Research traineeship veterinary medicine C.J.F. van den Akker (Clara)
Student number: 3538095 Supervisor: Herman Egberink
Utrecht University, department of virology August 10, 2015
Screening for different
coronaviruses in cats
ABSTRACT
Coronaviruses are enveloped RNA viruses, known for their high mutation rate and capacity to switch host. Infections with FCoV are very common in cats. To determine if infections with other coronaviruses than FCoV might occur in cats, 155 feline samples (sera, plasma and some ascites samples) were screened for reaction with 13 different S1-coronavirus proteins in an ELISA.
Many feline samples tested ELISA positive for S1-proteins from FCoV type 1 and 2, but also ELISA positive samples for the porcine coronaviruses PEDV, TGEV and SDCV and for human coronavirus 229E were seen. Twenty percent of the samples reacted with PEDV-S1-mFc, all of these samples also reacted with FCoV-S1-mFc. Cats that were experimentally infected with FCoV type 1 tested negative on PEDV-S1-mFc before infection, some of them tested PEDV-S1-mFc ELISA positive after infection with FCoV. A Virus Neutralization Test (VNT) on PEDV with the feline samples was carried out to further study the nature of the PEDV reacting antibodies in the ELISA. High neutralization titers were seen, but not always correlating with ELISA positive PEDV-S1-mFc results. Neutralization did however occur more among the PEDV-S1-mFc ELISA positive samples. VNT was also carried out on FCoV type 1 and 2 with porcine sera, to study the possibility of crossreactivity in more detail. Only one out of twenty porcine samples was able to neutralize FCoV type 1. Since sera of SPF cats infected with FCoV type 1 were positive on the PEDV-S1-mFc ELISA and that all of the S1-mFc ELISA positive cats are also FCoV-S1-mFc type 1 ELISA positive, crossreactivity between PEDV-S1 and FCoV-PEDV-S1 is likely, although the PEDV-S1-proteins only show 28,8% similarity. Neutralization of PEDV occurred more with the PEDV-S1-mFc positive samples, but also neutralizing samples which were ELISA negative were found. A possible explanation is that VNT is more sensitive than ELISA, but also aspecific inhibiting factors in the samples could have played a role in blocking virus replication. It could also be that the samples contained antibodies for neutralizing epitopes, but that these epitopes are not located on the S1 protein. More research is needed to differentiate between these theories. VNT on FCoV type 1 and 2 with porcine serum samples indicated no crossreactivity on neutralizing epitopes between PEDV and FCoV-S1, as nearly all of the PEDV-S1-mFc ELISA positive samples did not neutralize FCoV type 1. However, a
crossreaction between TGEV and FCoV type 2 was observed.
INTRODUCTION
Coronaviridae are a family of large RNA viruses with an
envelope, belonging to the order of Nidovirales (1,2). They have a broad host range and are able to infect many species, including humans, cats, dogs and pigs. Enteric infections and respiratory disease are the main clinical signs caused by these viruses (1,2). The envelope of the coronavirus is covered with spike
glycoproteins (S-proteins), which give the coronavirus its characteristic crown-like appearance (FIG. 1). The spike proteins make attachment to and entry of the cells possible, using receptors of the host cells that are specifically used by the virus. Where the rest of the virus is relatively well-conserved, the receptor binding domain is not (1). The differences in the receptor binding domain between coronaviruses that result from this, determine in part the cell and host specificity (3).
The receptor binding domain is located in the S1 domain of the spike protein. The S2 domain of the spike protein is responsible for making fusion possible (3-5). The specific binding of the spike protein with its receptor determines the tropism of the coronavirus. To make infection of a new species possible, the
coronavirus spike protein has to be adapted, so it can bind to the receptor of the new host. This adaptation can be the result of mutations within the gene encoding for the receptor binding domain including
recombination (3). Coronaviruses have a high mutation frequency, this can be contributed to three main reasons. Firstly, the RNA-dependent RNA polymerase has low fidelity, one mutation occurs in every 1000 to 10000 replicated nucleotides. Secondly, the virus uses the “copy choice” mechanism for RNA replication, which makes is possible to switch templates random. Thirdly, coronaviruses possess a genome of 26-32kb, the largest genome of the RNA viruses known (6,7).
Many different members of the Coronaviridae family exist. Based on their relationships and identic features, currently four genera of coronaviruses are distinguished: alphacoronaviruses, betacoronaviruses,
gammacoronaviruses and the newly characterized deltacoronaviruses (FIG. 2) (1,8).
Figure 2: genera of coronaviruses (1)
In cats, infections with the Feline Coronavirus (FCoV) are common (9). Two serotypes of this virus are known, based on the characteristics of their spike protein (3). FCoV belongs to the group of 1a alphacoronaviruses and most part of the feline population is known to be seropositive for FCoV type 1, especially cats living in catteries or multi-cat households (1,10). Feline coronavirus occurs in two biotypes: the feline enteric coronavirus (FeCV) causing an asymptomatic infection or mild diarrhoea and the feline infectious peritonitisvirus (FIP) causing a severe systemic disease (8,9). It is assumed that FIPV arises from mutations in the FeCV virus, but which specific mutations cause the switch of pathotype remains unknown (3). It is thought that a switch of tropism, from gut epithelial cells to macrophages and monocytes, plays an important role in this (11,12). So far no other coronavirus infections have been identified which infect cats (8).
In a recent pilot serological study cats were identified that were seropositive against S1 proteins of various other coronaviruses (Herman Egberink, personal communication, July 2015). One of these S1 proteins was from the 1b alphacoronavirus Porcine Epidemic Diarrhoea Virus (PEDV), causing diarrhoea in pigs. Also a few sera reacted positive against S1 proteins of coronaviruses known to cause respiratory infections in animals and man. The major goal of this research is to determine if infections with other coronaviruses belonging to the alphacoronaviruses, betacoronaviruses and deltacoronaviruses occur in cats. These might be viruses having a broader host-range or viruses genetically and antigenetically related to the known coronaviruses. To study the possible infection of cats with other coronaviruses, serological studies were performed. Recombinant S1 proteins were used in an enzyme-linked immunosorbent assay (ELISA) to screen feline sera for the presence of antibodies. This particular study focussed on the S1 proteins of swine (TGEV, PEDV, SDCV), bovine (BCoV) and human (OC43, HKU, 229E, SARS, MERS) coronaviruses.
MATERIALS AND METHODS
Samples of serum, plasma and ascites, which were retrieved from the databases at the Virology Department of Utrecht University, were tested. The samples originated from cats out of different populations or from
previous studies. The Kb-numbers are samples from cats of different age and origin that were collected at veterinary practices during a previous study on the prevalence of antibody titers against Feline panleukopenia virus. This makes this group of cats the most random group. The FIP/FIV numbers are blood samples from cats which have been sent in for diagnostics on Feline Infectious Peritonitis (FIP) or Feline Immunodeficiency Virus (FIV). A part of this group is called the FIP-numbers, most of these samples are derived from cats of which the diagnosis FIP has been confirmed. The cats from the FIP/FIV-numbers group are mostly housed in multi-cat circumstances. Also some samples from experimentally FCoV type 1 infected cats from previous studies have been tested.
An ELISA was performed on nearly all of the samples. Some of the samples that were positive for PEDV-s1-mFc were selected for Virus Neutralization Tests (VNT) on PEDV. Porcine serum samples from two populations were kindly provided by Wentao Li and used for a VNT on the two types of FCoV.
ELISA ON DIFFERENT CORONA S1 PROTEINS
ELISA’s have been performed following the improved ELISA protocol that was previously established in the laboratory of the Virology Institute Utrecht (Berend Jan Bosch and Joris de Jong, personal communication, July 2015).
Coating plates
ELISA-plates coated and frozen by Nancy Schuurman before and own plates coated according the same protocol were used. Thirteen S1-proteïns of twelve different coronaviruses were tested, eleven on one plate and the remaining two on a second plate. Eight different samples could be tested on each plate. 5µg of each protein was diluted in 20ml DPBS with mg/ca (Lonza, Lot#2MB227), to a final concentration of 0,25µg/ml. 100µl of the dilution was added to each well of the ELISA plate (Greiner bio-one REF# 655092), each column with another protein dilution. The plates were stored overnight at 4° Celsius to coat.
