Application of CD81 as a marker or target for PRRS or PRRSV

Abstract

The present application discloses the application of CD81 as a marker or target of PRRS or PRRSV, which provides assistance for detecting PRRS or PRRSV, provides a new target for preventing or treating PRRS or PRRSV, and provides technical support for breeding and screening new pig breeds (lines) resistant to PRRS or PRRSV.

Description

Technical Field

[0001] The present application relates to the technical field of PRRS or PRRSV, and specifically to the application of CD81 as a marker or target of PRRS or PRRSV. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the infectious diseases that poses the greatest threat to the healthy development of the global pig industry. Its pathogen is porcine reproductive and respiratory syndrome virus (PRRSV), which mainly infects pig alveolar macrophages, destroys the immune system, and causes immunosuppression, similar to the human immunodeficiency virus (HIV), so it is called the "pig HIV". PRRSV mainly causes reproductive disorders in sows, respiratory symptoms in pigs of different ages, especially piglets, and secondary infections such as Haemophilus parasuis and Streptococcus infections, causing huge economic losses to the global pig industry.

[0003] The biggest difference between PRRSV and other viruses is that it mainly infects pig alveolar macrophages and destroys the immune system. The virus has the following characteristics: extremely easy mutation and recombination, immunosuppression, persistent infection, and resistance-dependent enhancement (ADE), which makes the prevention and control of the disease very difficult and has become one of the most difficult diseases to prevent and control in the pig industry. Based on the above characteristics of the virus, the vaccine developed from the perspective of pathogen (PRRSV) has the following problems: slow or no induction of neutralizing antibodies, poor cross-protection against heterologous strains, long viremia of vaccine virus, easy recombination with field strains to produce recombinant viruses with stronger pathogenicity, resulting in the inability of vaccines to control the occurrence and spread of PRRS, and there is no effective antiviral drug. Therefore, it is necessary to conduct research from the perspective of host (pig) genetics, improve the host's resistance to PRRS from a genetic perspective, and cultivate new disease-resistant pig breeds (lines) by discovering PRRSV resistance / susceptibility genes, or screen antiviral drugs targeting susceptible genes. Currently, there are few reports on original molecular targets with significant breeding value, which seriously hinders the development of PRRS disease resistance breeding.

[0004] Biological breeding is the core of seed industry innovation. Cultivating strategic new varieties and achieving self-reliance in seed industry science and technology are the top priorities for solving the key problem of "seed sources" and winning the seed industry turnaround battle. Discovering original target genes with independent intellectual property rights is the basis and key to molecular disease resistance breeding.

[0005] Since genetic background is closely related to biological disease resistance, research from the perspective of host genetics, improving host resistance to PRRS at the genetic level, using genetic means to screen blue ear disease-resistant pigs, and finding genes related to PRRSV host resistance or susceptibility have incomparable advantages for the prevention and control of porcine reproductive and respiratory syndrome. It can not only study the infection mechanism of porcine reproductive and respiratory syndrome, but also provide new methods for the treatment and prevention of porcine reproductive and respiratory syndrome. Currently, there are few reports on original resistance molecular targets with great breeding value, which seriously hinders the development of PRRS disease-resistant breeding. By discovering PRRSV host resistance / susceptibility genes, and then breeding and screening new disease-resistant (PRRS) pig breeds (lines), exploring more related genes is of great significance for molecular resistance to porcine reproductive and respiratory syndrome. Summary of the invention

[0006] The application of CD81 as a marker or target of PRRS or PRRSV in this application provides assistance for the detection of PRRS or PRRSV, provides a new target for the prevention or treatment of PRRS or PRRSV, and also provides technical support for the breeding and screening of new disease-resistant (PRRS) pig breeds (lines).

[0007] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0008] In one aspect, the embodiment discloses a marker for a porcine reproductive and respiratory syndrome in vitro sample, wherein the marker is CD81.

[0009] In one aspect, the embodiment discloses a kit for detecting porcine reproductive and respiratory syndrome in vitro samples, which comprises a primer pair as shown in SEQ ID NO. 1 and 2.

