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Genetic analysis of bovine viral diarrhoea viruses from Australia
Veterinary Microbiology 106 (2005) 1–6 www.elsevier.com/locate/vetmic
Genetic analysis of bovine viral diarrhoea viruses from Australia Timothy J. Mahonya,*, Fiona M. McCarthya,1, Jennifer L. Gravela, Bruce Corneyb, Peter L. Younga, Stefan Vilcekc a
Department of Primary Industries and Fisheries, Level 6 North, Queensland Biosciences Precinct, 306 Carmody Road, Brisbane, Qld 4072, Australia b Animal Plant and Health Services, Department of Primary Industries and Fisheries, 665 Fairfield Road, Brisbane, Qld 4105, Australia c University of Veterinary Medicine, Kosice, Komenskeho 73, Slovakia Received 30 March 2004; received in revised form 7 October 2004; accepted 14 October 2004
Abstract Eighty-nine bovine viral diarrhoea viruses (BVDV) from Australia have been genetically typed by sequencing of the 50 untranslated region (50 -UTR) and for selected isolates the Npro region of the viral genome. Phylogenetic reconstructions indicated that all of the samples examined clustered within the BVDV type 1 genotype. Of the 11 previously described genetic groups of BVDV-1, 87 of the samples examined in this study clustered with the BVDV-1c, while two samples clustered with the BVDV-1a. Based on these analyses there appears to be limited genetic variation within the Australian BVDV field isolates. In addition, the phylogenetic reconstructions indicate that the clustering of Australian BVDV in the phylogenetic trees is not a result of geographic isolation. # 2005 Elsevier B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; 50 -UTR; Npro; Phylogenetic reconstruction
1. Introduction Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle worldwide. BVDV has been classified as a member of the genus * Corresponding author. Tel.: +61 7 3346 2705; fax: +61 7 3346 2727. E-mail address: [email protected] (T.J. Mahony). 1 Present address: College of Veterinary Medicine, Mississippi State University, MS 39762-6100, USA.
Pestivirus within the family Flaviviridae (Heinz et al., 2000). BVDV has a positive single-stranded RNA genome with a typical length 12.3 kbp. The BVDV genome encodes one open reading frame that is translated into a single polyprotein. The polyprotein is subsequently cleaved into the structural and non-structural proteins by viral and cellular proteases. The BVDV open reading frame, which starts with Npro viral autoprotease, is flanked at the 50 and 30 termini by untranslated regions (50 -UTR, 30 UTR) (Meyers and Thiel, 1996).
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.10.024
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T.J. Mahony et al. / Veterinary Microbiology 106 (2005) 1–6
Two recognised genotypes of BVDV, BVDV-1 and BVDV-2, have been distinguished on the basis of genome sequence variation (Heinz et al., 2000). BVDV-1 and 2 both cause acute and persistent infections and similar clinical diseases, however some hypervirulent BVDV-2 strains have been linked to a severe haemorrhagic syndrome (Corapi et al., 1989; Pellerin et al., 1994; Ridpath et al., 1994). BVDV-2 appears only in the Americas with sporadic outbreaks in Europe and Japan (Pellerin et al., 1994; Ridpath et al., 1994; Nagai et al., 1998; Wolfmeyer et al., 1997; Letellier et al., 1999; Vilcek et al., 2001). Both BVDV-1 and BVDV-2 have two recognised biotypes, non-cytopathic (ncp) and cytopathic (cp) which coexist in vitro (Moennig and Plagemann, 1992). Ncp-BVDV can cross the placenta and infect calves in utero. Depending on the stage of gestation the foetus may be reabsorbed, aborted or continue to develop. If development continues the calf may be born with no apparent clinical signs. The calf, however, will be persistently infected (PI) with BVDV and continue to shed the virus to susceptible animals (Roeder and Harkness, 1986). The PI calf may continue to develop to maturity but at some stage the ncp-BVDV may spontaneously mutate to the cp biotype, resulting in the onset of fatal mucosal disease (Brownlie et al., 1984; Meyers et al., 1991; Meyers and Thiel, 1996). The pestiviruses are considered to be both genetically and antigenically diverse. The diversity of BVDV needs to be considered when designing and constructing effective vaccination strategies for these viruses. The BVDV-1 viruses were initially divided into two major groups on the basis of 50 -UTR sequence variation, BVDV1a and BVDV1b (Pellerin et al., 1994; Ridpath et al., 1994). However, more extensive analyses indicate that there are at least 11 genogroups of BVDV-1 (Vilcek et al., 2001). A vaccine must accurately reflect the genotypes and antigenic types present in the country of use. To date phylogenetic analyses have been used to characterise the BVDV types of many regions and countries, however absent from this collection is the genotype types present in Australia. To address this situation, and as part of a BVDV vaccine development program the genetic diversity of BVDV viruses present in Australia was surveyed in this study.
