INTRODUCTION

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RNA virus replication is characterized by high mutation rates (10⁵ to 10³ misincorporations per nucleotide copied), short generation times, and high progeny yields (15). In addition to mutations introduced by their error-prone polymerases, RNA viruses also generate genomic variation by homologous and nonhomologous recombination and reassortment in those viruses with a segmented genome. Thus, RNA viruses exist as a heterogeneous population of closely related variants characterized by one or several dominant master nucleotide genome sequence(s) (quasispecies) (14, 15, 17, 35). The quasispecies nature of RNA viruses confers significant adaptive potential through selection of mutants with the highest fitness in a new environment, which allows for rapid evolution (40). Despite this potential advantage, arthropod-transmitted RNA viruses often evolve more slowly than nonarthropod-transmitted RNA viruses, likely because of restrictive pressures imposed during alternating passage in their vertebrate and invertebrate hosts (52, 60).

We investigated the evolution of bluetongue virus (BTV), the prototype member of the genus Orbivirus in the family Reoviridae (19). BTV is the causative agent of bluetongue, an insect-transmitted disease of sheep and some species of wild ruminants (37). In contrast, BTV infection of cattle is typically asymptomatic, and viremia is prolonged because of an interaction of BTV with bovine erythrocytes in which virus particles persist in cell membrane invaginations (6, 7, 38). The vectors of BTV are species of hematophagous midges in the genus Culicoides, and BTV infection occurs throughout tropical and temperate regions of the world (22). Culicoides sonorensis (formerly Culicoides variipennis) is the principal vector of BTV in North America (28, 54). Female Culicoides insects become persistently infected with BTV and can transmit the virus to susceptible ruminants after an extrinsic incubation period (EIP) of 10 to 14 days (20, 36, 41).

The BTV genome consists of 10 distinct double-stranded RNA genome segments that encode seven structural (VP1 to VP7) and four nonstructural (NS1, NS2, NS3, and NS3A) proteins (47, 48, 57). Genomic double-stranded RNA is surrounded in the virion by a double-layered protein capsid (56). The outer capsid consists of VP2 and VP5, encoded by genome segments 2 and 5, respectively (49). VP2 is responsible for adsorption and entry of BTV into mammalian cells, hemagglutination, neutralization, and serotype specificity, and multimers (dimers and/or trimers) of VP2 are layered upon a VP5 scaffold (25, 30). The VP2 and VP5 genes are especially variable among different serotypes and strains of BTV (5, 9, 11, 27, 49). Nonstructural proteins NS3 and NS3A are translated from genome segment 10 mRNA via two in-frame initiation codons (32). The NS3 and NS3A proteins localize to the cell plasma and intracellular smooth-surfaced vesicle membranes of BTV-infected cells and colocalize with extruding virus particles at the cell surface (33). NS3 and NS3A may be responsible for egress of virus particles from both mammalian and insect cells (2). The NS3/NS3A gene is relatively conserved among different serotypes and strains of BTV (5, 32, 49).

There is marked genetic variation of viruses within the BTV serogroup, with some 24 distinct serotypes and considerable strain variation within each serotype (4, 5, 9-11, 23, 24, 44, 46, 64). Although reassortment is central to the emergence of novel BTV variants (10, 43, 50, 51), the evolution of the BTV quasispecies that occurs as a result of genetic drift of individual genome segments during the natural cycle of BTV infection has yet to be characterized. We hypothesized that key viral genes that encode proteins involved in virus entry and egress (VP2 and NS3/NS3A genes, respectively) undergo genetic drift during sequential passage of BTV through its ruminant hosts and insect vector. Based on the data obtained in this study, we conclude that individual BTV genome segments evolve independently of one another by genetic drift in a host-specific fashion. In addition, we found that a unique viral variant was randomly ingested by C. sonorensis that fed on a sheep with low-titer viremia, thereby fixing a novel genotype (founder effect).

	MATERIALS AND METHODS

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Virus. A strain of BTV serotype 10 (BTV FI10O90Z) that originally was isolated from the blood of a sheep in California in 1990 was propagated as previously described (11, 27). Briefly, the virus was passaged once in embryonated chicken eggs, followed by plaque purification (three times) in Vero cells. Plaque-purified virus was passaged twice in baby hamster kidney (BHK-21) cells, and the virus titer was determined by microtitration assay (45). This virus stock was used to initiate the cycle of BTV infection depicted in Fig. 1.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Experimental transmission of BTV between sheep, cattle, and C. sonorensis. Virus RNA was directly amplified, cloned, and sequenced from the various hosts.

