pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two ...pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2—5 days?inoculation. Sequence analysis further confirmed the exis-tence of recovered and recombinant DEN2 viruses, whose 5′ termini had an additional non-virus nucleotides 揋? while the 3′ terminal sequences remained the same as natural. The neuroviru-lence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 105—102 TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.展开更多
基金The National Natural Science Foundation of China (Grant No. 30000144).
文摘pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infec-tious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2—5 days?inoculation. Sequence analysis further confirmed the exis-tence of recovered and recombinant DEN2 viruses, whose 5′ termini had an additional non-virus nucleotides 揋? while the 3′ terminal sequences remained the same as natural. The neuroviru-lence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 105—102 TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.
