Veterinary Microbiology 175 (2015) 1–6
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1) Gizem Alpay, Kadir Yes¸ilbag˘ * Department of Virology, Uludag University Faculty of Veterinary Medicine, Bursa, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2014 Received in revised form 26 September 2014 Accepted 30 October 2014
Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Bovine viral diarrhea virus BVDV-1 Cross neutralization Serological relation Subgroup
1. Introduction Bovine viral diarrhea virus (BVDV), which belongs to the Pestivirus genus of the family Flaviviridae, is an enveloped single-stranded RNA virus. Using sequences in a highly conserved region (50 UTR) of the viral RNA, BVDV strains are divided into two genotypes, namely BVDV-1 and BVDV-2 (Ridpath et al., 1994). To date, at least 18 genetic subgroups in BVDV-1 (BVDV-1a to BVDV-1r) and 4 subgroups in BVDV2 (BVDV-2a to BVDV-2d) have been described (Vilcek et al., 2001; Jackova et al., 2008; Nagai et al., 2008; Yesilbag et al.,
* Corresponding author. Tel.: +90 224 294 12 95; fax: +90 224 294 12 02. E-mail address: [email protected] (K. Yes¸ilbag˘). http://dx.doi.org/10.1016/j.vetmic.2014.10.034 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
2008; Giangaspero et al., 2008; Xue et al., 2010; Gao et al., 2013; Yesilbag et al., 2014). Acute infections by BVDV can lead to subclinical or mild disease. Diarrhea, coughing, poor performance, an increase in the frequency of other infections, persistent infection of fetuses and lethal outcomes with the development of mucosal disease are some of the frequently observed reasons for the economic losses caused by BVDV. With the consequence of these impacts, several European countries have organized BVDV eradication programs on a regional or national level (Houe et al., 2006; Presi et al., 2011; Sta˚hl and Alenius, 2012). The cross neutralization results against BVDV strains belonging to the same genotype may allow the characterization of antigenic and genomic differences
G. Alpay, K. Yes¸ilbag˘ / Veterinary Microbiology 175 (2015) 1–6
2
(Bachofen et al., 2008; Ridpath et al., 2010; Minami et al., 2011). Antigenic differences in field strains can affect not only diagnostic analysis but also protection against infection. It was demonstrated that previous infections or vaccination with a pre-defined BVDV strain may not create sufficient protection against different strains (Zimmer et al., 2002; Grooms et al., 2007). Variations in the geographical distribution of BVDV-1 subgroups have been well documented (Vilcek et al., 2001; Booth et al., 2013; Yesilbag et al., 2008). The serological differences among those viruses in the different genetic subgroups are important for epidemiology, prevention and vaccination programs. A clarification of the serological relationships among the BVDV subgroups will improve the design of effective immunization strategies. There are a limited number of studies (Couvreur et al., 2002; Becher et al., 2003; Bachofen et al., 2008; Minami et al., 2011; Ridpath et al., 2010) involving the serological relationship among the BVDV subgroups. In this study, the serological relatedness among representative BVDV-1 subgroups was investigated experimentally. This study was aimed at determining the serological similarity and between BVDV-1 subgroups that are either geographically restricted or commonly found around the world. 2. Material and methods 2.1. Viruses and virus propagation BVDV isolates representing 6 different BVDV-1 subgroups (BVDV-1a, -1b, -1d, -1f, -1h, -1l) were used for the immunization and cross neutralization assays. The viruses were non-cytopathogenic (ncp) field isolates, were selected according to their phylogenetic analyses (Yesilbag et al., 2008) and monoclonal antibody binding patterns (Yesilbag and Burgu, 2006) (Table 1). The selected isolates (TR-2, TR12, TR-11, TR-38, TR-23, TR-1) share the widest monoclonal antibody binding patterns in the subjected subgroups, respectively. The viruses used for the immunization were propagated in Sheep Fetal Thymus (SFT-R) cell line grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with (2%) serum replacement (Sigma, St. Louis, USA). Madin Darby Bovine Kidney (MDBK) cell line, grown in DMEM supplemented with (10%) fetal bovine serum, was used in the cross neutralization tests. The fetal bovine serum and cell lines were both free of BVDV antigen and antibodies when tested before use. Because all of the virus strains used in this study were non-cytopathogenic, virus
