A temperature-sensitive mutant of Sendai virus which establishes persistent infection in Vero cells without cell crisis

Eleven temperature-sensitive (ts) mutants of Sendai virus were examined for the ability to establish persistent infection in Vero cells at 37°. Only one mutant, ts-23, readily established persistent infection, while some of the mutants tested, like the parental wild virus, required long-term subcultivation for the establishment of persistent infection because of repeated cell crisis, and the remaining mutants failed to establish persistent infection. The ts-23 mutant replicated well in Vero cells, but infected cells showed no cytopathic effect and were readily subcultured. The fluid of these cultures contained neither defective interfering particles nor interferon, indicating that these factors are not involved in the establishment of persistent infection. In mixed infections, ts-23 virus markedly inhibited the development of cytopathic effect by the wild-type virus or the other ts mutants, and all or a part of the cells in the cultures beame persistently infected without showing cell crisis.     

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.     

Temperature-sensitive avian sarcoma viruses: a physiological comparison of twenty mutants

The behavior of 20 temperature-sensitive mutants of avian sarcoma virus has been examined. Three general parameters of infection were studied: virus replication, ability of the virus to induce cell focus formation on a monolayer and ability of the virus to induce cell colony formation in agar suspension. In addition, virion heat stability, morphology of infected cells at the nonpermissive temperature, and the ability of infected cells to promote plaque formation by avian leukosis virus were investigated. On the basis of these viral characteristics the mutants have been classified into 6 well-defined categories. Some differences between mutants within categories were also detected.     

A persistent infection of baby hamster kidney-21 cells with mumps virus and the role of temperature-sensitive variants.

A persistent infection of baby hamster kidney-21 (BHK-21) cells with mumps virus (BHKpi) was maintained for over 60 cell passages in the absence of antiserum. Viral persistence was demonstrated in the cultures by hemadsorption, immunofluorescence, multinucleate syncytia, and released mumps virus at the level of 10(2)--10(3) fluorescent focus-forming units/ml. No detectable levels of interferon were found in cultures persistently infected with mumps virus. Approximately 85--95% of the cells contained viral antigens. Nuclear fluorescence was observed in the persistently infected cells. Mumps virus from persistently infected clutures (MuVpi) was more heat-labile than wild-type mumps (MuVo) when subjected to 40 degrees C. BHKpi cells had a more rapid doubling time and a higher cloning efficiency in soft agar in comparison to BHK-21 cells. MuVpi was also found to be temperature-sensitive. The temperature-sensitivity of MuVpi was determined by the efficiency of plating at 33 degrees and 39 degrees C. MuVpi readily established a persistent infection in BHK-21 cells with less cytopathology than MuVo, and released temperature-sensitive virus.     

Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus

Replicate cultures of Aedes albopictus cells were infected with Sindbis virus and then maintained for long periods of time by weekly subculture. During the first week the viral titers ranged between 10⁸ and 10⁹ PFU/ml, but then gradually fell and within a few weeks stabilized at about 10⁵ to 10⁸ PFU/ml. Such cultures were followed for the appearance of temperature-sensitive (ts) virus, small-plaque virus, and for the appearance within the cells of small (12–15 S) double-stranded viral RNA (dsRNA). Cloning experiments carried out 6 months or more after the initial infection showed that persistently infected cultures gave rise to both virus-yielding and nonyielding clones. Similar results were obtained when virus-positive clones were recloned. Prolonged treatment of persistently infected cultures with anti-Sindbis virus serum resulted in curing of the virus infection. Cured cultures behaved in every way tested as normal uninfected A. albopictus cells. The ability to cure with anti-viral serum suggests that in this system extracellular virus is needed to perpetuate the long-term infection. After persistently infected cultures were subcultured, viral RNA synthesis and viral yields were maximal during the first 3–4 days. Thereafter, although the cell number continued to increase for several days, viral RNA synthesis and viral yields both decreased sharply. This result strongly suggests that A. albopictus cells have efficient means for the regulation of viral biosynthesis. Although the resistance of persistently infected cultures to superinfection could be accounted for by interfering temperature-sensitive nondefective virus, the presence in these cells of 12 S dsRNA suggests that defective viral genomes are also present.     

The role of defective interfering particles in persistent infection of Vero cells by measles virus.

