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

Abstract

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.

Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence

“…To our knowledge, all such reports show the loss to occur gradually, over several to many passages. For instance, the pathogenicity of the Cux-1 isolate of chicken anemia virus was substantially reduced after 50 to 175 passages (Todd et al 1995) (Hassan et al 1996). Similarly, Borrelia burgdorferi, the bacterial agent of Lyme disease, lost virulence after 17 to 21 passages (Moody et al 1990).…”

“…Repassing cultured parasites through a host to restore virulence in a cell line , Chang & Fish 1983, Stanley et al 1994, Todd et al 1995 did not work for Perkinsus marinus, because the virulence of P. marinus is lost as soon as the parasites are established in culture. We did not test virulence of the in vivo-passed parasites before they were reestablished in culture by immediately using them in challenge experiments, but the fact that the lethal density for both wild-type and cultured parasites (including the in vivo-passage experiment) was similar (10 6 to 10 7 parasites g -1 wwt) argues that cultured parasites, once inside the oyster, eventually become as virulent as those isolated directly from oysters.…”

“…Viruses, bacteria, and protozoans have all been reported to lose the ability to cause disease after a certain number of passages in culture (Giannini 1974, Chang & Fish 1983, Moody et al 1990, Kallinikova et al 1992, Hassan et al 1996, Mukhopadhyay et al 1998. For some pathogens, virulence can be maintained in a cultured line if it is repassed periodically through its host , Todd et al 1995. Chang & Fish (1983) cautioned against generalizing the results obtained using in vitro-cultured parasites until similarities and differences between cultured and wild-type parasites have been documented.…”

“…It is likely that the surviving parasites are better adapted to the new environment than those that died. It was not possible for us to determine whether there was a genetic component to the observed phenotypic change in the virulence of P. marinus, although it is worth noting that Sutherland et al (1996) found evidence that both phenotypic and genotypic changes were involved in the gradual loss of virulence in the cattle parasite Theileria annulata maintained in culture.Repassing cultured parasites through a host to restore virulence in a cell line , Chang & Fish 1983, Stanley et al 1994, Todd et al 1995 did not work for Perkinsus marinus, because the virulence of P. marinus is lost as soon as the parasites are established in culture. We did not test virulence of the in vivo-passed parasites before they were reestablished in culture by immediately using them in challenge experiments, but the fact that the lethal density for both wild-type and cultured parasites (including the in vivo-passage experiment) was similar (10 6 to 10 7 parasites g -1 wwt) argues that cultured parasites, once inside the oyster, eventually become as virulent as those isolated directly from oysters.…”

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

“…The virus can be grown in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells, a finding which has allowed the virus to be characterised (Todd et al, 1990) and its molecular biology elucidated (Noteborn & Koch, 1995). In a previous paper we described how recombinant DNA cloning and transfection methodologies have been used to select an attenuated CAV isolate following repeated passage of the virus in cell culture (Todd et al, 1995). In the present paper we describe more fully the effects of multiple cell culture passage on the biological behaviour of the Cux-1 isolate of CAV.…”