Temperature sensitivity of interferon susceptibility in L cells persistently infected with hemagglutinating virus of Japan (HVJ).

The present study shows that L cells persistently infected with HVJ (L-HVJ_ts cells) are less susceptible to the antiviral action of interferon as compared with the uninfected cells. L-HVJ_ts cells are also less susceptible to the anticellular action of interferon. It is noteworthy that the cells, when incubated at 38°, become fully susceptible to interferon at the expense of both hemadsorbing and cell-associated hemagglutinating activities. These findings suggest that lowered interferon susceptibility observed in the virus-carrier cells may be related to the virus maturation in the cells.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Induction of interferon by temperature-sensitive mutants of Newcastle disease virus

Spontaneously-selected and mutagen-induced temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were used to study interferon induction in chick embryo (CE) cells at temperatures permissive (37°) and nonpermissive (42°) for virus replication. Both infectious and UV-irradiated virus were tested for interferon-inducing ability in cells pretreated or not pretreated with homologous interferon. At 37°, only UV-irradiated NDV was capable of inducing interferon in cells not treated with interferon before infection. In cells pretreated with interferon, on the other hand, both unirradiated and UV-irradiated virus stimulated the production of interferon. At 42°, the interferon-inducing phenotype for some UV-irradiated ts mutants was dependent on whether or not cells were pretreated with interferon. For example, out of 10 mutants examined, one UV-irradiated ts mutant induced interferon in both untreated and interferon pretreated cells; 7 mutants failed to induce in untreated cells but induced from 25–100% of the wild-type level of interferon in cells pretreated with interferon; and two mutants failed to induce interferon in both types of cells. In addition, one mutant (NDV₀ts-100) induced low or undetectable levels of interferon at both 37° and 42°, conditions under which wild-type virus (NDV₀) produced significant levels of interferon. Co-infection of cells with UV-irradiated ts-100 and a preparation of NDV₀ exposed to prolonged irradiation resulted in considerable production of interferon. These results suggest the possibility that more than one virus function may be involved in interferon induction by NDV in CE cells.     

Host range mutants of an influenza A virus

Summary Temperature-sensitive(ts) mutants of fowl plague virus with ats-lesion in segment 1 (ts 3, polymerase 1 gene) or segment 2 (ts 90, transport gene) do not form plaques on MDCK cells at the permissive temperature, while the wild type andts-mutants of other groups are able to do so. This property is correlated with thets-lesion, since revertants for thets-lesion ofts 3 andts 90 again form plaques on MDCK cells. The block on MDCK cells—at least forts 3—may be located in a late function, since viral RNA polymerase and hemagglutinin are formed in almost normal yields. MDCK cells infected withts 3 orts 90 exhibit a retarded cytopathic effect at 33° C, but no cytopathic effect at 39° C, at which temperature the infected cells can be passaged and super-infected with the wild type strain. Cells surviving the infection withts 90 at 33° C sometimes grow out again to a normal monolayer. It is suggested that the spread of virus is inhibited under these conditions.With 1 Figure     

Reconstitution of herpes simplex virus type 1 ribonucleotide reductase activity from the large and small subunits

An assay for the presence of functional large (RR₁) and small (RR₂) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR₁, and ts1222, which has a lesion in RR₂. In cells infected with ts1207 at 39.5°C, the defective RR₁ is unable to associate with RR₂ to form an active enzyme, and, as a result, a pool of functional RR₂ and defective RR₁ accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31°C or 39.5°C accumulate a pool of functional RR₁, but do not contain detectable RR₂. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5°C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.     

Isolation and characterization of further defective clones of a temperature sensitive mutant (ts-1) of respiratory syncytial virus

Summary After exposure of the temperature sensitivets-1 mutant of respiratory syncytial virus to the chemical mutagen, nitrosoguanidine (NG), 2 clones of virus were recovered which were more temperature sensitive and stable genetically than thets-1 mutant. The initial criterion used for selection of the 2 clones was decreased ability to produce plaques at 36° C. Subsequently it was shown that the 2 clones grew less well at the restrictive temperatures of 37° and 38° C than did thets-1 parent. Peak titers of the NG derived clones were decreased 10–30 told at 37° C and over 100-fold at 38° C compared tots-1. Complementation analysis indicated that the NG mutants retained the same complementation pattern as thets-1 parent.With 2 Figures     

L929 cells infected with temperature sensitive mutants of vesicular stomatitis virus: virus replication is necessary for induction of changes in membrane permeability