[0010] In one aspect, the embodiment also discloses a kit for detecting porcine reproductive and respiratory syndrome virus, which comprises a primer pair as shown in SEQ ID NO. 1 and 2.

[0011] In some embodiments, the kit further comprises an RNA extraction reagent, a cDNA synthesis reagent, and a PCR amplification reagent. The RNA extraction reagent comprises: TRIZOL reagent, chloroform and isopropanol. The cDNA synthesis reagent comprises 1 μL of random primer Oligo (dT), 4 μL of 5× buffer, 1 μL of RNase inhibitor (20U / μL), 2 μL of dNTP Mix (10mM), 1 μL of M-MuLV reverse transcriptase (200U / μL), and the cDNA is stored at -20°C for standby use after synthesis. The PCR amplification reagent comprises a primer pair as shown in SEQ ID NO.1 and 2, dNTP, DNA polymerase and 10×PCR buffer.

[0012] On the one hand, the embodiment also discloses a kit for detecting porcine reproductive and respiratory syndrome virus in vitro samples, which includes a CD81 antibody and a secondary antibody.

[0013] On the one hand, the embodiment also discloses a kit for detecting porcine reproductive and respiratory syndrome virus, which includes a CD81 antibody and a secondary antibody.

[0014] In some embodiments, the primary antibody is CD81 antibody (ab155760, abcam) or a diluent thereof. The secondary antibody is goat anti-rabbit IgG (H+L) Alexa Flour 488 labeled secondary antibody or a diluent thereof.

[0015] On the one hand, the embodiments also disclose the use of CD81 as a target for preventing or treating porcine reproductive and respiratory syndrome in the preparation of an agent for preventing or treating porcine reproductive and respiratory syndrome.

[0016] In one aspect, the embodiments also disclose a drug for preventing or treating porcine reproductive and respiratory syndrome, comprising a vector overexpressing CD81.

[0017] In one aspect, the embodiment also discloses a drug for inhibiting porcine reproductive and respiratory syndrome virus, wherein the drug comprises a vector overexpressing CD81.

[0018] On the one hand, the embodiments also disclose the use of CD81 in breeding pig breeds resistant to porcine reproductive and respiratory syndrome.

[0019] In some embodiments, the application includes determining whether the pig individual is resistant or susceptible to porcine reproductive and respiratory syndrome virus based on the expression level of CD81 in the pig individual.

[0020] In these embodiments, the term "in vitro sample" refers to at least one of a pig blood sample, a pig saliva sample, a pig sweat sample, a pig urine sample, a pig throat swab sample, a pig milk sample, a pig semen sample, a pig skin swab sample, a pig feces sample, and a pig sputum sample that contains or is suspected of containing PRRSV, or refers to at least one of a pig blood sample, a pig saliva sample, a pig sweat sample, a pig urine sample, a pig throat swab sample, a pig milk sample, a pig semen sample, a pig skin swab sample, a pig feces sample, and a pig sputum sample that is infected with or suspected of having PRRS.

[0021] Compared with the prior art, this application has the following beneficial effects:

[0022] This application discovered for the first time that the expression of the four-transmembrane protein CD81 is inhibited after pigs are infected with the PRRSV virus, and the expression of CD81 will decrease significantly as the infection time increases. After further overexpression of CD81, it was shown that the CD81 protein level increased significantly, while the PRRSV-N protein expression level decreased significantly, and as the CD81 concentration increased, the degree of inhibition of the PRRSV-N protein became more obvious, indicating that CD81 inhibited PRRSV replication in a dose-dependent manner, and overexpression of CD81 could inhibit PRRSV replication. Silencing CD81 significantly promoted PRRSV replication. Therefore, promoting CD81 expression can improve pigs' resistance to blue ear virus, and CD81 can be used as a criterion for determining PRRSV susceptibility or resistance. Pigs with abnormally high CD81 expression levels are resistant to PRRSV, and vice versa. This application provides a new application of CD81. By discovering new disease resistance / susceptibility genes of PRRSV hosts, it is conducive to the breeding and screening of more new disease-resistant pig breeds (lines), laying a solid foundation for identifying pigs' susceptibility to PRRSV and the screening, identification and breeding of PRRSV-resistant pigs, which is conducive to the breeding of excellent pig strains and is of great significance for the prevention and control of porcine reproductive and respiratory syndrome.