Persistent infections by measles virus were rapidly established in the majority of Vero cells when monolayers were infected with virus stocks that had been passed three to five times from an undiluted inoculum. These virus stocks had low infectivity titres but normal haemagglutinin titres and were able to cause interference. The ability of such virus stocks to establish persistent infections seems to be due to the presence of defective interfering particles rather than of virus mutants. Measles virus released from a persistently infected Vero cell line at the 93rd passage had properties similar to the undiluted passage virus that generated persistent infections.     

Persistent infection with human cytomegalovirus in a lymphoblastoid cell line.

Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus (HCMV). The infected culture has persistently yielded HCMV with titers ranging from 2 × 10⁴ to 3 × 10⁵ PFU/ml over a period of 1 year. Infectious center and fluorescent antigen assays and electron microscopic examination indicated that 1–10% of the cells were infected. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells rather than to a defective or temperature-sensitive mutant of HCMV. The treatment of persistently infected cultures with anti-HCMV serum resulted in curing the virus infection. Cured cells in culture grew at the same rate as normal uninfected cells and became resistant to HCMV infection and relatively resistant to HSV infection.     

Temperature-sensitive mutants of Japanese encephalitis virus, isolated from chronically infected cultures of suckling mouse brain cells]

Reproduction of Japanese encephalitis virus isolated at various passage levels from chronically infected GMS-1-K33 cell cultures was maximum in chick embryo fibroblast cultures at a temperature of 28 degrees C. A temperature of 42 degrees C was found to be nonpermissive for the majority of variants and restrictive for the 20th passage virus. Some decline in titers was also observed at 37 degrees C. The ts mutants derived from the population of the persisting virus had lower pathogenicity for laboratory animals, were sensitive to RNA-ase and resistant to 4 M urea. The data obtained by us and other authors suggest that appearance and selection of ts mutants in persistent infection in cell cultures in a regular phenomenon playing a significant role both in establishment and maintenance of persistently infected systems.     

Neurovirulence Mutant of Vesicular Stomatitis Virus with an Altered Target Cell Tropism In Vivo

Intracerebral infection of weanling Swiss mice with a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), ts pi364, resulted in a unique neuropathological syndrome not previously described with other VSV mutants. Mice infected with wild-type VSV died from an acute encephalitis characterized by neuronal necrosis and efficient virus replication in both brain and spinal cord. In contrast, with VSV ts pi364, the most prominent histopathological feature was destruction of the ependyma of the lateral ventricles. Virus antigen was also limited to the leptomeninges and the lateral ventricles. Infected mice survived and developed hydrocephalus. Replication of ts pi364 in the brain was 10- to 100- fold less than that of wild-type VSV, and appearance of virus in the spinal cord was delayed. VSV ts pi364 was isolated from mouse cells persistently infected with VSV. Another VSV ts pi mutant, isolated from the same persistent infection, behaved in vivo like wild-type VSV, even though both mutants were very similar in plaque size, reversion frequency, cut-off temperature, and synthesis of virus-specific proteins at semipermissive temperature. These results strongly suggest that VSV ts pi364 has a second, non-ts mutation which results in a restricted target cell range in vivo; wild-type VSV can infect both neurons and ependymal cells, whereas ts pi364 does not replicate in neurons.     

Induction of an interferon-like substance in persistently infected Aedes albopictus cells

Summary The multiplication of Sindbis virus inSingh'S mosquito cell line derived from larvalA. albopictus was studied. Persistently infected cells are not able to support the growth of Sindbis virus to the same extent as cells infected for the first time. The maintenance of cell-virus equilibrium in persistently infected cells seems to be due to the presence of interferon-like antiviral substances. Mosquito cells which had been treated with media harvested from persistently infected cultures were protected from infection by Sindbis virus. The synthesis of these interferon-like substances is inhibited by actinomycin D. A possible implication of this observation is that in persistently infected mosquito cells the rate of replication of virus is controlled by the genome of the host cell.     

Complementation between temperature-sensitive mutants isolated from Aedes albopictus cells persistently infected with Western equine encephalitis virus.