Experimental transmission cycle of BTV infection. The cycle of experimental BTV transmission between sheep, cattle, and C. sonorensis is depicted in Fig. 1. Sheep and calves that were seronegative to BTV as determined by competitive enzyme-linked immunosorbent assay (cELISA) (Blueplate Special; DiagXotics) were obtained from northwestern California, a region that is free of BTV infection. A laboratory colony of C. sonorensis Wirth and Jones insects was established, using standard rearing methods (41), from a southern California field population that was susceptible to BTV infection (21). Larvae were reared to adults, and 1- to 4-day-old adult flies were orally infected with BTV by being fed on defibrinated sheep blood spiked with BTV FI10O90Z at a titer of 10^6.7 50% tissue culture infective doses (TCID₅₀) per ml (41). Insects (mixed females and males) fed on the infected blood through a paraffin membrane in a temperature-controlled feeding apparatus (31). Control insects were fed uninfected sheep blood. Engorged female C. sonorensis insects (plus some males) were held at 27°C for 10 days, and survivors (approximately 50) were used to infect a susceptible sheep by feeding them through a nylon mesh stocking affixed to a holding cage for 1 h on a shaved area of the back of the sheep. Female insects that engorged a second blood meal (n = 15; Table 1) were pooled and homogenized in grinding buffer (10 mM Tris-HCl, 10 mM NaCl, and 10 mM Na₂EDTA [pH 8]; 5 flies/100 µl) and stored at 70°C. Reverse transcriptase (RT)-nested PCR and virus isolation were used to confirm BTV infection of the insects.

Virus isolation. Virus isolation was performed on ruminant blood and homogenized insects. Blood samples were collected and processed as previously described (27). Briefly, confluent monolayers of BHK-21 cells in 24-well plates were inoculated with serial 10-fold dilutions of lysed, washed blood cells and incubated at 37°C for 10 days. Virus isolation was also done on homogenates of 15 to 30 pooled insects that were inoculated onto confluent monolayers of BHK-21 cells maintained in antibiotic medium (2.5 µg of amphotericin B/ml, 200 µg each of penicillin and streptomycin/ml, and 100 µg each of neomycin and gentamicin sulfate/ml) and incubated at 37°C for 8 days. Cultures that did not exhibit cytopathic effect were passaged a second time. Cytopathic agents isolated from ruminant blood and/or insect homogenates were confirmed as BTV by immunofluorescent staining of infected monolayers of BHK-21 cells grown on chamber slides using a fluorescein isothiocyanate-labeled monoclonal antibody to BTV core protein VP7 (61).

RNA extraction, RT-nested PCR amplification, and direct sequencing of BTV VP2 and NS3/NS3A genes in ruminant blood and insects. Total RNA was isolated directly from ruminant blood using RNA STAT-50 LS (Tel-Test, Inc., Friendswood, Tex.) according to the manufacturer's protocol. Total RNA was extracted from homogenized insects in grinding buffer as previously described (62). Total RNA isolated from ruminant blood and homogenized insects was screened for the presence of BTV RNA by amplification of a portion of the NS1 gene using a previously described RT-nested PCR protocol (62). Portions of the VP2 and NS3/NS3A genes were amplified directly from samples that were determined to contain BTV RNA by NS1 gene-specific RT-nested PCR. Viral RNA was reverse transcribed and nested PCR amplified using Superscript II reverse transcriptase (Gibco BRL) and Pfu DNA polymerase (Stratagene), respectively, and gene-specific oligonucleotide primers (see below). Pfu DNA polymerase was used to minimize artifactual substitutions (53). The first-strand cDNA was purified with a Qiaquick PCR purification kit after RNase digestion (Qiagen). VP2 gene cDNA was subjected to PCR (30 cycles) using primers that amplified nt 434 to 1653 (11). This first PCR product was used to seed the nested PCR (30 cycles), along with primers that amplified nucleotides (nt) 627 to 1548, resulting in a 922-bp portion of the VP2 gene that includes regions encoding the major neutralization determinants of BTV (12, 13). NS3/NS3A gene cDNA was synthesized and amplified with primers that amplified the entire gene (nt 1 to 822), which then was used as the template for the nested PCR with primers that amplified a 775-bp region [nt 25 to 799; numbering as described for the ATCC strain of BTV serotype 10 (44)]. Twelve separate RT-nested PCRs were done for each RNA sample, and the reactions were pooled, concentrated (Centricon-30; Amicon), and agarose gel purified using a commercial kit (Qiaquick; Qiagen). Purified RT-nested PCR products were directly sequenced using the described VP2 and NS3/NS3A gene nested PCR primers and internal gene-specific primers. Sequences obtained directly from RT-nested PCR products were designated consensus sequences. The pooling of multiple RT-nested PCR products was done to ensure the sequence data most accurately represented the true consensus sequence of the population and to control for artifactual substitutions.

