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 35n7 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

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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.

Amplification of avian paramyxovirus serotype 1 (APMV-1)-specific nucleic acid fragments, followed by 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 reverse transcriptase-polymerase chain reaction (RT-PCR) test for detection of a range of field cases and reference strains of APMV-1. Subsequent REA 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 the present paper, we report the development and application of a one-step RT-PCR test coupled with REA as a fast, specific method for both the detection and typing of APMV-1 from field samples.

A real-time polymerase chain reaction (PCR) assay was developed to specifically amplify infectious laryngotracheitis virus (ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism (RFLP) analysis of real-time PCR amplified fragments from a range of ILTV isolates using the restriction endonuclease MspI enabled differentiation between older ILTV isolates that were prevalent in the 1960s prior to the availability of vaccine strains and more recent isolates that predominantly are identical to vaccine strains. The assay, using real-time PCR, RFLP and sequence analysis, was used to characterize two recent field cases of infectious laryngotracheitis from Northern Ireland. One of the field cases was demonstrated to be similar to older ''wild-type'' isolates, while the other field case was identified to have a concurrent ILTV infection of both ''wild-type'' and vaccinal type origin. The assay described here using realtime PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.