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Variation in immunogenicity of ruminant pestiviruses as determined by the neutralisation assay
The Veterinary Journal The Veterinary Journal 169 (2005) 468–472 www.elsevier.com/locate/tvjl
Short communication
Variation in immunogenicity of ruminant pestiviruses as determined by the neutralisation assay J.R. Patel *, S. Didlick, J. Quinton Intervet UK Ltd., Walton Manor, Walton, Milton Keynes, Buckinghamshire, MK7 7AJ, UK Accepted 28 April 2004
Abstract Immunogenicity in na€ıve three-month-old Friesian bull calves of nine ruminant pestiviruses, three each of type 1 bovine virus diarrhoea virus (BVDV), type 2 BVDV and border disease virus (BDV) was directly compared in reciprocal cross-neutralisation tests using sera obtained eight weeks after intranasal and intravenous inoculation with live virus. Cytopathic (CP) type 1 BVDV strain C86, non-cytopathic (NCP) type 2 BVDV strain 890 and NCP BDV strain V2536/2 were found to elicit significantly broad cross-neutralising antibodies against viruses in other species whereas other virus strains in all three species produced a much more pronounced homologous and/or species specific response. Results are clearly relevant in the selection of strains for vaccines against diseases caused by these successful, economically important ubiquitous viruses. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Ruminant pestiviruses; Immunogenicity; Cross-neutralisation; Vaccines; Cattle
Bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens of ruminants worldwide. Although both cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV and BDV occur naturally, the reason for the success of pestiviruses is the ability of the NCP biotype to cross the placenta and establish a persistent infection of the immunologically na€ıve developing fetus. Recent sequencing data point to CP viruses arising from NCP viruses which have undergone recombination events involving insertion of host RNA and/or duplication of viral sequences (Becher et al., 1996). Classification based on phylogenetic analyses of 50 NTR, Npro , and E2 genes have placed ruminant pestiviruses into three main genotypes with 8–11 subgroups in type 1 BVDV, two in type 2 BVDV and two in BDV (Meyers and Thiel, 1996; Becher et al., 1997, 1999a,b; Becher and Thiel, 2002; Vilcek et al., 2001). Schirrmeier et al. (2002) placed BDV isolates into three subgroups as well as suggesting that an atypical
* Corresponding author. Tel.: +44-1480-464242; fax: +44-1480461541.
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isolate ‘Hobi’ should form a third genotype of BVDV. That BDV isolates be segregated into BDV-1, BDV-2 and BDV-3 was also a conclusion of Becher et al. (2003). Additionally, monoclonal antibodies to viral glycoproteins have been used to discriminate between pestivirus genotypes as well as between strains of one genotype (Wensvoort et al., 1989; Cay et al., 1989; Paton et al., 1994). There is a high degree of variability in the structural glycoprotein E2 with amino acid homology as low as 80% within one species and <60% between genotypes (Becher et al., 1994). Virus neutralising activity is predominantly directed against the glycoprotein E2 (Corapi et al., 1990; Xue et al., 1990) and is the main target in the development of vaccines against pestiviruses (Moennig, 1990; Van Oirschot et al., 1999). In this communication, we provide experimental evidence indicating that some BVDV and BDV strains within a genotype are significantly superior in mounting a broadly cross-neutralising pan genotype antibody response. The nine strains of ruminant pestiviruses investigated were: NCP strain WB (type 1 BVDV; Patel and Shilleto, 2003), CP strain C86 (type 1 BVDV; Patel et al., 2002), CP strain Oregon C24V (type 1 BVDV), NCP
J.R. Patel et al. / The Veterinary Journal 169 (2005) 468–472
strain 890 (type 2 BVDV; Bolin and Ridpath, 1992), NCP strains Giessen I and II (type 2 BVDV; Becher et al., 1999a,b, 2003), NCP strains T1802 and V2536/2 (BDV; Vilcek et al., 1997) and NCP strain L83/84 (BDV; Becher et al., 1994, Patel and Shilleto, 2003). Viruses were grown and titrated in bovine embryo lung (BEL) cells as previously described (Patel and Shilleto, 2003). BEL cells were grown in minimal essential medium supplemented with 10% donor horse serum (HS), 100 lg/mL streptomycin, 100 units/mL penicillin, two mM L -glutamine, 1.4 mg/mL L -arginine and 0.09% sodium bicarbonate (HSGM). This medium containing 2% HS (HSMM) was used for maintaining infected cell monolayers and diluting viruses and antisera in titrations. Viral antisera were raised in naturally reared na€ıve (pestivirus and pestivirus antibody free) Friesian-cross bull calves about three months of age. Virus inocula for calves were first cloned by a limiting dilution procedure in BEL cells grown in 96-well plates at 37 °C and further grown on initially in 24-well monolayers and then in 25 cm2 monolayers of BEL cells as described previously (Patel and Shilleto, 2003). Titres of the stocks administered to calves varied significantly (see Table 1) and each calf was bled and then inoculated with one millilitre of a virus stock intravenously (IV) and also intranasally (IN).