cDNA cloning and sequencing. VP2 and NS3/NS3A gene RT-nested PCR products amplified from ruminant blood and/or insects were cloned into the pCR 2.1-TOPO cloning vector according to the manufacturer's protocol (Invitrogen). The purification, screening, and sequencing of plasmid DNA were done as previously described (26). Sense and antisense strands were each sequenced with plasmid-specific and internal sequence-specific primers. Individual sequences of recombinant 2.1-TOPO vectors, representing gene sequences of individual members of the BTV quasispecies population, were designated clones. Twenty recombinant clones from each VP2 or NS3/NS3A gene RT-nested PCR product were sequenced unless otherwise stated.

Determination of mutations introduced by the methodology. To quantitate the frequency of any mutations introduced by the reverse transcription and nested PCR amplification processes, RNA transcripts derived from a single BTV FI10O90Z VP2 gene clone were RT-nested PCR amplified, and the cDNA products were cloned and analyzed for spurious mutations. Briefly, transcription of the partial VP2 gene was done using T7 RNA polymerase as previously described (1). Transcribed control RNA was agarose gel purified using a commercial kit (Qiaquick; Qiagen) and RT-nested PCR amplified. RT-nested PCR products were cloned, and 20 clones were sequenced and compared to the original VP2 gene clone. Direct nested PCR of the control RNA without prior reverse transcription failed to generate product, indicating that the control RNA preparation was not contaminated with residual plasmid DNA. T7 RNA polymerase infidelity was considered negligible, as the average error frequency of this enzyme is estimated at 5.0 × 10⁵ (29).

Sequence analysis. Computer analyses of DNA sequences were performed using the MacDNASIS Pro version 3.5 (Hitachi) and Sequencher 3.1.1 (Gene Codes Corp.) programs.

Nucleotide sequence accession numbers. The consensus sequences of BTV FI10O90Z NS3/NS3A and VP2 genes were submitted to GenBank and assigned accession numbers AY028210 and AY028211, respectively. The accession number for the entire VP2 gene of BTV serotype 10 isolate 10O90Z is U06785 (11).

	RESULTS

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Sequential BTV infection of a sheep, a calf, and C. sonorensis. BTV was successfully transmitted to a sheep, and then from the sheep to a calf by the bites of laboratory-raised C. sonorensis insects (Fig. 1). BTV infection of sheep, cattle, and insects was determined by virus isolation and RT-nested PCR (Table 1). Viremia persisted in the infected sheep for 2 to 9 days after exposure to infected insects as determined by virus isolation, and virus was not isolated at either 21 or 35 days p.i. BTV nucleic acid was detected in the sheep's blood by RT-nested PCR from 8 to 35 days p.i., consistent with the prolonged presence of BTV RNA in the absence of infectious virus that has previously been demonstrated in ruminants (34, 38, 55). The sheep seroconverted to BTV at 21 days p.i., as determined by cELISA. BTV infection of the insects used to infect the sheep, as well as those that fed on it at 8 and 21 days p.i., was confirmed by virus isolation and/or RT-nested PCR (Table 1). Viremia persisted in the infected calf from 7 to 19 days p.i., and BTV was not isolated at 35 days p.i. BTV nucleic acid was detected by RT-nested PCR in blood collected from the calf from 7 to 35 days p.i. The calf seroconverted to BTV at 19 days after exposure to the insects that had previously fed on the infected sheep. Insects fed on the calf at 7, 19, and 35 days p.i. did not contain BTV as determined by both virus isolation and RT-nested PCR.

