A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coli and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus

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SUMMARYAmino terminal sequences of the envelope protein E and the three largest nonstructural proteins NS1, NS3, and NS5 of the New Guinea C strain of dengue virus type 2 (DEN-2) were obtained by nucleotide and protein sequencing. Clones were prepared containing cDNA of DEN-2 virus in the plasmid pUC8. The nucleotide sequences of viral cDNA inserts were determined and the cDNA of each clone positioned on the flavivirus genomic map by comparison of the deduced amino acid sequence with that of yellow fever virus. Radiolabelled E, NS1, NS3 and NS5 were purified by lectin affinity chromatography and preparative gel electrophoresis. Purified proteins were subsequently analysed by Edman degradation to establish the origins of the amino termini of these proteins in the deduced DEN-2 amino acid sequence. Thus the amino acid sequences surrounding the likely proteolytic cleavage sites used in the formation of these four proteins were determined. Of particular interest was the sequence containing the amino terminus of NS3, namely Lys-Lys-GlnArg-Ala-Gly where Ala is the first amino acid of NS3. Cleavage following one basic residue in the flavivirus polyprotein has not been reported previously.

Five small round-structured viruses (SRSVs) associated with gastroenteritis in Victoria, Australia, from January to November 1994 were examined by sequencing cDNA prepared from faecal samples using RT-PCR. The sequence of the 3' half (3.8 kb) of the genome of one of these viruses, Camberwell, was determined. Camberwell virus was related most closely to Bristol and Lordsdale viruses, and belonged to the genetic group of SRSVs containing Bristol, Lordsdale, Toronto, OTH-25, Mexico, and Hawaii viruses. The amino acid identities between Camberwell and Bristol viruses for proteins encoded by ORF1 (partial), ORF2, and ORF3 were 99%, 98%, and 90%, respectively. A highly variable region in ORF3 corresponding to amino acid residues 123 to 169 (Bristol and Camberwell numbering) were identified. Short segments of ORF1 (polymerase region) and the highly variable ORF3 region was analysed for the other four viruses. The results obtained indicated the potential usefulness of the variable region in distinguishing between closely related viruses.

Five small round-structured viruses (SRSVs) associated with gastroenteritis in Victoria, Australia, from January to November 1994 were examined by sequencing cDNA prepared from faecal samples using RT-PCR. The sequence of the 3' half (3.8 kb) of the genome of one of these viruses, Camberwell, was determined. Camberwell virus was related most closely to Bristol and Lordsdale viruses, and belonged to the genetic group of SRSVs containing Bristol, Lordsdale, Toronto, OTH-25, Mexico, and Hawaii viruses. The amino acid identities between Camberwell and Bristol viruses for proteins encoded by ORF1 (partial), ORF2, and ORF3 were 99%, 98%, and 90%, respectively. A highly variable region in ORF3 corresponding to amino acid residues 123 to 169 (Bristol and Camberwell numbering) were identified. Short segments of ORF1 (polymerase region) and the highly variable ORF3 region was analysed for the other four viruses. The results obtained indicated the potential usefulness of the variable region in distinguishing between closely related viruses.