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Sera samples taken pre-inoculation (PI) and eight weeks after the IN and the IV inoculations were reciprocally titrated for virus neutralising (VN) antibody against each virus. For VN titrations, a standard constant virus (100–316 TCID50 )–serum dilution procedure was used. Inactivated (56 °C, 30 min) sera were serially diluted in HSMM in 96-well microtitration plates, followed by the addition to each well of an equal volume (50 lL) of virus suspension in HSMM containing 100– 316 TCID50 of virus. Mixtures were incubated at 37 °C for one hour in an atmosphere of 5% CO2 in air and then 100 lL of freshly dispersed suspension of 2–4 104 BEL cells in HSGM were added to each well and the plates incubated at 37 °C in an atmosphere of 5% CO2 in air. In order to establish the dose of each virus contained in reaction mixtures, three serial ten-fold dilutions of stock virus suspension added to reaction mixtures were incubated in replicates of four, in parallel to serum–virus mixtures, followed by the addition of the cell suspension. After five days’ incubation at 37 °C, ends points for VN antibodies to CP viruses were scored as monolayers completely free of cytopathic effect (CPE) while those for NCP viruses were scored as wells with complete CPE two–three days following superinfection with CP type 1 BVDV strain C86 as described previously (Patel and Shilleto, 2003). CP type 1 BVDV strain Oregon C24V
Table 1 Antibody titres by the neutralisation test of calf sera with homologous and heterologous ruminant pestivirus strains Antiserum to strain
Biotypea and titreb
Virus strain used as antigen Type 1 BVDV WB
Type 1 BVDV C86
CP, 5.4
Oregon C24V
CP, 6.0
WB
NCP, 6.2
Type 2 BVDV 890
NCP, 6.2
Giessen 1
NCP, 5.4
Giessen 2
NCP, 6.0
BDV V2536/2
NCP, 4.7
T1802/1
NCP, 4.4
L83/84
NCP, 6.6
a
11.5 2d 7.0 4 9.0 1 12.5 2.7 2.0 256 4.0 256 8.0 4 5.0 512 7.0 384
c
Type 2 BVDV
BDV
C86
C24V
890
Giessen 1
Giessen 2
L83/84
T1802/1
V2536/2
12.5 1 9.0 1 9.0 1 13.0 2 3.0 128 7.0 32 8.0 4 6.0 256 10.5 32
12.5 1 7.0 4 7.0 4 14.0 1 3.0 128 6.0 64 8.0 4 8.5 42.7 9.0 96
9.0 12 3.0 64 3.0 64 14.0 1.0 8.5 2.7 11.0 2 7.5 7.6 6.0 256 4.5 2063
9.0 12 4.0 32 3.0 64 12.0 4 10.0 1 12.0 1 7.0 8 5.5 341.3 5.0 1536
8.5 16 3.0 64 2.5 85.3 12.0 4 8.5 2.7 12.0 1 8.0 4 6.0 256 6.5 512
8.5 16 2.0 128 <2.0 >128 12.0 4 4.5 42.7 5.5 85.3 9.5 1.3 14.0 1 15.5 1
9.0 12 <2.0 >128 2.0 128 12.0 4 2.5 170.7 4.0 256 6.5 10.7 14.0 1 11.5 16
9.0 12 2.0 128 <2.0 >128 13.0 2 2.0 256 2.0 1024 10.0 1 12.5 2.7 11.0 24
CP denotes cytopathic and NCP denotes non-cytopathic. Infectivity (log10 TCID50 /mL) of inocula administered to calves at a rate of one mL intranasally and one mL intravenously. c VN antibody titres eight weeks after inoculations expressed as log2 of reciprocal of the highest dilution completely neutralising 100–316 TCID50 of virus. Figures with half digits represent complete neutralisation in one of two wells while lower dilutions gave complete neutralisation in both wells. d The lower figures in italics denote difference (folds) between the homologous and the heterologous VN antibody titres. b
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also gave closely similar (less than two-fold) VN titres in tests with homologous virus-serum pairs. VN antibody titre is expressed as log2 of reciprocal of the highest serum dilution completely neutralising 100–316 TCID50 of a virus. Values with half digits represent titres between dilutions. The VN antibody titre of each of the nine antisera with each pestivirus strain is given in Table 1. No activity was detected in any of the nine pre-inoculation sera in tests with homologous strains (data not shown). With the exception of antiserum to type 1 CP BVDV strain Oregon C24V, homologous titres were the highest and there were also examples of some antisera neutralising other strains in the same genotype to a similar or slightly (3–4 fold) lower extent but significantly (32– 512-fold) poorer to viruses belonging to other genotypes. This was the case with antisera to NCP BDV strain T1802/1, type 2 NCP BVDV strains Giessen 1 and 2 and type 1 NCP BVDV strain WB. Titres of an antiserum to members belonging to the same genotype were closely similar with an exception of the antiserum to BDV strain L83/84 and that to BDV strain V2536/2. The titre of the