Table 1 Properties of BVD virus isolates used for immunization and cross neutralization assay. Virus strain
BVDV-1 subgroups
Biotype
Year of isolation
BVDV-TR2 BVDV-TR12 BVDV-TR11 BVDV-TR38 BVDV-TR23 BVDV-TR1
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
n-cpe n-cpe n-cpe n-cpe n-cpe n-cpe
1999 2000 1999 1999 1997 1999
propagation was confirmed using the indirect immunoperoxidase monolayer assay (IIPMA), as described in this section. 2.2. Antigen preparation Each of viruses propagated in SFT-R cell line prepared in two cases of 150 cm2 flasks. Obtained supernatants were tested by indirect peroxidase linked antibody assay and a volume of 100 ml was concentrated for each of the viruses by centrifugation at 3000 rpm at +4 8C for 20 min using 100,000 MWCO ultrafiltration tubes (Vivaspin 20, Sartorius, Goettingen, Germany). The process was confirmed both by virus titration peroxidase linked antibody assay and the BVDV Ag ELISA (Herdcheck, Idexx, Bern, Switzerland). The amount of protein in the supernatants was measured using a spectrophotometer after completely removing phenol red by dialyzing (Sigma, St. Louis, USA). The protein concentrations of the dialyzed supernatants were calculated using the absorbances and the protein concentration determination nomogram. The virus suspensions were inactivated with binary ethyleneimine (0.8 g binary ethyleneimine, 1.5 ml b-naphthol violet, 0.01 g Na2S2O3 in 10 ml deionized water) for 24 h at 26 8C. The virus inactivation was stopped by sodium thiosulfate. To confirm viral inactivation, each of the inactivated virus suspension was inoculated on to MDBK cells for 3 consecutive blind passages each for 5 days. The success of the process was monitored by a peroxidase-linked antibody assay after each step of cell culture inoculation. A failure to detect positive signal indicating the presence of live virus in the inoculated cell cultures confirmed a total inactivation of the viruses. 2.3. Immunization of sheep Hyperimmunized sera against the studied viruses were raised in sheep. For that purpose, a total of 21 Awassi sheep aged 1–5 years old were divided into 7 groups that each included 3 sheep. All of the sheep were previously confirmed to be free of both the pestivirus antigen and antibodies using an antigen ELISA (Herdcheck, Idexx, Bern, Switzerland) and serum neutralization assays, respectively. Six experimental groups and one control group were arranged. The animals in the experimental groups were immunized with inactivated virus supernatants, which included 1 mg of protein in 0.5 ml phosphate buffer saline (PBS), while the control group of animals were mock inoculated with PBS. Four consecutive immunizations, the first 3 with an equal volume of Freund’s incomplete adjuvant (Sigma, St. Louis, USA) and the last one with Freund’s complete adjuvant (Sigma, St. Louis, USA), were performed via subcutaneous injections. The serum samples were collected at days 0, 15, 30, 45, 60 and 75 postimmunization and were heat inactivated at 56 8C for 30 min before testing. 2.4. Cross neutralization test Prior to comparing the cross neutralizing titers, each hyperimmune serum was tested against its homologous
G. Alpay, K. Yes¸ilbag˘ / Veterinary Microbiology 175 (2015) 1–6
BVDV strain that was given for immunization through a serum neutralization assay. Thus, the peak antibody titer among the 6 serum sampling days (0, 15, 30, 45, 60 and 75) was detected for each immunized animal in the group. The animal having the highest antibody titer in its own group was chosen, and its peak titered serum sample was used in the cross neutralization tests to determine the antibody titers against the other BVDV strains used in this study. For the antibody titration, 2-fold dilutions of heat inactivated hyperimmune sera were prepared in DMEM, and 400 ml was added to each well of 24-well plates as duplicates. After adding an equal amount of 100 TCID50 virus suspension, the plates were incubated for 1 h at 37 8C. An equal volume of the MDBK cell suspension (2 105 cells/ml) was dispensed, and the plates were incubated at 37 8C in 5% CO2 for 3 days before application of the IIPMA.
