Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDVinfected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes. Correspondence


Introduction
Bovine viral diarrhea virus (BVDV) is a small enveloped virus ubiquitous among cattle populations.The viral genome consists of a single-stranded, positive-sense RNA molecule of approximately 12.5 kb in length (1,2).The viral RNA genome contains a single open reading frame with a coding capacity of 3988 amino acids (1).Direct translation of the viral RNA genome yields a long polyprotein that is co-and post-translationally cleaved by cellular and virus-encoded proteases, giving rise to structural and non-structural viral proteins (1,2).A small and basic protein is thought to form the viral nucleocapsid, and three glycoproteins, namely E0/gp48, E1/gp25 and E2/gp53 are inserted into the viral envelope (2).The nonstructural protein NS23/p125 is considered the most important protein involved in virus replication (3).Because of unique features of the viral genome, the NS23/p125 protein may be occasionally cleaved originating two additional products, NS2/p54 and NS3/p80 (2,3).The production of NS3/p80 by some BVDV isolates is correlated with the production of cytopathology in cultured cells; such isolates are classified as cytopathic biotype.In contrast, most BVDV isolates express only the NS23/p125 polypeptide and do not produce cytopathology, being classified as non-cytopathic (3,4).In addition, according to the nucleotide sequence of the 5' untranslated region of the viral genome, BVDV isolates might be classified as genotype 1 and 2. Both cytopathic and non-cytopathic biotypes are found within each genotype (5,6).
BVDV is the type species of the genus Pestivirus, within the family Flaviviridae, along with the hog cholera virus (HCV) and border disease virus of sheep (7).Clinical manifestations of BVDV infection include fever, mild acute or chronic diarrhea, respiratory disease, and hemorrhagic disease (8).Infection of seronegative pregnant cows with a non-cytopathic BVDV biotype may lead to embryonic or fetal deaths, abortion or fetal mummification, congenital malformations, stillbirths or the birth of weak or apparently healthy calves, many of which may be persistently infected with the virus (8).Persistently infected animals are the main source of BVDV infection to other animals in that they continuously shed viruses.In addition, mucosal disease, one of the most severe clinical manifestations of BVDV infection, occurs in persistently infected animals usually within the first two years after birth (8).
The classification of BVDV isolates into distinct biotypes and genotypes has been unequivocal, yet the grouping of field isolates according to their antigenic profile has been quite difficult.Several cross-neutralization studies have demonstrated a marked antigenic diversity among geographically distinct isolates (9)(10)(11)(12).The antigenic variability among BVDV has been better defined by monoclonal antibody (mAb)-based studies (13,14).However, most existing mAbs have been produced against North American or European isolates of BVDV; consequently, it has been rather difficult to characterize South American BVDV isolates.In addition, the unavailability of mAb against Brazilian isolates has hampered the characterization and phenotypic studies of BVDV in Brazil.Thus, local isolates of BVDV that were antigenically distinct from the standard BVDV strains (12), as determined by characterization with a panel of internationally available mAbs, were used to immunize BALB/c mice with the main objective of producing hybridoma cells secreting anti-BVDV mAbs.

Antigen production and mouse immunization
The BVDV isolates were biologically cloned three times by limiting dilution and used to infect MDBK cells to produce antigen for mouse immunization.Cells were infected at a multiplicity of infection of 0.1 to 1.0 cell culture infectious dose (CCID 50 )/ cell and the cell supernatant was collected at 72 h postinfection.Following centrifugation at 25,000 g for 10 min to remove cell debris, the virus suspension was mixed with a solution of polyethylene glycol (PEG, MW 8000; Sigma) and NaCl to achieve a final concentration of 7% and 0.4 M, respectively, and stirred at 4 o C for 4 h.The viral particles were pelleted from the solution by centrifugation at 65.000 g for 30 min at 4 o C, and resuspended in MEM at 1/100 of the initial volume.The concentrated virus was titrated on MDBK cells and aliquots were stored at -70 o C. Concentrated virus was mixed with an equivalent volume of adjuvant and injected intraperitoneally (ip) into BALB/c mice (200 µl in Freunds complete adjuvant at day 0 and 200 µl in Freunds incomplete adjuvant at 14, 28, 42, 56 and 70 days after the first injection).Two hundred microliters of concentrated virus without adjuvant was injected ip 3 days prior to removal of the mouse spleen and cell fusion.