former was some 16–24fold higher against BDV strains V2536/2 and T1802/1; antiserum to BDV strain V2536/2 neutralised BDV strains V2536/2 and L83/84 to a similar extent and its titre was about 11-fold lower against BDV strain T1802/ 1. An interesting finding was the fact that an antiserum to one of three virus strains in each of the three genotypes tested was significantly more broadly cross-neutralising. Hence the antiserum to type 2 NCP BVDV strain 890 neutralised three type 2 BVDV, three type 1 BVDV and three BDV strains to a closely similar (two to four fold) extent. A slightly less (2–16-fold) but still remarkable cross-neutralisation was also recorded for antisera to type 1 CP BVDV strain C86 and BDV strain V2536/2. For the latter, a noteworthy observation was the fact that the magnitude of the homologous titre was 5–16-fold lower than the homologous activity of antisera to strains C86 and 890 and 16–48-fold lower than homologous titres of antisera to BDV strains T1802/1 and L83/84. The broad VN antibody response by strains 890, C86 and V2536/2 had not been recorded previously (Becher et al., 2003). This discrepancy is not readily explained and made difficult by the fact that the production of antisera to strains 890 and C86 were not described by Becher et al. (2003) whereas antiserum to strain V2536/2 was from a sheep, six weeks following IN and intramuscular (IM) inoculation with 7.0 log10 TCID50 of virus. It is however unlikely that the difference between the present and previous findings can be explained at the level of VN tests since test methods are closely similar except for the development of endpoints. Also we are not aware if Becher et al. (2003) used cloned viruses to raise antisera or not. Further-
more, passage histories of the strains could also influence their antigenicity. Although our approach is not novel, we believe that the finding that some strains in all three genotypes of ruminant pestiviruses elicit a significantly broader crossneutralising antibody response has not previously been reported. It should however be noted that the present data were generated using one calf per strain and there can be individual variation between animals. Since the VN titres to homologous and genotypically related strains were high, we do not consider individual variation between animals to be a factor in present investigation. In this regard a previous similar serological study (Howard et al., 1987) using one calf per BVDV strain has reported useful data. Also it should be stated that the antisera to nine strains were reciprocally tested against each strain twice, and for some strains three times, with a maximum two fold variation in VN titre. With the exception of BDV strains T1802/1 and V2536/2 all the other inocula administered to calves contained between 5.4 and 6.6 log10 TCID50 of virus per mL whereas those for strains T1802/1 and V2536/2 contained 4.4 and 4.7 log10 TCID50 per mL, respectively, since both the strains grew poorly in BEL cells. Nonetheless the VN antibody responses indicate that the amounts of strains T1802/1 and V2536/2 were sufficient to infect respective calves. Pellerin et al. (1994) used experimentally raised convalescent antisera to six BVDV strains (four type 1 strains – NADL, NY-1, C1, C3; and two type 2 strains – CD87 and waters) to compare neutralisation of 22 strains of BVDV. Their conclusion was division of the strains into classical strains (group I) and newly emergent strains also including the virulent thrombocytopenic isolates from Canada and USA (group II). In group II, cross-neutralisation of 10 strains including 890 by six antisera varied 85–512-fold and the variation in 11 strains in group I ranged 24–128-fold; an interesting result they reported was neutralisation of group 1 strain Q1808 by the six antisera varied by only 2–4-fold. In a cross-protection study in pregnant ewes immunised with experimental type 1 or type 2 BVDV vaccine, virus neutralising response for both vaccines was almost type specific (Paton et al., 1999). Since neutralisation of BVDV is predominantly mediated by antibody binding to glycoprotein E2 (Corapi et al., 1990; Xue et al., 1990) involving multiple epitopes (Bolin et al., 1988; Moennig et al., 1989) strain Q1808 is an interesting candidate for epitope and sequence analysis of its glycoprotein E2. We cannot explain at present why strains 890, C86 and V2536/2 elicit more broadly cross-neutralising antibodies. Since glycoprotein E2 of BVDV and CSFV have multiple epitopes involved in neutralisation (Bolin et al., 1988; Moennig et al., 1989; Wensvoort, 1989) a possible approach would be to analyse present convalescent antisera to strains 890, C86, V2536/2 and other strains for
