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.

A double antibody sandwich blocking ELISA, using a monoclonal antibody (MAb) against influenza A nucleoprotein (NP) was developed to detect antibodies against influenza. Collections of serum samples were obtained from human and various animal species. All influenza A subtypes induced antibodies against hemagglutinins and NP. A close correlation between titers of the hemagglutination inhibition (HI) test and the NP-ELISA was seen. Antibodies against influenza NP were demonstrated in serum samples from humans, ferrets, swine, horses, chickens, ducks, guinea pigs, mice, and seals. The serum samples were collected at intervals during prospective epidemiological studies, from experimental and natural infections, and vaccination studies. The decline of maternal antibodies was studied in swine and horses. The NP-ELISA enables rapid serological diagnosis and is suited for influenza A antibody screening, especially in species which harbor several influenza subtypes. The HI and neuraminidase inhibition tests, however, must still be used for subtyping.

The postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abundant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckel's diverticulum, and Peyer's patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.

SUMMARYThe purpose of the study was twofold First, using two groups of 22 foals each, we investigated the extent to which maternal antibodies interfere with the humoral response against equine influenza. The foals were born to mares that had been vaccinated twice yearly against influenza since 1982. Foals of group I were vaccinated three times at early ages (12 16, and 32 weeks of age), and foals of group II were likewise vaccinated but at later ages (24, 28, and 44 weeks of age). After the first and second vaccinations, neither group showed an increase in antibodies that inhibit haemagglutination. Group II foals, however, had a significantly stronger antibody response against nucleoprotein after the second vaccination than the foals ofgroup I. After the third vaccination, group II foals had a significantly stronger and longer lasting antibody response against haemagglutinin than the foals of group I. However, the antibody response to nucleoprotein was comparable in both groups.Second, the foals of group II were studied to determine the persistence of maternal antibodies directed against a common nucleoprotein and the haemagglutinin of two strains of equine influenza A virus. Biological half-lives of 39, 32, and 33 days were calculated for maternal antibodies directed against haemagglutinin of strains H7N7 Prague and H3N8 Miami, and against the nucleoprotein respectively. Maternal antibody titres at the time of vaccination were closely related to the degree of interference with the immune response. Because even small amounts of maternal antibodies interfered with the efficacy of vaccination, we conclude that foals born to mares vaccinated more than once yearly against influenza virus should not be vaccinated before 24 weeks of age.

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