St. Louis encephalitis virus temperature-sensitive mutants

Nine temperature-sensitive (ts) mutants of St. Louis encephalitis virus were isolated after "forced mutagenesis" with 5-fluorouracil or 5-azacytidine. The ts mutants could be grouped on the basis of RNA synthesis at 40 degrees C, the nonpermissive temperature and complementation analysis. Four complementation groups were identified. Members of two of the groups were negative for RNA synthesis at 40 degrees C while the remainder were positive.     

Principles of complementation interaction of ts mutants of orthomyxoviruses.

The possibility of complementation between ts mutants of fowl plague virus (FPV) belonging to 5 different complementation groups was studied using various time intervals between inoculation of the cells with two complementation partners. The structural proteins of virions formed on complementation of individual ts mutants with wild virus were analysed by polyacrylamide gel electrophoresis after amino acid pulse label followed by pulse chase. The features of complementation interactions between the mutants are discussed.     

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature-sensitive herpes simplex virus type 1 mutants defective in the shutoff of cellular DNA synthesis and host polypeptide synthesis

Two temperature-sensitive herpes simplex virus type 1 mutants, ts 1-8 and ts 199, belonging to different complementation groups, were isolated. Both mutants were defective in the shutoff of host DNA synthesis at 39.5 degrees C (nonpermissive temperature). ts 1-8 exhibited intermediate levels of viral DNA synthesis at 39.5 degrees C, while ts 199 was completely deficient in viral DNA synthesis at 39.5 degrees C. Comparative polyacrylamide gel electrophoresis of the ts 1-8, ts 199 and wild-type viral-coded polypeptides and cellular proteins produced in vivo at 34 degrees C and 39.5 degrees C during various periods post infection was performed. The results indicated that ts 1-8 and ts 199 were temperature-sensitive for the secondary suppression of host polypeptide synthesis. Production of the beta (early) and gamma (late) viral polypeptides was slightly delayed in the mutant-infected cells at early times post infection at both 34 degrees C and 39.5 degrees C. This delayed protein production was not evident at later times post-infection. The ts 1-8 and ts 199 mutants were distinct from the HSV-1 viron-associated host shutoff (vhs) mutants of Read and Frenkel (J. Virol. 46 (1983) 498).     

Temperature-sensitive (ts) mutants of an influenza A virus for which genetic synergism is required to express their ts phenotypes

Temperature-sensitive (ts) mutants of fowl plague virus (FU 67 and ts 7/36) have been isolated. They can be rescued to wild type by double infection with all standard ts mutants having single ts defects in any of the eight RNA segments. Rescue of FU 67 to wild type was not possible when ts mutants having simultaneous mutations in one of the RNA segments coding for the P proteins, the haemagglutinin, and nonstructural protein genes were used. After rescue with various prototype influenza A strains different RNA segments of FU 67 were replaced, depending on the strain used for rescue. The results are compatible with the idea that in FU 67 and ts the ts phenotype is caused by the synergism of mutations in several RNA segments, each of which by itself is not sufficient to exhibit the ts phenotype.     

Chemically-induced temperature sensitive mutants of dengue virus type 2

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.     

Temperature-sensitive mutants of herpes simplex virus type 2: description of three new complementation groups and studies on the inhibition of host cell DNA synthesis.

Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 degrees C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 degrees C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 degrees C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 degrees C) before the shift but which cannot be made at 38 degrees C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.     

Temperature-sensitive mutants of influenza virus: Identification of the loci of the two ts lesions in the Udorn-ts-1A2 donor virus and the correlation of the presence of these two ts lesions with a predictable level of attenuation

The influenza A/Udorn/72-ts-1A2 virus is currently being studied as a donor of its ts lesions to produce recombinants for use in a live virus vaccine. The Udorn/72-ts-1A2 virus, which possesses two ts lesions, was produced by mating two complementing, single-lesion ts mutants. One parent, the Udorn/72-ts-1 [A] clone 189 virus, bears a is lesion that belongs to our complementation-recombination group 1 and is located on RNA segment 1(P. Palese and M. B. Ritchey, 1977, Virology78, 183–191). The second parent, the Hong Kong/68-ts-315 virus, has a is mutation that belongs to our complementation-recombination group 5. The present study sought to determine the location of the complementation group 5 ts mutation. In addition, the RNAs of 6 is-1A2 recombinants bearing the surface antigens of influenza A/Vic/75 (H3N2) virus were analyzed by polyacrylamide gel electrophoresis in order to determine the parental origin of each gene. Finally, by correlating genotype with level of viral replication in the hamster it was possible to identify the genes of the ts-1A2 donor virus responsible for attenuation.     

Complementation with ts mutants of herpes simplex virus.

