Instituto de Virología, CICVyA, INTA-Castelar, CC77 (1708) Morón, Buenos Aires, Argentina1
Instituto de Microbiología y Zoología Agrícola, CICVyA, INTA-Castelar, CC77 (1708) Morón, Buenos Aires, Argentina2
Consejo Nacional de Investigaciones Científicas, Argentina3
Author for correspondence: Leandro Jones. Fax +54 11 4621 1743. e-mail ljones{at}cicv.inta.gov.ar
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Abstract |
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Introduction |
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Most of the infections caused by BVDV are of the acute type but around 0·11·8% of animals are persistently infected (Houe, 1999 ), representing the way in which the virus spreads and is maintained in natural populations. This persistent infection is the consequence of an in utero infection (around days 30150 of gestation), when the foetus is unable to mount an immune response. Unlike other RNA viruses in which antigenic evolution appears to act in the maintenance of persistence, persistence of BVDV is thought to be favoured by the existence of an antibody-resistant virus population (Hertig et al., 1995
; Paton et al., 1994
). Nevertheless, viral genetic change has been reported in viruses from persistently infected animals, although the biological significance of these changes still requires elucidation (Collins et al., 1999
).
During viral genome replication, variant mutants are spontaneously generated. Some of these variants may differ in replication efficiency or fitness (Holland et al., 1991 ; Martínez et al., 1991
; Domingo & Holland, 1997
). Knowledge of virus genetic heterogeneity in a single carrier individual may be fundamental in studying how genomic changes influence the ability of the virus to cause disease, which might be especially important to elucidate a pathogenic process caused by viral genomic variation. It may be also of value in understanding the evolution of the virus, designing vaccines and/or improving diagnostic assays. Intra-host genetic heterogeneity has been investigated widely in HCV, for which BVDV is a model (Oshima et al., 1991
; Martell et al., 1992
; Kato et al., 1992a
, b
; Enomoto et al., 1994
; Okamoto et al., 1992
; Okada et al., 1992
; Taniguchi et al., 1993
; Katayama et al., 1998
; Toyoda et al., 1998
; Jang et al., 1999
; Farci et al., 2000
).
The genome of BVDV is about 12 kb long; it has a single open reading frame and two untranslated regions (UTRs) at the 5' and 3' ends (reviewed by Meyers & Thiel, 1996 ). The 5'UTR is the most conserved region of the genome (De Moerlooze et al., 1993
). It has a highly structured internal ribosome entry site (IRES) (Poole et al., 1995
; Chon et al., 1998
; Le et al., 1998
), is involved in the regulation of genome replication and gene expression (Becher et al., 2000
; Yu et al., 2000
) and has some possible markers of virulence (Topliff & Kelling, 1998
). Furthermore, the 5'UTR has been used widely in studies of evolution, epidemiology and taxonomy (Harasawa & Giangaspero, 1998
; Hofmann et al., 1994
; Sakoda et al., 1999
; Baule et al., 1997
; Ridpath et al., 1994
; Pellerin et al., 1994
; Harpin et al., 1995; Jones et al., 2001
). In both picornaviruses and hepaciviruses, the 5'UTR is related to tropism and pathogenesis (Lerat et al., 2000
; Nakajima et al., 1996
; Funkhouser et al., 1999
).
Previous reports of BVDV quasispecies variability consist of studies on genomic regions that encode proteins E2 and NS3 of a virus from a persistently infected cow (Collins et al., 1999 ) and observations of the occurrence of stabilizing mutations over several cell passages of a recombinant BVDV (Becher et al., 2000
). We described the genetic heterogeneity in the 5'UTR from a cell-passaged isolate (Jones & Weber, 2001
). To our knowledge, the variability of the BVDV 5'UTR within a single individual has not been analysed. The aim of this work was to describe 5'UTR quasispecies in BVDV. Tissues from a field-infected foetus were used as a source of viral RNA to investigate the variability of the BVDV 5'UTR.
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Methods |
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Cloning.