[0023] In addition, the CD163 gene is the decisive receptor for PRRSV invasion, and edited pigs with this gene knocked out can completely resist PRRSV infection. The CD163 gene is the most valuable PRRSV resistance gene discovered so far, and it is of great significance in screening / identifying / breeding new varieties (lines) resistant to blue ear disease. CD81 and CD163 belong to the same CD molecule and have many similarities in biological functions. Therefore, CD81 can be used as an important molecular target for PRRS disease resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The expression levels of CD81 and PRRSV-N proteins in Marc-145 cells infected with PRRSV at different time points.

[0025] Figure 2 The expression levels of CD81 and PRRSV-N proteins after Marc-145 cells were infected with different multiplicity of infection (MOI) of PRRSV.

[0026] Figure 3 The expression levels of PRRSV-N protein at different time points after PRRSV infection after CD81 overexpression.

[0027] Figure 4 The effect of CD81 overexpression on PRRSV virus titer (plaque assay).

[0028] Figure 5This is the effect of CD81 overexpression on PRRSV-N protein expression (dose-dependent).

[0029] Figure 6 This is the effect of CD81 knockdown on PRRSV-N protein expression.

[0030] Figure 7 This is the effect of CD81 knockdown on the expression of PRRSV-N protein mRNA. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0032] Detection of CD81 gene expression

[0033] The embodiment provides a detection kit for in vitro samples infected with PRRS or PRRSV. The kit includes a primer pair as shown in SEQ ID NO.1 and 2. Specifically, the kit is an RT-PCR kit. The kit also includes: RNA extraction reagent, cDNA synthesis reagent, PCR amplification reagent. Among them, the RNA extraction reagent includes: TRIZOL reagent, chloroform and isopropanol. The cDNA synthesis reagent includes 1μL of random primer Oligo (dT), 4μL of 5× buffer, 1μL of RNase inhibitor (20U / μL), 2μL of dNTP Mix (10mM), 1μL of M-MuLV reverse transcriptase (200U / μL), and the cDNA is stored at -20°C for standby use after synthesis. The PCR amplification reagent includes a primer pair as shown in SEQ ID NO.1 and 2, dNTP, DNA polymerase and 10×PCR buffer.

[0034] Example RNA was extracted from in vitro samples infected with PRRS or PRRSV (e.g., Marc-145 cells infected with PRRS or PRRSV), cDNA was synthesized, and the expression of tetraspanin CD81 (Tetraspanin CD81, CD81, NM_001078679.1, NP_001072147.1) was detected using primers as shown in SEQ ID NO.1 and 2. It was found that as the virus infection multiplicity (MOI) increased, the expression level of CD81 protein decreased.

[0035] In some embodiments, Marc-145 cells (Shine Bio) were cultured in DMEM culture medium containing 10% fetal bovine serum, inoculated with the highly pathogenic PRRSV strain Li11 at an MOI of 0.5, and continued to be cultured at 37°C in DMEM culture medium containing 2% fetal bovine serum. The cells were collected after 12, 24, and 36 hours of culture at different time points as test samples for RT-PCR and Western-blot detection.

[0036] The RT-PCR detection process provided in some embodiments includes: taking 250 μL of cell fluid from 12h, 24h, and 36h, adding 750 μL of Trizol, mixing, and standing at room temperature for 5min; adding 0.2ml of chloroform to each 1mL of the sample lysed by the TRIZOL reagent, shaking vigorously for 15s, and incubating at 25°C for 2min. Centrifuge at 4°C and 12000rpm for 15min. After centrifugation, it will be divided into three layers: the lower red phenol chloroform phase, the middle layer, and the colorless upper aqueous phase, and all RNA is distributed in the aqueous phase; transfer the upper aqueous phase to a clean RNase-free centrifuge tube, add an equal volume of isopropanol to precipitate RNA, mix well, incubate at 25°C for 10min, and centrifuge at 4°C and 12000rpm for 10min. At this time, the RNA precipitate that was invisible before centrifugation will form a gelatinous precipitate on the bottom and side walls of the tube.