2. Materials and methods 2.1. Viral samples Ninety-one Australian BVDV isolates were included in this study (see Table 1). Of these, 75 were clinical submissions made to the Yeerongpilly Table 1 Description and the proposed genetic typing of the 91 Australian bovine viral diarrhoea virus (BVDV) samples utilised in this study Year
Sample identifier #
Genotype
VR1111 VR1112# VIAS1# VIAS2, VIAS3, VIAS4, VIAS5 VIAS6, VIAS7, VIAS8, VIAS9, VIAS10#, VIAS11#, VR95 VR3@, VR4@, VR5@, VR6@, VR39@, VR40@, VR41@, VR42@, VR43@, VR100^, VR102@, VR138, VR139, VR140, VR141, VR148, VR150, VR151, VR152, VR153, VR154, VR155, VR156, VR157, VR158, VR159
1c 1c 1c 1c 1c
1999 2000
VR149 VR448, VR449, VR450, VR451, VR920, VR921, VR922, VR923, VR924, VR925, VR926, VR927, VR977, VR979, VR980, VR981, VR982, VR983, VR987, VR988, VR989, VR990, VR991, VR992, VR994, VR995, VR996, VR997, VR1000, VR1001, VR1002, VR1003, VR1004, VR1005, VR1088, VR1089, VR1090, VR1091, VR1092, VR1093, VR1094, VR1095, VR1105a, VR1107a
1a 1c
2000 2000 Unknown Unknown Unknown
VR986 VR999, VR1007$ MD70# Trangieb Begab
1a 1c 1c 1c 1c
1989 1995 Unknown 1997 1998 1999
1c
Those samples for which Npro sequences were also determined are shaded. BVDV samples clustering to the genotype 1a are shown in bold type. Viral RNA was extracted from serum unless otherwise indicated; #cell culture supernatant, @blood, ^spleen, $intestine. a 0 5 -UTR sequence determined by direct sequencing of amplified product. b Australian sequences for 50 -UTR and Npro retrieved from GenBank using following accession numbers Trangie Y546: AF049222, Bega: AF049221. The sequences determine as part of this study have been submitted to GenBank and assigned the following accession numbers, 50 UTR: AY762988–AY763092, Npro: AY763093–AY763095.
T.J. Mahony et al. / Veterinary Microbiology 106 (2005) 1–6
Veterinary Laboratories, Department of Primary Industries, Brisbane, Queensland, Australia. These samples were initially tested for BVDV using an antigen capture ELISA. A further 11 isolates were kindly provided by Dr. Colin Wilks from the Victorian Institute of Animal Sciences. A further two isolates were kindly provided by Dr. Jan Smith from James Cook University. VR1007 was amplified from intestinal tissue taken from a feedlot bovine with suspected mucosal disease. The sequences for two Australian BVDV strains were retrieved from GenBank. A complete list of the BVDV samples examined in this study is shown in Table 1.
3
polymeraseR, FS (Applied Biosystems). The amplification product from some isolates were directly sequenced following gel purification. A 622 bp product encompassing the Npro region was amplified and sequenced for selected viral samples as described for the 50 -UTR region. The following PCR primers were used in PCR reaction: STARTfwd (50 - TGC TGT ACATGG CAC ATG GAG TTG-30 , nucleotide 371–394 of the NADL genome) and Nproseq1 (50 -CTA TCC TTC TCT GAT TCT CTG-30 , nucleotide 973–993 of the NADL genome). In all cases the DNA sequence data were analysed using ContiExpress from the Vector-NTI suite 6 of programs (Informax, USA).
2.2. RNA isolation Total RNA was extracted from sera samples using High Pure Viral RNA kits (Roche Molecular Biochemicals) according to the manufacturer’s instructions. RNA from tissues was extracted using RNeasy mini kits (Qiagen) in combination with QIAshredder homogenizer (Qiagen). 2.3. Reverse transcriptase PCR (RT-PCR) and sequencing A 292 bp DNA product was amplified from the 50 UTR using the Titan1 single tube reaction system (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Primers utilised were 5UTRfwd (50 -CTA GCC ATG CCC TTA GTA GGA CTA-30 , nucleotide 102–125 of the NADL genome, Genbank Accession number: BVI133739) and STARTrev (50 -CAA CTC CAT GTG CCA TGT AC AGC A30 , nucleotide 371–394 of the NADL genome). Results of PCR reactions were confirmed by 1% agarose gel electrophoresis. Where two or more DNA bands were evident the band corresponding to the expected size was excised from the agarose gel and recovered from the agarose using the QIAquick gel extraction kit (Qiagen). The purified PCR product was ligated into the pGemT-Easy (Promega) using T/A cloning. The clones (three of each sample) were sequenced in both strands with the T7 and SP6 sequencing primers. DNA sequencing was performed on an ABI automated A373 sequencer using dideoxy-sequencing chemistry utilising the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit, with AmpliTaq DNA
2.4. Phylogentic analysis Phylogenetic reconstructions for genetic typing of the viral samples were compiled using a 244 nucleotide region of the 50 -UTR (nucleotide 130– 374 of the NADL genome) and a 385 nucleotide region of Npro (nucleotide 386–770 of the NADL genome). Additional sequences from representative isolates of previously identified phylogenetic groups of BVDV-1 were included into phylogentic analysis. Nucleotide sequences from each region were aligned using the Clustal W program. Phylogenetic analysis was performed using the Neighbour program from the Phylip inference program package (Felsenstein, 1993). Statistical analysis of phylogenetic trees was determined by bootstrap analysis carried out on 100 replicates using Seqboot program and the resulting tree was drawn using the Consensus program (Felsenstein, 1993).
