Genetic diversification of field strains of bluetongue virus.

The considerable genetic heterogeneity of field strains of bluetongue virus (BTV) occurs as a consequence of both genetic drift and shift. Comparison of strains of BTV from the People's Republic of China and North America showed that viruses from the two regions were readily distinguished by sequence analysis of their S10 (which encodes the NS3/3A proteins) but not their L2 (which encodes the serotype-specific VP2 protein) genes. Subsequent laboratory studies showed that individual BTV genes evolve through a combination of genetic drift coupled with founder effect in vector insects. This model explains the diversification of BTV gene segments within each region, and can be extrapolated to explain diversification of BTV into distinct topotypes worldwide.

Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep.

The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)-nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111-222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTV as determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RT-nested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV.

Occurrence of genetic drift and founder effect during quasispecies evolution of the VP2 and NS3/NS3A genes of bluetongue virus upon passage between sheep, cattle, and Culicoides sonorensis.

Bluetongue virus (BTV) is the cause of an insect-transmitted virus infection of ruminants that occurs throughout much of the world. Individual gene segments differ between field strains of BTV; thus, we hypothesized that key viral genes undergo genetic drift during alternating passage of BTV in its ruminant and insect hosts. To test this hypothesis, variation in the consensus sequence and quasispecies heterogeneity of the VP2 and NS3/NS3A genes of a plaque-purified strain of BTV serotype 10 was determined during alternating infection of vector Culicoides sonorensis and a sheep and calf. Consensus sequences were determined after reverse transcriptase-nested PCR amplification of viral RNA directly from ruminant blood and homogenized insects, and quasispecies heterogeneity was determined by the sequencing of clones derived from directly amplified viral RNA. Comparison of these sequences to those of the original BTV inoculum used to initiate the cycle of BTV infection demonstrated, for the first time, that individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Furthermore, a unique viral variant was randomly ingested by C. sonorensis insects that fed on a sheep with low-titer viremia, thereby fixing a novel genotype by founder effect. Thus, we conclude that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV.

Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype.

Bluetongue virus (BTV) infection of ruminants is endemic throughout tropical and subtropical regions of the world. The S7 gene segments of prototype Chinese strains of BTV serotypes 1, 2, 3, 4, 12, 15, and 16 were sequenced and compared to the same genes of prototype strains of BTV from the US, Australia, and South Africa. The S7 genes and predicted VP7 proteins of the Chinese viruses were relatively conserved, with the notable exception of serotype 15. Furthermore, phylogenetic analysis of the S7 genes did not predict geographic origin of the various strains of BTV.

Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies.

Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.

Sequence comparison of the L2 and S10 genes of bluetongue viruses from the United States and the People's Republic of China.

Bluetongue virus (BTV) infection of ruminants is endemic throughout much of the US and China. The S10 and a portion of the L2 gene segments of Chinese prototype strains of BTV serotypes 1, 2, 3, 4, 12, 15, and 16 were sequenced and compared to the same genes of prototype and field strains of BTV from the US. Phylogenetic analysis of the S10 gene segregated the Chinese viruses into a monophyletic group distinct from the US viruses, whereas similar analysis of the L2 gene segregated strains of BTV according to serotype, regardless of geographic origin.

Identification of seven serotypes of bluetongue virus from the People's Republic of China.

Seven serotypes (1, 2, 3, 4, 12, 15 and 16) of bluetongue virus were isolated from the blood of sheep and cattle in the People's Republic of China between 1986 and 1996. Six of these viruses were isolated in Yunnan province. The sheep from which serotypes 1 and 16 were isolated showed obvious signs of bluetongue disease, whereas the cattle from which serotypes 2, 3, 4, 12 and 15 were isolated were clinically normal. Phylogenetic analyses of these viruses indicate that they are more closely related to one another, and to an Australian strain of serotype 1, than they are to prototype strains of bluetongue virus serotypes 2, 10, 11, 13 and 17 from the USA.