Viral RNA was partially purified from 10% homogenates of BVDV-positive foetal tissues in Eagles minimal essential medium. Virus suspensions were concentrated by adding 125 µl cold NaCl (4 M) and 0·1 g PEG8000 to 875 µl homogenate. Following thorough mixing, the preparation was incubated for 12 h at 4 °C and then pelleted by centrifugation at 4 °C in a microcentrifuge for 15 min. Pellets were washed with TE (0·01 M Tris, pH 7·8, and 0·005 M EDTA) and resuspended in 460 µl TE by gently pipetting up and down. The suspension was used immediately for RNA extraction by digestion with proteinase K followed by standard acid phenolchloroform extraction and ethanol precipitation (Sambrook et al., 1989 ). Pellets were resuspended in 5 µl ddH2O and used immediately for cDNA synthesis. cDNA synthesis and PCR were carried out as described above but PCR was conducted using Pfu DNA polymerase (Promega) and the number of cycles was 20 instead of 35 in order to minimize the frequency of in vitro-generated variability. Reactions were performed following the manufacturers instructions, with the above-mentioned cycling conditions. It must be stated that RNA dilutions of up to 1:100 still give positive amplification bands in RTPCR. This ensures that an amplification bottleneck is not biasing the complexity of the mutant spectrum. PCR products were purified from agarose gels using a commercial kit. Purified DNA was adenylated by treating with Taq DNA polymerase and cloned in a pGem vector using the pGemT-Easy System II kit (Promega). Cloning in Escherichia coli, strain J109, was carried out as suggested by the manufacturer except that the 37 °C incubation step prior to plating in ampicillin-containing medium was only for 5 min to avoid duplicated clones. Bacterial colonies were double screened by blue/white standard selection followed by insert-specific PCR testing. Recombinant plasmids were extracted from white colonies by the Turboprep method (Woodford & Usdin, 1991
) and used as templates for PCR amplification with primers 324 and 326. PCR conditions were as described above.
Single-strand conformational polymorphism (SSCP) analysis.
PCR products from the plasmid preparation described above were analysed by SSCP, as described previously (Jones & Weber, 2001 ). Briefly, DNA was denatured using formamide and electrophoresed immediately in both 15% acrylamide and acrylamideglycerol (15 and 5%, respectively) minigels at 20 °C, applying 200 V for 3 h. Gels were silver stained.
Sequence analysis.
Direct sequencing of both strands of the purified PCR products from variant clones was performed by the dideoxy-terminator method. Sequencing was performed using an ABI373 sequencer. Sequences were aligned using CLUSTALW software (Thompson et al., 1994 ). Clustering of sequences was analysed using the partition analysis of quasispecies (PAQ) program (Baccam et al., 2001
). Phylogenetic analysis was performed using the DNAPARS and DNAML programs of the PHYLIP package (Felsenstein, 1993
). Resampling of the original alignment for bootstrap analysis was performed with the SEQBOOT routine from PHYLIP.
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Results |
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Virus from the lung was analysed thoroughly by RTPCRSSCP as follows: four different RTPCRs (AD) were achieved using viral RNA extracted from a 10% lung homogenate. PCR products from these reactions were cloned and inserts from 158 clones were submitted to SSCP analysis. A total of 11 different banding patterns was detected (Fig. 1a, b
). There was one major pattern, Lung_M (Fig. 1a
, b
), represented by 125 clones (Fig. 1c
). Patterns Lung_Q1, _Q4 and _Q5 had 11, 5 and 9 representative clones, respectively (Fig. 1c
). These SSCP patterns were independent of the RTPCR from which each clone was derived (Table 1
). SSCP profiles Lung_D39, _D44, _D13, _A30, _C15, _C31, _C23 and _C17 were represented by just one clone each.
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Discussion |
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The 5'UTR was chosen for this analysis of quasispecies for several reasons: (i) it is the most conserved region of the pestivirus genome (therefore, RTPCR amplification is efficient and almost free of subpopulation selection due to the differential annealing of primers); (ii) the use of more variable regions for this type of analysis is not straightforward, as the presence of a large number of variants may induce artefactual polymorphisms related to sampling error; and (iii) the highly structured nature of the 5'UTR allows the randomness of the substitutions among variant sequences to be tested.