3. Results and discussion A survey of the genetic diversity of BVDV field isolates in Australia was conducted by determining the nucleotide sequences of the 50 -UTR of 89 strains, as well the Npro region for three strains. The 50 -UTR and Npro sequences for a further two Australian strains were retrieved from GenBank and added to the data sets. These analyses indicate that all of the BVDV field strains examined in this study were of the BVDV-1 genotype. On the basis of this study it appears as
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though the BVDV-2 genotype is absent from Australia. Single tube reverse transcription reactions were successfully utilised to amplify a 292 bp region of the pestivirus 50 -UTR from the samples listed in Table 1 (data not shown). Sequences from these clones were used in the analyses. Eighty-nine samples were sequenced in 50 -UTR and sequencing revealed their length varied from 292 to 295 bp (data not shown). The 50 -UTR phylogenetic reconstructions using sequences for Australian BVDV isolates illustrated that all 91 viruses analysed belonged to the BVDV-1 genotype (see insert in Fig. 1). As this tree was strongly deformed due to the large number of isolates clustered within one group, a separate tree was generated for phylogenetic analysis using twelve representative Australian isolates. This tree demonstrated that only two of the isolates, VR149 and VR986, clustered to the BVDV-1a subtype (Fig. 1). This subtype includes the highly characterised NADL strain. All other sequences formed a unique cluster in the phylogenetic tree (labelled in Fig. 1 as ‘‘?’’), which appeared to represent a potentially new genetic group
of BVDV-1 genotype with no reference strain so far identified. To identify the group designated ‘‘?’’ in the 50 -UTR analysis (Fig. 1), three isolates from this collection (MD70, VR1007, and VR999) and two additional sequences for Australian BVDV strains retrieved from GenBank (BVDV strain Bega and strain Trangie Y546, see Table 1) were regenotyped using the Npro coding region. It was not possible to sequence the Npro region of all isolates of interest due to instability of some samples. The resulting phylogenetic trees were constructed using a 385 bp sequences derived from the Npro region and indicated that the isolates in this study were not members of a new genetic group of the BVDV-1 genotype. The majority of the Australian BVDV strains are members of the previously described BVDV-1c genotype (Fig. 1). The BVDV1c branch was supported by high bootstrap scores. The BVDV-1c branch had not previously been identified using 50 -UTR sequences prior to the analyses presented here. The previously recognised members of this subgenotype were strains DeerNZ1 and 519 isolated from New Zealand and Germany respectively
Fig. 1. Phylogenetic trees showing the typing of Australian BVDV-1 isolates in 50 -UTR and Npro regions. The unrooted trees were based on analysis of partial 244 nt (50 -UTR) or 385 nt (Npro) long sequences. They were prepared using the Neighbor-joining method (Kimura 2-parameter method, transition/transversin 2.0). Tree in the insert shows the grouping of all Australian isolates analysed in 5-UTR. Australian isolates are labelled in bolt. Phylogenetic groups are labelled according to Vilcek et al. (2001). The group labelled as ‘‘?’’ could not be exactly identified as there was not a reference strain typed in 50 -UTR. Other nucleotide sequences were taken from Vilcek et al. (2001). The Npro sequences for the strains 519, 721, Deer NZ1 and Deer GB1 were taken from Becher et al. (1997) (GenBank numbers – AF144464, AF144463, U80903 and U80902). Numbers over branches indicate the percentage of 100 bootstrap replicates that support each phylogenetic branch.
