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In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status
The Veterinary Journal 192 (2012) 126–128
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status Barbara Lucchini, Wilma Ponti, Lauretta Turin, Valerio Bronzo, Licia Scaccabarozzi, Camilla Luzzago ⇑ Università degli Studi di Milano, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Via Celoria 10, 20133 Milano, Italy
a r t i c l e
i n f o
Article history: Accepted 5 May 2011
Keywords: Pestivirus Bovine viral diarrhoea virus In vitro infection Re-infection Peripheral blood mononuclear cell
a b s t r a c t The in vitro permissivity to infection with homologous and heterologous bovine viral diarrhoea virus (BVDV) strains of bovine peripheral blood mononuclear cells (PBMCs) from eight naïve and eight BVDV-1b immune animals was studied. Four reference strains (BVDV-1a NADL, BVDV-1b NY-1, BVDV2 125 and BVDV-2 890) were selected, based on genotype, prevalence and biotype. Virus neutralizing antibody titres were determined at bleeding and the viral loads were measured in PBMCs by end point titration in cell culture and by real-time PCR. PBMCs from both naïve and immune animals became infected by all BVDV strains tested, although virus titres were lower for immune heifers than naïve ones; the differences were significant for NADL (P < 0.05) and 890 (P < 0.001) strains. The in vitro model used in this study showed that PBMCs from immune animals are susceptible to re-infection with both homologous and heterologous BVDV strains, albeit at a lower extent than naïve cattle. Ó 2011 Elsevier Ltd. All rights reserved.
Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle belonging to the Pestivirus genus of the Flaviviridae family. Genetic typing of BVDV isolates has distinguished two species, BVDV-1 and BVDV-2, and a putative BVDV-3 (Liu et al., 2009) has been recently proposed. While BVDV-1 includes at least 11 subtypes, BVDV-1a to -1k (Vilcˇek et al., 2004), BVDV2 viruses are classified into two subtypes (Ridpath et al., 2000). BVDV infects a wide variety of cell types but has a preferential tropism for immune cells. Infection by both non-cytopathic (ncp) and cytopathic (cp) biotypes has been shown, in vitro and in vivo, to affect the activity of macrophages, alter the expression of immune cell receptors and deplete both CD4+ and CD8+ T-lymphocytes (Chase et al., 2004). It is still unclear whether cells from immune animals can prevent heterologous strains of the virus from achieving a productive infection. BVDV antigens have been detected in peripheral blood mononuclear cells (PBMCs) from seropositive animals in commercial herds (Gogorza et al., 2005); however, no estimate of when infection had occurred could be made and, because they did not genetically type the virus they detected, the authors were unable to rule out re-infection with a heterologous strain of BVDV. The aim of the present study was to investigate the in vitro permissivity of PBMCs from naïve and BVDV-1b immune animals to infection with both homologous and heterologous BVDV strains, belonging to both ncp and cp biotypes. Sera and PBMCs were collected from eight BVDV naïve Holstein–Friesian heifers from a BVDV-free dairy herd, and eight im⇑ Corresponding author. Tel.: +39 02 50318068. E-mail address: [email protected] (C. Luzzago). 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.05.001
mune Holstein–Friesian heifers from an infected herd (Luzzago et al., 2008). The experimental protocol was ethically approved by the Italian Ministry of Health (protocol number 2006070977003). The immune animals had been housed together with a heifer that was persistently infected (PI) with BVDV-1b which had been removed 6 months earlier. Four viral strains were used for in vitro infection and serological tests: the homologous strain (BVDV-1b ncp NY-1, ATCC VR-524) and three heterologous strains (BVDV-1a cp NADL, ATCC VR-534; BVDV-2 cp 125 and BVDV-2 ncp 890, National Veterinary Service Laboratories, USDA). PBMCs (1.2 106 cells) were infected at one multiplicity of infection (MOI) and incubated for 18 h. The BVDV stocks and the virus from infected and negative control PBMCs were titrated in Madin Darby Bovine Kidney cells (MDBK ATCC CCL-22) by observation of cytopathic effects for cp strains and by immunoperoxidase monolayer assay for ncp strains (Meyling, 1984) using the broad cross-reactivity monoclonal antibody 20.10.06 (Corapi et al., 1990). Sera were tested for antibody titres by virus neutralization (VN) tests. In order to exclude the hypothesis of a lower or absence of permissivity of PBMCs to re-infection with the homologous strain, cells infected with the BVDV-1b strain underwent viral RNA extraction, reverse transcription and quantification by TaqMan real-time PCR assays (Bhudevi and Weinstock, 2001). The virus quantity was calculated in relationship to the viral strain calibrator (c-DNA obtained from the dilution of the BVDV-1a Singer strain, corresponding to 2000 TCID50), expressing as TCID50/assay. The data on viral loads were tested for normality using a Shapiro–Wilk test and then with a non-parametric Kruskal–Wallis test.