Genetic stability of BTV during sequential transmission between a sheep, calf, and C. sonorensis The consensus sequence of portions of the VP2 and NS3/NS3A genes was determined from RNA extracted directly from the original BTV FI10O90Z inoculum, from insects used to transmit infection to the sheep, from blood collected from the infected sheep at 8 and 21 days p.i., from insects that fed on the sheep at 8 and 21 days p.i., and from blood collected from the infected calf at 7 and 19 days p.i. Portions of the VP2 (922 bp) and NS3/NS3A (775 bp) genes were directly RT-nested PCR amplified from these samples, sequenced, and compared to the consensus sequence of the original plaque-purified BTV FI10O90Z inoculum. With the notable exception of the insects that fed on the sheep at 8 days p.i., there were no nucleotide substitutions in the consensus sequences, regardless of the host or time of sample collection. Importantly, however, a single synonymous transition (A to G) at nt 1102 in the VP2 gene was present in virus contained in a pool of 20 insects that fed on the sheep at 8 days p.i. The consensus sequences of the VP2 gene contained in a second pool of insects that fed on the sheep at 8 days p.i. (n = 5) and from blood of the infected calf at 7 and 19 days p.i. were identical to that of the parental strain, BTV FI10O90Z. Thus, the variant virus in the first insect pool was not transmitted to the calf, or it was transmitted but replicated more slowly than other variants and hence was not detected.

Microheterogeneity of the BTV quasispecies population in ruminants and insects. To determine the type and number of mutations acquired in BTV FI10O90Z during transmission between ruminant and insect hosts, portions of the VP2 and NS3/NS3A genes were RT-nested PCR amplified from RNA extracts and were cloned, and 19 to 21 clones were sequenced for each sample. Clones were obtained directly from the BTV FI10O90Z inoculum, from insects used to infect the sheep, from sheep blood at 8 and 21 days p.i., from insects that fed on the sheep at 8 and 21 days p.i., and from calf blood at 7 and 19 days p.i. VP2 and NS3/NS3A gene clones were compared to the consensus sequences of the VP2 and NS3/NS3A genes of the original BTV FI10O90Z inoculum (Table 2). Of all nucleotide substitutions in the VP2 gene clones, 88% were transitions and 28% were nonsynonymous. A single nucleotide deletion in one VP2 gene clone obtained from sheep blood at 21 days p.i. and a nonsynonymous change in one clone obtained from calf blood at 19 days p.i. introduced premature stop codons at amino acid positions 297 and 277, respectively. The total number of nucleotide substitutions in the various VP2 gene clones ranged from 0 in virus present at 7 days p.i. of the calf to 22 for virus present in insects fed on the sheep at 8 days p.i. (20 clones sequenced for each). A temporal increase in the total number of mutations in the VP2 gene clones occurred in the course of infection of the sheep. Specifically, three mutations were present among the 21 VP2 gene clones directly amplified from blood at 8 days p.i. of the sheep, whereas eight mutations were present among the 20 VP2 gene clones at 21 days p.i. Furthermore, this trend toward an accumulation of mutations also was accompanied by an increase in the proportion of clones with the A-to-G mutation at nt 1102 of the VP2 gene. Two clones had this mutation at 8 days p.i. of the sheep, whereas four clones had this change at 21 days p.i.

Transmission of minor variants in the viral quasispecies between sheep and insects and mutational bias. Insects that fed on the sheep at 8 days p.i. clearly fixed a VP2 gene mutation. Specifically, an A-to-G mutation at nt 1102 of the VP2 gene was present in all 20 of the VP2 gene clones derived from this insect pool (Table 2). The variant with this mutation was selected from the quasispecies population present in sheep blood at 8 days p.i. and then was exclusively amplified in one or more insects in this pool. This same mutation was not present in a second pool of insects that fed on the sheep at the same time (n = 5) nor in virus present in insects that fed on the sheep at 21 days p.i. Thus, genetic changes in the VP2 gene that arose as a minor variant of the VP2 gene quasispecies in the sheep were fixed in one group of insects. Similarly, a single clone of the NS3/NS3A gene amplified from insects fed on the sheep at 21 days p.i. had a G-to-T mutation at nt 78, and a single clone from the sheep blood collected at the time of insect feeding had the identical mutation. A transition (G to A) at nt 1245 of the VP2 gene was present in one clone amplified from insects that fed on the sheep at 8 days p.i. and in two clones amplified from insects that fed on the sheep at 21 days p.i. This mutation was not present in clones obtained from sheep blood at 8 and 21 days p.i., suggesting that either the mutation arose in the insects or occurred only at a very low level in the blood of the viremic sheep and was selectively maintained in the insects.

