Studies on overwintering of bluetongue viruses in insects

David M. White1,{dagger}, William C. Wilson1, Carol D. Blair2 and Barry J. Beaty2

1 USDA, ARS, Arthropod-borne Animal Diseases Research Laboratory, Dept 3354, 1000 E. University Avenue, Laramie, WY 82071, USA
2 Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA

Correspondence
David M. White
chz8{at}cdc.gov


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection.

{dagger}Present address: CDC/Special Pathogens Branch, 1600 Clifton Rd NE, Building 15-SB, Mailstop G14, Atlanta, GA 30333, USA.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bluetongue viruses (BTVs; family Reoviridae, genus Orbivirus) are a group of economically important, arthropod-borne viruses that are maintained in the USA in a natural transmission cycle involving the haematophagous insect vector Culicoides sonorensis and sheep, cattle or wild ruminants (Holbrook et al., 2000). In temperate regions with cold winters, the vectors survive severe weather as larvae and the transmission cycle is interrupted, whereas BTVs may be maintained in year-round transmission cycles in temperate regions with mild winters (Gerry & Mullens, 2000), as well as in the tropics. However, even in these regions, the cycles may be interrupted by dry seasons or other adverse environmental conditions (Gibbs et al., 1992). Clearly, a mechanism(s) is required for the viruses to survive periods of inclement climatic conditions.

A number of hypotheses have been proposed for the seasonal recurrence of BTVs (Griot, 2000; Nevill, 1971; Tabachnick, 1996). Overwintering in the vertebrate host was the leading hypothesis for many years, but has not been proven (Caporale et al., 2004). Reintroduction of BTVs by infected vertebrate hosts or vectors could be another possible explanation. However, with random reintroduction, a greater diversity of serotypes than we have observed would be expected in an endemic area. We have regularly detected genetically similar BTV serotype 11 at the same premises in northern Colorado, USA (D. M. White, C. D. Blair & B. J. Beaty, unpublished data). The potential for local overwintering of BTVs in their vector needs to be investigated. Overwintering of Culicoides as adults has been demonstrated in mild climates with well-established hibernacula (Gerry & Mullens, 2000), but is exceptional in more severe climates, such as northern Colorado (F. Holbrook, personal communication), where overwintering as larvae is usual. Several arboviruses are transmitted vertically by infected female arthropods. This provides a mechanism for virus maintenance, as infected progeny emerge after periods of adverse environmental conditions to reinitiate the transmission cycle (Beaty & Calisher, 1991; Comer et al., 1990; Watts et al., 1973). A hallmark of arboviruses that are transmitted vertically by their arthropod vectors is their regular reappearance in a distinct geographical region. Overwintering in the vector could condition the annual, focal reappearance of BTVs, such as we have observed in northern Colorado (D. M. White, C. D. Blair & B. J. Beaty, unpublished data).

Although BTVs were not found to be transmitted transovarially in colonized C. sonorensis when assayed by virus isolation using vertebrate cells, viral antigen was detected in ovarian and reproductive-tract tissues, which could condition vertical transmission (Jones & Foster, 1971; Nunamaker et al., 1990). Orungo virus, another member of the genus Orbivirus, has been isolated from wild-caught male mosquitoes in the Ivory Coast (Cordellier et al., 1982), suggesting vertical transmission of that virus. Our previous studies suggested that BTVs are maintained transseasonally in C. sonorensis larvae in Colorado (Deines, 1995; Raich, 1995; White, 2001). BTV RNA and antigen were detected in a number of pools of early-season, field-collected larvae (Raich, 1995; White, 2001), but virus isolation was rare and achieved only when larvae were reared to adults in the laboratory (Deines, 1995). Indeed, virus isolation from field-collected vectors is infrequent, especially when using vertebrate cell cultures and even when inoculating chick embryos (the ‘gold standard’ for BTV isolation).