Tests to detect complementation between ts mutants of herpes simplex virus types 1 and 2 infectious centre and yield of virus assay were investigated. Progeny analysis of both intratypic and intertypic complementation showed a considerable proportion of recombinant or ts+ virus in the progeny; this was more marked in the infectious centre tests. Virus of intermediate temperature-sensitivity was produced in intratypic as well as intertypic complementation. Reduction in the input multiplicity of one of the two mutants in the test to extremely low levels did not prevent complementation, suggesting that non-infectious particles probably contribute to complementation. Demonstration of virus DNA synthesis in mixedly-infected cells at the non-permissive temperature was used to detect complementation between DNA-negative mutants.     

Analysis of ts mutations of cold-adapted influenza A/Leningrad/134/57 virus variants

By recombination of ts mutants of fowl plague virus belonging to different complementation groups with two cold-adapted variants of human influenza virus, the number and gene localization of ts mutations occurring in these variants was determined. In the course of passaging of human influenza virus at lowered temperature, the number of genes with ts mutations increased.     

Ts mutations in the genome of cold-adapted variants of human influenza virus A/Krasnodar/101/59 (H2N2) at different passage levels and their reproduction in lungs of hamsters

Human influenza virus A/Krasnodar/101/59 (H2N2) was passaged in chick fibroblast cultures in the presence of trypsin at suboptimal temperature. The virus which underwent 16 passages at 28 degrees C possessed cold-adapted (ca) and temperature sensitive (ts) phenotypes and formed larger plaques at the optimal temperature (33 degrees C). Its reproduction in the lungs of hamsters was decreased as evidenced by approximately 2.5 log10 lower titres; only one of 9 virus isolates from the lungs of hamsters acquired the ts +/- phenotype, although it had retained a ca phenotype. Recombination of this variant with ts mutants of fowl plague virus (FPV) revealed a ts mutation only in gene 4 of this variant coding for haemagglutinin (HA). The virus which had had 25 passages at 28 degrees C possessed the same properties as the previous variant, but all eight virus isolates from the lungs of hamsters retained the ts phenotype; the genome of this variant contained ts mutations in genes 1, 3, 4, 5 and 6. The mutation found in gene 8 was not a ts mutation. The virus, which underwent 25 passages at 28 degrees C and additional 15 passages at 27 degrees C, formed large plaques and alike to the previous variants it possessed the ca and ts phenotypes; however, its reproduction in the lungs of hamsters was decreased by 4.0 log10 and occurred in the lungs only of 4 out 16 infected animals. This variant contained ts mutations in genes 1, 3, 4, 5, 6 and 7 and a non-ts mutation in gene 8.     

Cellular quiescence modulates and replication of L15 mutants of vesicular stomatitis virus

Infectious particle production by temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) was measured in a variety of different host cell types maintained in a state of quiescence or stimulated to proliferate. At permissive temperatures, all ts mutants and the wild-type virus replicated equally well and with the same kinetics in both quiescent and proliferating cells. At semi-permissive temperatures, however, Lts mutants, with temperature-sensitive virion polymerases, showed a delay of about 6 h in infectious particle production relative to wild-type virus in proliferating cells and greater than 16 h in quiescent cells. The effect was specific for the Lts class of mutants and was not seen for representative mutants in any of the four other complementation groups of VSV. Regarding cellular determinants, the effect was correlated only with the growth phase and not with the species of origin, interferon inducibility or with malignant transformation.     

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.     

Recombination between temperature-sensitive mutants of simian virus 40

Seven temperature-sensitive (ts) SV40 mutants have been isolated and characterized. All of the mutants were defective in a late function. Four of the mutants were assigned to complementation group B, one to a new group designated C and two could not be assigned to a complementation group. Recombination occurred between mutants in the B and C complementation groups and between mutants in the B group. The recombination frequency (RF) between tsB302 and tsB306 was about 2.0 × 10^?4 when virus was used for infection, but was 10-fold higher when infectious ts SV40 DNAs were used. Treatment of doubly infected cells with 1-β-d-arabinofuranosylcytosine (ara-C) to interrupt DNA synthesis increased the RF 3- to 14-fold. Ultraviolet irradiation of viral inocula to 1–5% survival resulted in a 25- to 40-fold increase in RF. However, only a 2-fold increase in RF was obtained when uv-irradiated ts SV40 DNAs were used for infection. Ultraviolet irradiation of host CV-1 cells or pretreatment of host CV-1 cells with nonirradiated or uv-irradiated CV-1 DNA prior to infection failed to increase the RF between tsB302 and tsB306.     

Host dependence of naturally occurring temperature-sensitive influenza A viruses and location of their genetic lesions.