After overnight coating, the plates were washed with PBS0+tween20(0,05%) in the Biorad ELISA washer and blocked for one hour at 37°C with 200µ/well of Protifar blocking buffer (Protifar 5% + 0,05 Tween20 in PBSO). After blocking, the plates were cooled till room temperature, sealed and stored at -20°C for further use.
Performing ELISA
First, pre-coated ELISA plates, samples and negative control sera are put in the 37°C stove to thaw. Protifar blockbuffer is prepared ( 500ml PBS0+tween20 (0,05%) + 25g Protifar (Nutricia) or put out of the cooling if already made. 1:200 serum dilutions are made by mixing 8µl serum with 1592µl blockbuffer. Pre-coated ELISA plates are emptied and washed with PBS0+tween20(0,05%) in the Biorad ELISA washer. 100µl/well of each serum is pipetted on the plates, one serum each row. The plates are placed in the 37°C stove to incubate for one hour. Present antibodies in the sera can now bind to viral proteins on the plate. After incubating, the plates are again washed with the ELISA washer, so unbound proteins are washed away. A 1:4000 conjugate dilution is made with 12,5µl goat anti-cat conjugate (IgG, HRPO, 5-3-2013, Rockland) and 50ml Protifar blockbuffer. 100µl of this dilution is added to each well with the multichannel. The plates are put back in the incubator for an hour, so the conjugate can bind to the feline antibodies. Binding occurs due to the anti-cat antibodies. A horseradish peroxidase is bound to them, which makes a color signal possible in a later stadium. After incubation the plates are washed again, so unbound conjugate will be rinsed away and give no signal. 100µl TMB super slow substrate is added to each well, this will react with the horseradish peroxidase component of the conjugate if present in the well. The plates are stored in the dark and the reaction is stopped after ten minutes, by adding 100µl 2M H2SO4 to each well. Optical density (OD) values are readout at 450nm with the gen5 ELISA-reader.
Interpretation
OD-values are noted in excel and compared with the mean OD-values of the negative control samples (also called the background). Most samples have been tested in duplo, when enough material was available. The average OD-values of the two tests were calculated and compared with the background. OD-values higher than five times the background were considered positive, OD-values higher than three times the background were considered weakly positive. When several samples on the same ELISA-plate showed abnormal results (eg very high of low OD-values), these data were left out of consideration and only one dataset was used for that particular sample.
VIRUS NEUTRALIZATION TESTS (VNT)
VNT’s were performed with PEDV and with FCoV type 1 and type 2. VNT on PEDV was performed on Vero cells using the PEDV/GFP recombinant virus (13). Catus Whole Fetus-cells (FCWF-cells) were used for the VNT with both types of FCoV.
VNT with PEDV
Subculturing of Vero cells CCL-81
At first, the medium was prepared. One flask of 500ml Dulbecco's Modified Eagle Medium (DMEM) without pyruvate was mixed with 50ml fetal calf serum (FCS). To avoid bacterial growth, 0,5ml of antibiotics (penicillin and streptomycin) was added. Both the medium and trypsin were placed in the 37°C water bath. Supernatant was removed from a T75 flask containing a monolayer of Vero cells and the flask was rinsed with 4ml PBS0. This was to ensure the FCS in the residual medium would not inactivate the trypsin. 2,5ml trypsin was added to the T75 flask, to re-suspend the cells which adhered to the flask. The flask was placed back at 37°C, the optimal operating temperature of trypsin, for twelve minutes. Loosening of the cells was checked regularly by
microscope. For a 1:8 dilution, 5,5ml medium was added to the flask (so the total volume in the flask would be 8 ml). 1ml was transferred to another flask, 17ml medium was added and the flask is labelled and put back in the incubator.
Seeding plates with Vero cells CCL-81
dilution and placed in a 37°C 5% CO2 incubator. The cells were checked daily, seeding the plates was possible when a solid monolayer of cells is present. Subculturing procedures are followed as described before. After subculturing, the remains of the two flasks were put together, after which 0,2ml of the cell-mixture was used for the cell counter. For this, 10ml of isotonic was added to the cells and a cell count was performed.
The plates were seeded one day before the VNT, at a concentration of 200.000cells/ml . Using a multichannel pipette, 100µl was added to each well, after which the plates were labeled and put back in the incubator for a day.
Performing the VNT on PEDV
Because a genetically modified PEDV-strain was used, proceedings were carried out in a ML-2 lab. The modification of the virus makes infected cells express fluorescence, which can be made visible with a
fluorescence microscope. At first, the medium was prepared. 0,5ml pencillin/streptromycin and 55ml Tryptose Phosphate Broth (TPB) were added to a 500ml bottle of Alpha Modification of Eagle's Medium (aMEM). The medium was taken out of the cooling, to minimize the risk of cell damage by the low temperature. After this, the cat sera and plasma dilutions were made. For a 1:16 dilution, 103,1µl medium was added to the first row of a 96-well round bottom plate. To the second till eight row, 55µl of medium was added. 6,9µl of each heat inactivated (30 minutes at 56°C to inactivate the complement) serum/plasma was added to the first row, in duplo. To some wells an extra 6,9µl medium was added instead of serum, to serve as a control. Serial 2-fold dilutions are made by transferring 55µl from well to well with a multichannel pipette, starting with row 1 up to row 8. 55ul/well of row 8 is discarded. Just before using it, 16,33µl rPEDV-DR13-S-dORF3/GFP virus is added to 40ml medium, so the final concentration of the virus is 2000TCID50/ml. 55µl was added to each well with the multichannel pipette, with changing of pipette points after each step. The plates were put in the 37°C 5% CO2 incubator to incubate for an hour. 100µl co-incubated serum-virus was transferred to the wells of the Vero cell plates seeded before, mixing was done by resuspending 5 times. Before adding the serum-virus to the cells, the plates were emptied of medium. The plates were put in the incubator for 48 hours, after which the result could be readout with a fluorescence microscope.
Interpretation
All wells were checked with the EVOS-microscope for fluorescence. Infected cells express Green Fluorescent Protein (GFP), which was used as a read-out parameter. When no fluorescence was present, it was checked whether the cells were still alive. If no fluorescence was visible and cells were still alive, it was assumed that the sample blocked virus replication. When fluorescence was visible, it was scored as <10% of cells fluorescent, 10-70% of cells fluorescent and >70% of cells fluorescent. Each sample was tested in duplo, and the
neutralization titer was calculated by the mean of these two samples. The highest dilution of the serum at which no fluorescence was seen in at least one well, but with living cells, was classified as the neutralizing antibody titer.
VNT WITH FCOV TYPE 1 AND TYPE 2
Subculturing of Felis Catus Whole Fetus (FCWF)-cells
Cells were subcultured like the Vero cells (described above), but instead of leaving trypsin with the cells, it was removed before the flask was put in the incubator. After rinsing with PBS0, 4ml of trypsin was added to the flask. This was left in place for approximately a minute, after which it was removed. The cells were put in the 37°C incubator for a couple of minutes. FCWF-cells were subcultured in a 1:3 dilution, so 6ml of medium was added. After mixing, 2ml was transferred to a new flask, together with 16ml of medium.
Seeding plates with FCWF-cells
Three or four days before seeding the plates, two T75-flasks of FCWF-cells were subcultured in a 1:3 dilution and placed in a 37°C 5% CO2 incubator. The cells were checked daily, seeding the plates was possible when a solid monolayer of cells is present. Subculturing procedures are followed as described before. After
subculturing, the remains of the two flasks were put together, after which 20µl of the cell-mixture was used for counting the cells in a counting chamber under the light microscope.
When the plates were seeded one day before the VNT, a concentration of 300.000cells/ml was needed. The needed amount of cell suspension is calculated, this was made up to the desired volume needed for 100µl/well (10ml each plate).