B. Lucchini et al. / The Veterinary Journal 192 (2012) 126–128 Table 1 VN antibody titres of naïve and BVDV-1b immune animals vs. the BVDV homologous and heterologous strains, evaluated at timing of in vitro infection.
Reference strains BVDV-1b ncp NY1 BVDV-1a cp NADL BVDV-2 cp 125 BVDV-2 ncp 890 *
Naïve animals
Immune animals
log2 VN aba titre (geometric mean ± SD) 1±0
log2 VN aba titre (geometric mean ± SD) 8.00 ± 2.00*
1±0
3.00 ± 1.00
1±0
2.78 ± 0.05
1±0
3.01 ± 1.39
P < 0.001 vs. NADL, 125 and 890. Virus neutralizing antibody.
a
Fig. 1. Comparison of viral loads in PBMCs from naïve and immune animals following in vitro infection. PBMCs were infected with 1 MOI of BVDV strains and collected at 18 h post infection. Quantification was performed by end point titration in MDBK cells. The mean values (±standard error) of viral loads in PBMCs from eight animals/group are shown; , P = 0.036; , P < 0.001; Kruskal–Wallis test.
The comparison between VN antibody titres against homologous and heterologous strains in immune animals was assessed using univariate analysis of variance (PASW 18.0, SPSS). BVDV immune animals showed a significant difference in VN antibody titres between the homologous and the heterologous strains (P < 0.001); the latter three strains all had similar titres. BVDV-free animals were seronegative to all viral strains. Overall mean VN antibody titres are shown in Table 1. PBMCs from both naïve and immune animals were efficiently infected by all four BVDV strains. Quantification by end point titration in cell culture showed lower virus titres in PBMCs from immune animals than naïve animals for all the strains tested (Fig. 1). This difference was greater for heterologous strains than homologous ones and it was significant for the NADL (P < 0.05) and 890 (P < 0.001) strains. A similar trend was seen for the NY-1 strain by titration in cell culture and by real-time PCR. The latter had mean values (±standard deviation, SD) of 0.15 ± 0.32 TCID50/assay in immune animals and 1.24 ± 2.12 TCID50/assay in BVDV free animals. In both groups of animals, the ncp strains tested showed lower viral loads than the cp strains; this difference was statistically significant (P < 0.05), except for the comparisons between 890 and NY1 and NADL. In both groups of animals the lowest and the highest viral titres were observed in PBMCs infected with NY-1 and 125 strains, respectively. BVDV has been detected in PBMCs from seropositive animals (Gogorza et al., 2005) and long term persistent infection has been demonstrated after experimental infection in immune animals (Collins et al., 2009). Our study has highlighted the permissivity of PBMCs from immune animals to BVDV re-infection by homolo-
127
gous or heterologous strains in an in vitro infection model. Naïve animals showed higher viral loads than those from the immune group, irrespective of the inoculated viral strain. However, our results show that immune animals are incompletely protected against re-infection; this outcome has to be considered in terms of its potential impact on BVDV survival on-farm. A previous in vitro investigation demonstrated that lymphocytes from BVDV naïve cattle were more efficiently infected than those from vaccinated animals (Beer et al., 1997). In our study, the immune animals had been housed with a BVDV-1b PI animal up to 6 months before the present experiment. Although we could not rule out that the selected animals had been previously infected by BVDV strains unrelated to those tested, PBMCs may have received at least one natural stimulation by BVDV-1b, which could have produced an even more efficient lymphocyte reactivation. Nevertheless, the differences in viral loads in immune and naïve animals were greater for the heterologous strains (BVDV-2 strains and BVDV-1a) than for the homologous one (BVDV-1b), despite the weak cross-neutralizing activity. This suggests the involvement of a cell-mediated immune response in determining viral load. The BVDV-1b NY-1 strain showed the lowest viral loads irrespective of the status of the source of the PBMCs. As the assays were performed 18 h post-infection and the virus eclipse period was reported to be maximally completed in 12 h (Gong et al., 1996), it is likely that the virus replication cycle was complete at the time of the assay. Therefore, the low viral load probably resulted from less efficient adsorption and/or production of mature progeny virus particles. Moreover, Ridpath et