To obtain temperature-sensitive (ts) mutants, Aedes albopictus cells were persistently infected with wild-type Western equine encephalitis virus. Many leaky and a few stable ts mutants were found among small plaque-forming progeny virus from the cultures at the early stage (up to 30 days) after initial infection. At later times (80–170 days) the persistently infected cultures gave rise to small plaque variants with stable ts mutations. Twenty-four stable ts isolates from early and late passages were classified into three groups by RNA phenotype and complementation. They consisted of three RNA⁺ and one RNA^? mutants with single-site mutation from the early cultures. The remaining 20 ts mutants isolated from the late cultures all had multiple-site mutations; 19 of these were double mutants and may be identical, and one was a triple mutant. We also demonstrated positive complementation between single and multiple mutants. All ts mutants isolated from persistently infected cells possess either the group III RNA^? mutation or the group IV RNA⁺ lesion or the double mutation of group III/IV. The defect in the structural protein is likely to be in one of the envelope proteins, the E2 protein. It is possible that a control mechanism that favors the accumulation of such multiple mutants exists in persistently infected mosquito cells.     

Influence of cellular functions on the evolution of persistent infections with Junin virus

Summary Vero cell cultures persistently infected with Junin virus and subjected to different cultural conditions were established. The production of infectious plaque-forming virus,ts mutants and interfering viral particles was determined at different times during 110 days after infection. Carrier cultures maintained in stationary conditions continuously released PFU while proliferating persistent cultures exhibited a cyclical pattern which tends to a rapid PFU disappearance. Concomitantly, in stationary cultures the production of interfering particles was delayed and was lower than in actively growing persistent cells. The metabolic state of the infected cells did not affect the release ofts mutants. The results suggest that a cellular function is involved on the regulation of Junin virus persistent infections.With 2 FiguresMember of Research Career from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).     

Isolation and preliminary characterization of temperature-sensitive mutants of the murine sarcoma leukaemia virus complex.

Five temperature-sensitive mutants (ts I to 5) were isolated from a stock of the Moloney strain of murine sarcoma leukaemia virus complex which had been mutagenized by ultraviolet irradiation or N-methyl-N'-nitro-N-nitrosoguanidine. In mouse cells at the non-permissive temperature the mutants formed fewer foci of transformed cells than at the permissive temperature. The ts mutants were characterized by testing: (I) murine leukaemia virus (MuLV) clones from the ts complex, (2) the effect of additional wild type MuLV on focus formation, (3) focus formation in rat cells and (4) focus formation with pseudotypes rescued from non-producer cells. Two mutants (ts 1 and ts 3) were found to be ts MuLVs which did not possess heat labile virion proteins and were not ts in post-penetration helper functions necessary for the fixation of sarcoma virus transformation. The remaining three mutants (ts 2, ts 4 and ts 5) were ts murine sarcoma viruses which, however, showed no temperature-sensitive effect on the maintenance of transformed cell morphology nor on colony forming efficiency in soft agar.     

Further characterization of herpes virus persistence.

Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.     

Recombinant Sendai viruses with L1618V mutation in their L polymerase protein establish persistent infection, but not temperature sensitivity

The Sendai virus pi strain (SeVpi) isolated from cells persistently infected with SeV shows mainly two phenotypes: (1) temperature sensitivity and (2) an ability of establishing persistent infection (steady state). Three amino acid substitutions are found in the Lpi protein and are located at aa 1088, 1618, and 1664. Recombinant SeV(Lpi) (rSeV(Lpi)) having all these substitutions is temperature sensitive and is capable of establishing persistent infection (steady state). rSeVs carrying the fragment containing L1618V show both phenotypes. rSeV(L1618V), in which leucine at aa 1618 is replaced with valine, has the ability of establishing persistent infection, but is not a temperature-sensitive mutant, indicating that the ability of a virus to establish persistent infection can be separated from temperature sensitivity. The amino acid change at 1618(L-->V) coexisting with aa 1169 threonine is required for acquirement of a temperature-sensitive phenotype. Three amino acid substitutions are also found in the Ppi protein, but rSeV(Ppi) does not show these phenotypes.     

Ts mutants of the Eastern equine encephalomyelitis virus. Their generation, isolation and preliminary characterization

Chick embryo fibroblast cultures of the C/O phenotype (leukemia--free) infected with eastern equine encephalomyelitis (EEE) virus were incubated in the presence of 15 micrograms/ml N-methyl-N-nitro-N-nitrosoguanidine in the culture medium. Seven (5%) temperature-sensitive mutants were isolated from cell homogenates only in those cases where cell cultures before infection had been treated with actinomycin D. The recovered ts mutants are characterized by the marked ts- phenotype and genetic stability. The method of obtaining EEE virus ts mutants under the effect of N-methyl-N-nitro-N-nitrosoguanidine in C/O phenotype (leukemia-free) chick embryo fibroblast cultures treated before virus inoculation with actinomycin D is discussed.     

