trypsin) were added respectively; after adsorption for 2h, the supernatant was discarded, and then DMEM culture medium containing a certain concentration of trypsin was added, and cultured in a 37℃ incubator to observe the cell pathological changes. If there was no pathological change, the virus was collected after 5d; after freezing and thawing twice, the above process was repeated again for continuous subculture.

Take 250 μl of cell culture at each generation and perform RT-PCR detection to verify whether the virus passage is successful.

3.4 Indirect immunofluorescence detection

The third generation of PSV2019CM was inoculated into PK15 cells, and after adsorption for 1 hour, the cells were replaced with virus culture medium and continued to be cultured at 37°C. After 72 hours, the cell supernatant was discarded, washed once with PBST, fixed on ice with 70% ethanol for 15 minutes; washed 3 times with PBST and dried, added with 1:1000 diluted PSV monoclonal antibody, and incubated in a 37°C wet box for 1 hour; washed as above, added with 1:3000 diluted FITC-labeled goat anti-mouse IgG, and incubated at 37°C for 30 minutes; after washing, the cells were observed under an inverted fluorescence microscope (Axio Observer).

3.5 TCID ₅₀ determination

PK15 cells were plated in 96-well cell plates 24 hours in advance, and cytotoxic PSV2019CM of different generations was diluted 10-fold and then added to each well of the cells, 100 μl/well, and each dilution was repeated 8 wells. After adsorption for 1 hour, the supernatant was discarded, and PSV virus culture medium was added, 200 μl/well, and then placed in a 37°C 5% CO ₂ incubator for continued culture.

Then the TCID ₅₀ was determined by indirect immunofluorescence test: after 120 hours of culture, the supernatant was discarded, the cells were washed once with PBS, 100 μl of 75% ethanol was added to each well, and the cells were fixed at -20°C for 15 minutes; the fixative was discarded, the cells were washed three times with PBS, and then a 1:1500 dilution of PSV monoclonal antibody was added, and the cells were incubated in a 37°C wet box for 1 hour; the supernatant was discarded, the cells were washed as above, and a 1:3000 dilution of FITC-labeled goat anti-mouse IgG was added, and the cells were incubated in a 37°C wet box for 30 minutes; the supernatant was discarded, the cells were washed as above, and then 100 μl of PBS was added to each well, and the cells were observed under a fluorescence microscope. The TCID ₅₀ was calculated according to the Reed-Muench method.

3.6 Recombinant virus analysis

Reference information (Meng Li. Establishment of a multiplex PCR detection method for swine diarrhea pathogens and analysis of the evolution of major viruses [D]: Shanghai Ocean University; 2017), 23 pairs of primers were designed (see Table 2), the whole genome sequence of the obtained PSV2019CM strain was determined, and genetic evolution analysis was performed. The whole genome recombination analysis was further performed using RDP4.1 recombination software. In addition, the 5’ RACE Kit was used to determine its 5’ terminal sequence.

Table 2 Primers for PSV SHCM2019 whole genome amplification

3.7 Animal experiments

3.7.1 Generation screening of strains

Six 5-day-old PSV-negative piglets were randomly divided into 3 groups and observed for 3 days after grouping to allow the piglets to adapt to the new environment and determine their health. No food or water was allowed for 2 hours before the challenge. After the challenge, all piglets were guaranteed sufficient drinking water and fed commercial feed. The specific grouping and challenge conditions are shown in Table 3:

Table 3 Animal groups and treatments

After the virus attack, the clinical manifestations of the piglets were observed, including eating, drinking, spirit, feces, diarrhea, etc., and anal swabs were collected from the piglets every day to detect the viral load in the feces. If the piglets became ill, the time and condition of the onset were recorded. Six days after the virus attack, all piglets were dissected, and tissues such as brain, heart, liver, lung, kidney, duodenum, jejunum, ileum, blind, colon, rectum and intestinal lymph were collected for quantitative nucleic acid detection, and the intestines and lung tissues of the sick pigs were fixed with 4% paraformaldehyde, and then immediately sent to Shanghai Weiao Biotechnology Co., Ltd. for sectioning and HE staining.