J.R. Patel et al. / The Veterinary Journal 169 (2005) 468–472
their reactivity in competitive binding assays against neutralising monoclonal antibodies (Mabs) to different epitopes on glycoprotein E2. We did not investigate cross-neutralisation by combination of antisera to different virus strains but previous findings using anti E2 Mabs gave opposite results. Hence no synergistic enhancement was observed with anti E2 Mabs for BVDV (Moennig et al., 1989) while those for CSFV reacted synergistically (Wensvoort, 1989) It is however likely that neutralising epitopes on E2 of pestiviruses vary since some Mabs which neutralised one strain bound to another strain without neutralising its infectivity (Moennig et al., 1989). Variation in neutralising epitopes on E2 is also indicated by the observation of Pellerin et al. (1994) with type 1 BVDV strain Q1808. It is unlikely that differences in neutralisation kinetics account for our results and we assume from a previous study with BVDV (Chen and Johnson, 1986) that in our tests neutralisation occurs by a multi-hit mechanism requiring at least two antibody molecules to neutralise one infectious virus particle, as has also been documented for several other animal viruses (Della-Porta and Westaway, 1978). The present and the previous (Pellerin et al., 1994; Avalos-Ramirez et al., 2001; Becher et al., 2003) findings show a marked variation in immunogenicity of type 1 and type 2 BVDV strains and this variation is also reflected in currently available BVDV vaccines. Hence, a neutralisation-based analysis in na€ıve Holstein dairy heifers directly comparing three commercially available vaccines containing one type 1 and one type 2 BVDV strains, two type 1 strains and one type 1 strain respectively gave seroconversion rates of 100%, 69% and 38% against a BVDV type 1 strain and of 100%, 46% and 0% against a type 2 strain (Des Coteaux et al., 2003). Cross-neutralising responses due to BVDV vaccines, even against virus strains belonging to the same species, can vary significantly (Fulton and Burge, 2001). This point was clearly made by Fulton (Oklahoma State University) who presented live vaccine efficacy data at the annual Conference of Research Workers on Animal Diseases held in Chicago in November 2003. Hence a herd of 250 BVDV na€ıve heifers that was vaccinated three times with a modified live commercially available vaccine containing BVDV type 1a (strain Oregon C24V) and type 2a (strain 296) produced 31 persistently infected (PI) and 10 dead calves; interestingly, five virus isolates from the PI calves were all type 2a. In this regard it is also noteworthy that in Spain the type 1b subgroup predominates among BVDV isolates despite use of BVDV vaccines containing type 1b strains (Arias et al., 2003). The variation in cross-neutralising activity is also reflected in vaccine mediated cross-protection against trans-placental infection by type 1 and type 2 BVDV strains. Hence, some type 1 BVDV vaccines are protec-
471
tive against both type 1 and type 2 BVDV infections as has been the case with strain C86 (Patel et al., 2002; Makoschey et al., 2001) and WRL (Dean and Leyh, 1999) while some are not (Paton et al., 1999). While it is highly likely that neutralising antibodies play a major role in protecting against ruminant pestivirus infections (Howard et al., 1989; Howard, 1990; Bolin and Ridpath, 1995; Cortese et al., 1998; Beer et al., 2000), experimentally there is scanty information on the protective role for cellular immunity in infections by these viruses. That cellular immunity is important in protection is indicated by T lymphocyte depletion studies in gnotobiotic calves using Mabs which demonstrated that depletion of CD4 T cells resulted in prolonged and increased viraemia (Howard et al., 1992). However, our results indicate that, at least at the level of cross-neutralising antibody responses, some strains of BVDV and BDV are far superior candidates for development as vaccines.