3
Table 2 Calculated pre-inactivation infective titers of the virus strains used for immunization. Virus strain
BVDV-1 subgroups
Virus titers
BVDV-TR2 BVDV-TR12 BVDV-TR11 BVDV-TR38 BVDV-TR23 BVDV-TR1
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
105.45/0.1 ml 107.7/0.1 ml 106.45/0.1 ml 107.7/0.1 ml 106.7/0.1 ml 108.45/0.1 ml
virus strains. By this formula, a ratio (R) that is equal to or close to 100 indicates less antigenic differences.
3. Results
2.5. Indirect immunoperoxidase monolayer assay (IIPMA)
3.1. Virus propagation and antigen preparation
The detection of the propagated virus or the virus neutralized wells was achieved using the IIPMA (Ozkul et al., 2002). For that purpose, MDBK cells in 24-well plates used in the cross neutralization tests as described above were heat fixed at +80 8C for 3 h. To each well, 200 ml of 0.5% O-D-glucopyranoside (Sigma, St. Louis, USA) solution in w-PBS was added and incubated for 10 min at room temperature. The rinse and antibody dilution was performed with Tween-PBS (0.05% Tween 20 in PBS). After 3 washes, the monoclonal anti-BVDV mouse antibody specific to NS3 (pool 1/4/7) (Cedillio, 2004) was added and incubated for 90 min at room temperature. The same steps were repeated for the biotin labeled antimouse antibody (Pierce, Rockford, USA) and the streptavidin-biotinylated-HRPO conjugates (Pierce, Rockford, USA), respectively. After adding the substrate solution (2 mg AEC in 0.3 ml DMF, 4.7 ml Na-acetate buffer [pH 5.5] and 0.05% H2O2), an intracellular reddish staining developed in 30 min. The highest serum dilution that inhibited the virus propagation in 50% of the wells tested in duplicates was considered to be the virus neutralization titer. The coefficient of antigenic similarity (R) was calculated using the formula given below (Archetti and Horsfall, 1950; Bachofen et al., 2008).
As detected by the IIPMA, all of the virus strains were successfully propagated both in the SFT-R and MDBK cell lines. The virus titers which were determined before inactivation are shown in Table 2. After dialyzing the protein concentration of non-infected SFT-R cell (negative control) which was used as base line for the amount of protein calculation was 1 mg/ml. The value of concentrated virus suspensions ranged between 7.8 and 18 mg/ml. No virus was detected in the filtrate obtained by ultrafiltration, indicating the success of the concentration process. After virus inactivation procedure no staining was obtained by PLA for each of the three blind passages from all the virus concentrates. This data showed that there were no residual infective virus particles in the prepared antigen. On the other side positive results were also obtained by antigen capture ELISA confirming the presence of inactivated viruses in the concentrate.
R ¼ 100
p
AB BA=AA BB
In the formula, BA is A antiserum titer against B strain and AB is B antiserum titer against A virus strain. AA and BB are the homologous serum titers against their homologous
3.2. Cross neutralizing antibody titers The cross neutralizing antibody titers among the studied BVDV-1 subgroups are presented in Table 3. The highest titers were observed in the homologous pairs compared to the viruses from different subgroups. Except for the control groups, the sera from all of the experimental groups neutralized all of the viruses with some remarkable differences in the neutralization titers. The antiserum developed with the BVDV-1f strain showed a low neutralization activity (1:20) against the BVDV-1a and BVDV-1b strains. Additionally, the BVDV-1a and BVDV-1b
Table 3 Virus neutralization titers of hyperimmunized antiserums against studied BVDV-1 subgroups. BVDV-1 viruses
Antiserums
Control group
BVDV-1a
BVDV-1b
BVDV-1d
BVDV-1f
BVDV-1h
BVDV-1l
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
1:1280 1:1280 1:1280 1:20 1:320 1:80
1:640 1:2560 1:80 1:20 1:1280 1:160
1:320 1:1280 1:1280 1:40 1:160 1:320
1:20 1:20 1:1280 1:1280 1:640 1:320
1:640 1:2560 1:1280 1:320 1:5120 1:1280
1:1280 1:640 1:640 1:1280 1:1280 1:1280
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1) Gizem Alpay, Kadir Yes¸ilbag˘ * Department of Virology, Uludag University Faculty of Veterinary Medicine, Bursa, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2014 Received in revised form 26 September 2014 Accepted 30 October 2014
Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Bovine viral diarrhea virus BVDV-1 Cross neutralization Serological relation Subgroup
1. Introduction Bovine viral diarrhea virus (BVDV), which belongs to the Pestivirus genus of the family Flaviviridae, is an enveloped single-stranded RNA virus. Using sequences in a highly conserved region (50 UTR) of the viral RNA, BVDV strains are divided into two genotypes, namely BVDV-1 and BVDV-2 (Ridpath et al., 1994). To date, at least 18 genetic subgroups in BVDV-1 (BVDV-1a to BVDV-1r) and 4 subgroups in BVDV2 (BVDV-2a to BVDV-2d) have been described (Vilcek et al., 2001; Jackova et al., 2008; Nagai et al., 2008; Yesilbag et al.,
* Corresponding author. Tel.: +90 224 294 12 95; fax: +90 224 294 12 02. E-mail address: [email protected] (K. Yes¸ilbag˘). http://dx.doi.org/10.1016/j.vetmic.2014.10.034 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
2008; Giangaspero et al., 2008; Xue et al., 2010; Gao et al., 2013; Yesilbag et al., 2014). Acute infections by BVDV can lead to subclinical or mild disease. Diarrhea, coughing, poor performance, an increase in the frequency of other infections, persistent infection of fetuses and lethal outcomes with the development of mucosal disease are some of the frequently observed reasons for the economic losses caused by BVDV. With the consequence of these impacts, several European countries have organized BVDV eradication programs on a regional or national level (Houe et al., 2006; Presi et al., 2011; Sta˚hl and Alenius, 2012). The cross neutralization results against BVDV strains belonging to the same genotype may allow the characterization of antigenic and genomic differences
G. Alpay, K. Yes¸ilbag˘ / Veterinary Microbiology 175 (2015) 1–6
2
(Bachofen et al., 2008; Ridpath et al., 2010; Minami et al., 2011). Antigenic differences in field strains can affect not only diagnostic analysis but also protection against infection. It was demonstrated that previous infections or vaccination with a pre-defined BVDV strain may not create sufficient protection against different strains (Zimmer et al., 2002; Grooms et al., 2007). Variations in the geographical distribution of BVDV-1 subgroups have been well documented (Vilcek et al., 2001; Booth et al., 2013; Yesilbag et al., 2008). The serological differences among those viruses in the different genetic subgroups are important for epidemiology, prevention and vaccination programs. A clarification of the serological relationships among the BVDV subgroups will improve the design of effective immunization strategies. There are a limited number of studies (Couvreur et al., 2002; Becher et al., 2003; Bachofen et al., 2008; Minami et al., 2011; Ridpath et al., 2010) involving the serological relationship among the BVDV subgroups. In this study, the serological relatedness among representative BVDV-1 subgroups was investigated experimentally. This study was aimed at determining the serological similarity and between BVDV-1 subgroups that are either geographically restricted or commonly found around the world. 2. Material and methods 2.1. Viruses and virus propagation BVDV isolates representing 6 different BVDV-1 subgroups (BVDV-1a, -1b, -1d, -1f, -1h, -1l) were used for the immunization and cross neutralization assays. The viruses were non-cytopathogenic (ncp) field isolates, were selected according to their phylogenetic analyses (Yesilbag et al., 2008) and monoclonal antibody binding patterns (Yesilbag and Burgu, 2006) (Table 1). The selected isolates (TR-2, TR12, TR-11, TR-38, TR-23, TR-1) share the widest monoclonal antibody binding patterns in the subjected subgroups, respectively. The viruses used for the immunization were propagated in Sheep Fetal Thymus (SFT-R) cell line grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with (2%) serum replacement (Sigma, St. Louis, USA). Madin Darby Bovine Kidney (MDBK) cell line, grown in DMEM supplemented with (10%) fetal bovine serum, was used in the cross neutralization tests. The fetal bovine serum and cell lines were both free of BVDV antigen and antibodies when tested before use. Because all of the virus strains used in this study were non-cytopathogenic, virus