T.J. Mahony et al. / Veterinary Microbiology 106 (2005) 1–6
(Becher et al., 1997). The genotyping of DeerNZ1 and 519 was based solely on Npro analysis, as the 50 -UTR sequences of these isolates were not determined. Vilcek et al. (2001) did not identify any BVDV-1c genotypes based on 50 -UTR analyses using seventeen isolates from a previous study of New Zealand isolates (Vilcek et al., 1998). Nagai et al. (2004) have also utilised the 50 -UTR and Npro sequences from the Bega and Trangie Y546 in their analyses, which demonstrated clustering with strains DeerNZ1, 519 and some Japanese viruses to the subgenotype BVDV-1a0 . When combined with the analyses presented here, the subgenotype BVDV-1a0 described by Nagai et al. (2004) is analogous to BVDV-1c. When considered in the context of the study the distribution of genotypes does not appear to be a result of geography. It has been proposed that the non-coding region of the BVDV is unsuitable for phylogenetic studies (Becher et al., 1997). However, Vilcek et al. (2001) clearly demonstrated that 50 -UTR based studies give meaningful phylogenetic inferences. By sequencing both non-coding region (50 -UTR) and part of the coding region (Npro) analysis of these data sets as either separate or combined phylogenetic reconstructions yielded identical results. Although slightly higher bootstrap values were recorded for codingbased analyses, the results and overall topography of the phylogenetic trees were similar when compared to the non-coding based analyses. The results presented here were initially limited to the analysis of 50 -UTR sequences. This region is flanked by motifs that are highly conserved amongst the all pestiviruses. The high conservation of these motifs in all genotypes of BVDV permits the easy amplification of this region from diverse pestiviruses ensuring ready analysis of a wide diversity of virus samples with a high confidence of successful amplification, particularly from samples of unknown status. Furthermore, the comparatively short intervening sequence permits rapid and accurate acquisition of sequence data. These characteristics make the 50 -UTR an excellent sequence target for initial phylogenetic reconstructions of BVDV of unknown genotypes. It also permits the rapid primary genotyping of large numbers of viral samples in a timely fashion. Subsequently, selected isolates can be characterised further by determining larger sequence regions of coding sequence. Recently, Nagai et al.
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(2004) conducted extensive phylogenetic analyses of BVDV sequences using five separate genomic regions. For most of the isolates examined there was agreement between the subgenotype classifications as determined using sequences from each region. The isolates where discrepancies were identified were considered to be a result of recombination between subgroups as opposed to inaccurate phylogenetic inferences due to unsuitable sequence selection. As the first step in the development of an effective BVDV vaccine for the Australian cattle industry, the genetic diversity of a large number of BVDV field isolates has been examined. The isolates were examined in both untranslated and coding regions and the majority were typed as BVDV-1c. These results indicate the BVDV viruses in Australia at the time of this study were genetically homogeneous. When considered in the context of other studies it is reasonable to conclude that there is likely to be limited antigenic diversity in these viruses as well. The results presented here indicate it should be possible to develop effective vaccines to the BVDV strains currently circulating within Australia. However, it would be prudent to continue similar studies following the adoption of any vaccination program to ensure that more virulent genotypes do not emerge as a consequence of immune selection. Further any future genotyping studies conducted in tandem with vaccination programs should also include analysis of the antigen coding regions, such as glycoprotein E2, along with the 50 UTR and Npro regions. Such a comparative studied would enable monitoring of the effects of vaccination on the genotypes and antigenic variants within the population.
Acknowledgments The authors acknowledge Professor Colin Wilks of Victorian Institute for Animal Science and Dr. Jan Smith of James Cook University for provision of virus samples. The authors thank Mr Mark Kelly for performing the antigen capture ELISA analyses. The authors also thank Dr. Lee Taylor, Dr. Nigel Nichols and Dr. Steve Mathers for continued support and provision of viral samples. This work was supported by grant Flot.203 from Meat and Livestock Australia. S. V. was supported by VEGA grant (1/9014/02) and SP (51/0280803).
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References Becher, P., Orlich, M., Shannon, A.D., Horner, G., Konig, M., Thiel, H.J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366. Brownlie, J., Clarke, M.C., Howard, C.J., 1984. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535–536. Corapi, W.V., French, T.W., Dubovi, E.J., 1989. Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus. J. Virol. 63, 3934–3943. Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Heinz, F.X., Collett, M.S., Purcell, R.H., Gould, E.A., Howard, C.R., Houghton, M., Moormann, R.J.M., Rice, C.M., Thiel, H.-J., 2000. Family Flaviviridae. In: van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., Mcgeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, pp. 859–878. Letellier, C., Kerkhofs, P., Wellemans, G., Vanopdenbosch, E., 1999. Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5’ untranslated region. Vet. Microbiol. 64, 155–167. Meyers, G., Rumenapf, T., Tautz, N., Dubovi, E.J., Thiel, H.J., 1991. Insertion of cellular sequences in the genome of bovine viral diarrhea virus. Arch. Virol. Suppl. 3, 133–142. Meyers, G., Thiel, H.J., 1996. Molecular characterization of pestiviruses. Adv. Virus Res. 47, 53–118.