Cell fusion, hybridoma selection and screening
Three days prior to fusion, the mice were boosted with an ip injection of adjuvant-free concentrated virus.The spleen was removed and minced and the obtained lymphocytes were mixed with Sp2 cells at a proportion of 10:1.Cell fusion was induced with 50% PEG (MW 1500; Sigma) for 1 min at 37 o C, followed by slow addition of RPMI medium.The cells were then plated onto 96-well plates containing hypoxanthine-aminopterin-thymidine medium (Sigma) supplemented with 15% FBS and 20% conditioned media.Expanding hybridomas were detected 7 to 10 days after fusion and the supernatant was tested for the presence of mAbs by immunoperoxidase (IPX) or indirect fluorescent antibody (IFA) staining.Hybridoma cells secreting BVDV-specific mAbs were then cloned and their reactivity to several BVDV isolates was determined by IFA.

IPX and IFA staining
The presence of mAbs in the supernatant of expanding hybridomas was tested on MDBK cells infected with BVDV isolate UFSM-1.IPX was performed on 96-well plates containing BVDV-infected cell monolayers fixed with 35% acetone.One hundred microliters of undiluted hybridoma supernatant was incubated with infected and noninfected cells for 1 h at 37 o C.After washing, horseradish peroxidase-conjugated antimouse immunoglobulin (Sigma) was added and incubated as above.After removal of the conjugate, color development was observed following the addition of the substrate solution (3-amino-9-ethylcarbazole; Sigma).For IFA staining, MDBK cells infected with BVDV isolate UFSM-1 and non-infected MDBK cells were individualized by trypsinization and dropped on 12-spot Teflon slides.Following adhesion, cells were fixed with acetone, dried and incubated with undiluted medium collected from expanding hybridomas for 1 h at 37 o C. Fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin (Sigma) was added and incubated as above.

Production of ascitic fluid
For each hybridoma secreting anti-BVDV mAbs, 10 BALB/c mice were primed with Freunds complete adjuvant (Sigma) and 7 days later were injected ip with approximately 10 6 hybridoma cells.Ten days later the ascitic fluid was collected from the mice, cleared by low speed centrifugation, titrated, aliquoted and stored at -70 o C.

Characterization of the mAbs
The BVDV-specific mAbs secreted by hybridoma clones 3.1C4 and 6.F11 were characterized regarding immunoglobulin class, capacity to neutralize virus infectivity, spectrum of reactivity with different BVDV isolates, and ability to react with BVDV antigens in routinely fixed histological sections and in Western immunoblot.The immunoglobulin class and subclass of mAbs 3.1C4 and 6.F11 were determined using a commercially available kit, according to the manufacturers recommendations (Mouse Type Isotyping kit, Bio-Rad, Hercules, CA, USA).To investigate the capacity of the mAbs to neutralize virus infectivity, serial 2fold dilutions of the respective ascitic fluid were mixed with 100 CCID 50 of BVDV UFSM-1 isolate and incubated for 1 h at 37 o C. A suspension of MDBK cells was then added to the wells and the plates were incubated at 37ºC in the presence of 5% CO 2 .Virus neutralization or growth was monitored by IFA staining of cells, as described above.The spectrum of reactivity of mAbs 3.1C4 and 6.F11 was assessed by staining cell monolayers infected with BVDV field isolates and reference strains and by staining 2 HCV isolates with IFA (Table 1).The ability of the mAbs to react with BVDV antigens after routine formalin fixation and paraffin embedding was investigated by IPX staining according to previously described protocols, using mAb 15c5 as positive control (15).The ability of these mAbs to recognize viral proteins resolved by SDS-PAGE and immobilized on nitrocellulose membranes was investigated by Western immu-noblot analysis of BVDV-infected cell lysates, performed according to standard protocols (16).

Protein specificity of mAbs 3.1C4 and 6.F11
To determine the mAb protein specificity, CV-1 cells transfected with plasmid constructs that express BVDV proteins (see below) were fixed with acetone:methanol (1:1) and incubated with ascitic fluid diluted 1:100 or 1:500 for 1 h at room temperature.The ascitic fluid was then washed and the cells were flooded with FITC-labeled goat antimouse antibody (Sigma).Staining of infected cells was observed by fluorescence microscopy.

