Detection and differentiation of pathogenicity of avian paramyxovirus serotype 1 (APMV-1) from field cases using one-step real-time RT-PCR.

A real-time fluorescent RT-PCR assay was developed to amplify avian paramyxovirus serotype 1 (APMV-1)-specific nucleic acid fragments from field samples. Subsequent restriction endonuclease analysis (REA) using BglI was carried out to type strains according to their virulence. Primer sequences were used to amplify a 202 base-pair fragment, encompassing the fusion protein cleavage site, in a one-step RT-PCR test for detection of a range of field cases and reference strains of APMV-1. Subsequent restriction endonuclease analysis of the amplified fragments enabled differentiation of low virulent lentogenic field and vaccine strains from more virulent mesogenic and velogenic field strains of APMV-1, including pigeon PMV-1. In 2004, seven cases of pigeon PMV-1 in Northern Ireland were diagnosed and differentiated more rapidly using the fluorescent RT-PCR assay when compared with the use of virus isolation. We report the development and application of a one-step real-time RT-PCR test coupled with REA as a fast, specific method for both the detection and typing of APMV-1 from field samples.

Evaluation of strain-specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus (Chalmydia psittaci) for the detection of EAE by PCR.

Strain-specific primer sequences derived from the helicase gene of an ovine abortifacient strain (S26/3) of Chlamydophila abortus (Chlamydia psittaci) were evaluated for the diagnosis of enzootic abortion in ewes (EAE) by polymerase chain reaction (PCR). C abortus DNA was amplified from tissues submitted from ovine abortion cases using genus-specific and strain-specific primers in a standard thermal cycler. Amplification was followed by Southern blotting and hybridisation with a strain-specific probe. Real-time PCR was also evaluated using strain-specific primers in a microvolume fluorimeter-based thermal cycler (LightCycler). Detection using both PCR methods was compared with other diagnostic methods against the standard of McCoy cell culture isolation. In this paper we report the application of strain-specific PCR as a fast, sensitive, specific method for the detection of EAE.

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.

Characterisation of strain-specific sequences from an abortifacient strain of ovine Chlamydia psittaci using subtraction hybridisation.

Enzootic abortion in ewes (EAE) is caused by strains of Chlamydia psittaci which have the ability to invade and colonise the placenta of sheep. In an attempt to improve diagnostic methods for the detection of EAE, subtraction hybridisation was used to isolate unique fragments of the genome of an abortifacient strain (S26/3) of C. psittaci. One S26/3 strain-specific sequence identified was shown to encode a putative helicase which is repeated throughout the EAE genome. The labelled strain-specific helicase gene fragment was used in the dot-blot hybridisation test for the detection of EAE DNA in ovine placental samples. We report the identification of C. psittaci S26/3 strain-specific sequence with potential as diagnostic probes for the detection of EAE.

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.

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.

Sequence of porcine circovirus DNA: affinities with plant circoviruses.

The complete nucleotide sequence (1759 nt) of the ssDNA genome of porcine circovirus (PCV) was determined from a cloned dsDNA replicative form isolated from PCV-infected cells. Sequence analysis detected no significant nucleic acid or protein similarity with another animal circovirus, chicken anaemia virus (CAV) but, surprisingly, the highest protein similarity was obtained between the product of the largest predicted PCV ORF (ORF1; encoding a potential protein of 35.7 kDa) and a putative protein encoded by the plant circovirus banana bunchy top virus (BBTV). High protein similarity was also detected with the other plant circoviruses subterranean clover stunt virus (SCSV) and coconut foliar decay virus (CFDV). This region of protein identity corresponds with the putative plant circovirus replication-associated protein (Rep). The presence of a nonanucleotide sequence at the apex of a potential-stem loop structure, identical to that found in the plant circoviruses CFDV and SCSV and similar (one mismatch) to that found in the plant circovirus BBTV and in the geminiviruses, suggests that rolling-circle replication may operate during PCV DNA replication. These findings show that PCV is unique in that it bridges the gap between animal and plant circoviruses. The taxonomic relationship of PCV with other members of the Circoviridae is discussed.

Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments.

Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family, Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were "circularised" within the same 105 bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts.

Molecular cloning of an attenuated chicken anaemia virus isolate following repeated cell culture passage.