Performing the VNT on FCoV type 1 and type 2
This experiment was performed on the FCoV-sensitive FCWF cells. Two different viruses were used, the FCoV type 1 TN406HP (106 TCID50/ml) , and the type 2 FIPV-79-1146 (107,93 TCID50/ml). Porcine sera were tested, plus some feline sera/ascites as controls. The porcine sera were divided in two groups, based on a positive or negative result for the PEDV-S1-mFc protein in the ELISA. Each serum was tested on both viruses, also a virus control (without testing samples) was performed on both viruses. Prior to testing, sera and ascites were first inactivated for 30 minutes in a 56°C water bath, to eliminate complement. 5% DMEM penicillin/streptomycin + gentamycin was used to dilute the sera. In a micronic block, 60µl of each serum was mixed with 240µl
DMEM5% for a 1:5 dilution. 3-fold dilutions were made by transferring 100µl to the next row, up to the highest dilution of 1:10935. To keep the volume equally, 100µl was discarded from the last row. The virus was thawed and 200µl 100TCID50 added to each tube. Subsequently, the virus-serum mixture was incubated for 1 hour at 37°C 5% CO2, so present antibodies could neutralize the virus. Plates seeded with FCWF-cells were emptied and flushed with PBS DEAE. The co-incubated virus-serum mixture was transferred to the plates 100ul/well in triplo, and afterwards put back at 37°C 5% CO2. One plate was used for a virus- and cellcontrol. For the cellcontrol, 100µl medium was used without virus. The virus control as made with a tenfold dilution, ranging from 100TCID50 till 0,1TCID50. 50µl of each dilution was added to a row of wells, together with 50µl medium. Cells were checked daily by microscope and checked for cytopathologic effect (CPE). When CPE at the virus controls was complete, the plates were fixated and stained. This was done with 100µl 0,75% crystalviolet 9% formaldehyde each well. Formaldehyde fixes the cells to the plate, where crystalviolet stains intact cells.
Interpretation
All wells were checked by microscope for CPE, results could be readout when CPE at the virus controls was complete. CPE was scored as >50%, <50% but spread through the well and as only a local spot. Neutralization titer was determined as the dilution where at least two out of three wells had >50% CPE.
RESULTS
ELISA
In this project, a total of 155 samples were screened for the presence of antibodies against the S1-proteins of 12 different coronaviruses. The S1-protein was chosen, because this is the most variable protein and thus the most specific for the virus.
Figure 3 shows the percentages of samples that were positive in the ELISA against the different S1 proteins. This is also displayed for the two specific populations (Kb-numbers and FIP/FIV-numbers). For an overview of all ELISA results, see appendix 1.
No samples tested positive for the proteins derived from the human coronaviruses HKU, MERS, SARS and OC43. Twelve percent of the samples tested positive on the 229E-S1-mFc protein, which is also derived from a human corona virus. Some samples reached high OD-values for 229E-S1-mFc. For the human coronavirus protein NL63-S1-mFc, one percent of the samples tested positive. OD-values for this protein were lower than
OD-values seen for other proteins.
Thirteen percent of the samples tested positive for the porcine coronavirus protein TGEV-S1-mFc.
As expected, many samples tested positive for the feline coronavirus proteins FCoV-S1-mFc type 1, FCoV-S1 type 1 and FCoV-S1 type 2. Respectively 55%, 52% and 21% tested positive for this proteins. When only the FIP/FIV-numbers were taken into account, even 85%, 81% and 58% respectively tested positive. OD-values for the type 1 FCoV proteins were higher than OD-values for type 2 FCoV proteins.
20% of the samples tested positive for PEDV-S1-mFc, a great proportion of this belonged to the FIP-group. Accounting for all the proteins, the FIP/FIV-numbers reached the highest percentages of samples testing positive.
Figure 3: Feline samples (n=155) testing positive on the different coronavirus S1-proteins. The total of all tested samples is displayed, as well as the group of Kb-cats and FIP/FIV-cats
Several samples tested positive on more than one protein. In the figures 4-9 is shown which fraction of samples also tested positive on another protein.
Of the FCoV-S1-mFc type 1 positive sera, 95% tested also positive on FCoV-S1 type 1 (FIG. 4). All of the FCoV-S1 type 1 positive sera tested also positive for FCoV-S1-mFc (FIG. 5). A good correlation between these two does exist, although a few sera seemed to respond only on the S1 protein still containing the part. This mFc-part was used to purity the protein and was removed later to create the FCoV-S1 type 1 protein. The mFc mFc-part has a murine origin, so it was questioned if cats that have been in contact with mice before, would react on the mFc-part. Both FCoV-S1-mFc and FCoV-S1 were tested, to ascertain that these two gave comparable results and that therefore also the other proteins with mFc gave reliable results.
12% 1% 55% 20% 6% 52% 21% 13% 1% 7% 1% 48% 11% 3% 45% 13% 5% 1% 35% 4% 85% 58% 15% 81% 58% 50% 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Total Kb FIP/FIV
Figure 4: samples testing positive on the FCoV-S1-mFc type 1 protein (n=84) also tested positive on S1 proteins derived from other coronaviruses.
Figure 5: samples testing positive on the FCoV-S1 type 1 protein (n=80) also tested positive on S1 proteins derived from other coronaviruses.
The 229E-S1-mFc positive tested samples, except one, also showed high OD-values for the FCoV-S1-mFc protein, which is also from an alphacoronavirus (FIG. 6). In most cases, they also tested positive for other S1-proteins. One cat serum (Kb021) tested positive twice for 229E-S1-mFc with OD-values of 0,449 and 0,555 and for none of the other proteins in this assay.
95% 37% 39% 19% 24% 0% 20% 40% 60% 80% 100%
FCoV-S1 type 1 FCOV-S1 type 2
PEDV-S1-mFc 229E-S1-mFc TGEV-S1-mFc
FCoV-S1-mFc type 1
100% 38% 40% 19% 24% 0% 20% 40% 60% 80% 100% FCoV-S1-mFc type 1 FCOV-S1 type 2PEDV-S1-mFc 229E-S1-mFc TGEV-S1-mFc
FCoV-S1 type 1
Figure 6: samples testing positive on the 229E-S1-mFc protein (n=17) also tested positive on S1 proteins derived from other coronaviruses.
All of the PEDV-S1-mFc positive cats were also positive on FCoV-S1-mFc1 type1 (FIG. 7), but the FCoV-S1-mFc type 1 positive cat sera were positive for PEDV-S1-mFc in only 39% of the cases (FIG. 4).
Figure 7: samples testing positive on the PEDV-S1-mFc protein (n=33) also tested positive on S1 proteins derived from other coronaviruses.
Most sera that showed high OD-values for FCoV-S1 type 2 also showed high OD values for the two FCoV type 1 S1-proteins (FIG. 8). The majority of them showed higher OD values for the type 1 proteins, only three sera (FIP323, 329 and Kb101) showed higher values for the type 2 protein.
94% 88% 59% 65% 41% 0% 20% 40% 60% 80% 100% FCoV-S1-mFc type 1
FCoV-S1 type 1 FCoV-S1 type 2 PEDV-S1-mFc TGEV-S1-mFc
229E-s1-mFc
100% 97% 48% 33% 36% 0% 20% 40% 60% 80% 100% FCoV-S1-mFc type 1FCoV-S1 type 1 FCOV-S1 type 2
229E-S1-mFc TGEV-S1-mFc
PEDV-S1-mFc
Figure 8: samples testing positive on the FCoV-S1 type 2 protein (n=32) also tested positive on S1 proteins derived from other coronaviruses.
The sera that were positive for the TGEV-S1-mFc protein, were also positive for the FCoV-S1 type 2 protein (FIG. 9). Not all FCoV-S1 type 2 positive samples were also positive for the TGEV-S1-mFc protein on the other hand (FIG. 8).
Figure 9: samples testing positive on the TGEV-S1-mFc protein (n=21) also tested positive on S1 proteins derived from other coronaviruses.
Experimental cats:
The sera of “Kat89”, “Kat129” and “Kat131” were derived from cats in another experiment. In this experiment, these Specific Pathogen Free (SPF) cats were inoculated oronasally with respectively a dose of undiluted, 10-1 diluted and 10-2 diluted faecal extract, containing the FCoV type 1 strain UCD (14). Both sera before inoculation (the t=-5 samples) and sera after inoculation were tested in the ELISA.
