Molecular Basis of the Attenuation Exhibited by Molecularly Cloned Highly Passaged Chicken Anemia Virus Isolates

Abstract

Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.Chicken anemia virus (CAV) has a circular, single-stranded 2.3-kb DNA genome contained within an icosahedral capsid, 25 nm in diameter (9), and is the only member of the genus Gyrovirus of the virus family Circoviridae (6). The virus genome encodes 1 structural (VP1) and 2 nonstructural (VP2 and VP3) proteins ( Fig. 1a) (5). To date, all naturally occurring CAV isolates belong to the same serotype, and all are pathogenic when tested experimentally (2). We previously reported that molecularly cloned virus isolates that were selected from the Cuxhaven-1 (Cux) CAV isolate, which had received 310 cell culture passages (P310) in MDCC-MSB1 cells, showed variation with regard to pathogenicity and reactivity with a neutralizing monoclonal antibody (MAb), 2A9 (7,8). Of these, the attenuated P310-cloned isolate 34, which reacts weakly with MAb 2A9, differed from the pathogenic P310-cloned isolate 33, which reacts strongly with MAb 2A9, at two amino acid residues, namely, VP1 residue 89 and VP3 residue 41. In this study the significance of the VP1 amino acid change at residue 89 as a determinant of pathogenicity was investigated by producing and biologically characterizing chimeric and in-vitromutagenized viruses.The cloned low-passage Cux isolate and the P310-cloned isolates 33 and 34 were produced as described previously (4, 7). Indirect immunofluorescence (IIF) was used to determine the reactivities of the cloned, mutated, and chimeric CAV isolates with CAV-specific MAb 2A9 (7). Chimeric CAV replicative form (RF) DNAs were constructed from BamHI-PstI (BP), PstI-StuI (PS), and StuI-BamHI (SB) restriction fragments, which were produced by restricting cloned P310 RF 33 and 34 DNAs and were purified from agarose gel after electrophoretic fractionation. Following ligation, mixtures containing approximately equimolar amounts of the three fragments were used to transfect MDCC-MSB1 cells to generate the chimeric and reconstructed cloned isolate 34 (Fig. 1a and b). An additional chimeric virus isolate was produced by ligating the SB fragment derived from P310 RF 34 to the complementary BS fragment, derived from the recombinant CAV plasmid pCAA5 (4), which specifies the pathogenic cloned low-passage Cux isolate (Fig. 1c). In this case, PCR methods were used to amplify the BS and SB fragments prior to ligation and transfection. The PCR-ligation-PCR method was used to introduce a site-specific mutation by which amino acid 89 in VP1 of the cloned low-passage Cux isolate was changed from threonine to alanine to produce the VP1 aa89 Cux mutant (1). Experimental infections of 1-day-old specific-pathogen-free chicks were used to evaluate the pathogenicities of chimeric and muta...

Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains

“…Although there is evidence in other viruses such as rabies virus (Flamand et al, 1984), chicken anaemia virus (Scott et al, 2001;Todd et al, 2002), Newcastle disease virus (Meulemans et al, 1987), dengue fever virus (Lim & Ng, 1999) and tickborne encephalitis virus (Holzmann et al, 1990) that virulence (and attenuation) can be correlated, at least partly, to loss or modification of neutralizing epitopes, this was not supported for IBDV in our study. A role for the variable region of VP2 in virulence has been suggested by some authors (van Loon et al, 2002;Brandt et al, 2001), but it has never been shown to be critical in the sense that the presence of basic residues in the cleavage site of the F protein of Newcastle disease virus are for pathogenicity (Collins et al, 1993).…”

Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009: genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China

“…The subgroups were not designated, therefore, we have used the subgroup terms 1A/1B, 1C, and 2A-2E developed by Olvera et al [6]. The variable structural protein, Cap, is considered most important due to its potential association with pathogenicity [21,22]. Some previous reports suggested that PCV2b viruses might be more pathogenic than those of PCV2a [7,20,23,24].…”

Epidemiology and molecular characterization of chicken anaemia virus from commercial and native chickens in Taiwan

“…Therefore, the effects of these amino acid substitutions on CAVs need to be tested in the future. Tood, Scott, Ball, Borghmans, and Adair () reported that five amino acid changes at positions 75I, 89T, 125L, 141L and 144E of VP1 can induce attenuation. Furthermore, a marker for virulence was located at the amino acid position 394 of VP1 (Yamaguchi et al., ).…”