Inhibition of sendai virus-induced hemolysis by long chain fatty acids

A persistent infection (persistent infection I) of baby hamster kidney (BHK) cells with the WSN (H1N1) strain of influenza A virus was established using a virus stock which contained a high proportion of defective-interfering (DI) particles. Virus recovered from passage 92 (388 days) of persistent infection I was used to establish a second persistent infection (persistent infection II) in BHK cells. A number of phenotypic changes were identified in the virus isolated during the first 50 passages of persistent infection I (early pi virus). These included a decrease in the size of plaques, the appearance of temperature-sensitive mutants, and a decreased ability of amplified pi virus to agglutinate chicken erythrocytes. The decreased ability to cause hemagglutination was associated with a 20- to 30-fold increase in viral neuraminidase activity. Virus isolated after passage 63 of persistent infection I could not be amplified in eggs or in a number of cell lines. Although very little infectious virus was produced when cells were infected with these late pi viruses, cytopathology frequently occurred and an unusual pattern of viral protein synthesis was observed. The NP protein was the predominant protein synthesized, while the synthesis of M protein was drastically reduced relative to its synthesis in cells infected with parental WSN virus. The HA, NS1, and NS2 proteins were not detected; however, a virus-specific protein which migrates faster than NS2 was observed. Virus recovered from persistent infection II interfered with the replication of parental WSN virus in a mixed infection. The pattern of protein synthesis in such mixed infections resembled that in cells singly infected with late pi virus. DI particles did not appear to play a significant role either in the maintenance of the persistent infection, in the expression of the pi protein synthesis phenotype, or in the pi virus-mediated interference.     

Production of temperature-sensitive and pathogenic virus fromAedes albopictus cells (Singh) persistently infected with chikungunya virus

Summary WhenA. albopictus, clone C6/36, cells were infected with chikungunya (CHIK) virus, high virus yield accompanied by a cytopathic effect in the acute stage of infection was followed by a relatively low yield of virus over a long period of time. Virus produced from persistently infected cultures became gradually of smaller plaque size and more temperature-sensitive; however, such virus still retained pathogenicity for suckling mice even after one year of infection.When the persistently infected cells were subcultured, a dissociation was observed between the time course of cell growth and that of virus production, suggesting some intracellular mechanisms that turn off virus production. The greater part of the interference against CHIK virus by the culture medium of the persistently infected cells appeared to be mediated by the infective virus in the medium. The infective virus was easily removed from the persistently infected cells either by subculture or by cloning in the presence of anti-CHIK serum, yielding cured cultures or virus-negative clones.With 5 Figures     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Vero cells persistently infected with Tacaribe virus: role of interfering particles in the establishment of the infection

Eight Vero cell sublines (Vero T) persistently infected with wild type Tacaribe virus replicated in different hosts were established. In order to unravel the mechanism involved in the initiation and maintenance of persistence, the properties of virus shed by the sublines and the presence of interfering particles (IP) were analyzed. During the course of infection, persistent virus (Tac-pi) underwent mutations although no consistent pattern of virus evolution was observed. ts mutants were isolated from two Vero T sublines, whereas a slow growth variant was shed by another. The remaining sublines released virus resembling wt parental virus. Except for Vero T1 sublines, Vero T cultures shed no detectable IP. These results emphasize the point that neither the emergence of virus mutants nor the synthesis of IP is essential for the maintenance of the persistent state. To define the role of IP in the initiation of persistence, coinfection experiments with a characterized inoculum were performed. For that purpose, attempts were made to obtain IP stocks free from pfu by serial transfers of undiluted virus. Neither enrichment nor amplification of IP occurred, and virus stocks were freed of infectious virus by UV irradiation. If normal Vero cells were infected with Tac-pi virus released by Vero T2, Vero T3, Vero T4, Vero T5, Vero T6, Vero T7 and Vero T10 sublines, a complete destruction of the monolayer without cell recovery was observed. In contrast, parental and Vero T1 viruses always originated persistently infected sublines. Similarly, the addition of IP to virus inocula constituted by Tac-pi viruses released by Vero T2, Vero T3, Vero T4, Vero T5, Vero T6, Vero T7 and Vero T10 sublines gave rise to persistently infected cultures. These results suggest that although IP are not important by themselves in the maintenance of persistence, they play a major role in initiation.