[0037] The RT-PCR detection process provided in some embodiments also includes: the reaction system is 20 μL RNA (the viral RNA obtained in step 1 above), 2 μL universal primers are added, the reaction is placed in a 70°C water bath for 5 minutes, an ice water bath is placed for 5 minutes, 5×Buffer 8 μL, dNTPMixture 4 μL, RNase Inhibitor 1 μL, reverse transcriptase 1 μL are added, RNase free H2O is added to make the volume 40 μL, and the reaction is carried out at 42°C for 1 hour.

[0038] The RT-PCR detection process provided in some embodiments also includes: template cDNA 2 μL, 10×PCR buffer 2 μL, dNTP (2.5 mmol / L) 1.5 μL, upstream and downstream primers 25 mmol / L 0.5 μL each, rTaq DNA polymerase 0.5 μL, ddH2O 13 μL; reaction conditions: 95°C / 5 min, 94°C / 1 min, 50°C / 30 s, 72°C / 30 s, 35 cycles; finally 72°C / 10 min. The upstream primer-F of the CD81 gene is used: 5′-TACATCCTCATTGCCGTGGG-3′ (SEQ ID NO.1); the downstream primer-R: 5′-CATCCTTGGCAATCTGGTCCT-3′ (SEQ ID NO.2). GAPDH gene internal reference primers: upstream primer-F: 5′-TGACAACAGCCTCAAGATCG-3′ (SEQ ID NO. 3); downstream primer-R: 5′-GTCTTCTGGGTGGCAGTGAT-3′ (SEQ ID NO. 4).

[0039] The Western-blot detection process provided in some embodiments includes: preparing protein samples, and the principles of protein sample preparation are: ① extract as completely as possible or reduce the complexity of the sample to focus on extracting the target protein; ② ensure that the protein is in a soluble state; ③ prevent protein degradation, aggregation, precipitation, modification, etc. during the extraction process; ④ remove interfering molecules such as nucleic acids, polysaccharides, lipids, etc. as much as possible; ⑤ sample packaging and storage at -80°C to avoid repeated freezing and thawing. The protein samples were subjected to gel electrophoresis, and the bands were separated and then transferred to the membrane and blocked. CD81 antibody (sc-166029, proteintech) was diluted to a concentration of 1:1000 and incubated. GAPDH antibody (RM2002L, Beijing Ruikang Biotechnology Co., Ltd.) or α-Tubulin antibody (66031-1-lg, proteintech) was used as an internal reference at 4°C overnight. The washed membrane was incubated in secondary antibody (goat anti-rabbit IgG (H+L) Alexa Flour488 labeled secondary antibody) at room temperature for two hours. After washing, ECL chemiluminescence and exposure development were performed, and grayscale value analysis was performed using Quality One software.

[0040] like Figure 1 As shown, gel electrophoresis was performed on the amplified products obtained by RT-PCR, and the expression level of CD81 protein was significantly reduced 12, 24, and 36 hours after PRRSV infection.

[0041] like Figure 2 As shown, WB detection was performed on proteins extracted from the cell fluid at 12h, 24h, and 36h. As the virus infection multiplicity (MOI) increased, the decrease in CD81 protein expression level became more obvious.

[0042] CD81 gene overexpression

[0043] On the one hand, the embodiment also discloses a drug for inhibiting porcine reproductive and respiratory syndrome virus, the drug comprising a vector overexpressing CD81. By injecting the vector overexpressing CD81 into cells or individuals infected with porcine reproductive and respiratory syndrome virus, the replication of porcine reproductive and respiratory syndrome virus can be effectively inhibited.

[0044] On the one hand, the embodiment also discloses a drug for preventing or treating porcine reproductive and respiratory syndrome, the drug comprising a vector overexpressing CD81. By injecting the vector overexpressing CD81 into blue ear disease pigs, the replication of porcine reproductive and respiratory syndrome virus can be effectively inhibited.