Summary Infection of L₉₂₉ murine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were infected with each of fivets mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 °C) and non-permissive temperatures (39 °C). Protein synthesis in the presence and absence of Hygromycin B (Hyg.B) was analyzed during virus infection via incorporation of³⁵S-methionine in acid-precipitable material and SDS-polyacrylamide gel electrophoresis.Data indicate that mutants belonging to groups I (L protein), II (NS protein) and IV (N protein) do not inhibit host protein synthesis and do not induce any membrane changes when grown at the non-permissive temperature. Mutants of group III (M protein) and V (G protein), instead, do inhibit cell protein synthesis and induce membrane changes also when grown at the non-permissive temperature; this suggests that these effects do not correlate with the biological activity of these proteins and their interaction with the cellular membrane. On the other hand, mutants exhibiting defective steps of nucleocapsid replication are apparently unable to induce these effects once more suggesting that virus replication per se is essential, as also indirectly shown by experiments employing cycloheximide to mimic shut-off.This work was supported in part by grant from Consiglio Nazionale delle Ricerche, Rome, Italy, No. 85.00842.04 (Gruppo di Virologia).     

Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Temperature-sensitive mutants of influenza virus

Summary Six temperature-sensitive (ts) mutants were isolated from the progeny of wildtype influenza A virus grown in the presence of 5-fluorouracil. All mutants had the efficiency of plating lower than 10^–4 at 38° C compared with 31° C. One mutant (ts- 5) failed to produce hemagglutinin at the nonpermissive temperature. Temperature-shift experiments revealed that the temperature-sensitive step ofts-5 resides in the process normally taking place at 4–6 hours post infection. After mixed infection ofts-5 andts-9, different from each other in physiological defect, the occurrence of both complementation and recombination was demonstrated, confirming that they carry genetic defects at different genes.     

A temperature-sensitive mutant of western equine encephalitis virus with an altered envelope protein E1 and a defect in the transport of envelope glycoproteins

A temperature-sensitive (ts) mutant, ts-39, of western equine encephalitis virus had been shown to have a defect in hemagglutinating activity. The hemagglutinating protein E1 synthesized in ts-39-infected cells migrated faster than that of wild type of SDS-polyacrylamide gel electrophoresis. The altered E1 protein (designated as El¹) was synthesized in cells infected with the mutant even at the permissive temperature and incorporated into mature virus. In addition to E1¹, the B protein synthesized in ts-39-infected cells appeared to migrate faster than that made during wild type infection. This suggests that the altered mobility of E1¹ protein is not due to an altered cleavage from its precursor. The difference in electrophoretic mobility was not ascribed to incomplete or altered glycosylation since the unglycosylated form of E1¹ synthesized in the presence of tunicamycin also had a greater mobility compared with that of E1. In peptide mapping analyses, we could not detect any deleted peptide spots in the maps of E1¹ protein as compared with those of E1 protein.Ten ts+ revertants of the mutant were isolated from plaques formed at the restrictive temperature. The revertants occurred at a frequency of about 10^?6 and restored the thermostability of hemagglutinating activity as well. All of the revertants synthesized E1 and B proteins indistinguishable from those of wild type virus, suggesting that the alteration of E1 protein is genetically linked to the is lesion of the mutant. E1¹ protein could be immunoprecipitated with antiserum for purified E1 protein. Immunofluorescence studies showed that in the cells infected with ts-39 at the restrictive temperature E1¹ protein was not transported to the cell surface; neither was PE2 (or E2) protein. When the cultures were shifted to the permissive temperature, those glycoproteins came to reach the cell surface. Thus ts-39 virus was shown to have a reversible defect in the transport of the envelope glycoproteins.     

Temperature-sensitive mutants of influenza A virus. Transfer of the twots-1 A 2ts lesions present in the Udorn/72-ts-1 A 2 donor virus to the influenza A/Alaska/6/77 (H 3 N 2) wild type virus