Naturally occurring temperature-sensitive (ts) strains have been found in large number in human influenza A viruses of all subtypes (J. Virol. 41 (1982) 353). Further studies have demonstrated that the ts phenotype of these viruses is host-dependent in that they are highly ts in chick embryos and chick embryonic cells, but are ts+ in MDCK cells. Previous studies have located by complementation tests the ts lesion of two H3N2 viruses (HK/8/68 and Ningxia/01/72, also known as Xia-ts) on the NP gene and that of two H1N1 viruses (Tianjin/78/77 and Beijing/1/79) on the M protein gene. By recombination and polyacrylamide electrophoresis migration of the RNA segments of its ts+ recombinant with PR8, the ts lesion of a later H3N2 virus A/Qi/39/79 has now been located on the M protein gene. The possibility for Qi/39/79 of acquiring the M gene lesion by reassortment with concurrently circulating Tianjin/78/77-like (H1N1) virus which also has ts lesion on the M gene was investigated. In contrast to Tianjin/78/77 (H1N1), however, Qi/39/79 complemented well with ts 51, a WSN ts strain with a single M gene lesion. Qi/39/79 and Tianjin/78/77 also complemented each other. Thus, there are two intra-segmental complementation groups of the M gene: Qi/39/79 belongs to one complementation group, while WSN ts 51 and Tianjin/78/77 belong to another. At present, there is no evidence of reassortment involving the genes concerned in the ts lesions of H3N2 and H1N1 viruses under natural conditions.     

Establishment of persistent infection in BHK-21 cells by temperature-sensitive mutants of Sindbis virus

Twelve temperature-sensitive (ts) mutants of Sindbis were examined for their ability to establish persistent infection in BHK-21 cells at 39 degrees C. Five of these mutants were able to initiate colony formation in infected cultures, which followed an extensive c.p.e. Two of the mutants were able to establish persistent infections which survived beyond the fifth cell passage p.i. The ability to initiate colony formation was correlated with low reversion of the ts mutation, or with ability to interfer with the multiplication of the wild-type virus. Virus released from persistently infected cultures was not temperature-sensitive. The restriction of virus multiplication in persistently infected cells operated prior to virus-specified RNA synthesis. It is concluded that in this system establishment of persistent infection depends on an inhibition of virus multiplication early in infection and occurs in only a small proportion of infected cells.     

Complementation analysis of the dales collection of vaccinia virus temperature-sensitive mutants

A collection of randomly generated temperature-sensitive (ts) vaccinia virus (strain IHD-W) mutants were reported by S. Dales et al., (1978, Virology, 84, 403-428) in 1978 and characterized by electron microscopy. We have performed further genetic analysis on the Dales collection of mutants to make the mutants more useful to the scientific community. We obtained the entire Dales collection, 97 mutants, from the American Type Culture Center (ATCC). All 97 mutants were grown and reassessed for temperature sensitivity. Of these, 16 mutants were either very leaky or showed unacceptably high reversion indices even after plaque purification and therefore were not used for further analysis. The remaining 81 ts mutants were used to perform a complete complementation analysis with each other and the existing Condit collection of ts vaccinia virus (strain WR) mutants. Twenty-two of these 81 Dales mutants were dropped during complementation analysis due to erratic or weak behavior in the complementation test. Of the 59 mutants that were fit for further investigation, 30 fall into 13 of Condit's existing complementation groups, 5 comprise 3 previously identified complementation groups independent of the Condit collection, and 24 comprise 18 new complementation groups. The 59 mutants which were successfully characterized by complementation will be accessioned by and made available to the scientific community through the ATCC.     

Cross-reactive, cell-associated antigen on L929 cells infected with temperature-sensitive mutants of sindbis virus.

Temperature-sensitive (ts) mutants of Sindbis virus (SIN) were used to aid in the identification of alphavirus cross-reactive proteins on the surface of infected cells by antibody-dependent, complement-mediated cytolysis. Antisera prepared in rabbits against purified SIN or Semliki Forest viruses were highly cytotoxic for cells infected with wild-type SIN and for cells infected at the permissive temperature with maturation-defective, ts mutants of SIN belonging to several distinct complementation groups. When these SIN mutants were analyzed by antibody-dependent, complement-mediated cytolysis at the restrictive temperature only cells infected with the SIN mutant of complementation group E, ts20, participated in both homologous (with anti-SIN serum) and heterologous (with anti-Semliki Forest virus serum) antibody-dependent, complement-mediated cytolysis reactions. These data and the known defect of ts20 suggested that the cell-associated viral E1 glycoprotein was a functional target antigen for homologous and cross-immunoreactivity in alphavirus-infected cells. At the restrictive temperature there were quantitative differences in antibody-dependent, complement-mediated cytolysis reactivity of ts20- versus wild type-infected cells consistent with the suggestion that ts20-infected cells do not fully express all of the homologous or the cross-reactive antigenic determinants found in wild-type infection. Additional potential sites for antigenic determinants involved in alphavirus-immune cross-reactivity are discussed in relation to events in virus maturation.