Acknowledgements We thank Drs. S. Edwards (VLA, Weybridge, UK), P. Nettleton (Moredun Institute, Edinburgh, UK) and H-J. Thiel (Institute for Virology, Justus-Liebig University, Giessen, Germany) for the kind provision of viruses.
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Short communication
Variation in immunogenicity of ruminant pestiviruses as determined by the neutralisation assay J.R. Patel *, S. Didlick, J. Quinton Intervet UK Ltd., Walton Manor, Walton, Milton Keynes, Buckinghamshire, MK7 7AJ, UK Accepted 28 April 2004
Abstract Immunogenicity in na€ıve three-month-old Friesian bull calves of nine ruminant pestiviruses, three each of type 1 bovine virus diarrhoea virus (BVDV), type 2 BVDV and border disease virus (BDV) was directly compared in reciprocal cross-neutralisation tests using sera obtained eight weeks after intranasal and intravenous inoculation with live virus. Cytopathic (CP) type 1 BVDV strain C86, non-cytopathic (NCP) type 2 BVDV strain 890 and NCP BDV strain V2536/2 were found to elicit significantly broad cross-neutralising antibodies against viruses in other species whereas other virus strains in all three species produced a much more pronounced homologous and/or species specific response. Results are clearly relevant in the selection of strains for vaccines against diseases caused by these successful, economically important ubiquitous viruses. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Ruminant pestiviruses; Immunogenicity; Cross-neutralisation; Vaccines; Cattle
Bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens of ruminants worldwide. Although both cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV and BDV occur naturally, the reason for the success of pestiviruses is the ability of the NCP biotype to cross the placenta and establish a persistent infection of the immunologically na€ıve developing fetus. Recent sequencing data point to CP viruses arising from NCP viruses which have undergone recombination events involving insertion of host RNA and/or duplication of viral sequences (Becher et al., 1996). Classification based on phylogenetic analyses of 50 NTR, Npro , and E2 genes have placed ruminant pestiviruses into three main genotypes with 8–11 subgroups in type 1 BVDV, two in type 2 BVDV and two in BDV (Meyers and Thiel, 1996; Becher et al., 1997, 1999a,b; Becher and Thiel, 2002; Vilcek et al., 2001). Schirrmeier et al. (2002) placed BDV isolates into three subgroups as well as suggesting that an atypical
* Corresponding author. Tel.: +44-1480-464242; fax: +44-1480461541.
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isolate ‘Hobi’ should form a third genotype of BVDV. That BDV isolates be segregated into BDV-1, BDV-2 and BDV-3 was also a conclusion of Becher et al. (2003). Additionally, monoclonal antibodies to viral glycoproteins have been used to discriminate between pestivirus genotypes as well as between strains of one genotype (Wensvoort et al., 1989; Cay et al., 1989; Paton et al., 1994). There is a high degree of variability in the structural glycoprotein E2 with amino acid homology as low as 80% within one species and <60% between genotypes (Becher et al., 1994). Virus neutralising activity is predominantly directed against the glycoprotein E2 (Corapi et al., 1990; Xue et al., 1990) and is the main target in the development of vaccines against pestiviruses (Moennig, 1990; Van Oirschot et al., 1999). In this communication, we provide experimental evidence indicating that some BVDV and BDV strains within a genotype are significantly superior in mounting a broadly cross-neutralising pan genotype antibody response. The nine strains of ruminant pestiviruses investigated were: NCP strain WB (type 1 BVDV; Patel and Shilleto, 2003), CP strain C86 (type 1 BVDV; Patel et al., 2002), CP strain Oregon C24V (type 1 BVDV), NCP
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strain 890 (type 2 BVDV; Bolin and Ridpath, 1992), NCP strains Giessen I and II (type 2 BVDV; Becher et al., 1999a,b, 2003), NCP strains T1802 and V2536/2 (BDV; Vilcek et al., 1997) and NCP strain L83/84 (BDV; Becher et al., 1994, Patel and Shilleto, 2003). Viruses were grown and titrated in bovine embryo lung (BEL) cells as previously described (Patel and Shilleto, 2003). BEL cells were grown in minimal essential medium supplemented with 10% donor horse serum (HS), 100 lg/mL streptomycin, 100 units/mL penicillin, two mM L -glutamine, 1.4 mg/mL L -arginine and 0.09% sodium bicarbonate (HSGM). This medium containing 2% HS (HSMM) was used for maintaining infected cell monolayers and diluting viruses and antisera in titrations. Viral antisera were raised in naturally reared na€ıve (pestivirus and pestivirus antibody free) Friesian-cross bull calves about three months of age. Virus inocula for calves were first cloned by a limiting dilution procedure in BEL cells grown in 96-well plates at 37 °C and further grown on initially in 24-well monolayers and then in 25 cm2 monolayers of BEL cells as described previously (Patel and Shilleto, 2003). Titres of the stocks administered to calves varied significantly (see Table 1) and each calf was bled and then inoculated with one millilitre of a virus stock intravenously (IV) and also intranasally (IN).