Summary The Udorn/72-ts-1 A 2 temperature-sensitive influenza A virus has a 37° C shutoff temperature and ats mutation on the genes coding for the P1 and P3 proteins. Thists donor virus was produced with the expectation that the transfer of its twots genes would regularly and predictably attenuate each new variant of influenza A virus. It had previously been mated with the A/Victoria/75 (H 3 N 2) virus and five Vic/75-ts-1 A 2 recombinants were isolated that had bothts-1 A 2ts genes andin vitro andin vivo genetic and biological properties similar to their Udorn/72-ts-1 A 2 parent. The present study was designed to determine if the acquisition of the twots-1 A 2ts genes would also confer a specific level of attenuation on the influenza A/Alaska/6/77 (H 3 N 2) wild type virus.Fifteen recombinant Alaska/77-ts-1 A 2 viruses were isolated and characterized genetically for the number and location ofts mutations. These clones were also studied for their level of replication and genetic stability in hamsters. Four recombinants possessed both of thets-1 A 2 mutations and had a 37° C shutoff temperature for plaque formation. Two recombinants possessed only ats P1 gene and had either a 38° C or 39° C shutoff temperature. The remaining nine clones had thets P3 gene and a shutoff temperature of 37° C, 38° C or 39° C.Each of the four 37° C shutoff temperature recombinants that possessed bothts P1 and P3 genes were restricted at least 10,000-fold in replication in the hamster's lung and approximately 100-fold in the nasal turbinates compared to the level of replication of wild type virus in these sites. All isolates from these animals retained thets phenotype. The level of replicationin vivo of thets P1 and P3 segregants was related to their shutoff temperature of plaque formationin vitro, e.g. the 38° Cts P3 segregant was less restricted in replication in the lungs than a 37° Cts P3 segregant. All isolates from animals infected with thets P3 segregants werets, whereas a low level of genetic instability was detected for one of thets P1 segregants. Since ten independentts-1 A 2 recombinants (one Udorn/72, 5 Victoria/75 and 4 Alaska/77) with bothts-1 A 2 mutations exhibited the same genetic and biologic properties, it is likely that thesets P1 and P3 genes were the prime determinants of attenuation and could effect a similar level of attenuation in other influenza A viruses within the H 3 N 2 subtype.With 2 FiguresThe work of J. G. M. constitutes partial fulfillment of the requirements for a Doctor of Philosophy degree from George Washington University, Washington, D. C.     

Viral interference phenomena induced by foot-and-mouth disease temperature-sensitive mutants in bovine kidney cells

Summary Cultures of bovine kidney (BK) cells infected with temperature-sensitive(ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5°C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when super-infected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of thets mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by thets mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected withts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. Thets mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of tsBN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [³H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5°. However, when higher (41°–42°) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of tsBN75 shifted to 42° at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When tsBN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5° C), the restart of DNA synthesis preceeded the appearance of mitotic cells. These data suggest that the tsdefect of tsBN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Comparative biological characterization of mouse adenovirus strains FL and K 87 and seroprevalence in laboratory rodents

Summary The growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37° C in liquid medium. The K 87 strain was completely inactivated after 5–15 minutes at 56° C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony.With 2 Figures     

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ.

This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.     

The effect of temperature on production and function of bovine interferons

Summary Subnormal temperatures were found to depress the production of interferon by bovid herpesvirus 2 (BHV-2)-infected bovine testicular cells, bovine peripheral blood leukocytes, and bovine monocytes, as well as by BHV-2 antigen-stimulated immune peripheral blood leukocytes. Interferon titers generated at 30° C were approximately 10 percent of those at 40° C. Also, subnormal temperatures depressed interferon function. Bovine testicular cells treated at 40° C for 24 hours with high concentrations of BHV-2-induced bovine monocyte interferon or BHV-2 antigen-stimulated immune peripheral blood leukocyte interferon, and then infected with BHV-2 and retreated with interferon at 40° C, had little or no viral growth or cytopathic effect after 72 hours. Cultures without interferon, or those treated with the same amount of interferon at 30° C, had significantly more cytopathic effect and had viral titiers up to 10⁷ TCID₅₀ higher than cultures at 40° C. Earlierin vitro studies done without exogenous interferon showed that BHV-2 replicated to high titers at 30° but not at 40° C. Thus, at low temperatures (30° C)in vitro, BHV-2 induced little interferon, was not highly suppressed by interferon, and replicated to high titers. At higher temperatures (40° C), BHV-2 replication induced high interferon levels, was strongly suppressed by interferon, and replicated poorly. This may help explain the restriction of BHV-2 lesions to skin despite the presence of virus in both skin and internal organs in infected cattle.With 5 Figures     

Temperature-sensitive mutants of influenza A virus: Evaluation of the Alaska/77-ts-1 A 2 temperature-sensitive recombinant virus in seronegative adult volunteers