The unique structural characteristics of the BTV virion may account for these observations. The BTV outer capsid is composed of virion proteins (VPs) 2 and 5, which are encoded by genome segments 2 and 5, respectively. VP2 is the vertebrate cell-receptor ligand (Hassan & Roy, 1999). The inner capsid is composed of VP7 and VP3, encoded by genome segments 7 and 3, respectively. VP7 has been shown to be the invertebrate cell-receptor ligand (Tan et al., 2001; Xu et al., 1997). Chymotrypsin digestion of intact virions cleaves VP2 and generates infectious subviral particles. These particles are more infective for cultured Culicoides cells and insects than the intact virions, presumably due to unmasking of VP7. In addition, core particles, completely devoid of VP2 and -5, are significantly less infective for both cultured mammalian cells and C6/36 (Aedes albopictus) cells than for Culicoides cells (Mertens et al., 1996). Interestingly, persistent infection of plants results in downregulation of certain genes of Wound tumour virus (WTV; family Reoviridae, genus Phytoreovirus) and reduced infectivity for insect vector cells (Reddy & Black, 1974, 1977). Persistent infection of C. sonorensis larvae could result in downregulation of VP2 and -5 expression, thereby complicating BTV isolation from insects in conventional vertebrate cell cultures. Other viral components that could be important in a persistent infection are the membrane-binding proteins NS3/3a (encoded by genome segment 10), which have a role in non-cytopathic release of the virus from infected cells (Beaton et al., 2002; Hyatt et al., 1991).

These studies examined the potential for BTV overwintering in larval Culicoides. We investigated the presence of the BTV genome in field-collected larvae of C. sonorensis and other Culicoides spp. and in persistently infected cell cultures. Whilst provocative observations were made, they were based entirely on detection of nucleic acid sequences. A mechanism that could explain these observations is proposed to form hypotheses for future studies.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Insect cell-culture lines and viruses.
The KC cell line was derived from embryonic AK colony Culicoides variipennis (now sonorensis) at the US Department of Agriculture – Arthropod-Borne Animal Diseases Research Laboratory (ABADRL), as described by Wechsler et al. (1989). Similar methods were used to generate the W3 and W8A cell lines from field-collected Culicoides; however, the larvae were collected from a BTV-endemic site in northern Colorado in September (W3) and October (W8A) of 1995, reared to adults in the laboratory, fed a blood meal, mated and allowed to lay eggs, which were subsequently processed to generate the cell-culture lines (McHolland & Mecham, 2003).

The AK colony of C. sonorensis was started from insects collected in Idaho, USA, in 1973 in the midst of a BTV outbreak. Two virus isolates made at that time and deposited in the ABADRL collection, 600520 (from a pool of adult Culicoides) and 600015 (from a sheep), were also included in these analyses.

Collection of Culicoides from BTV-endemic sites.
Larval Culicoides were collected from two endemic sites less than 30 miles apart in northern Colorado in the spring and summer of 1996 and 1998. In 1996, collected larvae were reared to adulthood in the laboratory and identified to species level by wing-vein patterns. Insects were pooled, homogenized and assayed for virus by: (i) intravascular inoculation of embryonating chicken eggs (ECEs) followed by blind passage in vertebrate cell cultures; (ii) intrathoracic inoculation of Aedes triseriatus mosquitoes followed by blind passage in vertebrate cell cultures; and (iii) direct inoculation of vertebrate cell cultures (Deines, 1995). All insects were negative by all three virus-isolation methods. The remaining insect homogenates were stored at 4 °C until RNA isolation, up to 2 years later. In the summer of 1998, all stages of immature Culicoides (larvae and pupae) were collected, pooled and placed into ddH2O, then frozen at –80 °C for RNA isolation and nested RT-PCR without attempting virus isolation.