3.7.2 Fluorescence quantitative PCR

Take 100 mg of the animal tissue collected in 3.2.1, add 1 mL of sterile PBS and grind it, centrifuge it at 4°C 4,000 rpm for 10 min, and then take 250 μL of supernatant into a new RNase-free EP tube. Resuspend the anal swabs collected daily with 1 mL of PBS, vortex and mix, and take 250 μL of supernatant into an RNase-free EP tube after centrifugation. The total viral nucleic acid in the supernatant of the diseased material was extracted using the TRIzol method, and the extraction concentration was determined using a nucleic acid protein analyzer. According to the PrimeScript ^TM RT manual, genomic DNA removal and subsequent reverse transcription reaction were performed, and then the cDNA template chain was absolutely quantitatively determined using qPCR technology. The primers used to detect the number of viral copies in this embodiment are a pair of specific primers PSV-1/PSV-2 designed by Chen J et al. for PSV 5′-UTR (see Table 4).

Table 4 PSV fluorescence quantitative PCR amplification primers

Premix Ex Taq^TMII说明书对已知浓度的重组质粒和反转录为cDNA的病料样品进行qPCR检测，每样品3复孔。本试验利用ABI 7500荧光定量PCR仪进行数据测定分析，标准曲线及样品中病毒拷贝数的计算公式为：“Ct＝斜率*lgx+截距”。具体计算方法是先把各稀释度质粒浓度及其Ct值代入以获得标准曲线，之后将测得的各样品的Ct值分别代入标准曲线所在公式，利用线性外推法获得样品中的病毒拷贝数。Blunt-zero plasmid containing PSV 5′-UTR fragment was diluted 10 times and its concentration was measured.

Premix Ex Taq ^TM II Instructions: qPCR detection was performed on recombinant plasmids of known concentrations and disease samples reverse transcribed into cDNA, with 3 replicates per sample. This experiment used ABI 7500 fluorescence quantitative PCR instrument for data measurement and analysis, and the standard curve and the calculation formula for the number of viral copies in the sample were: "Ct = slope * lgx + intercept". The specific calculation method was to first substitute the concentration of each dilution plasmid and its Ct value to obtain the standard curve, and then substitute the measured Ct values of each sample into the formula of the standard curve, and use linear extrapolation to obtain the number of viral copies in the sample.

Each well was coated with 100TCID50PSV virus at 4℃ overnight, the solution in the well was removed, 200μl PBST washing solution was added to each well, shaken on a shaker for 3min, poured out, patted dry, and washed 5 times; 250μl PBST blocking solution containing 5% skim milk was added to each well, blocked in a 37℃ incubator for 2h, poured out, and washed 5 times; serum samples were diluted with PBS, 100μl samples were added to each well, incubated in a 37℃ incubator for 1.5h, poured out, and washed 5 times; 100μl PSV monoclonal antibody (1:2000 dilution) was incubated in a 37°C incubator for 1 hour, the supernatant was discarded, and the cells were washed 5 times. 100 μl of enzyme-labeled secondary antibody diluted in PBST was added to each well, the cells were incubated in a 37°C incubator for 1 hour, the cells were discarded, and the cells were washed 5 times. 50 μl of color developing solution prepared by mixing solution A and solution B in a ratio of 1:1 was added to each well, and the cells were developed at 37°C in the dark for 10 minutes. 50 μl of stop solution was added to each well, and the results were determined within 10 minutes.

4. Results

4.1 RT-PCR test results of samples

The results of RT-PCR of the first six generations of viruses are shown in Figure 1. Generations G1 to G6 all had a single band, and no band was amplified from uninoculated cells. Further sequencing results confirmed that the amplified fragment was PSV, with a size of 624 bp.