Moennig, V., Plagemann, P.G., 1992. The pestiviruses. Adv. Virus Res. 41, 53–98. Nagai, M., Sato, M., Nagano, H., Pang, H., Kong, X., Murakami, T., Ozawa, T., Akashi, H., 1998. Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV 2) in the 50 untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan. Vet. Microbiol. 60, 271–276. Nagai, M., Hayashi, M., Sugita, S., Sakoda, Y., Mori, M., Murakami, T., Ozawa, T., Yamada, N., Akashi, H., 2004. Phylogenetic analysis of bovine viral diarrhea viruses using five different genetic regions. Virus Res. 99, 103–113. Pellerin, C., van den Hurk, J., Lecomte, J., Tussen, P., 1994. Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 203, 260–268. Ridpath, J.F., Bolin, S.R., Dubovi, E.J., 1994. Segregation of bovine viral diarrhea virus into genotypes. Virology 205, 66–74. Roeder, P.L., Harkness, J.W., 1986. BVD virus infection: prospects for control. Vet. Rec. 119, 143–147. Vilcek, S., Bjorklund, H.V., Horner, G.W., Meers, J., Belak, S., 1998. Genetic typing of pestiviruses from New Zealand. New Zealand Vet. J. 46, 35–37. Vilcek, S., Paton, D.J., Durkovic, B., Strojny, L., Ibata, G., Moussa, A., Loitsch, A., Rossmanith, W., Vega, S., Scicluna, M.T., Paifi, V., 2001. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch. Virol. 146, 99–115. Wolfmeyer, A., Wolf, G., Beer, M., Strube, W., Hehnen, H.R., Schmeer, N., Kaaden, O.R., 1997. Genomic (50 UTR) and serological differences among German BVDV field isolates. Arch. Virol. 142, 2049–2057.
Genetic analysis of bovine viral diarrhoea viruses from Australia Timothy J. Mahonya,*, Fiona M. McCarthya,1, Jennifer L. Gravela, Bruce Corneyb, Peter L. Younga, Stefan Vilcekc a
Department of Primary Industries and Fisheries, Level 6 North, Queensland Biosciences Precinct, 306 Carmody Road, Brisbane, Qld 4072, Australia b Animal Plant and Health Services, Department of Primary Industries and Fisheries, 665 Fairfield Road, Brisbane, Qld 4105, Australia c University of Veterinary Medicine, Kosice, Komenskeho 73, Slovakia Received 30 March 2004; received in revised form 7 October 2004; accepted 14 October 2004
Abstract Eighty-nine bovine viral diarrhoea viruses (BVDV) from Australia have been genetically typed by sequencing of the 50 untranslated region (50 -UTR) and for selected isolates the Npro region of the viral genome. Phylogenetic reconstructions indicated that all of the samples examined clustered within the BVDV type 1 genotype. Of the 11 previously described genetic groups of BVDV-1, 87 of the samples examined in this study clustered with the BVDV-1c, while two samples clustered with the BVDV-1a. Based on these analyses there appears to be limited genetic variation within the Australian BVDV field isolates. In addition, the phylogenetic reconstructions indicate that the clustering of Australian BVDV in the phylogenetic trees is not a result of geographic isolation. # 2005 Elsevier B.V. All rights reserved. Keywords: Bovine viral diarrhoea virus; 50 -UTR; Npro; Phylogenetic reconstruction
1. Introduction Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle worldwide. BVDV has been classified as a member of the genus * Corresponding author. Tel.: +61 7 3346 2705; fax: +61 7 3346 2727. E-mail address: [email protected] (T.J. Mahony). 1 Present address: College of Veterinary Medicine, Mississippi State University, MS 39762-6100, USA.
Pestivirus within the family Flaviviridae (Heinz et al., 2000). BVDV has a positive single-stranded RNA genome with a typical length 12.3 kbp. The BVDV genome encodes one open reading frame that is translated into a single polyprotein. The polyprotein is subsequently cleaved into the structural and non-structural proteins by viral and cellular proteases. The BVDV open reading frame, which starts with Npro viral autoprotease, is flanked at the 50 and 30 termini by untranslated regions (50 -UTR, 30 UTR) (Meyers and Thiel, 1996).