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Sera samples taken pre-inoculation (PI) and eight weeks after the IN and the IV inoculations were reciprocally titrated for virus neutralising (VN) antibody against each virus. For VN titrations, a standard constant virus (100–316 TCID50 )–serum dilution procedure was used. Inactivated (56 °C, 30 min) sera were serially diluted in HSMM in 96-well microtitration plates, followed by the addition to each well of an equal volume (50 lL) of virus suspension in HSMM containing 100– 316 TCID50 of virus. Mixtures were incubated at 37 °C for one hour in an atmosphere of 5% CO2 in air and then 100 lL of freshly dispersed suspension of 2–4 104 BEL cells in HSGM were added to each well and the plates incubated at 37 °C in an atmosphere of 5% CO2 in air. In order to establish the dose of each virus contained in reaction mixtures, three serial ten-fold dilutions of stock virus suspension added to reaction mixtures were incubated in replicates of four, in parallel to serum–virus mixtures, followed by the addition of the cell suspension. After five days’ incubation at 37 °C, ends points for VN antibodies to CP viruses were scored as monolayers completely free of cytopathic effect (CPE) while those for NCP viruses were scored as wells with complete CPE two–three days following superinfection with CP type 1 BVDV strain C86 as described previously (Patel and Shilleto, 2003). CP type 1 BVDV strain Oregon C24V
Table 1 Antibody titres by the neutralisation test of calf sera with homologous and heterologous ruminant pestivirus strains Antiserum to strain
Biotypea and titreb
Virus strain used as antigen Type 1 BVDV WB
Type 1 BVDV C86
CP, 5.4
Oregon C24V
CP, 6.0
WB
NCP, 6.2
Type 2 BVDV 890
NCP, 6.2
Giessen 1
NCP, 5.4
Giessen 2
NCP, 6.0
BDV V2536/2
NCP, 4.7
T1802/1
NCP, 4.4
L83/84
NCP, 6.6
a
11.5 2d 7.0 4 9.0 1 12.5 2.7 2.0 256 4.0 256 8.0 4 5.0 512 7.0 384
c
Type 2 BVDV
BDV
C86
C24V
890
Giessen 1
Giessen 2
L83/84
T1802/1
V2536/2
12.5 1 9.0 1 9.0 1 13.0 2 3.0 128 7.0 32 8.0 4 6.0 256 10.5 32
12.5 1 7.0 4 7.0 4 14.0 1 3.0 128 6.0 64 8.0 4 8.5 42.7 9.0 96
9.0 12 3.0 64 3.0 64 14.0 1.0 8.5 2.7 11.0 2 7.5 7.6 6.0 256 4.5 2063
9.0 12 4.0 32 3.0 64 12.0 4 10.0 1 12.0 1 7.0 8 5.5 341.3 5.0 1536
8.5 16 3.0 64 2.5 85.3 12.0 4 8.5 2.7 12.0 1 8.0 4 6.0 256 6.5 512
8.5 16 2.0 128 <2.0 >128 12.0 4 4.5 42.7 5.5 85.3 9.5 1.3 14.0 1 15.5 1
9.0 12 <2.0 >128 2.0 128 12.0 4 2.5 170.7 4.0 256 6.5 10.7 14.0 1 11.5 16
9.0 12 2.0 128 <2.0 >128 13.0 2 2.0 256 2.0 1024 10.0 1 12.5 2.7 11.0 24
CP denotes cytopathic and NCP denotes non-cytopathic. Infectivity (log10 TCID50 /mL) of inocula administered to calves at a rate of one mL intranasally and one mL intravenously. c VN antibody titres eight weeks after inoculations expressed as log2 of reciprocal of the highest dilution completely neutralising 100–316 TCID50 of virus. Figures with half digits represent complete neutralisation in one of two wells while lower dilutions gave complete neutralisation in both wells. d The lower figures in italics denote difference (folds) between the homologous and the heterologous VN antibody titres. b
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also gave closely similar (less than two-fold) VN titres in tests with homologous virus-serum pairs. VN antibody titre is expressed as log2 of reciprocal of the highest serum dilution completely neutralising 100–316 TCID50 of a virus. Values with half digits represent titres between dilutions. The VN antibody titre of each of the nine antisera with each pestivirus strain is given in Table 1. No activity was detected in any of the nine pre-inoculation sera in tests with homologous strains (data not shown). With the exception of antiserum to type 1 CP BVDV strain Oregon C24V, homologous titres were the highest and there were also examples of some antisera neutralising other strains in the same genotype to a similar or slightly (3–4 fold) lower extent but significantly (32– 512-fold) poorer to viruses belonging to other genotypes. This was the case with antisera to NCP BDV strain T1802/1, type 2 NCP BVDV strains Giessen 1 and 2 and type 1 NCP BVDV strain WB. Titres of an antiserum to members belonging to the same genotype were closely similar with an exception of the antiserum to BDV strain L83/84 and that to BDV strain V2536/2. The titre of the former was some 16–24fold higher against BDV strains V2536/2 and T1802/1; antiserum to BDV strain