The sera “91type1” and “93type1” were also derived from SPF cats after inoculation with a FCoV type 1 strain. 91type1 t=0 and 93type1 t=0 are sera of the same cats, but before inoculation. All four sera have been tested in the ELISA. 97% 94% 50% 31% 63% 0% 20% 40% 60% 80% 100% FCoV-S1-mFc type 1
FCoV-S1 type 1 PEDV-S1-mFc 229E-S1-mFc TGEV-S1-mFc
FCoV-S1 type 2
95% 90% 95% 57% 33% 0% 20% 40% 60% 80% 100% FCoV-S1-mFc type 1FCoV-S1 type 1 FCoV-S1 type 2 PEDV-S1-mFc 229E-S1-mFc
TGEV-S1-mFc
The results of “Kat89 t=-5”, “Kat129 t=-5” and “Kat131 t=-5” show that the sera did not react with any of the corona S1-proteins before inoculation, which was expected as these cats are SPF cats. The three sera after inoculation with the UCD extract, “Kat89”, “Kat129” and “Kat131” all reacted with the FCoV-S1 and FCoV-S1-mFc. “Kat129” and “Kat131” however showed also a high OD for the PEDV-S1-FCoV-S1-mFc. “Kat89” showed a low OD on PEDV-S1-mFc one time, the duplo showed mildly positive (three times background) for this protein. Roughly the same accounted for the “91type1” and “93type1” sera. At t=0, both sera tested negative on all the corona S1-proteins, as might be expected from SPF cats. The sera after inoculation showed high OD-values for FCoV-S1-mFc and FCoV-S1, but “91type1” also showed a high OD-value for PEDV-S1-mFc.
VNT ON PEDV
Since several feline sera tested positive for antibodies against the S1 protein of PEDV, it was decided to screen some of these samples in a virus neutralization assay with PEDV also (FIG. 10). A total of 88 different feline samples (52 PEDV-S1-mFc ELISA-positive, 35 ELISA-negative and one unknown) and two porcine samples were tested. 51 of the feline samples neutralized the PEDV virus to some extent. Neutralizing titers varied greatly, from 16 to 72576. Mainly the FIP-number cat samples gave high neutralization titers. For the complete results, see appendix 2.
Figure 10: Neutralization titers on PEDV compared with OD-values on ELISA on PEDV-S1-mFc. Cut-off point for mildly positive (3-5 times background) and positive (>5 times background) from ELISA are marked with the dotted lines.
From the PEDV-S1-mFc ELISA-negative samples, 17% was positive in the neutralization assay (TABLE 1). These 6 PEDV-S1-mFc negative samples which neutralized PEDV reached low titers in most cases, but two of them reached titers of 512 and 2112 respectively. 84,21% of the samples that were mildly ELISA-positive (>3 but <5 times background) neutralized the virus. Of the PEDV-S1-mFc ELISA positive samples, 88% was able to neutralize the virus.
0 500 1000 1500 2000 0,000 0,500 1,000 1,500 2,000 2,500 3,000 VN T PE D V titer s
OD-values in ELISA on PEDV-S1-mFc
FIP-cats
experimental cats and controls
Kb-cats and FIP not confirmed Background x3 Background x5
PEDV-S1-mFc ELISA
% samples that
neutralized PEDV
<3x background
17%
3-5x background
84%
>5x background
88%
Table 1: PEDV neutralization, divided in three groups based on PEDV-S1-mFc ELISA results
>
When the possibility to neutralize is compared with a positive or negative ELISA result on FCoV-S1-mFc, it is seen that 4% of the negative samples is able to neutralize, and 86% of the positive samples (TABLE 2). The ELISA negative sample that is able to neutralize is the only ascites sample and reaches a titer of 16.
FCoV-S1-mFc ELISA
% samples that
neutralized PEDV
<3x background
4%
3-5x background
0%
>5x background
86%
Table 2: PEDV neutralization, divided in three groups based on FCoV-S1-mFc ELISA results
VNT ON FCOV
Twenty pig samples, together with four cat samples as control, were tested on their capacities to neutralize the FCoV type 1 and type 2 virus (TABLE 3). All of the PEDV-S1-mFc ELISA negative porcine samples did not neutralize the FCoV type 1 virus. One PEDV-S1-mFc ELISA positive sample was able to neutralize the FCoV type 1 virus.
Four PEDV-S1-mFc ELISA positive and six PEDV-S1-mFc negative pig samples were able to neutralize the type 2 virus to some extent. For one pig sample, not enough serum was available to calculate a titer for the type 2 FCoV virus.
FCoV type 1 FCoV type 2
TN406HP
titer on
t=72h
FIPV70-1146
titer on
t=48h
#3114
pig
15
5
2,32
0,15
3,75
1,06
#22561
pig
15
<5
1,69
0,13
1,26
0,24
#10435
pig
15
<5
2,90
0,18
2,11
0,29
#7148
pig
5
5
2,59
0,11
3,37
0,51
#13922
pig
5
<5
3,09
0,16
1,93
0,26
#13785
pig
5
<5
3,26
0,20
2,77
0,46
#34797
pig
15
15
1,07
0,25
3,77
0,38
#3585
pig
15
<5
2,58
0,16
2,47
0,55
#1569
pig
45
30
3,45
0,15
3,62
1,45
#3522
pig
15
<40
2,90
0,18
1,82
0,61
A1
pig
15
135
0,07
3,15
no data
0,35
B1
pig
15
45
0,10
2,57
no data
0,19
C1
pig
15
45
0,09
2,04
no data
0,10
D1
pig
15
5
0,10
1,70
no data
0,19
E1
pig
15
<5
0,09
0,25
no data
0,08
F1
pig
15
<5
0,07
0,08
no data
0,09
G1
pig
15
<5
0,10
0,09
no data
0,09
H1
pig
15
<5
0,08
0,10
no data
0,06
B2
pig
15
135
0,08
2,63
no data
0,20
A2
pig
5
135
0,07
1,89
no data
0,18
TN406HP
titer on
t=72h
FIPV70-1146
titer on
t=48h
PEDV-S1-mFc
TGEV-S1-mFc
FCOV-S1
type1
FCOV-S1-mFc type1
3645
<5
0,84
0,78
3,53
3,44
1215
<5
135
1215
0,28
2,25
0,07
0,15
15
<5
0,06
0,06
0,05
0,05
sample species
FIP714
ELISA (OD values)
VNT
PEDV-S1-mFc
TGEV-S1-mFc
FCOV-S1
type1
FCOV-S1-mFc type1
te
st
sa
mp
le
s
PE
D
V
-S
1-mF
c
n
eg
ati
ve
in
E
LI
SA
PE
D
V
-S
1-mF
c
p
os
iti
ve
in
E
LI
SA
Control-samples (cats)
VNT
ELISA (OD values)
SPF
sample
type
FCoV type 1 +
FCoV type 1 +
FCoV type 2 +
G317
cat12day70147
Table 3: results of the virus neutralization test on FCoV type 1 and 2, together with the OD-values in the different ELISA’s. Pig sera were selected on PEDV-S1-mFc ELISA results. The PEDV-S1-mFc positive pig sera originated from an Asian population, the negative pig sera were from a Dutch population. Cat sera serve as a control.
VNT titers lower or the same as negative controls are highlighted in red, titers higher than negative controls are highlighted in green. Higher titers are darker green. ELISA values are considered positive when OD is >5 times the OD of the SPF cat.
DISCUSSION
ELISA
The purpose of this study was to screen feline sera against several coronaviruses. As expected, many of the feline samples reacted with the FCoV proteins (10). More cats seemed to recognize the FCoV type 1, than FCoV type 2, which also corresponds with findings from earlier studies (15-17). The majority of the sera that both reacted with FCoV type 1 and 2, had higher OD-values for the FCoV type 1 proteins than for the type 2 protein and should therefore be classified as type 1 FCoV (L. Tuinte, unpublished results). Three sera show highest OD-values for FCoV-S1 type 2 and should be classified as type 2 sera, but this should be confirmed by
crossneutralization tests (L. Tuinte, unpublished results).
A substantial amount of the tested feline samples also seemed to react with S1-proteins derived from human and porcine coronaviruses. This could be due to cross-reactivity, or due to previous infections with these porcine or human viruses or with a yet unknown virus which shows similarities with these porcine and human viruses. The different observations will be commented in more detail below.
