Sequence comparison of pigeon circoviruses.

The genome sequences of eight pigeon circoviruses (PiCV) were determined and compared with four previously published sequences. The viruses compared were from the USA, five European countries, China and Australia and included PiCVs from racing, feral, ornamental and meat pigeons and a Senegal dove (Streptopelia senegalensis). The 12 PiCV genomes, ranging from 2032 to 2040 nucleotides in length, displayed similar organizations. Pairwise comparisons showed that the genome nucleotide sequence identities ranged from 85.1% to 97.8% and that the amino acid identities of the putative replication associated (Rep) and putative capsid (Cap) proteins displayed ranges of 91.5-99.1% and 73.0-99.3%, respectively. Comparative analyses identified conserved nucleotide sequences within the Rep gene and 3' intergenic regions, which would be suitable for diagnostic PCR primers, and variable amino acid sequences within the capsid proteins, which should be considered when selecting virus isolates for vaccine development.

Diagnosis of goose circovirus infection in Hungarian geese samples using polymerase chain reaction and dot blot hybridization tests.

A polymerase chain reaction (PCR) and dot blot hybridization (DBH) test have been developed for the diagnosis of infection by a novel circovirus of geese (GoCV). These tests were applied to samples of bursae of Fabricius from sick and dead birds from commercial goose farms in Hungary. In this second report of the occurrence of circovirus infection in diseased geese, 103 of 214 (48.1%) and 37 of 150 (24.6%) birds, and 49 of 76 (64.5%) and 18 of 76 (23.7%) flocks were positive by PCR and DBH respectively. The sensitivity of the PCR test was such that 0.10 fg of virus DNA was detectable. The DBH test was less sensitive, only detecting larger amounts (40 pg) of DNA, but was used as a semi-quantitative method for detecting the presence of virus. The incidence of infection was affected by factors such as the age of the birds and rearing methods.

Immunopathologic investigations with an attenuated chicken anemia virus in day-old chickens.

The immunopathologic effects induced by two attenuated chicken anemia virus (CAV) isolates, known as cloned isolate 34 (CI 34) and cloned revertant isolate 18 (CRI 18), that were derived from highly passaged pools of Cux-1 CAV isolate, were compared with those induced by a pathogenic, molecularly cloned, low-passage Cux-1 isolate (CI Cux). This comparison involved the intramuscular inoculation of 1-day-old specific-pathogen-free chicks with each of the viruses and investigation of birds at selected days postinoculation for gross pathology and depletions in the thymic T-cell populations as determined by flow cytometry. Whereas infection with the pathogenic CI Cux produced severe anemia and pronounced bone marrow and thymus lesions, infections with the attenuated CRI 18 and CI 34 isolates produced no anemia, no or mild lesions, respectively, and moderate T-cell depletion. The results suggest that, with CAV, reduced pathogenicity for 1-day-old chicks correlates with reduced depletion of T-cell populations in the thymus and with reduced severity of lesions in the thymus and bone marrow.

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.

Nucleotide sequence-based identification of a novel circovirus of canaries.

Circovirus-like, spherical particles measuring 16 to 18 nm in diameter were detected in organ homogenates from adult canaries that had died after a short illness characterized by dullness, anorexia, lethargy and feather disorder. A polymerase chain reaction method, based on degenerate primers specific to conserved amino acid sequences in the circovirus replication-associated protein, was used to amplify DNA specific to a novel circovirus, tentatively named canary circovirus (CCV). Sequence analysis of a 510 nucleotide genomic fragment indicated that CCV exhibited 67.4, 64.9, 53.7 and 53.9% nucleotide identities and 70.0, 61.8, 40.4 and 40.1% amino acid identities with columbid (pigeon) circovirus (CoCV), beak and feather disease virus (BFDV), porcine circovirus type 1 and porcine circovirus type 2, respectively. CCV therefore represents a new avian virus of the genus Circovirus of the family Circoviridae, and is more closely related to CoCV than BFDV.The availability of nucleotide sequence data will facilitate the development of DNA-detecting diagnostics with which the prevalence of CCV infections can be assessed.

Biochemical characterization of salmon pancreas disease virus.

Salmon pancreas disease virus (SPDV) has been shown to cause severe economic losses in farmed Atlantic salmon (Salmo salar) and has been reported to occur in Europe, Scandinavia and the United States. This paper describes the biochemical characterization of SPDV in terms of its RNA and protein composition. SPDV was purified by precipitation from infected Chinook salmon embryo (CHSE-214) cell-culture supernatant and sucrose density-gradient centrifugation. Fractions containing virus were identified by an immunodot blot assay using an SPDV-specific MAb. Two major proteins with molecular masses of approximately 55 and 50 kDa, putatively identified as the E1 and E2 alphavirus glycoproteins respectively, were detected when purified virus preparations were analysed by PAGE. Radiolabelling experiments indicated that SPDV infection of CHSE-214 cells did not shut-off host-cell protein synthesis, making attempts to identify virus-specific proteins unsuccessful. However, radioimmunoprecipitation assay (RIPA) experiments showed that two SPDV-specific MAbs reacted with a protein in the 50-55 kDa range. Northern blot hybridization with cloned cDNA probes indicated that infected cells contained RNA species of approximately 11.4 and 4 kb, which correspond to the genomic and subgenomic RNAs specified by SPDV. The results described are consistent with SPDV being characterized as an alphavirus.

Effect of multiple cell culture passages on the biological behaviour of chicken anaemia virus.

The Cux-1 isolate of chicken anaemia virus (CAV) was passaged over 320 times in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells. Comparison of the infectivity titres of virus pools derived from viruses that had received 0 (P0), 49 (P49), 170 (P170) and 320 (P320) passages in our laboratory indicated that the yields of infectious virus increased over 100-fold with passage number from P0 to P170. P320 exhibited unusual cell culture growth characteristics in that, unlike its lower passage counterparts, virus-specific immunofluorescence (IF) and cytopathic effect were detected at very low levels at 2 days post infection, with an additional passage of infected cells into fresh medium being required to produce high levels of infectious virus. Experimental infection of 1-day-old SPF chicks showed that P170 and P320 were substantially attenuated compared to P49 and a pathogenic, low-passage isolate used as control. When assessed by the indirect IF test, infection of 1-day-old chicks with the P49 and P170 isolates elicited similar levels of CAV-specific antibody to those elicited by infection with the pathogenic, low-passage virus and higher than those elicited by infection with the P320 isolate. Experimental infection of 5-week-old chicks indicated that the attenuated P170 and P320 isolates invoked similar CAV-specific antibody levels to those invoked by the pathogenic P49 and control isolates.