Nucleic acid detection and sequence analysis.
Total RNA was extracted from sample pools by using a PureScript kit (Gentra Systems) and 5 µl of the total volume (50 µl) was used as the template for cDNA generation. Reverse transcription was performed by using Moloney murine leukemia virus reverse transcriptase and 10 µl of the 20 µl reaction was used as template for the first round of PCR. After 35 (45 for genome segment 10) cycles of 1 min at 95 °C, 1 min at 56 °C and 2 min at 72 °C (final extension of 10 min at 72 °C), 10 µl of the first-round PCR was used as the template for the nested PCR, which followed the same program. Primers were designed from published sequences (Table 1) and the same primers were used for both RT and the first round of PCR (Huismans & Van Dijk, 1990; Lee & Roy, 1986; Purdy et al., 1985).


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Table 1. Primers used for the detection of BTV genome segments by nested RT-PCR

All primers were designed from published sequences.

 
Nested RT-PCR products were subjected to automated sequencing and contigs were assembled in ContigExpress and then aligned by using CLUSTAL W (Thompson et al., 1994; provided by InforMax). Variability in the sequences was observed in the PCR products, but for the purposes of this analysis, the majority (consensus) sequence for each sample was used. Phylogenetic trees were inferred by neighbour-joining using distance matrices generated with the Tamura–Nei method in MEGA with 1000 bootstrap replicates (Kumar et al., 1993).

Statistical analysis of the frequency of genome-segment detection was performed by using McNemar's test for matched pairs (Agresti, 1996). P values were calculated by using the binomial distribution rather than the {chi}2 approximation.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of genome segment 7 sequences from field-collected material and cell lines
By RT-PCR analysis, BTV genome segment 7 sequences were detected in 10 % (31/319) and 30 % (17/56) of field-collected Culicoides pools in 1996 (Table 2) and 1998 (Table 3), respectively, and in all Culicoides cell-culture lines (Table 3). Nested RT-PCR bands of the appropriate size were detected in four pools from 1996 and are included in the total in Table 2, but were not sequenced due to technical difficulties. It was not known whether the lower detection rate of BTV RNA in the 1996 collections was due to storage of the samples for 2 years before processing. The prevalence of BTV nucleic acid in insects from 1998 was probably a better estimate of the true prevalence in field-collected samples, but this could vary annually. Phylogenetic analyses grouped all of the larval, Culicoides cell-culture line and viral isolate segments in clade 1 of the five recognized groupings of BTV segment 7 sequences worldwide (Wilson et al., 2000), suggesting that differences in sequences between the samples were probably negligible from a biological viewpoint (Fig. 1). The number of differences between the most dissimilar sequences was small (17 changes in the 286 bp sequence) and they were all silent, except for one resulting in a conservative amino acid change.


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Table 2. RT-PCR for BTV RNA-positive Culicoides spp. larval pools collected in 1996

Insect species were determined based on wing-vein patterns and geographical distribution of Culicoides spp. in North America (Holbrook et al., 2000). Mixed samples were pools of insects that were unidentifiable by wing-vein patterns (wings were absent or damaged). +, RT-PCR product obtained; –, no product; Band, PCR product of the appropriate size present upon agarose gel electrophoresis, but could not be sequenced; ND, RT-PCR not done due to lack of starting material (RNA was used up completely in other tests).

 

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Table 3. RT-PCR for BTV RNA-positive Culicoides spp. pools collected in 1998 and embryonic Culicoides cell lines

Adults were collected incidentally during sampling of mud for larvae and were included with larvae and pupae in subsequent tests. +, RT-PCR product obtained; –, no product obtained; Band, PCR product of the appropriate size present upon agarose gel electrophoresis, but could not be sequenced; ND, test not done due to the lack of starting material (RNA was used up completely in other tests).

 


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Fig. 1. Phylogenetic analysis of segment 7 sequences amplified from field-collected larvae, Culicoides cell-culture lines and two virus isolates from Idaho, 1973. A ‘GB’ suffix indicates a sequence from GenBank. Bootstrap values represent percentages of inclusion in 1000 bootstrap replicates. Data from the embryonic Culicoides cell-culture lines and viral isolates are in bold.

