Persistent infection of a bovine kidney cell line with Newcastle disease virus
Louza, Armando Carvalho
Continuous passage of undiluted supernatant fluids from cultures of a bovine kidney cell line MDBK infected with the Herts 33 strain of NDV, led to the establishment of a carrier state [MDBKcs].The general properties of this virus-cell system were compared with those of a regulated-type of NDV persistent infection of MDBK cells [MDBKpi] which was accidentally induced in this laboratory in 1962 [ Edwards, 1972] .The intracellular polypeptide composition of the cell associated virus in MDBKpi was different from that in MDBKcs cells. In MDBKcs virus, the usual pattern of composition and distribution of NDV polypeptides was observed whereas the MDBKpi virus lacked the M protein which is necessary for viral assembly.Experiments using both RNA and protein synthesis inhibitors [actinomycin D and cycloheximide, respectively] failed to induce an increased release of infectious virus from MDBKpi cell cultures. This indicates that the blockage is not dependent on cellular RNA or protein synthesis.The presence of a cellular control mechanism of NDV replication was detected in the persistently infected MDBKcs cultures as well as in MDBK cells primarily infected with NDV and there is evidence that a similar mechanism occurs in MDBKpi cells. It is emphasised, however, that the cellular block does not prevent the release of fully infectious virus from primary or carrier state cultures.Although the virus released from MDBKpi does not undergo a productive replication cycle in permissive cell culture systems or in embryonated eggs, it is able to attach, replicate, haemadsorb and induce cell fusion at the first passage level. The available evidence suggests that a defect in the synthesis of M protein miight account for failure to assemble complete viral particles at the cell surface.Besides promoting the release of non -infectious particles, the absence of the viral Iii polypeptide may also be responsible for the intracellular accumulation of viral nucleocapsids. These structures were found to be of two different types, namely granular and smooth, according to the presence or absence of a sheath - covering the RNP component. The accumulation' observed in the nucleus of some cells in aged cultures was always restricted to smooth nucleocapsids. Furthermore, only the granular nucleocapsids were shown conclusively to be NDV-specific by immunoperoxidase techniques.Biochemical experiments on Herts NDV and MDBKcs virus, propagated in embryonated eggs, showed that both types consist of a mixture of two distinct kinds of virus particles. The first of these sedimented at a density of 1.12.in sucrose or tartrate gradients and revealed an unusual polypeptide composition including a significantly reduced NP/F peak, low haemagglutinin and neuraminidase activities, and law infectivity titres; whereas the virus sedimenting at a density of 1.18 was of the standard type.A viral inhibitory factor [VIA was detected in the supernatant fluids of MDBKcs cell cultures. This viral-induced component was NDV-specific, could not be sedimented by ultracentrifugation, and was able to protect indicator cell monolayers against infection by homologous virus but not by related or unrelated viruses.On the other hand, absence of the viral inhibitory factor in MDBKpi cell culture fluids suggested that a different mechanism is involved in the maintenance of the regulated type of infection.Unsuccessful attempts were made to transfect the putative integrated viral information in DIVA from MDBK cells persistently infected with NDV. However, the results of several experiments involving DNA analogues and the fact that a transient "cure" of MDBKpi monolayers was obtained after prolonged propagation seem to suggest that viral integration is a possible hypothesis, at least so far as regulated infections are concerned.