The pathogenicity of the Cux-1 isolate of chicken anaemia virus (CAV) was substantially reduced following large numbers (50 to 173) of passages in MDCC-MSBl cells. Restriction endonuclease analysis of polymerase chain reaction (PCR)- amplified DNAs and recombinant plasmids containing DNA inserts specified by CAV that had been passaged 173 times, indicated that the population of high-passage virus was genetically diverse. A 210-base pair (bp) insertion, containing a 19-bp sequence identical to four repeated sequences that are located in the putative non-coding region of the genome was shown to have become established in the virus population by passage number 30. Individual virus isolates that were selected from the high-passage virus population using recombinant DNA cloning and transfection methodologies varied in their pathogenicities. One cloned virus isolate, designated number 10, produced virtually no anaemia and substantially reduced levels of aplasia of the bone marrow and thymus atrophy. The pathogenicity of this isolate was restored following 10 passages in young chicks.

Characterization of viral DNAs from cells infected with chicken anaemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments.

The viral DNAs induced by the unclassified animal virus, chicken anaemia agent (CAA), during replication in MDCC-MSB 1 cells have been investigated. Analyses after S1 nuclease, restriction endonuclease and denaturation treatments indicated that infected cell extracts contained genome-size, single-stranded DNA (M(r) 2.3 kb), closed and open circular, double-stranded replicative form (RF) DNAs (M(r) 2.3 kbp) and a population of smaller double-stranded DNAs (M(r) 0.8 kbp). Recombinant plasmids containing 2.3 kbp CAA RF fragments cloned at the PstI, BamHI and EcoRI sites failed to transfect MDCC-MSB 1 cells. However, one plasmid, which contained two 2.3 kbp CAA RF fragments ligated in tandem at the PstI site, and cloned 2.3 kbp PstI, BamHI and EcoRI fragments, excised from their respective plasmids by restriction endonuclease digestion, were capable of transfection. The nucleotide sequence of the circular genome (2298 bp) of the Cux-1 isolate of CAA has indicated the presence of three overlapping open reading frames (ORFs) of 52 kDa, 24 kDa and 13 kDa on one strand. The existence of these ORFs was corroborated by analyses of partial sequences from three other isolates. The non-coding region of the CAA genome contained sequences with putative regulatory function. These results are discussed in relation to the "rolling circle" model of DNA replication.

Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe.

A dot blot hybridization assay capable of detecting chicken anemia agent (CAA)-specific DNA in tissues from infected birds has been developed. The assay uses a 32P-labeled DNA probe prepared from cloned CAA-specific fragments representing the entire virus genome and has a sensitivity limit of between and 1 and 10 pg. DNAs from CAA isolates originating in the Federal Republic of Germany, Japan, the United States, the United Kingdom, and Australia were detected. Investigation of specimens from experimentally infected chicks indicated that virus-specific DNA was detected in the tissues of birds from 5 through 42 days after infection and that greater amounts were usually detected in the thymus than in the spleen, liver, feces, or blood. Tissues from specific-pathogen-free and broiler chicks which had become infected at an older age through contact with experimentally infected anemic chicks also contained CAA-specific DNA detectable by the assay. Thymuses from 1- to 2-week-old chicks from eight commercial broiler flocks which had been showing clinical signs characteristic of anemia-dermatitis syndrome were found positive by the hybridization technique, but thymuses from chicks obtained from broiler flocks which did not show such signs were found negative. Of the 35 positive samples (from 46 samples tested), 19 (54%) contained virus-specific DNA in sufficiently great amounts to permit 4-h autoradiography exposures and sample throughput times of 2 days. When compared with virus isolation, the CAA dot blot hybridization assay is time- and labor-saving.

Purification and biochemical characterization of chicken anaemia agent.

Chicken anaemia agent (CAA) was purified using differential centrifugation and successive cycles of equilibrium density gradient centrifugation using sucrose and CsCl. The purification method was dependent on the use of an antigen-detecting ELISA based on a CAA-specific monoclonal antibody. Virus particles banded at a density of 1.33 to 1.34 g/ml in CsCl and measured 23.5 +/- 0.8 nm in diameter. Purified preparations contained one major polypeptide (Mr 50,000) and a single-stranded, circular DNA (2.3 kb). CAA shares some of the biochemical characteristics possessed by porcine circovirus and the virus associated with psittacine beak and feather disease.