229E
From all tested feline samples, twelve percent reacted with the human coronavirus protein 229E-S1-mFc. Most 229E-positive samples also reacted with one or more of the other tested coronaviruses. Interestingly, one feline serum only reacts with the 229E-S1-mFc protein. The fact that in this cat no reaction was seen with the other tested (alpha)coronaviruses, makes cross-reactivity less likely.
Barlough et al (18) exposed minimal-disease cats to live 229E virus, but little to no replication of the virus was reported. No clinical signs related to 229E-infected were noted either. However, when these cats were again exposed to FCoV, antibody titers were boosted to high levels indicating that 229E did cause a priming effect (18). No naturally infected cats with 229E are known so far (8).
229E is able to use feline aminopeptidase N as its cellular receptor. Feline aminopeptidase N is a glycoprotein that is expressed on the cell surface of various feline cells (19). Also FCoV type 2, TGEV and CCoV are able to use this receptor, FCoV type 1 is not (20-22). Probably these alphacoronaviruses have all evolved from the same virus which infected cats using feline aminopeptidase N. Mutations caused different host-specificity in the different viruses, but they all remained the ability to use the feline cellular receptor (8).
It seems therefore possible that the positive ELISA-reaction for 229E-S1-mFc is due to a natural with 229E in this cat, or with a 229E-like virus.
TGEV
A total of thirteen percent of all tested cats seemed to recognize the TGEV-S1 protein, all of these cats also recognized FCoV-S1. TGEV-S1 and FCoV-S1 type 2 are highly related (6,8), which is reflected by their 66% agreement in amino acid sequence (FIG 11). It is believed that FCoV type 2 originated from a recombination event between FCoV type 1 and Canine Coronavirus (CCoV) type 2. CCoV type 2b on its turn is thought to originate from recombination between CCoV type 2a and TGEV (6). Similarity between the S1-proteins of TGEV and FCoV type 2 are likely to have resulted in cross-reactivity.
Figure 11: amino acid sequences of the different S1-proteins compared with the amino acid sequences of other S1-proteins, similarities are displayed in percentages (Wentao Li, personal communication, July 2015).
Swine Delta Coronavirus (SDCV)
SDCV is the only deltacoronavirus tested in this study and has therefore the least similarities with the other tested coronaviruses. It is remarkable that 6% of the samples tested positive for this porcine virus. Most S1-mFc positive samples also reacted with another S1-protein, but two samples only reacted with the SDCV-S1-mFc protein. Crossreactivity is less likely with this quite unrelated coronavirus, specificity of these observations should be further investigated.
PEDV
The most striking observation is that many of the tested feline sera reacted with the PEDV-S1-mFc protein. This observation might be explained by either crossreactivity between PEDV-S1 and FCoV-S1 or by the occurrence of a PEDV(-like) virus in cats.
No cat sample recognizes the PEDV-S1-mFc antigen without also recognizing FCoV-S1-mFc type 1. This could fit with cross reactivity, the cat immune system made antibodies against the FCoV-S1 protein, but for one or more epitopes which also exist on the PEDV-S1 protein. This could explain why the samples that react in the ELISA with the PEDV-S1-mFc protein, were also always positive for the FCoV-S1-mFc protein. Not all of the samples reacting with the FCoV-proteins also reacted with the PEDV-protein. The immune system of every cat varies, it could be that some cats make antibodies to epitopes specific for the FCoV-virus only.
Another explanation for the PEDV-S1-mFc positivity could be that these cats actually had antibodies for a PEDV(-like) virus. Truong et al (23) found in their research one out of 24 cats PCR-positive on PEDV. This was however found in a sample of the tonsils, no PEDV-positive results were found for the viscera, where PEDV is expected. No other literature is found in which PEDV-positive cats are reported. Nonetheless, if an infection with PEDV or a PEDV-like virus would be responsible for the PEDV-S1-mFc positive ELISA results, also some cats reacting only on the PEDV-S1-mFc protein and not always also on the FCoV-S1-mFc protein would be expected. The fact that some experimental SPF cats which were infected with FCoV type 1, also showed high OD-values for the PEDV-S1-mFc protein, provides more steady proof that crossreactivity played an important role in the positive PEDV-S1-mFc results. These cats were seronegative against all tested S1 proteins before infection with FCoV and next were only infected with FCoV. Not all of the FCoV-inoculated experimental SPF cats tested positive on PEDV-S1-mFc protein, probably this is because these cats made antibodies against epitopes that did not cross-react with epitopes on the PEDV-S1 protein.
When comparing the amino acid sequences between the different S1 proteins it is evident that the homology between the different S1 proteins is low. The receptor binding PEDV-S1 and FCoV type 1-S1 proteins show 28,8% similarity (FIG 11). When the amino-acid sequences of the S1-proteins are compared in more detail, a
few regions with minor sequence similarity can be identified, but only at a maximum of seven subsequent amino-acids (Berend Jan Bosch, personal communication, July 2015).
Not all of the ELISA-samples have been tested in duplo, which makes the test results of these specific samples less reliable. This mainly finds its effect on individual test results and will be of less importance for the
interpretation of the whole. It also has to be noted that high OD-values correlate with high antibody titers, but not in a linear way. At some point a plateau phase occurs and high OD-values will not increase any further.
VNT ON PEDV
Since virus neutralization is more specific than ELISA, VNT with PEDV was performed to further study the nature of the PEDV reacting antibodies in the ELISA. VNT titers were compared with the OD-values of the PEDV-S1-mFc ELISA result, to see whether a correlation between these values exists. No linear correlation was found between these two values, but as mentioned before OD-values do not correlate in a linear way with the amount of antibodies, especially with high OD-values. Also VNT will test for other antibodies than ELISA does. However, it is noteworthy that virus neutralization of PEDV occurred with the feline sera and also more with the PEDV-S1-mFc ELISA-positive samples. This was even more the case with the FCoV-S1-mFC ELISA positive samples, a majority of these samples neutralized PEDV in the VNT. Only one sample that tested negative in the ELISA on this protein was able to neutralize the virus, and only with the lowest measurable titer. This sample was the only ascites sample, some factors in it might have interfered with the VNT.
Another observation that can be made, is that the nine highest neutralizing antibody titers were all from FIP-number cats. Eleven out of fourteen tested FIP-cats displayed titers of at least 768. Thirteen out of fourteen FIP-cat samples neutralized the PEDV virus. The one sample (FIP329) that did not neutralize had also a much lower OD-value in the ELISA than the other FIP-cat samples. The background of this cat was checked and it appeared to be a healthy cat without FIP.
These FIP-number samples had on average also a higher PEDV-S1-mFc OD-value, but there were also quite a lot of them which had low PEDV OD-values and still neutralized the PEDV strongly. The eleven samples with the highest titers for neutralization all had a nearly maximal OD-value for PEDV-S1-mFc.
Some samples with high PEDV-S1-mFc OD values did not neutralize PEDV in the VNT. This could be because of VNT being more specific. Probably the feline sera contained some antibodies which reacted in the PEDV-S1-mFc ELISA, but no neutralizing antibodies.
On the other hand, some samples which did not react with PEDV-S1-mFc in the ELISA, did reach high neutralization titers. An explanation could be that these antibodies react on epitopes that are not on the S1-protein. As the ELISA in this research project only focusses on the S1-protein, PEDV neutralizing antibodies for other epitopes would not be notified in the ELISA. Also neutralization might be more sensitive than ELISA. Another explanation could be that factors other than antibodies aspecifically inhibit viral growth. This would also elucidate why the majority of the FIP-numbers neutralize PEDV infection at high titers, as it is known that FIP-infected cats have many inflammation factors such as cytokines and interferons circulating in the blood (10).
To determine if other factors than antibodies might have an effect on the outcome of the test, some
experiments for the future can be proposed. VNT could also be repeated with the same type of Vero cells and the same sera, but with another virus that also grows on Vero cells and is unknown for cats. If inhibition also occurs with this other virus, aspecific inhibition of virus replication is more likely.
prior to the VNT. This will remove the antibodies from the serum and if still neutralizing supports the role of other factors in inhibiting virus replication. Also the purified antibodies can be tested again in the VNT to determine if neutralization is indeed antibody mediated. Another option is to add specific S-proteins to the neutralization assay, to see if neutralization is then blocked, indicating that inhibition of viral replication is due to specific antibody reaction.