	DISCUSSION

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At least 24 serotypes of BTV exist worldwide (22), and there is extensive genetic variation among field and laboratory strains of the virus (5, 9-11, 23, 24, 44, 46, 64). Reassortment of gene segments has been repeatedly demonstrated among strains of BTV (10, 27, 43, 50, 51); however, the additional contribution of genetic drift of individual gene segments to the evolution of BTV has not been investigated. We evaluated the molecular evolution of BTV by direct RT-nested PCR amplification, cloning, and sequencing of VP2 and NS3/NS3A genes at key points during transmission of the virus between sheep, cattle, and vector C. sonorensis, an experimental transmission scheme intended to mimic natural BTV infection. The VP2 and NS3/NS3A gene consensus sequences remained stable throughout the transmission cycle, with the notable exception of the founder effect event in insects that fed on the sheep at 8 days p.i., consistent with a central role for purifying selection in BTV evolution. This occurred despite the fact that the region of the VP2 gene analyzed encodes the major neutralization determinants of BTV (12) and thus presumably would be subjected to immune selection in ruminants. The data clearly confirm that variation does occur through genetic drift of individual BTV genome segments, generating mutant spectra in sheep, cattle, and C. sonorensis. Furthermore, our data prove that vector insects can randomly acquire and amplify minor variants from the quasispecies virus populations that occur in the blood of BTV-infected ruminants (founder effect).

Arthropod-borne RNA viruses evolve more slowly than single-host RNA viruses, as selective pressures encountered by arboviruses in their vertebrate and insect hosts are predicted to minimize both genetic drift and the occurrence of founder effect (42, 52, 58-60). The low mutation frequencies of the VP2 and NS3/NS3A genes of BTV during transmission between ruminants and insects, however, did not preclude quasispecies evolution, as both genes acquired both random and biased mutations while the consensus sequence was conserved. A variety of variants characterized the BTV quasispecies in insects and ruminants, including point mutations, a deletion mutant, and a termination mutant (Table 2). Random sampling during genetic bottleneck allowed specific amplification of a variant with an A-to-G mutation at nt 1102 of the VP2 gene in the group of insects that fed on the sheep at 8 days p.i. Clearly, BTV exhibits characteristics of quasispecies evolution during its natural cycle of transmission, because evolution of the RNA virus quasispecies is not simply a consequence of an accumulation of mutations as the virus replicates. Rather, quasispecies evolution occurs as disequilibrium of the population of multiple variants in response to variations in population size and environmental selection (3).

Founder effect, or genetic bottlenecking, promotes rapid genotypic and phenotypic changes in RNA viruses (8, 16, 18). Founder effect can overcome selective pressures and promote evolutionary change when populations are small by allowing random sampling accidents that result in the fixation of specific genotypes to occur (60, 63). Titers of BTV were low in the sheep and calf at the times of insect feeding (mean titers, 10^3.7 and 10^1.9 TCID₅₀/ml of blood, respectively). C. sonorensis insects imbibe approximately 10⁴ ml at each blood meal (39); thus, the insects that fed on the viremic sheep and calf each ingested approximately 0.5 and 0.008 TCID₅₀ of BTV, respectively. These are ideal conditions for founder effect, as evidenced by the specific amplification in one pool of insects of a variant strain of BTV with an A-to-G mutation at nt 1102 of the VP2 gene. Fixation of the synonymous A-to-G mutation at nt 1102 of the VP2 gene in the insects that fed on the sheep at 8 days p.i. demonstrates either that random drift played a significant role in the evolution of BTV FI10O90Z or that the VP2 RNA itself was subjected to positive selection. The mechanism of founder effect affords a highly effective means for genetic diversification of individual BTV genome segments and potentially explains the considerable sequence divergence that occurs among field strains of BTV in nature, including viruses that cocirculate in the same region (9-11, 27, 44).

In summary, sequence analysis of the VP2 and NS3/NS3A genes during sequential transmission between sheep, cattle, and vector C. sonorensis confirmed that BTV exists as a quasispecies in both its ruminant and insect hosts. Despite conservation of the consensus sequence of the VP2 and NS3/NS3A genes during transmission, one group of insects randomly selected and amplified a minor viral variant within the quasispecies population of BTV in the blood of the infected sheep and thereby fixed a mutant genotype that changed the VP2 gene consensus sequence (founder effect). It is likely that BTV generates genetic strain diversity and overcomes evolutionary constraints encountered during sequential replication in its ruminant and insect hosts through a combination of reassortment of gene segments and utilization of the process of founder effect that was demonstrated for the first time in this study.