[0045] Some embodiments provide a construction process of the vector for overexpressing CD81. The process includes: amplifying the CD81 gene CDS sequence (SEQ ID NO.5) with the primer pair shown in SEQ ID NO.6 and SEQ ID NO.7; inserting the CDS sequence into pcDNA3.1 (V79020, ThermoFisher), transforming the ligation product into Escherichia coli DH5α competent cells; screening positive clones, and obtaining the vector for overexpressing CD81 by colony PCR and sequencing.

[0046] Some test cases measured the inhibitory effect of the vector overexpressing CD81 on the replication of porcine reproductive and respiratory syndrome virus. Specifically, cells were cultured in two 6-well plates with DMEM culture medium containing 10% fetal bovine serum to 80% density, and one 6-well plate was used with Lipofectamine TM 3000 (purchased from Thermo Fisher Scientific) were used to transfer pcDNA3.1-CD81-myc and pcDNA3.1-myc empty vectors into cells, respectively, and cultured in a 37°C, 5% CO2 incubator for 6 h. The supernatant was discarded, the cells were washed once with PBS, and cultured for another 24 h with DMEM culture medium containing 10% fetal bovine serum. Cell samples were collected, and the expression of CD81 was detected by using the WB kit provided in the above embodiment, and the virus titer of the cell supernatant was observed by a plaque assay.

[0047] like Figure 3 As shown, after CD81 overexpression, the CD81 protein level was significantly increased (using myc tag antibody), and the PRRSV-N protein expression level was significantly decreased at different time points after PRRSV infection.

[0048] like Figure 4 As shown, after CD81 overexpression, the viral titer in the cell supernatant was significantly reduced.

[0049] like Figure 5 As shown, with the increase of pcDNA3.1-CD81-myc plasmid concentration, the degree of inhibition of PRRSV-N protein became more obvious, suggesting that CD81 inhibits PRRSV replication in a dose-dependent manner. The above results further prove that CD81 overexpression inhibits PRRSV replication.

[0050] CD81 knockdown

[0051] To further detect the inhibitory effect of CD81 on PRRSV replication, the embodiment also designed and synthesized CD81 interfering RNA (siRNA) according to the CDS sequence of the CD81 gene, and designed a pair of control siRNA as a negative control (Negative Control, NC), named si-CD81 and si-NC, respectively, both of which consisted of a sense chain (sense) and an antisense chain (antisense), and were synthesized by Suzhou Jima Gene Co., Ltd. The nucleotide sequence (5'-3') is as follows:

[0052] si-CD81-sense: GCGCUGUGAUGAUGUUCGUdTdT (see SEQ ID NO. 8);

[0053] si-CD81-antisense: ACGAACAUCAUCACAGCGCdTdT (see SEQ ID NO. 9);

[0054] si-NC-sense: UUCUCCGAACGUGUCACGUdTdT (see SEQ ID NO. 10);

[0055] si-NC-antisense: ACGUGACACGUUCGGAGAAdTdT (see SEQ ID NO. 11).

[0056] Use of Lipofectamine TM PAMs cells were cultured in a six-well plate in 1640 culture medium containing 10% fetal bovine serum to a confluence of 60-80%, and the above-mentioned si-CD81, si-NC and Lipofectamine TM 2000 with Opti- Reduced Serum Medium (purchased from Thermo Fisher Scientific, catalog number: 31985062) was diluted; after dilution, the mixture was mixed at a volume ratio of 1:1, incubated at room temperature for about 5 minutes, and then added to PAMs cells, cultured in a 37°C, 5% CO2 incubator for 6-8 hours, the supernatant was discarded, the cells were washed 1-2 times with PBS, and then cultured with complete medium for another 48 hours, the cells were collected, and the CD81 knockdown effect was detected by western-blot.

[0057] Take another six-well plate, select CD81 knockdown PAMs cells and culture them for 48 hours, then inoculate PRRSV (MOI = 0.1), and then culture for another 36 hours or 48 hours. Collect the cells for western-blot to verify the effect of CD81 knockdown on intracellular PRRSV proliferation.

[0058] The results are as follows Figure 6 As shown, compared with the control group (addition of si-NC treatment, CD81 gene expression was not affected), silencing CD81 gene expression by siRNA significantly promoted the expression of PRRSV-N protein, indicating that the level of PRRSV replication was increased. "+" and "-" indicate that the corresponding plasmid was added or not added during transfection, and Si-CD81 and si-NC represent CD81 siRNA and negative control siRNA, respectively.