It has to be noted that the results in the VNTs were sometimes inconsistent. In a couple of samples debris, precipitates, bacteria and fungus were seen, which made it difficult to interpret the results. Some of the samples are more than 30 years old and might have been thawed many times, which could contribute to the pollution. However most of the samples were of good quality as determined by visual examination. Also blood plasmas have been used, it is possible than clothing has occurred. For future research, also antifungals could be added during the VNT to rule out fungal inhibition.
Virus neutralization was determined by visual inspection of the wells for reduction of number of fluorescent cells. PEDV does also induce a CPE. A possibility for future studies is staining the cells for metabolic activity, this could provide an objective parameter to determine cell viability in cells inoculated with the different samples with and without virus. This could give also more information on whether or not the samples have some degree of toxicity.
The earlier described samples “Kat89”, “Kat129”, “Kat131”, “91type1” and “93type1” have also been tested in this VNT, as well as the samples from these cats before infection. The samples from before infection with the FCoV type 1 virus all show no neutralization, which is expected with these SPF-cats. Noteworthy, all of the samples after infection show neutralization, however with low titers of maximum 32. This is also the case for the samples after infection which tested negative in ELISA for PEDV-S1-mFc. Unfortunately, these VNT results were sometimes quite ambiguous and difficult to readout, with duplo’s which do not correspond well with each other and neutralization seems to “skip” some dilutions. Nevertheless, it is evident that sera after infection with FCoV type 1 have an effect on neutralization. Also this effect could be due to aspecific inhibiting factors which are released after FCoV type 1 infection, or could be due to crossreactivity.
VNT ON FCOV
VNT with porcine sera is carried out on both types of FCoV, to determine whether there was also crossreaction at the level of neutralization. VNT titers were compared with ELISA results that were determined in previous studies (Joris de Jong, personal communication, July 2015). It is noticeable that with these porcine sera, ELISA results for FCoV-S1 and FCoV-S1-mFc do not correlate, in contrast to the findings with the feline samples. The protein with mFc still attached to it reacted much less with the porcine samples and lower OD-values were achieved. The presence of the mFc part seemed to block the binding of the antibodies from the PEDV positive swine sera, which might be due to some kind of steric hindrance. This seemed not to be the case for all proteins with mFc, as for PEDV-S1-mFc and TGEV-S1-mFc high OD-values were reached. The porcine sera which reacted with PEDV-S1-mFc, also reacted with FCoV-s1-mFc type 1. This makes crossreactivity of antibodies between FCoV and PEDV again more likely.
VNT on FCoV type 1
No correlation was seen between the PEDV-S1-mFc ELISA scores and the ability of the sera to neutralize the TN406HP FCoV type 1 virus strain. Most PEDV-S1-mFc ELISA positive sera showed similar results as the SPF control feline serum and the ELISA PEDV-S1-mFc negative samples and were thus considered as negative on FCoV type 1 neutralizing activity. For positive as well as negative samples low neutralization titers of up to 15 were found, probably this can be attributed to aspecific factors in these sera.
When high titers in the VNT with feline sera against PEDV were solely attributable to crossreactivity, it was expected this would also occur vice versa and that all of the PEDV-S1-mFc ELISA positive porcine samples would reach high titers in VNT on FCoV type 1. This is not the case, which raises the presumption that VNT results are against PEDV are not completely contributable to crossreactivity. However, it has to be noted that porcine sera were selected based on ELISA results and ELISA-positive results do not always have to correlate with the presence of neutralizing antibodies.
VNT on FCoV type 2
Between the PEDV-S1-mFc ELISA scores and the ability of the sera to neutralize the FIPV70-1146 FCoV type 2 strain no correlation was found. Some ELISA PEDV-S1-mFc negative sera showed high titers of neutralization of FCoV type 2, although not as high as the type 2 positive control.
When type 2 virus neutralization is compared with ELISA results on the TGEV-S1-mFc protein, these two do correlate. ELISA PEDV-S1-mFc negative sera that had high OD values for the TGEV-S1 protein (at least five times the background, but even way more in these cases) showed neutralization capacities for the FCoV type 2 virus. As mentioned before, TGEV and FCoV type 2 are highly related. It is very plausible that epitopes from TGEV are conserved in FCoV type 2 and crossreactivity occurs in VNT.
The ELISA PEDV-S1-mFc positive pig sera did not neutralize the FCoV type 2 virus or only in small extent. All of these sera tested also negative in the ELISA for TGEV-S1-mFc protein. This probably has to do with the fact that the PEDV-positive pigs are from a different (Asian) population than the (Dutch) pigs which are PEDV-negative on ELISA. Other diseases are endemic in Asia than in the Netherlands.
SCREENING FOR DIFFERENT CORONAVIRUSES IN CATS
In conclusion, serological screening of feline sera revealed positive results against S1 protein of different coronaviruses. The PEDV-S1 positive sera most likely represent cross-reactivity with the FCoV-S1 proteins and not an infection with a PEDV(-like) virus. The basis of the neutralization of PEDV to high titers with some of the feline sera needs further investigation. VNT on FCoV type 1 and 2 with porcine samples did not reveal
crossreactivity on the level of neutralizing epitopes. Interestingly, by ELISA a few sera were detected that only reacted with the S1 protein of 229E or SDCV, which might indicate infection with these or related
coronaviruses in cats. Further studies are needed to explain these results.
ACKNOWLEDGEMENT
This report has been established thanks to the time and guidance of Herman Egberink. Also I would like to thank Nancy Schuurman for her help with the practical aspects of this study. Wentao Li, Berend-Jan Bosch and Joris de Jong are thanked for providing results, sharing their ideas and help with the laboratory work.
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APPENDIX 1: ELISA ON DIFFERENT CORONA S1 PROTEINS
Results of the feline sera screened for S1-proteins from 12 different coronaviruses. Alphacoronaviruses are in yellow, betacoronaviruses are in green and deltacoronaviruses are in blue.
Samples are tested in duplo, the average OD-value of both tests is shown. This is not the case for the samples in grey, only one value is shown for those samples.
Negative controls, used to calculate background, are shown in green.