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.10.024
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T.J. Mahony et al. / Veterinary Microbiology 106 (2005) 1–6
Two recognised genotypes of BVDV, BVDV-1 and BVDV-2, have been distinguished on the basis of genome sequence variation (Heinz et al., 2000). BVDV-1 and 2 both cause acute and persistent infections and similar clinical diseases, however some hypervirulent BVDV-2 strains have been linked to a severe haemorrhagic syndrome (Corapi et al., 1989; Pellerin et al., 1994; Ridpath et al., 1994). BVDV-2 appears only in the Americas with sporadic outbreaks in Europe and Japan (Pellerin et al., 1994; Ridpath et al., 1994; Nagai et al., 1998; Wolfmeyer et al., 1997; Letellier et al., 1999; Vilcek et al., 2001). Both BVDV-1 and BVDV-2 have two recognised biotypes, non-cytopathic (ncp) and cytopathic (cp) which coexist in vitro (Moennig and Plagemann, 1992). Ncp-BVDV can cross the placenta and infect calves in utero. Depending on the stage of gestation the foetus may be reabsorbed, aborted or continue to develop. If development continues the calf may be born with no apparent clinical signs. The calf, however, will be persistently infected (PI) with BVDV and continue to shed the virus to susceptible animals (Roeder and Harkness, 1986). The PI calf may continue to develop to maturity but at some stage the ncp-BVDV may spontaneously mutate to the cp biotype, resulting in the onset of fatal mucosal disease (Brownlie et al., 1984; Meyers et al., 1991; Meyers and Thiel, 1996). The pestiviruses are considered to be both genetically and antigenically diverse. The diversity of BVDV needs to be considered when designing and constructing effective vaccination strategies for these viruses. The BVDV-1 viruses were initially divided into two major groups on the basis of 50 -UTR sequence variation, BVDV1a and BVDV1b (Pellerin et al., 1994; Ridpath et al., 1994). However, more extensive analyses indicate that there are at least 11 genogroups of BVDV-1 (Vilcek et al., 2001). A vaccine must accurately reflect the genotypes and antigenic types present in the country of use. To date phylogenetic analyses have been used to characterise the BVDV types of many regions and countries, however absent from this collection is the genotype types present in Australia. To address this situation, and as part of a BVDV vaccine development program the genetic diversity of BVDV viruses present in Australia was surveyed in this study.
2. Materials and methods 2.1. Viral samples Ninety-one Australian BVDV isolates were included in this study (see Table 1). Of these, 75 were clinical submissions made to the Yeerongpilly Table 1 Description and the proposed genetic typing of the 91 Australian bovine viral diarrhoea virus (BVDV) samples utilised in this study Year
Sample identifier #
Genotype
VR1111 VR1112# VIAS1# VIAS2, VIAS3, VIAS4, VIAS5 VIAS6, VIAS7, VIAS8, VIAS9, VIAS10#, VIAS11#, VR95 VR3@, VR4@, VR5@, VR6@, VR39@, VR40@, VR41@, VR42@, VR43@, VR100^, VR102@, VR138, VR139, VR140, VR141, VR148, VR150, VR151, VR152, VR153, VR154, VR155, VR156, VR157, VR158, VR159
1c 1c 1c 1c 1c
1999 2000
VR149 VR448, VR449, VR450, VR451, VR920, VR921, VR922, VR923, VR924, VR925, VR926, VR927, VR977, VR979, VR980, VR981, VR982, VR983, VR987, VR988, VR989, VR990, VR991, VR992, VR994, VR995, VR996, VR997, VR1000, VR1001, VR1002, VR1003, VR1004, VR1005, VR1088, VR1089, VR1090, VR1091, VR1092, VR1093, VR1094, VR1095, VR1105a, VR1107a
1a 1c
2000 2000 Unknown Unknown Unknown
VR986 VR999, VR1007$ MD70# Trangieb Begab
1a 1c 1c 1c 1c
1989 1995 Unknown 1997 1998 1999
1c
Those samples for which Npro sequences were also determined are shaded. BVDV samples clustering to the genotype 1a are shown in bold type. Viral RNA was extracted from serum unless otherwise indicated; #cell culture supernatant, @blood, ^spleen, $intestine. a 0 5 -UTR sequence determined by direct sequencing of amplified product. b Australian sequences for 50 -UTR and Npro retrieved from GenBank using following accession numbers Trangie Y546: AF049222, Bega: AF049221. The sequences determine as part of this study have been submitted to GenBank and assigned the following accession numbers, 50 UTR: AY762988–AY763092, Npro: AY763093–AY763095.
T.J. Mahony et al. / Veterinary Microbiology 106 (2005) 1–6
Veterinary Laboratories, Department of Primary Industries, Brisbane, Queensland, Australia. These samples were initially tested for BVDV using an antigen capture ELISA. A further 11 isolates were kindly provided by Dr. Colin Wilks from the Victorian Institute of Animal Sciences. A further two isolates were kindly provided by Dr. Jan Smith from James Cook University. VR1007 was amplified from intestinal tissue taken from a feedlot bovine with suspected mucosal disease. The sequences for two Australian BVDV strains were retrieved from GenBank. A complete list of the BVDV samples examined in this study is shown in Table 1.
3
polymeraseR, FS (Applied Biosystems). The amplification product from some isolates were directly sequenced following gel purification. A 622 bp product encompassing the Npro region was amplified and sequenced for selected viral samples as described for the 50 -UTR region. The following PCR primers were used in PCR reaction: STARTfwd (50 - TGC TGT ACATGG CAC ATG GAG TTG-30 , nucleotide 371–394 of the NADL genome) and Nproseq1 (50 -CTA TCC TTC TCT GAT TCT CTG-30 , nucleotide 973–993 of the NADL genome). In all cases the DNA sequence data were analysed using ContiExpress from the Vector-NTI suite 6 of programs (Informax, USA).