 
Analysis of genome segment 2 sequences from field-collected material and cell lines
By RT-PCR analysis, BTV genome segment 2 was detected in <1 % (1/319) and 16 % (9/56) of field-collected Culicoides pools in 1996 (Table 2) and 1998 (Table 3), respectively. Nested RT-PCR products of the correct size were detected in two pools from 1998, but were not sequenced due to technical difficulties. Phylogenetic analyses indicated that all of the sequences were very similar to a BTV-11 genome segment 2 (Fig. 2), consistent with our findings for virus isolates from the same endemic focus. There were two changes in the most dissimilar sequences over the 150 bp analysed: one was silent and one caused a conservative amino acid change. Genome segment 2 was also detected in two of the embryonic cell-culture lines (W3 and W8A), but with only one of the two primer sets (L2-2) that showed similar sensitivities in non-quantitative tests (Table 3). Primers for genome segment 2 of other BTV serotypes were not used to test the pools.



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Fig. 2. Phylogenetic analysis of segment 2 sequences amplified from field-collected larvae and Culicoides cell-culture lines. A ‘GB’ suffix indicates a sequence from GenBank. Bootstrap values represent percentages of inclusion in 1000 bootstrap replicates. Data from the embryonic Culicoides cell-culture lines are in bold.

 
Analysis of genome segment 3 sequences from field-collected material and cell lines
BTV genome segment 3 was detected by nested RT-PCR in 7 % (21/319) and 27 % (15/56) of field-collected Culicoides pools from 1996 (Table 2) and 1998 (Table 3), respectively. A nested RT-PCR product of the correct size was detected in one pool from 1996 and one pool from 1998, but was not sequenced due to technical difficulties. Phylogenetic analyses indicated that all of the sequences grouped within the North American topotype (Fig. 3) (Gould & Pritchard, 1991). The number of differences between the two most dissimilar sequences was small: there were 10 changes in the 194 bp sequence and all were silent except for one that resulted in a conservative amino acid change. Genome segment 3 was also detected in all embryonic cell-culture lines tested (Table 3, Fig. 3).



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Fig. 3. Phylogenetic analysis of segment 3 sequences amplified from field-collected larvae, Culicoides cell-culture lines and two virus isolates from Idaho, 1973. A ‘GB’ suffix indicates a sequence from GenBank. Bootstrap values represent percentages of inclusion in 1000 bootstrap replicates. Data from the embryonic Culicoides cell-culture lines and viral isolates are in bold. African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV) were included as outgroups.

 
Sequence analysis of genome segment 10 from field-collected material and cell lines
BTV genome segment 10 was detected by nested RT-PCR in 2 % (6/319) and 13 % (7/56) of field-collected Culicoides pools in 1996 (Table 2) and 1998 (Table 3), respectively. Nested RT-PCR products of the correct size were detected in two pools from 1996 and one pool from 1998, but were not sequenced due to technical difficulties. Phylogenetic analyses indicated that all of the sequences clustered within US group 2 (Bonneau et al., 1999), suggesting that the differences are probably negligible from a biological viewpoint (Fig. 4). The number of differences between the two most dissimilar sequences was larger than seen for the other segments; there were 53 changes over the 702 bp sequence. All of the changes were silent except for 15 resulting in non-conservative and four resulting in conservative amino acid changes, which were grouped at the ends of the sequence. Genome segment 10 was also detected in all embryonic cell-culture lines tested (Table 3, Fig. 4).



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Fig. 4. Phylogenetic analysis of segment 10 sequences amplified from field-collected larvae, Culicoides cell-culture lines and two virus isolates from Idaho, 1973. A ‘GB’ suffix indicates a sequence from GenBank. Bootstrap values represent percentages of inclusion in 1000 bootstrap replicates, as assigned by Bonneau et al. (1999). Data from the embryonic Culicoides cell-culture lines and viral isolates are in bold. Epizootic hemorrhagic disease virus (EHDV) was included as an outgroup.