HKU1-
S1-mFc
MERS-
S1-mFc
SARS-
S1-mFc
229E-
S1-mFc
OC43-
S1-mFc
NL63-
S1-mFc
FCOV-
S1-mFc
type1
PEDV-
S1-mFc
SDCV-
S1-mFc
FCoV-S1
type 1
FcoV-S1
type
2
TGEV-
S1-mFc
BCOV-
S1-mFc
24/11/86 hap. 0,076 0,060 0,083 0,080 0,094 0,062 0,057 0,114 0,122 0,059 0,062 0,099 0,134 91type1 0,058 0,060 0,075 0,210 0,099 0,077 2,693 1,164 0,186 2,812 1,383 0,262 0,079 91type1 t=0 0,104 0,047 0,099 0,071 0,122 0,086 0,068 0,126 0,158 0,056 0,053 0,088 0,087 93type1 0,066 0,056 0,071 0,132 0,080 0,063 3,020 0,119 0,095 3,013 0,088 0,077 0,068 93type1 t=0 0,078 0,044 0,077 0,063 0,080 0,062 0,057 0,101 0,125 0,055 0,046 0,070 0,067 Boosterserum kropveld 0,059 0,050 0,079 0,062 0,084 0,072 0,067 0,080 0,095 0,057 0,057 0,091 0,127 C11572 0,070 0,068 0,075 0,211 0,088 0,100 2,390 0,612 0,104 2,349 0,283 0,337 0,086 C13299 0,060 0,053 0,067 0,146 0,122 0,094 0,107 0,123 0,092 0,047 0,239 0,297 0,062 C14155 0,094 0,067 0,106 0,100 0,147 0,240 3,313 0,686 0,206 3,497 1,103 0,482 0,166 C14162 0,075 0,063 0,077 0,223 0,121 0,117 3,113 0,407 0,465 2,474 0,510 0,328 0,071 C16122 0,064 0,113 0,089 0,082 0,086 0,074 0,068 0,082 0,109 0,178 0,141 0,116 0,081 C16240 0,100 0,127 0,143 0,348 0,172 0,154 3,785 1,050 0,125 3,536 0,239 0,159 0,108 C16564 0,051 0,051 0,087 0,066 0,084 0,064 0,062 0,092 0,114 0,045 0,051 0,079 0,075 C16679 0,087 0,087 0,136 0,127 0,132 0,095 1,649 0,213 0,334 2,028 0,091 0,158 0,152 C16689 0,053 0,057 0,079 1,091 0,075 0,126 3,215 1,412 0,174 3,525 0,217 0,181 0,088 C16690 0,077 0,077 0,095 0,757 0,138 0,204 3,375 1,286 0,161 3,481 0,699 0,443 0,098 CV777 eind postserum 0,214 0,193 0,178 0,232 0,239 0,225 1,083 3,095 0,270 0,462 0,264 0,637 0,658 CV777 postserum 0,236 0,235 0,234 0,243 0,265 0,243 0,539 2,969 0,306 0,511 0,290 0,777 0,714 FIP1013 0,112 0,104 0,152 0,218 0,125 0,116 2,253 0,126 0,130 1,522 0,240 0,228 0,120 FIP1019 0,070 0,092 0,164 0,270 0,129 0,102 3,334 0,397 0,159 3,402 0,868 1,055 0,091 FIP1026 0,050 0,045 0,061 0,113 0,068 0,139 3,330 2,194 0,397 3,386 0,367 0,397 0,062 FIP1042 0,085 0,067 0,086 0,069 0,067 0,061 3,344 2,664 1,574 3,469 1,793 1,049 0,064 FIP323 0,070 0,063 0,110 0,097 0,086 0,070 0,513 0,132 0,121 0,377 3,036 1,904 0,073 FIP329 0,065 0,051 0,092 0,332 0,109 0,074 0,420 0,537 0,168 0,194 3,170 2,403 0,074 FIP654 0,113 0,078 0,093 0,097 0,091 0,080 3,287 0,502 0,139 3,404 0,096 0,102 0,089 FIP660 0,077 0,084 0,151 0,101 0,101 0,070 3,290 0,855 0,103 3,422 0,083 0,068 0,064 FIP679 0,101 0,086 0,140 1,151 0,158 0,142 3,397 0,372 0,561 3,420 1,890 0,922 0,086 FIP714 0,104 0,086 0,189 1,020 0,176 0,677 3,440 0,838 0,152 3,525 2,585 0,780 0,099 FIP750 0,118 0,135 0,127 0,427 0,146 0,168 3,135 0,342 0,271 3,161 0,125 0,098 0,097 FIP797 0,093 0,061 0,070 2,390 0,098 0,073 3,267 1,319 0,132 3,391 1,368 0,347 0,075 FIP807 0,122 0,147 0,215 0,235 0,158 0,160 3,644 2,748 0,168 3,606 0,357 0,192 0,100 FIP902 0,085 0,077 0,087 0,491 0,101 0,129 3,401 0,272 0,686 3,133 0,073 0,076 0,094 FIP980 0,063 0,059 0,102 0,172 0,137 0,142 3,518 0,185 0,232 3,356 1,118 0,958 0,078 G317 0,045 0,044 0,063 0,113 0,057 0,049 0,147 0,283 0,103 0,070 3,106 2,249 0,062Kat129 0,053 0,052 0,073 0,125 0,062 0,057 3,242 0,914 0,107 2,918 0,140 0,113 0,059 Kat129 t=-5 0,075 0,046 0,084 0,062 0,078 0,066 0,054 0,092 0,136 0,048 0,047 0,069 0,062 Kat131 0,059 0,057 0,069 0,100 0,070 0,062 3,220 0,610 0,104 2,748 0,137 0,106 0,058 Kat131 t=-5 0,069 0,043 0,081 0,062 0,083 0,064 0,065 0,124 0,138 0,052 0,050 0,079 0,070 Kat89 0,055 0,049 0,058 0,112 0,058 0,050 2,500 0,248 0,079 2,351 0,087 0,079 0,058 Kat89 t=-5 0,086 0,050 0,098 0,068 0,078 0,068 0,062 0,111 0,124 0,046 0,049 0,070 0,065 Kb001 0,060 0,079 0,101 0,065 0,084 0,088 0,054 0,076 0,098 0,060 0,062 0,074 0,078 Kb002 0,097 0,093 0,177 0,088 0,184 0,100 0,099 0,150 1,033 0,058 0,068 0,099 0,127 Kb003 0,079 0,076 0,103 0,071 0,099 0,068 0,280 0,094 0,106 0,107 0,074 0,105 0,084 Kb006 0,122 0,105 0,108 0,081 0,139 0,090 0,943 0,109 0,140 0,166 0,173 0,125 0,478 Kb011 0,077 0,076 0,089 0,081 0,078 0,068 0,067 0,094 0,090 0,052 0,072 0,071 0,076 Kb013 0,084 0,108 0,150 0,070 0,105 0,087 0,074 0,103 0,132 0,075 0,111 0,080 0,095 Kb014 0,100 0,093 0,121 0,102 0,152 0,090 0,448 0,118 0,137 0,200 0,062 0,071 0,081 Kb015 0,077 0,055 0,080 0,068 0,102 0,073 0,071 0,095 0,098 0,041 0,049 0,067 0,077 Kb018 0,063 0,058 0,076 0,083 0,081 0,074 3,556 0,099 0,087 2,314 0,076 0,078 0,064 Kb019 0,087 0,065 0,072 0,073 0,093 0,054 0,110 0,081 0,090 0,051 0,053 0,070 0,075 Kb020 0,100 0,089 0,104 0,101 0,137 0,097 1,732 0,145 0,116 0,461 0,083 0,082 0,096 Kb021 0,069 0,089 0,104 0,502 0,090 0,049 0,055 0,087 0,112 0,049 0,053 0,066 0,081 Kb022 0,062 0,048 0,083 0,081 0,097 0,056 3,083 0,261 0,099 2,184 0,279 0,143 0,069 Kb023 0,073 0,076 0,084 0,200 0,112 0,101 2,693 0,175 0,120 1,942 1,550 0,727 0,109 Kb024 0,125 0,091 0,152 0,128 0,210 0,151 0,146 0,182 0,448 0,055 0,094 0,105 0,195 Kb025 0,089 0,064 0,079 0,073 0,092 0,066 0,063 0,098 0,112 0,070 0,077 0,080 0,090 Kb026 0,080 0,068 0,097 0,098 0,144 0,072 0,067 0,150 0,164 0,063 0,080 0,090 0,100 Kb027 0,077 0,072 0,099 0,107 0,158 0,087 3,208 0,177 0,176 2,969 1,038 0,650 0,121 Kb028 0,127 0,081 0,101 0,629 0,130 0,117 3,460 0,356 0,238 3,043 0,335 0,275 0,197 Kb029 0,090 0,061 0,085 2,233 0,192 0,438 3,363 0,651 0,130 3,105 0,125 0,090 0,121 Kb030 0,097 0,091 0,131 0,130 0,124 0,092 1,408 0,123 0,106 0,355 0,129 0,096 0,084 Kb031 0,049 0,048 0,061 