Summary An influenza A virus recombinant bearing the surface antigens of the A/Alaska/6/77 (H 3 N 2) wild type virus and the twots genes of the A/Udorn/72-ts-1 A 2 (H 3 N 2) virus was evaluated for attenuation, antigenicity, and transmissibility in 28 adult volunteers all of whom possessed a preinoculation serum hemagglutination-inhibiting (HAI) antibody titer of 1:8 and 18 of whom also possessed a serum neuraminidase-inhibiting (NI) antibody titer of 1:4. The Alaska/77-ts-1 A 2 recombinant, which had a 37° C shutoff temperature for plaque formation andts mutations on the genes thought to code for the P1 and P3 polymerase proteins, infected 71 percent of the vaccinees when administered at a dose of 10^6.5 TCID₅₀. Only 3 percent of the vaccinees developed symptoms in contrast to 50 percent of volunteers who received 10^4.2 TCID₅₀ of wild type virus. Vaccinees shed virus for a shorter interval and at a lower titer than the volunteers who received wild type virus. Eachts-1 A 2 isolate retained thets phenotype indicating that the recombinant was stable genetically in seronegative adults. An immunological response, as measured by a rise in serum HAI and/or NI antibody, was detected in 71 percent of the vaccinees and 87 percent of the recipients of wild type virus. Transmission of vaccine virus to susceptible contacts was not observed. The twots-1 A 2ts genes have now been transferred to two variants within the H 3 N 2 subtype, the Vic/75 and Alaska/77 viruses, and have rendered the viruses satisfactorily attenuated for adults. The level of infectivity of the Alaska/77-ts-1 A 2 virus appeared to be low, however.This work supported in part by contracts N01-A 1-42553 and N01-A1-22503 sponsored by the National Institute of Allergy and Infectious Diseases.     

Effects of freezing on African horse-sickness virus

Summary The infectivity of both neurotropic and viscerotropic African horsesickness virus decreased markedly when the viruses, suspended either in tissue-culture medium or phosphate buffered saline, were stored at temperatures between –20° C and –30° C. Using infectious tissue-culture fluids, the inactivation curves of the virus at –22° C and –30° C were compared. During the first 7 days' storage, 4.7 and 3.9 log units of infectivity were lost at the respective temperatures. It was established that salts such as NaCl, CaCl₂ and MgCl₂ contained in the solutions, were chiefly responsible for the inactivation of the virus. Without these salts, AHS virus was rather stable at –20° C. Infectivity of AHS virus was protected by adding approximately 5% lactose, sucrose, or glucose to the suspension before freezing at –20° C to –30° C. Glycerin, polyvinylpyrolidone, and a high concentration of serum also protected the virus infectivity. AHS virus was stable at –70° C even in the presence of salts.This work was undertaken at the Near East Animal Health Institute, a project established by the United Nations Development Program Special Fund through the Food and Agriculture Organization in cooperation with the Ministry of Agriculture of Iran.A summary of this work was reported at the 52nd Annual Meeting of the Federation of American Societies for Experimental Biology, April 15–20, 1968, Atlantic City, New Jersey.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) III. Involvement of SV40 gene A in the expression of TrAg in permissive cells.

Temperature-sensitive (ts) mutants of simian virus (SV40) which map in the early region of the SV40 genome were used to determine the role of the viral genome in the expression of SV40-specific transplantation rejection antigen (TrAg). The results indicated that tsA mutants (1612, 1637, 7, and 28) did not induce the expression of SV40-TrAg at the surface of infected permissive African green monkey kidney cells (TC-7) at 41° but did induce the expression of TrAg at the permissive temperature (33°) in TC-7 cells. Wild-type SV40 and late SV40 temperature-sensitive mutants (tsBC1602, tsBC1606, tsB8, and tsC219) induced SV40-TrAg in TC-7 cells at nonpermissive and permissive temperatures with equal efficiency. One of the mutants belonging to complementation group D (tsD1601) was defective in inducing SV40-TrAg at 41°. Kinetic studies indicated that SV40-TrAg appears by 18 hr after infection at 41° and 38 hr post-infection at 33°, paralleling closely the synthesis of T antigen. The synthesis of immunoreactive T antigen in TC-7 cells infected with tsA mutants at nonpermissive temperature did not correlate with the inability of tsA mutants to express TrAg at nonpermissive temperature. We conclude that the expression of TrAg in SV40-infected cells depends upon normal functioning of the A gene.     

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.