2.2. RNA isolation Total RNA was extracted from sera samples using High Pure Viral RNA kits (Roche Molecular Biochemicals) according to the manufacturer’s instructions. RNA from tissues was extracted using RNeasy mini kits (Qiagen) in combination with QIAshredder homogenizer (Qiagen). 2.3. Reverse transcriptase PCR (RT-PCR) and sequencing A 292 bp DNA product was amplified from the 50 UTR using the Titan1 single tube reaction system (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Primers utilised were 5UTRfwd (50 -CTA GCC ATG CCC TTA GTA GGA CTA-30 , nucleotide 102–125 of the NADL genome, Genbank Accession number: BVI133739) and STARTrev (50 -CAA CTC CAT GTG CCA TGT AC AGC A30 , nucleotide 371–394 of the NADL genome). Results of PCR reactions were confirmed by 1% agarose gel electrophoresis. Where two or more DNA bands were evident the band corresponding to the expected size was excised from the agarose gel and recovered from the agarose using the QIAquick gel extraction kit (Qiagen). The purified PCR product was ligated into the pGemT-Easy (Promega) using T/A cloning. The clones (three of each sample) were sequenced in both strands with the T7 and SP6 sequencing primers. DNA sequencing was performed on an ABI automated A373 sequencer using dideoxy-sequencing chemistry utilising the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit, with AmpliTaq DNA
2.4. Phylogentic analysis Phylogenetic reconstructions for genetic typing of the viral samples were compiled using a 244 nucleotide region of the 50 -UTR (nucleotide 130– 374 of the NADL genome) and a 385 nucleotide region of Npro (nucleotide 386–770 of the NADL genome). Additional sequences from representative isolates of previously identified phylogenetic groups of BVDV-1 were included into phylogentic analysis. Nucleotide sequences from each region were aligned using the Clustal W program. Phylogenetic analysis was performed using the Neighbour program from the Phylip inference program package (Felsenstein, 1993). Statistical analysis of phylogenetic trees was determined by bootstrap analysis carried out on 100 replicates using Seqboot program and the resulting tree was drawn using the Consensus program (Felsenstein, 1993).
3. Results and discussion A survey of the genetic diversity of BVDV field isolates in Australia was conducted by determining the nucleotide sequences of the 50 -UTR of 89 strains, as well the Npro region for three strains. The 50 -UTR and Npro sequences for a further two Australian strains were retrieved from GenBank and added to the data sets. These analyses indicate that all of the BVDV field strains examined in this study were of the BVDV-1 genotype. On the basis of this study it appears as
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though the BVDV-2 genotype is absent from Australia. Single tube reverse transcription reactions were successfully utilised to amplify a 292 bp region of the pestivirus 50 -UTR from the samples listed in Table 1 (data not shown). Sequences from these clones were used in the analyses. Eighty-nine samples were sequenced in 50 -UTR and sequencing revealed their length varied from 292 to 295 bp (data not shown). The 50 -UTR phylogenetic reconstructions using sequences for Australian BVDV isolates illustrated that all 91 viruses analysed belonged to the BVDV-1 genotype (see insert in Fig. 1). As this tree was strongly deformed due to the large number of isolates clustered within one group, a separate tree was generated for phylogenetic analysis using twelve representative Australian isolates. This tree demonstrated that only two of the isolates, VR149 and VR986, clustered to the BVDV-1a subtype (Fig. 1). This subtype includes the highly characterised NADL strain. All other sequences formed a unique cluster in the phylogenetic tree (labelled in Fig. 1 as ‘‘?’’), which appeared to represent a potentially new genetic group
of BVDV-1 genotype with no reference strain so far identified. To identify the group designated ‘‘?’’ in the 50 -UTR analysis (Fig. 1), three isolates from this collection (MD70, VR1007, and VR999) and two additional sequences for Australian BVDV strains retrieved from GenBank (BVDV strain Bega and strain Trangie Y546, see Table 1) were regenotyped using the Npro coding region. It was not possible to sequence the Npro region of all isolates of interest due to instability of some samples. The resulting phylogenetic trees were constructed using a 385 bp sequences derived from the Npro region and indicated that the isolates in this study were not members of a new genetic group of the BVDV-1 genotype. The majority of the Australian BVDV strains are members of the previously described BVDV-1c genotype (Fig. 1). The BVDV1c branch was supported by high bootstrap scores. The BVDV-1c branch had not previously been identified using 50 -UTR sequences prior to the analyses presented here. The previously recognised members of this subgenotype were strains DeerNZ1 and 519 isolated from New Zealand and Germany respectively
Fig. 1. Phylogenetic trees showing the typing of Australian BVDV-1 isolates in 50 -UTR and Npro regions. The unrooted trees were based on analysis of partial 244 nt (50 -UTR) or 385 nt (Npro) long sequences. They were prepared using the Neighbor-joining method (Kimura 2-parameter method, transition/transversin 2.0). Tree in the insert shows the grouping of all Australian isolates analysed in 5-UTR. Australian isolates are labelled in bolt. Phylogenetic groups are labelled according to Vilcek et al. (2001). The group labelled as ‘‘?’’ could not be exactly identified as there was not a reference strain typed in 50 -UTR. Other nucleotide sequences were taken from Vilcek et al. (2001). The Npro sequences for the strains 519, 721, Deer NZ1 and Deer GB1 were taken from Becher et al. (1997) (GenBank numbers – AF144464, AF144463, U80903 and U80902). Numbers over branches indicate the percentage of 100 bootstrap replicates that support each phylogenetic branch.