al. (2007) showed that inoculation with BVDV-1b NY-1 resulted in milder clinical signs than other BVDV-1 field strains. This is likely to reflect an efficient maintenance strategy for this subtype which was the first BVDV strain to be isolated in the 1950s and still remains one of the most prevalent BVDV strains worldwide (Ridpath et al., 2010). In conclusion, using an in vitro BVDV infection model of bovine PBMCs we showed that immune animals are susceptible to reinfection with both homologous and heterologous viral strains, although at a lower extent compared to naïve animals. Conflict of interest statement None of the authors has any financial or personal relationship that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Professor E.J. Dubovi for providing monoclonal antibody, Dr. M. Frigerio for help in collecting samples and Dr. S. Russo for the English revision. Work funded by the PRIN Grant 2006 from the Italian Ministry of Education, Scientific Research and Health. References Beer, M., Wolf, G., Pichler, J., Wolfmeyer, A., Kaaden, O.R., 1997. Cytotoxic Tlymphocyte responses in cattle infected with bovine viral diarrhea virus. Veterinary Microbiology 58, 9–22. Bhudevi, B., Weinstock, D., 2001. Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus. Veterinary Microbiology 83, 1– 10. Chase, C.C., Elmowalid, G., Yousif, A.A., 2004. The immune response to bovine viral diarrhea virus: A constantly changing picture. Veterinary Clinics of North America: Food Animal Practice 20, 95–114. Collins, M.E., Heaney, J., Thomas, C.J., Brownlie, J., 2009. Infectivity of pestivirus following persistence of acute infection. Veterinary Microbiology 138, 289–296. Corapi, W.V., Donis, R.O., Dubovi, E.J., 1990. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. American Journal of Veterinary Research 51, 1388– 1394.
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B. Lucchini et al. / The Veterinary Journal 192 (2012) 126–128
Gogorza, L.M., Morán, P.E., Larghi, J.L., Seguì, R., Lissarrague, C., Saracco, M., Braun, M., Esteban, E.N., 2005. Detection of bovine viral diarrhea virus (BVDV) in seropositive cattle. Preventive Veterinary Medicine 72, 49–54. Gong, Y., Trowbridge, R., Macnaughton, T.B., Westaway, E.G., Shannon, A.D., Gowans, E.J., 1996. Characterization of RNA synthesis during a one step growth curve and replication mechanism of bovine viral diarrhoea virus. Journal of General Virology 77, 2729–2736. Liu, L., Xia, H., Wahlberg, N., Belák, S., Baule, C., 2009. Phylogeny, classification and evolutionary insights into pestiviruses. Virology 385, 351–357. Luzzago, C., Frigerio, M., Piccinini, R., Daprà, V., Zecconi, A., 2008. A scoring system for risk assessment of the introduction and spread of bovine viral diarrhoea virus in dairy herds in Northern Italy. Veterinary Journal 177, 236– 241. Meyling, A., 1984. Detection of BVD virus in viraemic cattle by an indirect immunoperoxidase technique. In: McNulty, M.S., McFerran, J.B. (Eds.), Recent
Advances in Virus Diagnosis (CEC Seminar). Martinus Nijhoff, Belfast, UK, pp. 37–46. Ridpath, J.F., Neill, J.D., Frey, M., Landgraf, J.G., 2000. Phylogenetic, antigenic and clinical characterization of type 2 BVDV from North America. Veterinary Microbiology 77, 145–155. Ridpath, J.F., Neill, J.D., Peterhans, E., 2007. Impact of variation in acute virulence of BVDV1 strains on design of better vaccine efficacy challenge models. Vaccine 25, 8058–8066. Ridpath, J.F., Fulton, R.W., Kirkland, P.D., Neill, J.D., 2010. Prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in Australia and feedlots in the southwestern United States. Journal of Veterinary Diagnostic Investigation 22, 184–191. ˇ urkovicˇ, B., Kolesárová, M., Greiser-Wilke, I., Paton, D., 2004. Genetic Vilcˇek, S., D diversity of international bovine viral diarrhea virus (BVDV) isolates: Identification of a new BVDV-1 genetic group. Veterinary Research 35, 609–615.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status Barbara Lucchini, Wilma Ponti, Lauretta Turin, Valerio Bronzo, Licia Scaccabarozzi, Camilla Luzzago ⇑ Università degli Studi di Milano, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Via Celoria 10, 20133 Milano, Italy
a r t i c l e
i n f o
Article history: Accepted 5 May 2011
Keywords: Pestivirus Bovine viral diarrhoea virus In vitro infection Re-infection Peripheral blood mononuclear cell