 
Statistical analysis of the frequency of genome-segment detection
A statistical analysis of the frequency of genome-segment detection was performed, which assumed that all sequences should be detected when any are detected (H0: {pi}12={pi}21). This analysis showed that genome segment 7 was detected significantly more often than genome segment 2 in both the 1996 and 1998 datasets. In 1996, the core protein-encoding genome segments 7 and 3 were detected in 10 and 7 % of all field-collected pools, respectively, whereas the outer capsid-encoding genome segment 2 was detected in <1 % of the pools (P<0·0001). With the improved RNA-isolation methods that were utilized in 1998, the core-encoding genome segments were detected in 30 and 27 % of field-collected pools, respectively, whereas the outer capsid-encoding segment was detected in only 16 % of the pools (P=0·0308). Observed differences in detection could be due to sensitivity of primer binding or other technical issues, or could reflect a reduction in the abundance of the outer capsid-encoding sequences during the persistent infection of insects.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Overwintering represents a critical time of survival for any arbovirus, as well as its insect vector. Understanding the mechanism by which a virus overwinters is of crucial importance in defining the basic epidemiology of the virus. Whilst a number of overwintering mechanisms have been proposed for BTV, vertical transmission of a persistent, non-cytopathic infection of the invertebrate vector is the most straightforward means for the virus to survive periods of vector inactivity.

The detection of BTV nucleic acid in field-collected Culicoides spp. larval pools supports the hypothesis that the virus can overwinter in vertically infected immature life stages of the vector. Whether the BTV RNA-positive larvae in the pools would have emerged and been able to transmit infectious virus was not determined and cannot be extrapolated directly from these data. Previously, PCR-detectable BTV nucleic acids in bovine blood samples were shown to be epidemiologically irrelevant because they could not infect naïve, blood-feeding Culicoides, ECEs or naïve sheep (MacLachlan et al., 1994; Tabachnick et al., 1996). However, the observed maintenance of BTVs in endemic foci led to the hypothesis of overwintering in vertically infected progeny; these observations are not readily supported by any other overwintering mechanism. Introduction of infected Culicoides from geographically distant areas by wind and overwintering of infected adult insects could be responsible for outbreaks in some locations; however, these mechanisms cannot account for the consistent recurrence of genetically similar BTVs in regions with harsh winters, such as northern Colorado.

The finding of BTV-positive Culicoides crepuscularis is consistent with previous collections from this site (Table 2): although this species has been known as an avian feeder, previous work has described a subspecies or strain commonly caught in live-feeding traps on domestic ruminants (Raich et al., 1997). This suggests that C. crepuscularis might play a minor role in the epidemiology of BTV in northern Colorado. An overwintering mechanism similar to that in the primary North American vector, C. sonorensis, may occur in these insects, or these insects may simply be persistently infected with a virus that is incapable of infecting vertebrate hosts.

Taking the best estimates of the natural prevalence of BTV in Culicoides larvae, 30 % of field-collected pools had detectable core-encoding sequences, whereas only 16 % had detectable outer capsid-encoding sequences. These results suggest that expression of outer capsid-encoding genes may be downregulated (although probably not entirely absent) during persistent infections in the invertebrate host, as these gene products are not required for infection of Culicoides cells. This could lessen the metabolic burden of viral infection in the overwintering larvae. A precedent for this hypothesis exists in WTV, a reoviral pathogen of plants that is transmitted by leafhoppers. WTV can ‘lose’ genome segments during long-term infection of plants without alternative passage in insects (Reddy & Black, 1974, 1977). WTV segments that are necessary for insect-cell infection were not detectable in persistently infected plants by PAGE, which was reported to detect segments that are present in only 0·1 % of the total virions analysed (Reddy & Black, 1977). These studies demonstrated that ‘lost’ segments could be recovered by selecting for wild-type genomes, indicating that all segments were present in a viral subpopulation, even though some were undetectable (Reddy & Black, 1977).

