Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A:Impacts on rescue of sequence-modifying viruses

Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.

Construction of Full-length Infectious Clone for Encephalomyocarditis Virus BJC3 and Identification of the Rescued Virus

The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.

Susceptibility and tissue specificity of Spodoptera frugiperda to Junonia coenia densovirus

The fall armyworm,Spodoptera frugiperda,which destroys many economic crops such as rice and maize,has recently invaded China.Insect viruses as biological control agents play important roles in killing pests.One potential viral insecticide is the environmentally highly infective and virulent densovirus.We successfully rescued Junonia coenia densovirus(JcDV)using its infectious clone in different insect cell lines and larvae of three insect species.Results showed that the lysate of cultured insect cells transfected by the JcDV infectious clone killed the 2 nd instar S.frugiperda.The LD50 of homogenate from JcDV-infected Spodoptera litura to the 2 nd instar S.frugiperda(1.76×10^(8)viral genome copies per larva during 10 d post infection)was higher than that of the 2 nd instar S.litura(7.39×10^(7)Jc DV genome copies)or Helicoverpa armigera larvae(9.71×10^(7)JcDV genome copies).The LT50 of the S.litura homogenate(2.60×10^(9)viral genome copies each larva)to the 2 nd instar S.frugiperda was 6.96 d,longer than that of the S.litura(6.18 d)or the 2 nd instar H.armigera(5.94 d).JcDV could infect the fat body of H.armigera,but not S.frugiperda or S.litura.Although JcDV can infect all three lepidopteran species,their susceptibility to the virus differs.JcDV has great potential as a biological control agent against pests such as S.frugiperda.