Transient expression of BVDV proteins
Confluent CV-1 cells (African Green monkey kidney, ATCC-CCL 70) were infected with a recombinant vaccinia virus expressing phage T7 RNA polymerase (vTF7-3) (17) at an input multiplicity of 5 for 45 min at 37 o C. DNA-lipid complexes were prepared at room temperature using lipofectamine (Gibco-BRL) and DNA at a weight ratio of 12:1, as recommended by the manufacturer.Immediately following infection, CV-1 cells were washed three times with Dulbeccos minimal essential medium (DMEM, Gibco) without serum and transfected with 0.50 ng of each of five plasmid constructs that comprise the entire polyprotein of BVDV strain NADL (18).The DNAlipofectamine complexes were incubated with cells at 37 o C in the presence of 5% CO 2 for 4 h.After lipofection, the DNA-lipid complexes were removed and the cells were supplemented with DMEM and 10% FBS for an additional 13 h, when the cells were fixed for IFA analysis.

Results
The fusion experiments that yielded hy- bridoma cells secreting anti-BVDV mAbs were performed using virus antigens from cells infected with the isolate UFSM-1 (12).Following five fusions of lymphocytes and myeloma cells, several expanding hybridoma cells were found to be reactive to BVDVinfected cells.However, only two hybridoma clones (3.1C4 and 6.F11) were stabilized and found to secrete specific mAbs against BVDV proteins; several other hybridoma clones secreted mAbs that were also reactive to cellular components.These mAbs have not been further characterized.Both mAbs were reactive by IFA to all 32 South American isolates and several US isolates of BVDV, including viruses from both genotypes (BVDV type 1 and 2), but not to two HCV isolates (Table 1).The BVDVspecific mAb-secreting hybridoma cells were then cloned and were used to produce mAbs either in cell culture or by injection of the hybridoma cells into BALB/c mice to obtain ascitic fluid.
Hybridoma supernatant and ascitic fluid derived from clones 3.1C4 and 6.F11, that were found to react specifically with BVDVinfected cells by IFA and IPX, yielded a positive signal by IFA even when diluted up to 100 (supernatant) and 25,600 times (ascitic fluid).Both mAbs were shown to belong to the IgG2a isotype.Only mAb 3.1C4 had neutralizing activity (1:80) against BVDV parental virus (UFSM-1, data not shown).This mAb also showed neutralizing activity against the BVDV Singer strain and against a BVDV type 2 cytopathic virus, although at lower titers.The protein specificity of the mAbs could not be determined by Western immunoblot analysis of lysates obtained from BVDV-infected cells.The mAbs did not react with viral proteins separated by SDS-PAGE and transferred to nitrocellulose membranes (data not shown), suggesting they are probably directed to conformational-type epitopes.Likewise, the mAbs failed to react with viral proteins by IPX in tissues submitted to routine formalin fixa-tion and paraffin embedding.However, using a transient BVDV-protein expression system, it was demonstrated that 3.1C4  reacts with E2/gp53 and mAb 6.F11 reacts with E1/gp25 (Table 2 and Figure 1).

