Chicken Anemia Virus Isolates Research Articles

Vertical Induction of the Inclusion Body Hepatitis/Hydropericardium Syndrome with Fowl Adenovirus and Chicken Anemia Virus

The hypothesis that fowl adenovirus (FAV) and chicken anemia virus (CAV), transmitted vertically and simultaneously, induce the inclusion body hepatitis (IBH)/hydropericardium (HP) syndrome in progeny chickens was tested. Thus, 35-wk-old light brown layer breeders, showing absence of antibodies against FAV and variable titers against CAV, were intramuscularly singly infected with the FAV serotype 4 isolate 341 or dually infected with CAV (isolate 10343) and FAV. All hens (groups A [FAV alone], B [FAV + CAV], and C [noninfected]) were clinically healthy throughout the experimental period. Both infectious viruses FAV and CAV were isolated from progenies obtained as early as 5 days after infection of their breeders. Hematocrit, serum proteins, and aspartate-aminotransferase values showed a few statistical differences between the progeny groups. Most of these differences were detected in the progeny chickens of group B. However, almost all values met reference values for the species. The pathologic findings showed that progeny chickens obtained from both singly and dually infected breeders developed macroscopic and histopathologic changes of IBH/HP. The pathologic findings shown by progeny chickens of group A (FAV) were not expected because neither synergism nor prior immunodepression by CAV was concurrent. Chickens of group B (CAV + FAV) also developed IBH/HP. Although not many differences in the evaluated parameters between groups A and B were statistically significant, most pathologic findings of group B indicated a more severe manifestation of the disease. However, because FAV alone did reproduce the syndrome, the results shown by group B would not allow a definitive confirmation of the hypothesis that the association of FAV and CAV is necessary for the successful induction of the IBH/HP syndrome in chickens when transmitted vertically.

Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages.

The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13). Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus. In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1. Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage. One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9. Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool. It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity.

Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach.

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.

Gene Organization of Chicken Anemia Virus

The genomic DNA of chicken anemia virus (CAV) was cloned and sequenced from a Japanese isolate CAA82-2. The nucleotide sequence of CAA82-2 isolate was 98% identical with that of the European Cuxhaven-1 strain (Noteborn et al., J. Virol. 65, 3131-3139, 1991). Nine open reading frames (ORFs) consisting of more than 100 nucleotides were found, i.e., four ORFs (CA1-CA4) on the plus strand and five ORFs (CA1R-CA5R) on the minus strand. These ORFs with the exception of CA4 are conserved between the two CAV isolates. All of these ORPs were expressed in Escherichia coli as fusion proteins with β-galactosidase. By Western blot analysis, the CA2 and CA3 fusion proteins were found to react with CAV-infected chicken sera. Rabbit hyperimmune sera against the CA1, CA2, and CA3 fusion proteins were produced and tested their reactivity to CAV-infected cells. Two viral proteins with the apparent size of 54 and 16 kDa reacted with the antibodies against CA1 and CA3 fusion proteins, respectively. The 16-kDa protein, CA3, was suggested to be a major immunogen on CAV infection.

Chicken anaemia virus infection: Molecular basis of pathogenicity

Chicken anaemia virus (CAV) is a small virus of a unique type with a particle diameter of 23 to 25 nm and a genome consisting of a circular single-stranded (minus-strand) DNA. This DNA multiplies in infected cells via a circular double-stranded replicative intermediate, which was recently cloned. DNA analysis of CAV strains isolated in different continents revealed only minor differences among the various isolates. Apparently, all CAV isolates belong to a single serotype. CAV is not related to other known animal single-stranded circular-DNA viruses, such as porcine circovirus and psittacine beak-and-feather-disease virus. The major transcript from the CAV genome is an unspliced polycistronic mRNA of about 2100 nucleotides encoding three proteins of 51.6 kDa (VP1), 24.0 kDa (VP2) and 13.6 kDa (VP3 or apoptin). All three predicted CAV proteins are synthesized in CAV-infected cells. Immunization with (recombinant) VP1 and VP2 synchronously synthesized in the same cells elicits a protective response and can be used as subunit vaccine against chicken infectious anaemia. CAV causes clinical and subclinical disease in chickens, and is recognized as an important avian pathogen worldwide. In young chickens, CAV causes a transient severe anaemia due to destruction of erythroblastoid cells in the bone marrow and immunodeficiency due to depletion of cortical thymocytes. The depletion of the cortical thymocytes is considered to cause a (transient) immunodeficiency resulting in enhanced concurrent infections and to vaccination failures. The depletion of thymocytes and most likely also of erythroblastoid cells occurs via CAV-induced apoptosis. The CAV-encoded protein apoptin is the main inducer of this phenomenon.

Identification of the promoter region of chicken anemia virus (CAV) containing a novel enhancer-like element.

The single promoter region in the cloned genome [Noteborn et al., J. Virol. 65 (1991) 3131-3139] of chicken anemia virus (CAV) in chicken T-cells was analysed via CAT assays. A unique region containing four or five near-perfect direct repeats (DR) of 21 bp with one 12-bp insert was proven to be the main transcription-activation element, with enhancer-like characteristics. PCR studies revealed that CAV isolates from across the world all contained this promoter sequence. Electrophoretic mobility-shift assays (EMSA) showed that individual DR units, as well as the 12-bp insert, can bind to nuclear factors of chicken T-cells. Competition assays revealed that the DR units bound to factors other than the 12-bp insert. A synthetic oligodeoxyribonucleotide containing an SP1-box (5'-GGGCGG) could compete with factors binding to the 12-bp insert. Purified human SP1 was shown to have very strong affinity for the 12-bp insert.

Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction.

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.