It is also possible that differential sensitivity of the nested RT-PCR assay could account for the observed results. The detection of only one or two genome segments in these assays could be due to relative primer-binding efficiencies. By controlling for intra- and inter-reaction variability, the S7 primer set was shown to be the most sensitive, whereas primer sets L2, L2-2 and L3 were observed to have roughly similar (and lower) sensitivities, followed by a much lower sensitivity for the S10 primer set in non-quantitative tests (data not shown). Additionally, the observed frequencies could be due to sequence variation in genome segment 2 (mutations or deletions at primer-binding sites). This is unlikely, however, given the sequence stability that is seen in segment 2 over time (de Mattos et al., 1994) and the fact that the primer sets were designed to bind to stable regions of genome segment 2 (Ghiasi et al., 1987). The detection of genome segment 2 in less than half (40 %) of the field-collected larval pools where segments 7 and/or 3 were detected suggested an actual downregulation of expression of genome segment 2 during persistent BTV infection of insect cells. The results from the cell-culture lines derived from C. sonorensis embryos supported this finding. In the older cell line (KC, propagated for 16 years), genome segment 2 was undetectable, whereas in the newer cell lines (W3 and W8A, propagated for 9 years), only a fragment of segment 2 was detected, suggesting a selection process to eliminate ‘unnecessary’ genetic material (Table 3).

Genome segment 2 could be downregulated in transcriptional activity or absolute number in the persistently infecting virus population. As total RNA preparations were used, a downregulation of transcriptional activity could decrease the concentration of target molecules to below the threshold for detection. Conversely, a subpopulation of viral variants containing only the inner capsid or ‘invertebrate shell’ of the virus could emerge. As these variants would presumably have a shorter generation time, they would most probably become the predominant members of the population. This would decrease the absolute numbers of genome segment 2 in infected larvae to below the detection threshold of nested RT-PCR.

Predominance of core particles of BTVs in long-term, persistent vector infections could also explain the relatively low rate of isolation of BTVs from insects versus vertebrates. If the vertebrate cell-receptor ligand VP2 (which is encoded by segment 2) is expressed at low levels in the insect, traditional vertebrate cell-based isolation methods (i.e. mammalian cell culture and chicken embryos) would be inefficient until upregulation of expression of all virus genes required for vertebrate cell infection (e.g. following multiple blind passages).

Evidence of genome segment 2 in a subpopulation of BTV RNA-positive larvae indicates that recovery of viruses that are capable of infecting mammalian hosts is possible. Recovery of intact virus could be linked to metabolic or environmental signals that activate virus gene expression and/or change the selection pressures in the insect to favour rescue of a full genome complement, as apparently occurs with WTV. This ‘regenerated’ virus, present in the emerging adult, would then be able to enter the transmission cycle in the following season. Future studies to elucidate the details of the mechanism of virus persistence in vectors are crucial to the relevance of these data.

Whilst the evidence suggests that BTVs overwinter in vector insects in temperate regions, the epidemiological relevance of BTV nucleic acid persistence in field-collected larvae remains to be determined. Further studies are necessary to determine the likelihood, validity and relevance of the proposed overwintering mechanism.


   ACKNOWLEDGEMENTS
 
Cell-culture lines were received from Linda McHolland and Dr Jim Mecham at ABADRL. We thank Jessica Kern and Bailey Rapp for processing and testing of sample pools. We also thank Dr Teresa Raich and Susan Deines, who conducted the initial studies at the BTV-endemic sites in northern Colorado – this work would not have been possible without their findings. This work was supported by USDA Biotechnology Training Grant 96-38420-3039, Agricultural Experiment Station funds from the College of Veterinary Medicine and Biomedical Sciences Research Council, Colorado State University, and the USDA-ARS-ABADRL. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 19 May 2004; accepted 19 October 2004.



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