a b s t r a c t The in vitro permissivity to infection with homologous and heterologous bovine viral diarrhoea virus (BVDV) strains of bovine peripheral blood mononuclear cells (PBMCs) from eight naïve and eight BVDV-1b immune animals was studied. Four reference strains (BVDV-1a NADL, BVDV-1b NY-1, BVDV2 125 and BVDV-2 890) were selected, based on genotype, prevalence and biotype. Virus neutralizing antibody titres were determined at bleeding and the viral loads were measured in PBMCs by end point titration in cell culture and by real-time PCR. PBMCs from both naïve and immune animals became infected by all BVDV strains tested, although virus titres were lower for immune heifers than naïve ones; the differences were significant for NADL (P < 0.05) and 890 (P < 0.001) strains. The in vitro model used in this study showed that PBMCs from immune animals are susceptible to re-infection with both homologous and heterologous BVDV strains, albeit at a lower extent than naïve cattle. Ó 2011 Elsevier Ltd. All rights reserved.
Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle belonging to the Pestivirus genus of the Flaviviridae family. Genetic typing of BVDV isolates has distinguished two species, BVDV-1 and BVDV-2, and a putative BVDV-3 (Liu et al., 2009) has been recently proposed. While BVDV-1 includes at least 11 subtypes, BVDV-1a to -1k (Vilcˇek et al., 2004), BVDV2 viruses are classified into two subtypes (Ridpath et al., 2000). BVDV infects a wide variety of cell types but has a preferential tropism for immune cells. Infection by both non-cytopathic (ncp) and cytopathic (cp) biotypes has been shown, in vitro and in vivo, to affect the activity of macrophages, alter the expression of immune cell receptors and deplete both CD4+ and CD8+ T-lymphocytes (Chase et al., 2004). It is still unclear whether cells from immune animals can prevent heterologous strains of the virus from achieving a productive infection. BVDV antigens have been detected in peripheral blood mononuclear cells (PBMCs) from seropositive animals in commercial herds (Gogorza et al., 2005); however, no estimate of when infection had occurred could be made and, because they did not genetically type the virus they detected, the authors were unable to rule out re-infection with a heterologous strain of BVDV. The aim of the present study was to investigate the in vitro permissivity of PBMCs from naïve and BVDV-1b immune animals to infection with both homologous and heterologous BVDV strains, belonging to both ncp and cp biotypes. Sera and PBMCs were collected from eight BVDV naïve Holstein–Friesian heifers from a BVDV-free dairy herd, and eight im⇑ Corresponding author. Tel.: +39 02 50318068. E-mail address: [email protected] (C. Luzzago). 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.05.001
mune Holstein–Friesian heifers from an infected herd (Luzzago et al., 2008). The experimental protocol was ethically approved by the Italian Ministry of Health (protocol number 2006070977003). The immune animals had been housed together with a heifer that was persistently infected (PI) with BVDV-1b which had been removed 6 months earlier. Four viral strains were used for in vitro infection and serological tests: the homologous strain (BVDV-1b ncp NY-1, ATCC VR-524) and three heterologous strains (BVDV-1a cp NADL, ATCC VR-534; BVDV-2 cp 125 and BVDV-2 ncp 890, National Veterinary Service Laboratories, USDA). PBMCs (1.2 106 cells) were infected at one multiplicity of infection (MOI) and incubated for 18 h. The BVDV stocks and the virus from infected and negative control PBMCs were titrated in Madin Darby Bovine Kidney cells (MDBK ATCC CCL-22) by observation of cytopathic effects for cp strains and by immunoperoxidase monolayer assay for ncp strains (Meyling, 1984) using the broad cross-reactivity monoclonal antibody 20.10.06 (Corapi et al., 1990). Sera were tested for antibody titres by virus neutralization (VN) tests. In order to exclude the hypothesis of a lower or absence of permissivity of PBMCs to re-infection with the homologous strain, cells infected with the BVDV-1b strain underwent viral RNA extraction, reverse transcription and quantification by TaqMan real-time PCR assays (Bhudevi and Weinstock, 2001). The virus quantity was calculated in relationship to the viral strain calibrator (c-DNA obtained from the dilution of the BVDV-1a Singer strain, corresponding to 2000 TCID50), expressing as TCID50/assay. The data on viral loads were tested for normality using a Shapiro–Wilk test and then with a non-parametric Kruskal–Wallis test.