4.2 Virus isolation

PSV-positive samples were inoculated into PK-15 cells for virus isolation, and CPE was first observed when the cells were blindly propagated to the G3 generation. As shown in Figure 2, the uninoculated cells grew normally (A), and some cells became round, shrunken, and slightly fell off at about 30 hours after inoculation (B). At about 70 hours after inoculation, most cells had completely detached from the cell bottle (C).

4.3 Electron microscopy observation

To determine the morphological characteristics of the virus, transmission electron microscopy was used to image and observe the suspected PSV virus particles. The electron microscopy observation of the negatively stained sample is shown in Figure 3. As can be seen from Figure 3, the virus particles are microspherical, without an envelope, and have a diameter of about 30 nm, which is consistent with previous reports (Li Y, Du L, Jin T, Cheng Y, Zhang X, Jiao S, et al. Characterization and epidemiological survey of porcine sapelovirus in China [J]. Vet Microbiol 2019, 232: 13-21), and no other virus-like particles were observed, indicating that the virus was successfully isolated.

4.4 Determination of virus titer

The cell culture fluids of the isolates from generations G3 to G6 were collected, diluted 10-fold and inoculated into PK-15 cells for TCID50 determination. The results are shown in Tables 2-4. The TCID50 of the isolates increased with the number of passages, reaching a maximum of 10 ^6.6 1TCID50/mL at the 5th generation.

Table 5 TCID ₅₀ determination results of isolated strains

4.5 Whole genome sequence information

The cDNA of PSV2019CM isolate was used as a template, and the whole genome of the virus was amplified using specific primers. Each sequencing fragment was verified by BLAST. After confirming that the sequencing was correct, each fragment was sheared and spliced using DNAstar. Finally, the full length of the isolate was 7,567bp, with an ORF of 6,996bp in length, and a 465bp 5′-UTR and a 106bp 3′-UTR on both sides of the genome. The sequence has been uploaded to NCBI, and the specific information can be viewed by looking up the sequence number of Genbank No. MN685785.

4.6 Phylogenetic analysis of isolates

MEGA7.0 was used to analyze the evolutionary relationship of the protein coding regions of PSV2019CM isolates and other representative strains at home and abroad. The results are shown in Figure 4. PSV2019CM has the closest evolutionary relationship with YC2011 isolates and is clustered in the Chinese PSV group; Chinese PSV isolates are most closely related to Korean PSV isolates and are more distantly related to European isolates; PSV isolates from various countries are clustered in the same group and have obvious grouping with other species of Sapelo virus. Further, DNAstar was used to analyze the nucleotide and amino acid similarities of the above strains. The results showed that PSV2019CM had a high homology with YC2011 isolates, with a nucleotide similarity of 90.8% and an amino acid similarity of 98%; the nucleotide similarity between Chinese PSV strains and Korean PSV strains was 87.2% to 89.8%, and the nucleotide similarity with European PSV strains was 78.7% to 86.4%. In addition, phylogenetic analysis of the VP1 gene of PSV showed that the topological structure and evolutionary tree branches of the VP1 tree were different from those of the ORF tree.

4.6 Recombinant virus analysis

The recombinant analysis of the isolates was performed using SimPlot 3.5.1 and RDP 4.10 software. The results of the recombination events identified based on the polyprotein gene of the potential recombinant strains are shown in Figures 5-8. The similarity analysis of the polyprotein gene of the PSV recombinant strain was performed using SimPlot software. The similarity comparison results of the analyzed sequence PSV2019CM and the reference sequence ISU-SHIC (KX810820)/PoSav VIRESHeBo4 C1 (MK378925) are shown in Figure 5. Bootstrap analysis of the PSV gene structure and the identification of the recombination breakpoints of the recombinant strains by RDP software are shown in Figure 6. The phylogenetic tree analysis of the protein coding region of SHCM2019 and its potential parent strains is shown in Figure 7, in which trees were drawn according to different parts of the recombinant sequence (Potential recombinant parent part), only the confirmed recombinant region was drawn (i.e., minor parent part), and only the confirmed non-recombinant region was drawn (i.e., major parent part). patent part); the P values of the nine algorithms in the RDP software for detecting recombination events are shown in Figure 8, and the Confirmation table shows the number of strains that have detected the recombination event using the nine different methods and the degree of conformity with the currently detected recombination events.