V2536/2 neutralised BDV strains V2536/2 and L83/84 to a similar extent and its titre was about 11-fold lower against BDV strain T1802/ 1. An interesting finding was the fact that an antiserum to one of three virus strains in each of the three genotypes tested was significantly more broadly cross-neutralising. Hence the antiserum to type 2 NCP BVDV strain 890 neutralised three type 2 BVDV, three type 1 BVDV and three BDV strains to a closely similar (two to four fold) extent. A slightly less (2–16-fold) but still remarkable cross-neutralisation was also recorded for antisera to type 1 CP BVDV strain C86 and BDV strain V2536/2. For the latter, a noteworthy observation was the fact that the magnitude of the homologous titre was 5–16-fold lower than the homologous activity of antisera to strains C86 and 890 and 16–48-fold lower than homologous titres of antisera to BDV strains T1802/1 and L83/84. The broad VN antibody response by strains 890, C86 and V2536/2 had not been recorded previously (Becher et al., 2003). This discrepancy is not readily explained and made difficult by the fact that the production of antisera to strains 890 and C86 were not described by Becher et al. (2003) whereas antiserum to strain V2536/2 was from a sheep, six weeks following IN and intramuscular (IM) inoculation with 7.0 log10 TCID50 of virus. It is however unlikely that the difference between the present and previous findings can be explained at the level of VN tests since test methods are closely similar except for the development of endpoints. Also we are not aware if Becher et al. (2003) used cloned viruses to raise antisera or not. Further-
more, passage histories of the strains could also influence their antigenicity. Although our approach is not novel, we believe that the finding that some strains in all three genotypes of ruminant pestiviruses elicit a significantly broader crossneutralising antibody response has not previously been reported. It should however be noted that the present data were generated using one calf per strain and there can be individual variation between animals. Since the VN titres to homologous and genotypically related strains were high, we do not consider individual variation between animals to be a factor in present investigation. In this regard a previous similar serological study (Howard et al., 1987) using one calf per BVDV strain has reported useful data. Also it should be stated that the antisera to nine strains were reciprocally tested against each strain twice, and for some strains three times, with a maximum two fold variation in VN titre. With the exception of BDV strains T1802/1 and V2536/2 all the other inocula administered to calves contained between 5.4 and 6.6 log10 TCID50 of virus per mL whereas those for strains T1802/1 and V2536/2 contained 4.4 and 4.7 log10 TCID50 per mL, respectively, since both the strains grew poorly in BEL cells. Nonetheless the VN antibody responses indicate that the amounts of strains T1802/1 and V2536/2 were sufficient to infect respective calves. Pellerin et al. (1994) used experimentally raised convalescent antisera to six BVDV strains (four type 1 strains – NADL, NY-1, C1, C3; and two type 2 strains – CD87 and waters) to compare neutralisation of 22 strains of BVDV. Their conclusion was division of the strains into classical strains (group I) and newly emergent strains also including the virulent thrombocytopenic isolates from Canada and USA (group II). In group II, cross-neutralisation of 10 strains including 890 by six antisera varied 85–512-fold and the variation in 11 strains in group I ranged 24–128-fold; an interesting result they reported was neutralisation of group 1 strain Q1808 by the six antisera varied by only 2–4-fold. In a cross-protection study in pregnant ewes immunised with experimental type 1 or type 2 BVDV vaccine, virus neutralising response for both vaccines was almost type specific (Paton et al., 1999). Since neutralisation of BVDV is predominantly mediated by antibody binding to glycoprotein E2 (Corapi et al., 1990; Xue et al., 1990) involving multiple epitopes (Bolin et al., 1988; Moennig et al., 1989) strain Q1808 is an interesting candidate for epitope and sequence analysis of its glycoprotein E2. We cannot explain at present why strains 890, C86 and V2536/2 elicit more broadly cross-neutralising antibodies. Since glycoprotein E2 of BVDV and CSFV have multiple epitopes involved in neutralisation (Bolin et al., 1988; Moennig et al., 1989; Wensvoort, 1989) a possible approach would be to analyse present convalescent antisera to strains 890, C86, V2536/2 and other strains for