B. Lucchini et al. / The Veterinary Journal 192 (2012) 126–128 Table 1 VN antibody titres of naïve and BVDV-1b immune animals vs. the BVDV homologous and heterologous strains, evaluated at timing of in vitro infection.
Reference strains BVDV-1b ncp NY1 BVDV-1a cp NADL BVDV-2 cp 125 BVDV-2 ncp 890 *
Naïve animals
Immune animals
log2 VN aba titre (geometric mean ± SD) 1±0
log2 VN aba titre (geometric mean ± SD) 8.00 ± 2.00*
1±0
3.00 ± 1.00
1±0
2.78 ± 0.05
1±0
3.01 ± 1.39
P < 0.001 vs. NADL, 125 and 890. Virus neutralizing antibody.
a
Fig. 1. Comparison of viral loads in PBMCs from naïve and immune animals following in vitro infection. PBMCs were infected with 1 MOI of BVDV strains and collected at 18 h post infection. Quantification was performed by end point titration in MDBK cells. The mean values (±standard error) of viral loads in PBMCs from eight animals/group are shown; , P = 0.036; , P < 0.001; Kruskal–Wallis test.
The comparison between VN antibody titres against homologous and heterologous strains in immune animals was assessed using univariate analysis of variance (PASW 18.0, SPSS). BVDV immune animals showed a significant difference in VN antibody titres between the homologous and the heterologous strains (P < 0.001); the latter three strains all had similar titres. BVDV-free animals were seronegative to all viral strains. Overall mean VN antibody titres are shown in Table 1. PBMCs from both naïve and immune animals were efficiently infected by all four BVDV strains. Quantification by end point titration in cell culture showed lower virus titres in PBMCs from immune animals than naïve animals for all the strains tested (Fig. 1). This difference was greater for heterologous strains than homologous ones and it was significant for the NADL (P < 0.05) and 890 (P < 0.001) strains. A similar trend was seen for the NY-1 strain by titration in cell culture and by real-time PCR. The latter had mean values (±standard deviation, SD) of 0.15 ± 0.32 TCID50/assay in immune animals and 1.24 ± 2.12 TCID50/assay in BVDV free animals. In both groups of animals, the ncp strains tested showed lower viral loads than the cp strains; this difference was statistically significant (P < 0.05), except for the comparisons between 890 and NY1 and NADL. In both groups of animals the lowest and the highest viral titres were observed in PBMCs infected with NY-1 and 125 strains, respectively. BVDV has been detected in PBMCs from seropositive animals (Gogorza et al., 2005) and long term persistent infection has been demonstrated after experimental infection in immune animals (Collins et al., 2009). Our study has highlighted the permissivity of PBMCs from immune animals to BVDV re-infection by homolo-
127
gous or heterologous strains in an in vitro infection model. Naïve animals showed higher viral loads than those from the immune group, irrespective of the inoculated viral strain. However, our results show that immune animals are incompletely protected against re-infection; this outcome has to be considered in terms of its potential impact on BVDV survival on-farm. A previous in vitro investigation demonstrated that lymphocytes from BVDV naïve cattle were more efficiently infected than those from vaccinated animals (Beer et al., 1997). In our study, the immune animals had been housed with a BVDV-1b PI animal up to 6 months before the present experiment. Although we could not rule out that the selected animals had been previously infected by BVDV strains unrelated to those tested, PBMCs may have received at least one natural stimulation by BVDV-1b, which could have produced an even more efficient lymphocyte reactivation. Nevertheless, the differences in viral loads in immune and naïve animals were greater for the heterologous strains (BVDV-2 strains and BVDV-1a) than for the homologous one (BVDV-1b), despite the weak cross-neutralizing activity. This suggests the involvement of a cell-mediated immune response in determining viral load. The BVDV-1b NY-1 strain showed the lowest viral loads irrespective of the status of the source of the PBMCs. As the assays were performed 18 h post-infection and the virus eclipse period was reported to be maximally completed in 12 h (Gong