Neutralizing monoclonal antibodies directed against five antigenic sites on the spike (S) S 1 glycopolypeptide of avian infectious bronchitis virus (IBV) were used to select neutralization-resistant variants of the virus. By comparing the nucleotide sequence of such variants with the sequence of the IBV parent strain, we located five antigenic sites on the amino acid sequence of the S 1 glycopolypeptide. The variants had mutations within three regions corresponding to amino acid residues 24 to 61, 132 to 149 and 291 to 398 of the S1 glycopolypeptide. The location of three overlapping antigenic sites on the IBV spike protein was similar to the location of antigenic sites on the spike protein of other coronaviruses.

Monoclonal antibodies (MAbs) directed against structural proteins of infectious bronchitis virus (IBV) were produced to analyse the antigenic structure of this virus. Competitive binding of enzyme-labelled and unlabelled MAbs to IBV peplomer protein was analysed in an antibody binding assay to test the relatedness of the epitopes defined by the MAbs. Based on the competition groups, eight epitope clusters were defined (S-A to S-H); six of these clusters (S1-A to S I-F) were located on the S1 subunit and two (S2-G and S2-H) on the $2 subunit of the peplomer protein.Epitope clusters S 1-A and S1-B overlapped extensively. The biological activities of the MAbs were determined and correlated to the epitope clusters. Monoclonal antibodies directed against epitope clusters S 1-A to S1-E and one MAb directed against cluster S2-G moderately to strongly neutralized IBV at titres higher than 2 log~o, whereas the remaining MAbs, directed against S1 and $2, neutralized at titres lower than 2 log~o. One MAb, directed against cluster S1-D, inhibited the agglutination of chicken erythrocytes.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained doubleand single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

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