It is well known that experimentally generated variability is a major problem in studying virus quasispecies (reviewed by Smith et al., 1997 ). The low proportion of randomness in the data presented here may be attributable to two principal facts: (i) Pfu polymerase has a 90% reduction in the error rate when compared with Taq polymerase (Lundberg et al., 1991
); and (ii) the step of virus concentration, since using high concentrations of virus appears to reduce the likelihood of introducing experimental artefacts (Laskus et al., 1998
). A work presented by Arias et al. (2001)
, which compared the performance of molecular and biological cloning for studying foot-and-mouth disease virus quasispecies, showed similar results. These authors observed that biological and molecular clones provide indistinguishable definitions of the mutant spectrum. We chose to clone RTPCR products obtained directly from animal organs in order to avoid the selection of virus subpopulations by replication in cell culture.
The genomic sector analysed here spans residues 128372 of the NADL reference strain sequence (Colett et al., 1988 ). As expected, the observed nucleotide changes do not appear to affect structures essential for IRES function (Chon et al., 1998
). The BVDV IRES has been shown to act in RNA stability, RNA replication, translation of the viral polyprotein and encapsidation of the viral genome (Becher et al., 2000
; Yu et al., 2000
). 5'UTR variants could be related with the existence of different environmental constraints in different tissues, i.e. conditions in different tissues might favour the presence of different virus quasispecies acting as selective pressures over some 5'UTR sectors. However, we may not conclude that the changes observed in the 5'UTR have an adaptive function, as quasispecies evolution in the E2-encoding region may occur in the absence of an apparent selective force (Collins et al., 1999
). In addition, changes in any region of the genome may be linked with changes in another region (i.e. if changes occurred at the same time).
In HCV, phylogenetic analysis has been used to demonstrate the occurrence of compartmentalized virus replication through the identification of monophyletic groups exclusive to different cells (Roque Afonso et al., 1999 ). Although we were able to detect sequences exclusive to different organs, quasispecies did not form any kind of groups according to the organ of origin, neither by phylogenetic- (Fig. 3
) nor by similarity (PAQ)-clustering analysis (data not shown). These may indicate either the non-existence of clonal lineages replicating in different organs or its existence at proportions not detectable by the strategy implemented in this work. In any case, the detection of unique sequences in different organs is a proof of the existence of variants occurring in low frequencies. That is, representatives of some lineages are in minor numbers and thus have less chance of being detected. Thus, our data suggest that BVDV could replicate at high rates in some tissues (in blood cells, for example, as there are evidences of the presence and ability of replication of BVDV in white blood cells) (Truitt & Schechmeister, 1973
; Ohmann, 1983
; Brodersen & Kelling, 1998
), while replication rates in some cells may be very low. Low-kinetic 5'UTR subpopulations have been detected in HCV and have been suggested to act in avoiding immune surveillance (Jang et al., 1999
).
Immunity does not always protect the foetus from congenital infection (van Oirschot et al., 1999 ). Ability to elude antibodies may be an explanation for transplacental infections in seropositive dams. Previous works suggest that the genetic variability of the 5'UTR correlates with antigenic characteristics (van Rijn et al., 1997
; Flores et al., 2000
; Nagai et al., 2001
). The diversity observed by us in the 5'UTR might be an indirect evidence of a high variability in other genomic regions, which may be a strategy for excluding some virus variants from immune system recognition. Previous works have shown the existence of herd-specific BVDV strains (Paton et al., 1994
), apparently maintained by persistently infected animals (Hamers et al., 1998
). If these persistently infected animals are born from vaccinated cows, then the herd may be a focus for spreading vaccine-resistant BVDV variants.
BVDV has been used as a model for HCV (Weiner et al., 1991 ; Ruibal Brunet et al., 1999
; Prince et al., 2000
; Baginski et al., 2000
; Zitzmann et al., 1999
). Since BVDV is much easier to be passaged in cell culture than HCV, it may be a good candidate for studies of the relationship between virus quasispecies and virus behaviour under particular conditions; for instance, the presence of an antiviral compound. Results originating from this type of study may serve as a preliminary test prior to the analysis of HCV.
The strategies used in this work may be adapted easily both to analyse large numbers of samples of other genomic regions and for the study of BVDV quasispecies evolution in systems different from infected animals. Further work is necessary to elucidate the biological role of quasispecies in BVDV. A deeper knowledge of the genetic variability of BVDV will be of great importance for classification, diagnosis, vaccination and understanding some as yet unknown aspects of BVDV biology.
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Acknowledgments |
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Footnotes |
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Received 16 October 2001;
accepted 22 March 2002.