Intragenic Recombination as a Mechanism of Genetic Diversity in Bluetongue Virus

Bluetongue (BT), caused by Bluetongue virus (BTV), is an economically important disease affecting sheep, deer, cattle, and goats. Since 1998, a series of BT outbreaks have spread across much of southern and central Europe. To study why the epidemiology of the virus happens to change, it is important to fully know the mechanisms resulting in its genetic diversity. Gene mutation and segment reassortment have been considered as the key forces driving the evolution of BTV. However, it is still unknown whether intragenic recombination can occur and contribute to the process in the virus. We present here several BTV groups containing mosaic genes to reveal that intragenic recombination can take place between the virus strains and play a potential role in bringing novel BTV lineages.Bluetongue (BT) is an economically significant disease that seriously threatens sheep, some species of deer, and to a lesser extent cattle and goats. As a vector-borne viral disease of ruminants, BT is endemic in tropical and subtropical countries (46). However, a series of BT outbreaks have spread across much of southern and central Europe since 1998 (29). Thus, it is of great importance to fully understand the molecular basis driving the change of its epidemiology so as to prevent or limit future BT pandemics.Bluetongue virus (BTV), the pathogen of BT, belongs to the Orbivirus genus of the Reoviridae family (46). The virus has a segmented double-stranded RNA (dsRNA) genome that is packaged in a nonenveloped, icosahedral particle (46). Its 10 dsRNA segments encode 11 proteins, VP1 to VP7 (encoded by segments 1, 2, 3, 4, 6, 9, and 7, respectively), NS1 to SN3 (encoded by segments 5, 8, and 10, respectively), and NS3A (encoded by segment 10) (46). Two structural proteins, VP2 and VP5, form the outer layer of the virion particle and are responsible for cell attachment and virus entry (18, 31, 32), neutralizing epitope (14, 21), and virus virulence (36). Both of them are highly variable and generate 24 serotypes of the virus (44). The inner layers contain VP1, VP3, VP4, VP6, and VP7, and form the “core” of the BTV capsid. VP1 and VP6 are involved in RNA replication as the RNA-dependent RNA polymerase (54) and helicase/NTPase, respectively (49). VP7 forms the surface of the core and functions during the entry of the core into insect cells (44) and also can react with “core neutralizing” antibodies as a major serogroup-specific antigen (32, 44). These core proteins and two nonstructural proteins, NS1 and NS2, are thought to be relatively conservative, so that antigenic cross-reaction can take place between different BTV strains and serotypes, whereas NS3/N3a is more variable than the other nonstructural or core proteins (46).The genetic diversity and variation in sequences of different BTV genome segments were initially identified by RNA oligonucleotide fingerprint analysis of BTV field samples (47). Until now, reassortment and dynamic gene mutation, regarded as the key factors responsible for the genetic diversity of BTV, have been studied in details (46). The two mechanisms can result in both genetic drift and genetic shift and contribute to BTV evolution (47). It has been revealed that high-frequency genome segment reassortment occurs readily between different BTV serotypes (16). Thus, segment reassortment is an important factor in generation of genetic diversity in orbivirus populations in nature (45). In addition, it has been shown that homologous recombination can also play a role in the genetic diversity and evolution of some RNA viruses (24, 33) and bring on virulent variants of these viruses at last (8, 56). Although homologous recombination has been observed in rotavirus, a member of the Reoviridae (39, 40), it is still unknown whether the intragenic recombination can occur and play a role in the generation of genetic diversity in orbivirus populations.To determine whether homologous recombination shaped the evolution of BTV and to provide some insights into the recombination itself in the virus, we analyzed roughly 690 complete segments of BTV deposited in GenBank to see whether some of them underwent intragenic recombination event. Several BTV groups isolated at different time points and in different countries were found containing the same (or similar) mosaic segments, demonstrating that intragenic recombination had occurred in the field and that these viruses with mosaic segments had become prevailing strains. That is, intragenic recombination can play a potential role in generating genetic diversity of BTV and exert its influence on the change of BTV epidemiology.