The complete nucleotide sequence ofthe genome of the type 1 poliovirus vaccine strain (LSc,2ab) was determined by using molecular cloning and rapid sequence analysis techniques. The restriction fragments of double-stranded cDNA synthesized from the vaccine strain RNA were inserted into the adequate sites of cloning vector pBR322. Sequence analysis of the cloned DNAs revealed that the virion RNA molecule was 7,441 nucleotides long and polyadenylylated at the 3' terminus. When the nucleotide sequence was compared with that of the genome of the virulent parental strain (Mahoney), 57 base substitutions were observed to be scattered all over the genome. Of these, 21 resulted in amino acid changes in a number of viral proteins. A cluster of amino acid changes is located in the viral coat proteins, especially in the NH2-terminal halfofthe viral capsid protein VP1.These results may imply that the mutations in the VP1 coding region contribute to attenuation.The genome ofpoliovirus is a single-stranded RNA with positive polarity, in which all of the viral genetic information is stored (1). This genomic RNA is composed of =7,500 nucleotides, polyadenylylated at the 3' terminus (2) and covalently attached to a genome-linked protein (VPg) at the 5' terminus (3-6). Re PVl(Sab)] is a live vaccine strain derived from the PV1(M) by spontaneous mutations during the attenuation process (9, 10).To determine the molecular basis for the biological differences between virulent and attenuated poliovirus strains, the sequences of large and unique RNase Ti-and A-resistant ohigonucleotides of PV1(M) and PV1(Sab) have been compared. We have shown that mutations detected by oligonucleotide analysis were caused by single base substitutions and appeared to be scattered all over the genome (11).For further comparative sequence studies, the restriction fragments obtained from double-stranded cDNA of the PV1(Sab) genome have been cloned (12). We report here the complete 7,441-nucleotide sequence of the PV1(Sab) genome, and the mutation sites were identified by comparison of our sequence with the known sequence of the PV1(M) genome (7,8).

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.

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Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.

DNA fragments complementary to the Newcastle disease virus genome (strain D26) were cloned and sequenced. The sequence of 2,617 nucleotides from the 3' end of the genome was determined and an open reading frame (OP-1) consisting of 1,467 nucleotides, most likely encoding NP protein, was found in this region. This was followed by a second unfinished open reading frame (OP-2) of at least 729 nucleotides which continued beyond the 2,617th nucleotide. Another relatively short (312 nucleotides long) open reading frame (OP-2') was found overlapping with OP-2, but its significance is still unclear. The amino acid sequence deduced from the nucleotide sequence of OP-1 showed a moderate homology to that of the NP protein of Sendai virus in the central portion of the peptide. The leader sequence of 53 nucleotides was also identified. The 5' end of mRNAs synthesized in the infected cells was analyzed and found to be m7GpppA, suggesting that the transcription of viral mRNAs starts with A, but not with G residue.