Foot-and-mouth disease virus low-fidelity polymerase mutants are attenuated

Previous studies have shown that RNA viruses can be attenuated by either increased or decreased viral polymerase replication fidelity. Although foot-and-mouth disease virus (FMDV) high-fidelity RNA-dependent RNA polymerase (RdRp) variants with an attenuated phenotype have been isolated using mutagens, no FMDV mutant with a low-fidelity polymerase has been documented to date. Here, we describe the generation of several FMDV RdRp mutants using site-directed mutagenesis via a reverse genetic system. Mutation frequency assays confirmed that five rescued FMDV RdRp mutant populations had lower replication fidelity than the wild-type virus population, which allowed us to assess the effects of the change in replication fidelity on the virus phenotype. These low-fidelity FMDV RdRp mutants showed increased sensitivity to ribavirin or 5-fluorouracil (5-FU) treatment without a loss of growth capacity in cell cultures. In addition, decreased fitness and attenuated virulence were observed for the RdRp mutants with lower fidelity. Importantly, based on a quantitative analysis for fidelity and virulence, we concluded that lower replication fidelity is associated with a more attenuated virus phenotype. These results further contribute to our understanding of the replication fidelity of polymerases of RNA viruses and its relationship to virulence attenuation.     

Virulence of temperature-sensitive mutants of foot-and-mouth disease virus

A number of temperature-sensitive mutants isolated from two strains of foot-and-mouth disease virus were examined for their virulence in suckling mice. The majority of the mutants were found to be less virulent than the parent virus strains, ranging from slight to total attenuation, but two mutants retained parental levels of virulence. There was no correlation between mutant cut-off temperatures and virulence, or the revertant content of mutant preparations and virulence. It was not always possible to regain parental levels of virulence by isolating phenotypic revertants or wild-type recombinants from genetic crosses, but recombinants were significantly more virulent than either of the two input viruses in a genetic cross.     

Isolation and genetic characterization of temperature-sensitive mutants that define five additional recombination groups in simian rotavirus SA11

Nineteen independent temperature-sensitive (ts) mutants were isolated from SA11 following mutagenesis with proflavin or 5-azacytidine. Fourteen of the ts mutants fell into one or another of five mutant groups previously defined by recombination. Five of the ts mutants defined five recombination groups (F, G, H, I, and J) that had not been previously identified. Thus, 10 of the 11 expected mutant groups have been identified in SA11. The prototype mutants of the 10 mutant groups were tested for recombination at nonpermissive temperature to determine if any group had a lesion affecting recombination. Most mutant pairs recombined efficiently; however, the tsH mutant was restricted in its recombination with the tsB and tsI mutants and the tsG and tsJ mutants failed to recombine at detectable levels at nonpermissive temperature. The mutants of groups F-J did not complement, or did so inefficiently, and interfered with the growth of wild type at both permissive and nonpermissive temperatures. The growth properties of the mutants of groups F-J are described.     

Temperature-sensitive mutants of vaccinia virus. I. Isolation and preliminary characterization.

Fifty-seven temperature-sensitive (ts) mutants of a neurotropic variant of vaccinia virus were obtained. A comparison of the effectiveness of 6 different mutagens showed that in in vivo experiments nitrosomethylurea induced 10%, nitrosoguanidine 8%, and bromodeoxyuridine 2.5% ts mutants. In vitro, at the same degree of inactivation, UV irradiation, hydroxylamine and nitrous acid induced 2.5% ts mutants. Out of 49 mutants studied, 27 had a DNA+ phenotype, 19 a DNA+/-and 3 a DNA- phenotype. Under permissive conditions, two thirds of all the mutants tested formed thermolabile virions. In experiments on genetic recombination, the recombination frequency between different pairs was from 2.8 to 57%. Seven of the mutants were plotted on the virus genetic map in a certain sequence.     

Insertion and deletion mutants of vaccinia virus

Thirteen viable insertion mutants of vaccinia virus have been constructed. These mutants, containing coding sequences of the herpes simplex virus thymidine kinase (HSV-TK) gene, were generated by marker transfer via in vivo recombination. The mutants were identified using a replica filter plating technique by in situ hybridization using 32P-nick translated HSV-TK sequences and obtained as pure cultures by repeated plaque purification. Some of these insertion mutants were in turn used as substrates to generate viable deletion mutants of vaccinia virus in the presence of 5'-bromodeoxyuridine. An example of this approach resulting in a vaccinia virus deleted of approximately 1.5 kb of nonessential DNA is presented. Furthermore, the analysis of spontaneously occurring viable deletion mutants of vaccinia lacking approximately 21.4 kb of nonessential DNA is described.     

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.     

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.     

Factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11

A number of factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11 have been examined. (1) Statistical analyses of recombination frequency (RF) indicated that (a) the variability noted in RF was not related to variations in experimental conditions and (b) a linear map of the mutations could not be drawn. (2) The wild phenotype of recombinant progeny was stable on passage. (3) Aggregates of progeny virus or heterozygous progeny virus particles did not contribute significantly to the observed RF. (4) RF increased in parallel with multiplicity of infection. (5) A maximal, or near maximal, RF was obtained at the earliest time significant recombinants could be detected. (6) Recombination was efficient at nonpermissive temperature. (7) Complementation did not occur or was inefficient. (8) Mutants from all recombination groups interfered with the growth of wild-type virus at both permissive and nonpermissive temperatures.     

Selection and characterization of an acid-resistant mutant of serotype O foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.     

Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization.

Forty-nine temperature-sensitive mutants of fowl plague virus (FPV) strain Rostock and four is mutants of FPV-strain Dobson were isolated by utilizing two methods of plaque screening, after either spontaneous or chemically induced mutagenesis. Twenty-nine of the FPV-Rostock mutants were further characterized by genetic recombination studies and were found to fall into six high frequency recombination groups. The genome segment carrying the is mutation in each group was identified by analyzing the gene composition of ts⁺ recombinants generated from crosses between representatives of each group and ts mutants of FPV-Dobson. It was concluded that the six groups correspond to mutations in six different genome segments, namely, those coding for the P₁, P₂, P₃, HA, NP, and NS proteins.     

Isolation and characterization of temperature-sensitive mutants of fowl plague virus.

Twenty-five temperature-sensitive (ts) mutants of fowl plague virus have been isolated and tentatively characterized. Twenty-three of them can be arranged into six recombination groups. The classification of the two other ts mutants is still uncertain. RNA-negative mutants have been found in four recombination groups. One group harbors ts mutants with a lesion in the hemagglutinin gene, another group consists of mutants with a lesion in the neuraminidase gene. One group seems to have a maturation defect. Cells infected with still another group retain the RNA polymerase-template complex within the nucleus. The biological properties of the ts mutants of the various recombination groups suggest that at least some of the gene products might have more than one function.     

Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae)

Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41 degrees C as the nonpermissive temperature and 36 degrees C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus.     

Genetic studies of respiratory syneytial virus temperature-sensitive mutants

Summary Seven temperature-sensitive mutants of RS virus were analyzed genetically for evidence of complementation and recombination. The results of these tests suggested that there were 3 mutually exclusive complementation groups. No evidence of recombination was obtained; however, mixed infection of cells with mutants belonging to 2 different complementation groups yielded viruses which appeared to be complementing heterozygotes.     

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.     

Isolation and genetic characterization of temperature-sensitive mutants of simian rotavirus SA 11

Ten stable temperature-sensitive (ts) mutants have been isolated from the simian rotavirus SA11 following mutagenesis with nitrous acid or hydroxylamine. These mutants define five recombination groups between which recombination occurs at high frequency (>1.0%) and within which recombination does not occur at detectable levels (<0.2%). These observations support the interpretation that recombination in rotaviruses occurs by reassortment of genome segments during mixed infection. Four of the is mutants are members of the mutant group designated A, three is mutants are members of group B, and groups C, D, and E are each represented by single is mutants. The is mutants were unable to efficiently complement during mixed infection. Growth studies indicated that the ts lesions conditionally, and strongly, inhibit both virus yield and plaque formation.     

Characterisation of temperature-sensitive mutants of herpes simplex virus type 2. Growth and DNA synthesis

The growth of 10 temperature-sensitive (ts) mutants of type 2 herpes simplex virus has been studied at permissive (31 °) and restrictive (38 °) temperatures. At 31 °, maximum virus yields were attained by all mutants 18–24 hours after infection—some 8–12 hr later than for wild-type virus. At 38 °, the mutants failed to replicate, although they all adsorbed to cells as efficiently as at 31 °. Five mutants were slightly, but not markedly, more thermolabile than wild-type virus. Three mutants synthesised viral DNA at 38 °, the others did not. One DNA-negative mutant, ts9, did not shut off host cell DNA or histone synthesis at 38 °.     

A segmented form of foot-and-mouth disease virus interferes with standard virus: a link between interference and competitive fitness

Serial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome.     

Electron microscopic studies of respiratory syneytial temperature-sensitive mutants

Summary The A2 strain of respiratory syncytial (RS) virus and several temperature sensitive mutants derived from it were grown at the permissive (32° C) and restrictive temperature (39° C) in HeLa cells and thin sections of these cells were examined by electron microscopy. The mutants selected for this study were representative of 3 different complementation groups. The parent strain underwent the same sequence of morphogenesis at the permissive and restrictive temperatures. Morphogenesis of each of the mutants at the permissive temperature was similar to that of the parent strain. At the restrictive temperature cells infected with mutants belonging to two of the complementation groups developed a significantly reduced amount of one or more of the recognizable viral structures. In one instance (ts-4, complementation group A) budding and release of virus did not occur, while in another (ts-7, complementation group C) no virus specific structures were seen. Cells infected with the ts-2 mutant of complementation group B exhibited normal viral morphogenesis at restrictive temperature.