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VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

van de Water SG, van Gennip RG, Potgieter CA, Wright IM, van Rijn PA - J. Virol. (2015)

Bottom Line: Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics.Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins.African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Overview of mutations in Seg-10 of AHSV4LP. AUG and stop codons in the ORF of NS3 are indicated by * and ×, respectively. Seg-10 RNA sequences are indicated by lines, and putatively translated NS3-related ORFs are represented by boxes. Locations of the late domain (LD) and transmembrane regions 1 and 2 (TM1 and TM2, respectively) are indicated. Deletions are indicated by dashed lines.
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Figure 2: Overview of mutations in Seg-10 of AHSV4LP. AUG and stop codons in the ORF of NS3 are indicated by * and ×, respectively. Seg-10 RNA sequences are indicated by lines, and putatively translated NS3-related ORFs are represented by boxes. Locations of the late domain (LD) and transmembrane regions 1 and 2 (TM1 and TM2, respectively) are indicated. Deletions are indicated by dashed lines.

Mentions: NS3/NS3a of AHSV was studied by the introduction of several mutations into Seg-10. First, the start codon of NS3 (mutAUG1), the start codons of NS3 and NS3a (mutAUG1+2), and three additional in-frame stop codons downstream of these mutated start codons (mutAUG1+2&STOPS) were studied (Fig. 2). All mutations were successfully incorporated into Seg-10 of AHSV4LP. Immunostaining of infected monolayers with NS3-directed MAbs was still positive for mutAUG1, indicating expression of the NS3a protein. Immunostaining was negative for mutAUG1+2 and mutAUG1+2&STOPS (Table 2). Transient translation from DNA-dependent T7 RNA polymerase-driven transcription (see Materials and Methods) is more sensitive, and transient expression of mutAUG1+2 and mutAUG1+2&STOPS showed immunostaining with NS3 MAbs, which suggested weak NS3-related expression by viruses with the respective mutated Seg-10. To ensure the knockout of any NS3-related translation, mutAUG1+2 was combined with an out-of-frame deletion of 32 bp (positions 92 to 123) (delLD) encompassing the putative late domain (LD) of NS3 (49), with an in-frame deletion of 315 bp (positions 139 to 453) encompassing the putative transmembrane region 1 (delTM1), or with an out-of-frame deletion of transmembrane region 2 of 145 bp (positions 454 to 598) (delTM2) (Fig. 2). All deletion variants of AHSV4LP were successfully rescued. Cells infected with these mutants tested negative by IPMAs with NS3 MAbs (Table 2). However, transient NS3-related expression for delLD was positive, and a derivative of delTM2 with wild-type start codons of NS3 and NS3a showed weak immunostaining with NS3 MAbs. Finally, mutAUG1+2 was combined with delLD, delTM1, and delTM2 (Fig. 2). This deletion in Seg-10 of 266 bp (delLD&delTM1+2) was also stably incorporated into AHSV4LP, and immunostaining with NS3 MAbs was negative for both infected monolayers and NS3-related transient expression (Table 2). We conclude that NS3/NS3a of AHSV is not essential for virus replication in vitro, but Seg-10 RNA is essential, as virus rescue without this RNA failed. Apparently, Seg-10 of delLD&delTM1+2 contains RNA sequences sufficient but essential for in vitro replication of AHSV.


VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

van de Water SG, van Gennip RG, Potgieter CA, Wright IM, van Rijn PA - J. Virol. (2015)

Overview of mutations in Seg-10 of AHSV4LP. AUG and stop codons in the ORF of NS3 are indicated by * and ×, respectively. Seg-10 RNA sequences are indicated by lines, and putatively translated NS3-related ORFs are represented by boxes. Locations of the late domain (LD) and transmembrane regions 1 and 2 (TM1 and TM2, respectively) are indicated. Deletions are indicated by dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524073&req=5

Figure 2: Overview of mutations in Seg-10 of AHSV4LP. AUG and stop codons in the ORF of NS3 are indicated by * and ×, respectively. Seg-10 RNA sequences are indicated by lines, and putatively translated NS3-related ORFs are represented by boxes. Locations of the late domain (LD) and transmembrane regions 1 and 2 (TM1 and TM2, respectively) are indicated. Deletions are indicated by dashed lines.
Mentions: NS3/NS3a of AHSV was studied by the introduction of several mutations into Seg-10. First, the start codon of NS3 (mutAUG1), the start codons of NS3 and NS3a (mutAUG1+2), and three additional in-frame stop codons downstream of these mutated start codons (mutAUG1+2&STOPS) were studied (Fig. 2). All mutations were successfully incorporated into Seg-10 of AHSV4LP. Immunostaining of infected monolayers with NS3-directed MAbs was still positive for mutAUG1, indicating expression of the NS3a protein. Immunostaining was negative for mutAUG1+2 and mutAUG1+2&STOPS (Table 2). Transient translation from DNA-dependent T7 RNA polymerase-driven transcription (see Materials and Methods) is more sensitive, and transient expression of mutAUG1+2 and mutAUG1+2&STOPS showed immunostaining with NS3 MAbs, which suggested weak NS3-related expression by viruses with the respective mutated Seg-10. To ensure the knockout of any NS3-related translation, mutAUG1+2 was combined with an out-of-frame deletion of 32 bp (positions 92 to 123) (delLD) encompassing the putative late domain (LD) of NS3 (49), with an in-frame deletion of 315 bp (positions 139 to 453) encompassing the putative transmembrane region 1 (delTM1), or with an out-of-frame deletion of transmembrane region 2 of 145 bp (positions 454 to 598) (delTM2) (Fig. 2). All deletion variants of AHSV4LP were successfully rescued. Cells infected with these mutants tested negative by IPMAs with NS3 MAbs (Table 2). However, transient NS3-related expression for delLD was positive, and a derivative of delTM2 with wild-type start codons of NS3 and NS3a showed weak immunostaining with NS3 MAbs. Finally, mutAUG1+2 was combined with delLD, delTM1, and delTM2 (Fig. 2). This deletion in Seg-10 of 266 bp (delLD&delTM1+2) was also stably incorporated into AHSV4LP, and immunostaining with NS3 MAbs was negative for both infected monolayers and NS3-related transient expression (Table 2). We conclude that NS3/NS3a of AHSV is not essential for virus replication in vitro, but Seg-10 RNA is essential, as virus rescue without this RNA failed. Apparently, Seg-10 of delLD&delTM1+2 contains RNA sequences sufficient but essential for in vitro replication of AHSV.

Bottom Line: Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics.Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins.African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The Netherlands.

No MeSH data available.


Related in: MedlinePlus