et al., 1996), it is likely that the virus replication cycle was complete at the time of the assay. Therefore, the low viral load probably resulted from less efficient adsorption and/or production of mature progeny virus particles. Moreover, Ridpath et al. (2007) showed that inoculation with BVDV-1b NY-1 resulted in milder clinical signs than other BVDV-1 field strains. This is likely to reflect an efficient maintenance strategy for this subtype which was the first BVDV strain to be isolated in the 1950s and still remains one of the most prevalent BVDV strains worldwide (Ridpath et al., 2010). In conclusion, using an in vitro BVDV infection model of bovine PBMCs we showed that immune animals are susceptible to reinfection with both homologous and heterologous viral strains, although at a lower extent compared to naïve animals. Conflict of interest statement None of the authors has any financial or personal relationship that could inappropriately influence or bias the content of the paper. Acknowledgments We thank Professor E.J. Dubovi for providing monoclonal antibody, Dr. M. Frigerio for help in collecting samples and Dr. S. Russo for the English revision. Work funded by the PRIN Grant 2006 from the Italian Ministry of Education, Scientific Research and Health. References Beer, M., Wolf, G., Pichler, J., Wolfmeyer, A., Kaaden, O.R., 1997. Cytotoxic Tlymphocyte responses in cattle infected with bovine viral diarrhea virus. Veterinary Microbiology 58, 9–22. Bhudevi, B., Weinstock, D., 2001. Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus. Veterinary Microbiology 83, 1– 10. Chase, C.C., Elmowalid, G., Yousif, A.A., 2004. The immune response to bovine viral diarrhea virus: A constantly changing picture. Veterinary Clinics of North America: Food Animal Practice 20, 95–114. Collins, M.E., Heaney, J., Thomas, C.J., Brownlie, J., 2009. Infectivity of pestivirus following persistence of acute infection. Veterinary Microbiology 138, 289–296. Corapi, W.V., Donis, R.O., Dubovi, E.J., 1990. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. American Journal of Veterinary Research 51, 1388– 1394.
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B. Lucchini et al. / The Veterinary Journal 192 (2012) 126–128
Gogorza, L.M., Morán, P.E., Larghi, J.L., Seguì, R., Lissarrague, C., Saracco, M., Braun, M., Esteban, E.N., 2005. Detection of bovine viral diarrhea virus (BVDV) in seropositive cattle. Preventive Veterinary Medicine 72, 49–54. Gong, Y., Trowbridge, R., Macnaughton, T.B., Westaway, E.G., Shannon, A.D., Gowans, E.J., 1996. Characterization of RNA synthesis during a one step growth curve and replication mechanism of bovine viral diarrhoea virus. Journal of General Virology 77, 2729–2736. Liu, L., Xia, H., Wahlberg, N., Belák, S., Baule, C., 2009. Phylogeny, classification and evolutionary insights into pestiviruses. Virology 385, 351–357. Luzzago, C., Frigerio, M., Piccinini, R., Daprà, V., Zecconi, A., 2008. A scoring system for risk assessment of the introduction and spread of bovine viral diarrhoea virus in dairy herds in Northern Italy. Veterinary Journal 177, 236– 241. Meyling, A., 1984. Detection of BVD virus in viraemic cattle by an indirect immunoperoxidase technique. In: McNulty, M.S., McFerran, J.B. (Eds.), Recent
Advances in Virus Diagnosis (CEC Seminar). Martinus Nijhoff, Belfast, UK, pp. 37–46. Ridpath, J.F., Neill, J.D., Frey, M., Landgraf, J.G., 2000. Phylogenetic, antigenic and clinical characterization of type 2 BVDV from North America. Veterinary Microbiology 77, 145–155. Ridpath, J.F., Neill, J.D., Peterhans, E., 2007. Impact of variation in acute virulence of BVDV1 strains on design of better vaccine efficacy challenge models. Vaccine 25, 8058–8066. Ridpath, J.F., Fulton, R.W., Kirkland, P.D., Neill, J.D., 2010. Prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in Australia and feedlots in the southwestern United States. Journal of Veterinary Diagnostic Investigation 22, 184–191. ˇ urkovicˇ, B., Kolesárová, M., Greiser-Wilke, I., Paton, D., 2004. Genetic Vilcˇek, S., D diversity of international bovine viral diarrhea virus (BVDV) isolates: Identification of a new BVDV-1 genetic group. Veterinary Research 35, 609–615.