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their reactivity in competitive binding assays against neutralising monoclonal antibodies (Mabs) to different epitopes on glycoprotein E2. We did not investigate cross-neutralisation by combination of antisera to different virus strains but previous findings using anti E2 Mabs gave opposite results. Hence no synergistic enhancement was observed with anti E2 Mabs for BVDV (Moennig et al., 1989) while those for CSFV reacted synergistically (Wensvoort, 1989) It is however likely that neutralising epitopes on E2 of pestiviruses vary since some Mabs which neutralised one strain bound to another strain without neutralising its infectivity (Moennig et al., 1989). Variation in neutralising epitopes on E2 is also indicated by the observation of Pellerin et al. (1994) with type 1 BVDV strain Q1808. It is unlikely that differences in neutralisation kinetics account for our results and we assume from a previous study with BVDV (Chen and Johnson, 1986) that in our tests neutralisation occurs by a multi-hit mechanism requiring at least two antibody molecules to neutralise one infectious virus particle, as has also been documented for several other animal viruses (Della-Porta and Westaway, 1978). The present and the previous (Pellerin et al., 1994; Avalos-Ramirez et al., 2001; Becher et al., 2003) findings show a marked variation in immunogenicity of type 1 and type 2 BVDV strains and this variation is also reflected in currently available BVDV vaccines. Hence, a neutralisation-based analysis in na€ıve Holstein dairy heifers directly comparing three commercially available vaccines containing one type 1 and one type 2 BVDV strains, two type 1 strains and one type 1 strain respectively gave seroconversion rates of 100%, 69% and 38% against a BVDV type 1 strain and of 100%, 46% and 0% against a type 2 strain (Des Coteaux et al., 2003). Cross-neutralising responses due to BVDV vaccines, even against virus strains belonging to the same species, can vary significantly (Fulton and Burge, 2001). This point was clearly made by Fulton (Oklahoma State University) who presented live vaccine efficacy data at the annual Conference of Research Workers on Animal Diseases held in Chicago in November 2003. Hence a herd of 250 BVDV na€ıve heifers that was vaccinated three times with a modified live commercially available vaccine containing BVDV type 1a (strain Oregon C24V) and type 2a (strain 296) produced 31 persistently infected (PI) and 10 dead calves; interestingly, five virus isolates from the PI calves were all type 2a. In this regard it is also noteworthy that in Spain the type 1b subgroup predominates among BVDV isolates despite use of BVDV vaccines containing type 1b strains (Arias et al., 2003). The variation in cross-neutralising activity is also reflected in vaccine mediated cross-protection against trans-placental infection by type 1 and type 2 BVDV strains. Hence, some type 1 BVDV vaccines are protec-
471
tive against both type 1 and type 2 BVDV infections as has been the case with strain C86 (Patel et al., 2002; Makoschey et al., 2001) and WRL (Dean and Leyh, 1999) while some are not (Paton et al., 1999). While it is highly likely that neutralising antibodies play a major role in protecting against ruminant pestivirus infections (Howard et al., 1989; Howard, 1990; Bolin and Ridpath, 1995; Cortese et al., 1998; Beer et al., 2000), experimentally there is scanty information on the protective role for cellular immunity in infections by these viruses. That cellular immunity is important in protection is indicated by T lymphocyte depletion studies in gnotobiotic calves using Mabs which demonstrated that depletion of CD4 T cells resulted in prolonged and increased viraemia (Howard et al., 1992). However, our results indicate that, at least at the level of cross-neutralising antibody responses, some strains of BVDV and BDV are far superior candidates for development as vaccines.
Acknowledgements We thank Drs. S. Edwards (VLA, Weybridge, UK), P. Nettleton (Moredun Institute, Edinburgh, UK) and H-J. Thiel (Institute for Virology, Justus-Liebig University, Giessen, Germany) for the kind provision of viruses.
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