0,058 0,057 0,050 0,117 0,066 0,091 0,052 0,056 0,062 0,061 Kb032 0,073 0,062 0,085 0,083 0,086 0,068 0,082 0,094 0,114 0,075 0,063 0,077 0,160 Kb033 0,074 0,086 0,113 0,116 0,122 0,081 2,621 0,247 0,153 1,300 0,098 0,071 0,080 Kb034 0,086 0,077 0,102 0,075 0,087 0,067 0,071 0,075 0,092 0,052 0,064 0,083 0,097 Kb035 0,080 0,070 0,089 0,090 0,083 0,084 0,164 0,111 0,104 0,085 0,071 0,057 0,062 Kb036 0,056 0,058 0,069 0,068 0,077 0,057 0,065 0,070 0,087 0,056 0,065 0,066 0,073 Kb037 0,058 0,053 0,068 0,105 0,074 0,058 2,044 0,115 0,095 1,474 0,087 0,080 0,083 Kb039 0,144 0,138 0,174 0,351 0,221 0,142 2,369 0,718 0,282 2,268 0,231 0,317 0,206 Kb040 0,122 0,098 0,117 0,570 0,160 0,151 2,967 1,402 0,225 2,785 0,397 0,333 0,178 Kb041 0,078 0,080 0,093 0,085 0,105 0,082 0,985 0,116 0,145 0,743 0,083 0,126 0,100 Kb042 0,100 0,102 0,151 0,104 0,138 0,135 0,120 0,174 0,189 0,093 0,112 0,184 0,202 Kb043 0,074 0,072 0,242 0,078 0,100 0,077 0,071 0,098 0,130 0,076 0,097 0,088 0,106 Kb044 0,113 0,102 0,118 0,093 0,137 0,104 0,094 0,111 0,119 0,069 0,112 0,097 0,102 Kb045 0,054 0,054 0,061 0,069 0,064 0,055 0,989 0,084 0,095 0,725 0,124 0,082 0,061 Kb046 0,081 0,081 0,081 0,080 0,088 0,073 0,365 0,116 0,098 0,363 0,128 0,083 0,086 Kb047 0,061 0,066 0,105 0,319 0,088 0,218 3,105 1,234 0,284 3,031 1,045 0,159 0,082 Kb048 0,082 0,071 0,146 0,125 0,106 0,139 0,087 0,169 0,117 0,061 0,088 0,080 0,087 Kb049 0,077 0,116 0,150 0,142 0,169 0,163 0,155 0,149 0,104 0,060 0,069 0,073 0,072 Kb051 0,082 0,094 0,153 0,166 0,193 0,157 2,386 0,253 0,213 1,685 0,118 0,120 0,092
Kb052 0,072 0,066 0,082 0,083 0,157 0,124 0,530 0,143 0,167 0,231 0,077 0,080 0,078 Kb054 0,086 0,086 0,120 0,118 0,272 0,097 0,970 0,111 0,134 0,634 0,076 0,098 0,117 Kb056 0,088 0,086 0,117 0,117 0,100 0,097 0,151 0,121 0,134 0,109 0,083 0,104 0,104 Kb057 0,128 0,102 0,218 0,194 0,219 0,172 3,080 0,271 0,267 2,753 1,524 0,200 0,187 Kb058 0,076 0,099 0,107 0,101 0,090 0,091 0,090 0,115 0,119 0,102 0,141 0,094 0,102 Kb059 0,059 0,060 0,082 0,083 0,062 0,081 3,054 0,226 0,122 2,730 0,057 0,087 0,082 Kb060 0,090 0,076 0,105 0,093 0,116 0,079 1,714 0,176 0,124 1,220 0,080 0,080 0,102 Kb062 0,060 0,061 0,077 0,069 0,070 0,065 0,067 0,085 0,098 0,062 0,066 0,065 0,068 Kb064 0,088 0,081 0,103 0,091 0,081 0,085 0,088 0,095 0,117 0,081 0,078 0,096 0,094 Kb065 0,046 0,044 0,057 0,049 0,058 0,049 0,051 0,065 0,075 0,042 0,045 0,059 0,060 Kb066 0,054 0,049 0,068 0,060 0,071 0,066 0,075 0,073 0,076 0,054 0,050 0,069 0,083 Kb067 0,090 0,088 0,106 0,097 0,109 0,082 2,583 0,387 0,116 2,061 0,233 0,105 0,094 Kb068 0,069 0,059 0,083 0,076 0,097 0,087 0,542 0,109 0,153 0,482 0,070 0,083 0,105 Kb069 0,089 0,076 0,090 0,239 0,098 0,121 2,681 0,656 0,173 1,997 0,093 0,093 0,125 Kb070 0,155 0,186 0,144 0,141 0,149 0,125 2,952 0,224 0,146 1,914 0,345 0,133 0,148 Kb071 0,065 0,063 0,062 0,101 0,083 0,076 2,911 0,202 0,112 2,812 1,052 0,442 0,081 Kb072 0,094 0,078 0,092 0,086 0,115 0,101 0,081 0,128 0,148 0,142 0,209 0,091 0,109 Kb073 0,073 0,058 0,072 0,062 0,081 0,060 0,060 0,078 0,094 0,085 0,060 0,070 0,076 Kb074 0,075 0,083 0,070 0,065 0,081 0,072 0,068 0,080 0,108 0,136 0,170 0,076 0,092 Kb075 0,097 0,114 0,186 0,162 0,073 0,092 0,102 0,100 0,176 0,070 0,167 0,106 0,180 Kb076 0,107 0,106 0,117 0,091 0,133 0,106 0,084 0,126 0,140 0,080 0,082 0,116 0,135 Kb077 0,076 0,065 0,067 0,348 0,084 0,108 3,213 1,283 1,648 3,241 1,713 0,561 0,080 Kb079 0,086 0,068 0,099 0,098 0,083 0,069 1,393 0,103 0,113 1,409 0,060 0,072 0,079 Kb080 0,089 0,078 0,104 0,090 0,105 0,088 0,079 0,100 0,126 0,077 0,075 0,088 0,109 Kb081 0,074 0,077 0,110 0,113 0,109 0,088 2,202 0,180 0,155 2,258 0,097 0,080 0,131 Kb082 0,070 0,064 0,085 0,067 0,068 0,079 0,067 0,076 0,096 0,076 0,080 0,063 0,077 Kb083 0,064 0,066 0,088 0,073 0,079 0,066 0,056 0,084 0,105 0,054 0,059 0,064 0,068 Kb084 0,090 0,127 0,135 0,143 0,129 0,095 3,542 0,372 0,135 3,385 0,556 0,308 0,125 Kb085 0,124 0,110 0,120 0,114 0,142 0,107 0,092 0,122 0,140 0,080 0,081 0,121 0,146 Kb086 0,052 0,053 0,060 0,054 0,073 0,056 0,049 0,087 0,168 0,050 0,050 0,062 0,083 Kb087 0,076 0,066 0,082 0,168 0,201 0,081 3,477 0,312 0,156 2,887 0,398 0,109 0,119 Kb088 0,088 0,092 0,180 0,198 0,174 0,104 0,447 0,412 0,357 0,498 0,081 0,134 0,099 Kb089 0,071 0,049 0,080 0,071 0,091 0,061 0,970 0,177 0,122 0,657 0,052 0,066 0,089 Kb090 0,094 0,076 0,093 0,081 0,107 0,092 0,081 0,125 0,149 0,080 0,202 0,087 0,099 Kb091 0,080 0,055 0,121 0,065 0,091 0,076 0,069 0,124 0,144 0,054 0,054 0,070 0,079 Kb092 0,052 0,043 0,070 0,059 0,081 0,066 0,071 0,093 0,097 0,043 0,047 0,071 0,075 Kb093 0,077 0,047 0,077 0,113 0,196 0,138 0,079 0,187 0,127 0,055 0,057 0,066 0,069 Kb094 0,059 0,051 0,103 0,082 0,065 0,062 3,157 0,309 0,145 2,556 0,186 0,119 0,073 Kb095 0,049 0,044 0,097 0,110 0,127 0,075 0,118 0,126 0,101 0,045 0,045 0,061 0,064 Kb096 0,061 0,048 0,064 0,251 0,123 0,089 3,093 0,611 0,108 2,428 0,091 0,132 0,061 Kb097 0,060 0,054 0,061 0,127 0,143 0,064 2,071 0,083 0,106 1,731 0,085 0,115 0,089 Kb098 0,097 0,101 0,082 0,115 0,114 0,073 2,688 0,433 0,179 2,065 0,163 0,168 0,126 Kb099 0,064 0,072 0,063 0,071 0,091 0,057 0,193 0,115 0,129 0,107 0,122 0,089 0,102 Kb100 0,070 0,079 0,079 0,085 0,098 0,063 3,060 0,371 0,242 3,046 0,068 0,089 0,209 Kb101 0,132 0,074 0,092 0,156 0,151 0,167 0,797 0,291 0,111 0,409 1,929 1,472 0,104 Kb102 0,054 0,087 0,059 0,114 0,065 0,054 0,061 0,073 0,109 0,064 0,043 0,079 0,067