In SimPlot Figure 5, PSV2019CM was treated as an independent recombination event and subjected to standard similarity node analysis to find out whether a recombination event occurred. The results are shown in Figures 6-7. The PSV2019CM isolate was highly similar to the HeB04 nucleotide sequence in the VP1, VP2 and VP3 regions, and highly similar to ISU-SHIC in the 3D region. Subsequently, in RDP 4.10, nine algorithms, including RDP, Chimaera, Bootscan, 3Seq, Maxchi, Phylpro, GENECONV, LARD and SISCAN, were used to analyze the recombinant strains to find potential breakpoints of the recombinant strains. Bootscan analysis showed that the PSV2019CM isolate had a recombination breakpoint in the 3D region, and the evolutionary tree of the recombinant strain was constructed using the neighbor-joining method. The recombination region of the PSV2019CM isolate was located at 5,817-6,352bp, which further verified the recombination region of PSV2019CM. In addition, when the recombinant strains were analyzed using nine algorithms, six of them detected the recombination signal when the P value was < 0.05, as shown in Figure 8 .

4.7 PSV strain challenge test in piglets

After the virus attack, the piglets had poor appetite and were listless. Subsequently, they had mild abdominal distension and symptoms of feces attached around the anus. A piglet in Experimental Group 1 (low-dose group) developed watery diarrhea on the 5th day after inoculation, while a piglet in Experimental Group 2 (high-dose group) developed watery diarrhea on the 2nd day after inoculation. All piglets were artificially killed on the 6th day after the virus attack. Subsequently, they were autopsied and observed, and it was found that the intestinal and lung lesions of the sick pigs were obvious. The intestines of the piglets inoculated with PSV were inflated, thinned, and translucent. The intestinal contents were soft and thin, and there were local bleeding spots in the lungs. The lungs were swollen, and the lesions in the high-dose group were slightly more obvious than those in the low-dose group. The piglets in the control group had no obvious clinical symptoms, the area around the anus was clean, and the intestines and lungs were normal.

4.8 Tissue viral load detection

After autopsy, 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 showed that the viral content in the intestine and lung was high, and the content in the cecum, colon, and rectum was slightly higher than that in the duodenum, jejunum, and ileum; the viral content in the brain and lymph was low, and no viral nucleic acid was detected in the heart, liver, and kidney (Figure 9).

PSV2019CM isolate is pathogenic to piglets:

Different doses of PSV2019CM were used to inoculate 9-day-old piglets, and the incidence of the piglets was observed. Anal swabs were collected every day, and the viral load in the feces was detected by qPCR. If any piglets were sick, pathological observation and nucleic acid quantitative detection were performed on the sick piglet tissues. The results showed that one piglet in experimental group 1 had watery diarrhea on the 5th day after inoculation, and one piglet in experimental group 2 had watery diarrhea on the 2nd day after inoculation. All piglets were dissected on the 6th day, and it was found that the lesions mainly occurred in the intestines and lungs of the piglets. The pathological manifestations were: thinning of the intestinal tract, rotten intestinal contents, atrophy and rupture of intestinal villi, edema of the intestinal submucosa; swelling of the lungs, local bleeding, thickening of the alveolar septa and serous exudation, and infiltration of inflammatory cells. PSV is mainly colonized in the intestines and lungs, and there is also a small amount of distribution in the brain and intestinal lymph, but no viral nucleic acid was detected in the heart, liver, and kidneys. Animals infected with PSV can excrete toxins through feces, and the viral load in the feces is high 3 to 6 days after the virus is attacked.

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.

Images