Identification of the Genome Segments of Bluetongue Virus Type 26/Type 1 Reassortants Influencing Horizontal Transmission in a Mouse Model

Abstract

Bluetongue virus serotypes 1 to 24 are transmitted primarily by infected Culicoides midges, in which they also replicate. However, “atypical” BTV serotypes (BTV-25, -26, -27 and -28) have recently been identified that do not infect and replicate in adult Culicoides, or a Culicoides derived cell line (KC cells). These atypical viruses are transmitted horizontally by direct contact between infected and susceptible hosts (primarily small ruminants) causing only mild clinical signs, although the exact transmission mechanisms involved have yet to be determined. We used reverse genetics to generate a strain of BTV-1 (BTV-1 RGC7) which is less virulent, infecting IFNAR(−/−) mice without killing them. Reassortant viruses were also engineered, using the BTV-1 RGC7 genetic backbone, containing individual genome segments derived from BTV-26. These reassortant viruses were used to explore the genetic control of horizontal transmission (HT) in the IFNAR(−/−) mouse model. Previous studies showed that genome segments 1, 2 and 3 restrict infection of Culicoides cells, along with a minor role for segment 7. The current study demonstrates that genome segments 2, 5 and 10 of BTV-26 (coding for proteins VP2, NS1 and NS3/NS3a/NS5, respectively) are individually sufficient to promote HT.

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells

“…BTV-1RG C7 is a clone of BTV serotype 1, previously derived by reverse genetics based on the genome sequence of the BTV-1 reference strain [ 67 ]. BTV-8RSArrrr/08 was obtained from the orbivirus reference collection at the Pirbright Institute, UK.…”

“…The rescue of BTV-1 in BSR cells from synthetic ssRNAs has previously been described [ 67 ]. A BTV-1RG C7 genetic backbone was used to engineer viruses with deletion mutations for NS4, and NS5 and a double deletion mutant for NS4 and NS5.…”

“…A BTV-1RG C7 genetic backbone was used to engineer viruses with deletion mutations for NS4, and NS5 and a double deletion mutant for NS4 and NS5. The plasmid used to generate BTV-1ΔNS5 (containing NS5 deletion mutations) was prepared by PCR amplification of the plasmid pGEX-4T-2 (which contains a full-length cDNA copy of BTV-1 Seg-10 [ 67 ]). Primers containing deoxy-uracil nucleotides replacing the deoxy-thymidine nucleotides ( Table S2 ) were designed to mutate positions 109 (T to C) and 136 (T to C), thus changing both in-frame ATG codons to ACG ( Figure S24 ).…”

“…Single (BTV-1ΔNS4 or BTV-1ΔNS5) and double (BTV-1ΔNS4/ΔNS5) deletion mutants were rescued by transfecting BSR cells with the relevant ssRNAs, as previously described [ 67 , 79 ].…”

Redirecting Imipramine against Bluetongue Virus Infection: Insights from a Genome-wide Haploid Screening Study

“…In general, the global distribution of BTV links closely to the favorable ecosystems of different midge species in temperate or tropical regions [ 4 ]. Currently, 36 distinct BTV serotypes have been identified in different ruminants [ 5 , 6 , 7 , 8 ]. Sheep are more vulnerable to BTV infection with high mortality rates, whereas cattle and goats are less affected.…”