Table 1 Properties of BVD virus isolates used for immunization and cross neutralization assay. Virus strain
BVDV-1 subgroups
Biotype
Year of isolation
BVDV-TR2 BVDV-TR12 BVDV-TR11 BVDV-TR38 BVDV-TR23 BVDV-TR1
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
n-cpe n-cpe n-cpe n-cpe n-cpe n-cpe
1999 2000 1999 1999 1997 1999
propagation was confirmed using the indirect immunoperoxidase monolayer assay (IIPMA), as described in this section. 2.2. Antigen preparation Each of viruses propagated in SFT-R cell line prepared in two cases of 150 cm2 flasks. Obtained supernatants were tested by indirect peroxidase linked antibody assay and a volume of 100 ml was concentrated for each of the viruses by centrifugation at 3000 rpm at +4 8C for 20 min using 100,000 MWCO ultrafiltration tubes (Vivaspin 20, Sartorius, Goettingen, Germany). The process was confirmed both by virus titration peroxidase linked antibody assay and the BVDV Ag ELISA (Herdcheck, Idexx, Bern, Switzerland). The amount of protein in the supernatants was measured using a spectrophotometer after completely removing phenol red by dialyzing (Sigma, St. Louis, USA). The protein concentrations of the dialyzed supernatants were calculated using the absorbances and the protein concentration determination nomogram. The virus suspensions were inactivated with binary ethyleneimine (0.8 g binary ethyleneimine, 1.5 ml b-naphthol violet, 0.01 g Na2S2O3 in 10 ml deionized water) for 24 h at 26 8C. The virus inactivation was stopped by sodium thiosulfate. To confirm viral inactivation, each of the inactivated virus suspension was inoculated on to MDBK cells for 3 consecutive blind passages each for 5 days. The success of the process was monitored by a peroxidase-linked antibody assay after each step of cell culture inoculation. A failure to detect positive signal indicating the presence of live virus in the inoculated cell cultures confirmed a total inactivation of the viruses. 2.3. Immunization of sheep Hyperimmunized sera against the studied viruses were raised in sheep. For that purpose, a total of 21 Awassi sheep aged 1–5 years old were divided into 7 groups that each included 3 sheep. All of the sheep were previously confirmed to be free of both the pestivirus antigen and antibodies using an antigen ELISA (Herdcheck, Idexx, Bern, Switzerland) and serum neutralization assays, respectively. Six experimental groups and one control group were arranged. The animals in the experimental groups were immunized with inactivated virus supernatants, which included 1 mg of protein in 0.5 ml phosphate buffer saline (PBS), while the control group of animals were mock inoculated with PBS. Four consecutive immunizations, the first 3 with an equal volume of Freund’s incomplete adjuvant (Sigma, St. Louis, USA) and the last one with Freund’s complete adjuvant (Sigma, St. Louis, USA), were performed via subcutaneous injections. The serum samples were collected at days 0, 15, 30, 45, 60 and 75 postimmunization and were heat inactivated at 56 8C for 30 min before testing. 2.4. Cross neutralization test Prior to comparing the cross neutralizing titers, each hyperimmune serum was tested against its homologous
G. Alpay, K. Yes¸ilbag˘ / Veterinary Microbiology 175 (2015) 1–6
BVDV strain that was given for immunization through a serum neutralization assay. Thus, the peak antibody titer among the 6 serum sampling days (0, 15, 30, 45, 60 and 75) was detected for each immunized animal in the group. The animal having the highest antibody titer in its own group was chosen, and its peak titered serum sample was used in the cross neutralization tests to determine the antibody titers against the other BVDV strains used in this study. For the antibody titration, 2-fold dilutions of heat inactivated hyperimmune sera were prepared in DMEM, and 400 ml was added to each well of 24-well plates as duplicates. After adding an equal amount of 100 TCID50 virus suspension, the plates were incubated for 1 h at 37 8C. An equal volume of the MDBK cell suspension (2 105 cells/ml) was dispensed, and the plates were incubated at 37 8C in 5% CO2 for 3 days before application of the IIPMA.