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(Becher et al., 1997). The genotyping of DeerNZ1 and 519 was based solely on Npro analysis, as the 50 -UTR sequences of these isolates were not determined. Vilcek et al. (2001) did not identify any BVDV-1c genotypes based on 50 -UTR analyses using seventeen isolates from a previous study of New Zealand isolates (Vilcek et al., 1998). Nagai et al. (2004) have also utilised the 50 -UTR and Npro sequences from the Bega and Trangie Y546 in their analyses, which demonstrated clustering with strains DeerNZ1, 519 and some Japanese viruses to the subgenotype BVDV-1a0 . When combined with the analyses presented here, the subgenotype BVDV-1a0 described by Nagai et al. (2004) is analogous to BVDV-1c. When considered in the context of the study the distribution of genotypes does not appear to be a result of geography. It has been proposed that the non-coding region of the BVDV is unsuitable for phylogenetic studies (Becher et al., 1997). However, Vilcek et al. (2001) clearly demonstrated that 50 -UTR based studies give meaningful phylogenetic inferences. By sequencing both non-coding region (50 -UTR) and part of the coding region (Npro) analysis of these data sets as either separate or combined phylogenetic reconstructions yielded identical results. Although slightly higher bootstrap values were recorded for codingbased analyses, the results and overall topography of the phylogenetic trees were similar when compared to the non-coding based analyses. The results presented here were initially limited to the analysis of 50 -UTR sequences. This region is flanked by motifs that are highly conserved amongst the all pestiviruses. The high conservation of these motifs in all genotypes of BVDV permits the easy amplification of this region from diverse pestiviruses ensuring ready analysis of a wide diversity of virus samples with a high confidence of successful amplification, particularly from samples of unknown status. Furthermore, the comparatively short intervening sequence permits rapid and accurate acquisition of sequence data. These characteristics make the 50 -UTR an excellent sequence target for initial phylogenetic reconstructions of BVDV of unknown genotypes. It also permits the rapid primary genotyping of large numbers of viral samples in a timely fashion. Subsequently, selected isolates can be characterised further by determining larger sequence regions of coding sequence. Recently, Nagai et al.
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(2004) conducted extensive phylogenetic analyses of BVDV sequences using five separate genomic regions. For most of the isolates examined there was agreement between the subgenotype classifications as determined using sequences from each region. The isolates where discrepancies were identified were considered to be a result of recombination between subgroups as opposed to inaccurate phylogenetic inferences due to unsuitable sequence selection. As the first step in the development of an effective BVDV vaccine for the Australian cattle industry, the genetic diversity of a large number of BVDV field isolates has been examined. The isolates were examined in both untranslated and coding regions and the majority were typed as BVDV-1c. These results indicate the BVDV viruses in Australia at the time of this study were genetically homogeneous. When considered in the context of other studies it is reasonable to conclude that there is likely to be limited antigenic diversity in these viruses as well. The results presented here indicate it should be possible to develop effective vaccines to the BVDV strains currently circulating within Australia. However, it would be prudent to continue similar studies following the adoption of any vaccination program to ensure that more virulent genotypes do not emerge as a consequence of immune selection. Further any future genotyping studies conducted in tandem with vaccination programs should also include analysis of the antigen coding regions, such as glycoprotein E2, along with the 50 UTR and Npro regions. Such a comparative studied would enable monitoring of the effects of vaccination on the genotypes and antigenic variants within the population.
Acknowledgments The authors acknowledge Professor Colin Wilks of Victorian Institute for Animal Science and Dr. Jan Smith of James Cook University for provision of virus samples. The authors thank Mr Mark Kelly for performing the antigen capture ELISA analyses. The authors also thank Dr. Lee Taylor, Dr. Nigel Nichols and Dr. Steve Mathers for continued support and provision of viral samples. This work was supported by grant Flot.203 from Meat and Livestock Australia. S. V. was supported by VEGA grant (1/9014/02) and SP (51/0280803).
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