[0059] Real-time PCR was further used for verification. Figure 7 As shown, after CD81 gene knockdown, the mRNA expression level of PRRSV-N protein was significantly increased.

[0060] CD8 1. Breeding for resistance to porcine reproductive and respiratory syndrome Applications

[0061] On the one hand, the embodiments also disclose the use of CD81 in breeding pig breeds resistant to porcine reproductive and respiratory syndrome.

[0062] In some embodiments, the application includes: judging whether the pig individual is resistant or susceptible to porcine reproductive and respiratory syndrome virus based on the expression level of CD81 in the pig individual.

[0063] The applications provided in some embodiments include: using the local Chinese breeds Tongcheng pigs, Meishan pigs and foreign breeds Landrace pigs and Large White pigs as breeding samples. Among them, it is known that Tongcheng pigs and Meishan pigs are not susceptible to PRRSV, that is, they are resistant; while Landrace pigs and Large White pigs are susceptible to PRRSV, that is, they are not resistant. 24 Tongcheng pigs, Meishan pigs, Landrace pigs, and Large White pigs aged 4-6 weeks that are double negative for PRRSV antigen and antibody were selected, 8 pigs / group, that is, divided into 3 groups: PRRSV challenge group (nasal drops + intramuscular injection, challenge dose: 3×10 5 TCID50 / head), DMEM control group (negative control, nasal drops + intramuscular injection of equal amount of DMEM), and no treatment group (blank control). Blood was collected 3 days before the challenge and on the 7th, 14th, 21st, and 28th days after the challenge. The control group was blood-collected simultaneously, and the animals were killed on the 28th day to collect blood, lung, lymph node, and tonsil tissues. A portion of the serum was taken for virus TCID50 determination (tissue half infection dose), and the remaining serum and lung, lymph node, and tonsil tissues were extracted for RNA. The RT-PCR kit provided in the above embodiment was used to detect the mRNA expression level of the CD81 gene. In addition, the mRNA expression level of PRRSV-N protein was also detected by qRT-PCR (Effects of Nsp9 genes of strong and weak strains of PRRSV on PRRSV replication, Zhao Mengmeng et al., Acta Agriculturae Boreali-Sinica, Issue 1, 2017), and pathological sections were made on the lungs and lymph nodes. The resistance / susceptibility of local breeds Tongcheng pigs, Meishan pigs, and foreign breeds Landrace pigs and Large White pigs to PRRSV was determined based on TCID50, PRRSV-N protein expression levels, and lung and lymph node tissue pathological sections. The relationship between the expression level of CD81 in pigs and the resistance / susceptibility of the above four breeds of pigs to PRRSV was studied. According to the results, it can be judged that Tongcheng pigs and Meishan pigs are not susceptible to PRRSV (i.e., they are resistant), while Landrace pigs and Large White pigs are susceptible to PRRSV (i.e., they are not resistant). At the same time, the results of the detection of CD81 gene expression levels in pigs before infection (gene background expression levels in pigs not infected) showed that the expression of CD81 gene in Tongcheng pigs and Meishan pigs was significantly higher than that in Landrace pigs and Large White pigs, further proving that Tongcheng pigs and Meishan pigs are resistant to PRRSV compared with Landrace pigs and Large White pigs, indicating that the resistance / susceptibility of pigs to PRRSV can be identified by detecting the expression level of CD81 gene. After infection, the expression of CD81 gene in pigs was significantly reduced, which is consistent with the experimental results at the cell level (in vitro).

[0064] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Use of a vector overexpressing CD81 in the preparation of an agent for preventing or treating porcine reproductive and respiratory syndrome, wherein the vector overexpressing CD81 carries the nucleotide sequence shown in SEQ ID NO.

5.

2. The use of CD81 as a target in the preparation of a reagent for detecting whether a pig individual is resistant or susceptible to porcine reproductive and respiratory syndrome virus, including judging whether the pig individual is resistant or susceptible to porcine reproductive and respiratory syndrome virus based on the expression level of CD81 in the pig individual.

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