3
Table 2 Calculated pre-inactivation infective titers of the virus strains used for immunization. Virus strain
BVDV-1 subgroups
Virus titers
BVDV-TR2 BVDV-TR12 BVDV-TR11 BVDV-TR38 BVDV-TR23 BVDV-TR1
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
105.45/0.1 ml 107.7/0.1 ml 106.45/0.1 ml 107.7/0.1 ml 106.7/0.1 ml 108.45/0.1 ml
virus strains. By this formula, a ratio (R) that is equal to or close to 100 indicates less antigenic differences.
3. Results
2.5. Indirect immunoperoxidase monolayer assay (IIPMA)
3.1. Virus propagation and antigen preparation
The detection of the propagated virus or the virus neutralized wells was achieved using the IIPMA (Ozkul et al., 2002). For that purpose, MDBK cells in 24-well plates used in the cross neutralization tests as described above were heat fixed at +80 8C for 3 h. To each well, 200 ml of 0.5% O-D-glucopyranoside (Sigma, St. Louis, USA) solution in w-PBS was added and incubated for 10 min at room temperature. The rinse and antibody dilution was performed with Tween-PBS (0.05% Tween 20 in PBS). After 3 washes, the monoclonal anti-BVDV mouse antibody specific to NS3 (pool 1/4/7) (Cedillio, 2004) was added and incubated for 90 min at room temperature. The same steps were repeated for the biotin labeled antimouse antibody (Pierce, Rockford, USA) and the streptavidin-biotinylated-HRPO conjugates (Pierce, Rockford, USA), respectively. After adding the substrate solution (2 mg AEC in 0.3 ml DMF, 4.7 ml Na-acetate buffer [pH 5.5] and 0.05% H2O2), an intracellular reddish staining developed in 30 min. The highest serum dilution that inhibited the virus propagation in 50% of the wells tested in duplicates was considered to be the virus neutralization titer. The coefficient of antigenic similarity (R) was calculated using the formula given below (Archetti and Horsfall, 1950; Bachofen et al., 2008).
As detected by the IIPMA, all of the virus strains were successfully propagated both in the SFT-R and MDBK cell lines. The virus titers which were determined before inactivation are shown in Table 2. After dialyzing the protein concentration of non-infected SFT-R cell (negative control) which was used as base line for the amount of protein calculation was 1 mg/ml. The value of concentrated virus suspensions ranged between 7.8 and 18 mg/ml. No virus was detected in the filtrate obtained by ultrafiltration, indicating the success of the concentration process. After virus inactivation procedure no staining was obtained by PLA for each of the three blind passages from all the virus concentrates. This data showed that there were no residual infective virus particles in the prepared antigen. On the other side positive results were also obtained by antigen capture ELISA confirming the presence of inactivated viruses in the concentrate.
R ¼ 100
p
AB BA=AA BB
In the formula, BA is A antiserum titer against B strain and AB is B antiserum titer against A virus strain. AA and BB are the homologous serum titers against their homologous
3.2. Cross neutralizing antibody titers The cross neutralizing antibody titers among the studied BVDV-1 subgroups are presented in Table 3. The highest titers were observed in the homologous pairs compared to the viruses from different subgroups. Except for the control groups, the sera from all of the experimental groups neutralized all of the viruses with some remarkable differences in the neutralization titers. The antiserum developed with the BVDV-1f strain showed a low neutralization activity (1:20) against the BVDV-1a and BVDV-1b strains. Additionally, the BVDV-1a and BVDV-1b
Table 3 Virus neutralization titers of hyperimmunized antiserums against studied BVDV-1 subgroups. BVDV-1 viruses
Antiserums
Control group
BVDV-1a
BVDV-1b
BVDV-1d
BVDV-1f
BVDV-1h
BVDV-1l
BVDV-1a BVDV-1b BVDV-1d BVDV-1f BVDV-1h BVDV-1l
1:1280 1:1280 1:1280 1:20 1:320 1:80
1:640 1:2560 1:80 1:20 1:1280 1:160
1:320 1:1280 1:1280 1:40 1:160 1:320
1:20 1:20 1:1280 1:1280 1:640 1:320
1:640 1:2560 1:1280 1:320 1:5120 1:1280
1:1280 1:640 1:640 1:1280 1:1280 1:1280