Discussion
The clinical syndromes associated with BVDV infection result in severe economic losses to the cattle industry around the world.In endemic areas, the prevalence of BVDV antibodies among adult cattle may reach up to 70 to 80% (19).Because of their RNA genome and ubiquitous nature, a marked antigenic and genetic heterogeneity is observed among field BVDV isolates, repre-senting a problem for diagnosis and vaccine development (10).Thus, to identify potential vaccine candidate strains, it is necessary to perform phenotyping studies in order to identify the isolates that are representative of the viruses circulating among a given cattle population.These local isolates are more likely to elicit an immune response that is protective to most isolates prevalent in the area than viral strains derived from other geographic locations.Phenotyping studies of BVDV were made possible only after the production of mAbs to laboratory reference strains.Furthermore, mAbs are considered a powerful tool for research and diagnostic purposes.
Recently, several reports have demonstrated that BVDV is highly prevalent in Brazilian cattle (12,16,20), and as such, it is probably associated with significant economic losses to the national livestock industry.These reasons prompted us to initiate epidemiological studies on BVDV infection followed by antigenic and molecular characterization of BVDV circulating in Brazilian cattle.Although the production of mAbs to several BVDV isolates has already been reported, most of these mAbs have been produced against North American and European strains (9,13,14,21).In addition, most of these mAbs were produced against laboratory reference strains rather than against field virus isolates.In a recent study, we have demonstrated that the BVDV viruses isolated in Brazil display marked antigenic differences when compared to North American reference strains (12,16).We understand that the production of mAbs to Brazilian BVDV field isolates, antigenically distinct from the standard strains, will help in better defining the antigenic properties of these isolates and in designing more effective diagnostic tools and vaccines.
The low number of hybridoma clones secreting BVDV-specific mAbs obtained in the present study may be attributed to problems inherent to the technique per se, and to the peculiar biological properties of the pestiviruses as well.These include the inability of some viral isolates to replicate to high titers in cell culture, the poor antigenicity of BVDV to BALB/c mice and the difficulties in achieving a pure and concentrated suspension of viral particles for mouse immunization.The two mAbs obtained so far failed to recognize viral protein following SDS-PAGE and immunoblot analysis of infected cells, suggesting that they recognize conformational-type epitopes.Consequently, the identification of the protein specificity of these mAbs was made possible only by using a transient protein expression system in which BVDV proteins were expressed individually on CV-1 cells.Both mAbs reacted with viral structural proteins: mAb 6.F11 reacted with glycoprotein E1/gp25 and mAb 3.1C4 reacted with glycoprotein E2/gp53.
Most mAbs against BVDV and other pestiviruses produced to date fall into two major groups: the first group comprises the pan-pestivirus mAbs, which recognize most pestivirus isolates and are mostly directed at the non-structural polypeptide NS23/p125 (9,13,14,21).NS23/p125 is a multifunctional protein ultimately involved in viral RNA replication, and is highly conserved among pestiviruses (3).The other group comprises the type-specific mAbs, which are able to distinguish between strains or virus clusters possessing slight antigenic differences.Most of these mAbs are directed to the envelope glycoproteins E0/gp48 and E2/gp53 (9,13,14,22).Glycoprotein E2/gp53 is the major envelope glycoprotein and is believed to play a major role in the initial interactions of virions with cell membrane proteins during virus attachment and penetration (3).This viral glycoprotein contains at least three highly variable regions and is a major target for neutralizing antibodies (3,21,22).
Cellular receptor-binding proteins usually contain highly conserved domains that mediate specific interactions with cellular components (23,24).These conserved re-gions, usually inaccessible to antibody binding, are often surrounded by variable regions that allow viruses to escape from the immune response (23,24).Thus, mAbs directed at these variable regions usually fail to recognize a considerable number of field isolates (9,13,14,21,22).Interestingly, mAb 3.1C4 was able to recognize all BVDV isolates tested so far.This suggests that it binds to a highly conserved epitope within E2/ gp53, which is possibly involved in an important biological function and therefore is under strong variability constraint.Furthermore, the epitope recognized by mAb 3.1C4 seems to be required for initiation of infection since viral infectivity was substantially reduced following virus-neutralization assays.To date, only a few broadly reactive mAbs against E2/gp53 have been described (14).Nevertheless, testing mAb 3.1C4 with a higher number of BVDV isolates will be necessary in order to unequivocally ascertain whether the epitope recognized is indeed thoroughly conserved.
The binding specificity of mAb 6.F11 was directed to the structural protein E1/ gp25, which is an envelope protein covalently attached to the E2/gp53 glycoprotein (3).The role of E1/gp25 remains to be determined.Nonetheless, because mAb 6.F11 was also broadly reactive to BVDV isolates, it is conceivable that this protein contains epitopes that are thoroughly conserved and might be exploited as an important diagnostic tool.
Although mAbs 3.1C4 and 6.F11 recognized all BVDV isolates tested, they failed to react with a few HCV isolates tested so far.If this property holds true for other HCV isolates, it may be exploited to distinguish pestivirus infection of pigs in areas where HCV eradication programs are underway and rapid diagnosis is required.In addition, because the epitopes recognized by these mAbs are located in viral structural proteins, they might be useful to develop immunology-based diagnostic tests such as antigen capture ELISA or IPX with the main objec-tive of identifying persistently infected animals in a herd.

Figure 1 -
Figure1-Indirect fluorescent antibody (IFA) assay of CV-1 cells transfected with a plasmid encoding the BVDV glycoprotein E2/gp53 gene under the control of the phage T7 polymerase promoter.CV-1 cells were infected with a recombinant vaccinia virus expressing the phage T7 polymerase and then transfected with the plasmid.Cells were fixed 16 h after transfection and stained with IFA using mAb 3.1C4 as primary antibody.