Susan B. Spring

Temperature-sensitive mutants of influenza virus: VI. Transfer of ts lesions from the Asian subtype of influenza a virus (H2N2) to the Hong Kong subtype (H3N2)

Abstract Temperature-sensitive genetic lesions were transferred from the ts -1 (H2N2) and ts -2 (H2N2) mutants of influenza A virus to wild-type influenza A (H3N2) virus by genetic reassortment. The ts -2 (H3N2) recombinants appeared to be homogeneous and did not undergo recombination with one another, suggesting that the original ts -2 mutant of influenza A (H2N2) contained a ts lesion(s) on only one RNA segment of its genome. In contrast the ts -1 (H3N2) recombinants fell into three phenotypic subsets which differed in degree of temperature sensitivity. Initially, the three subsets of ts -1 (H3N2) recombinants were thought to represent distinct complementation-recombination groups. However, complementation-recombination between the three subsets of ts -1 (H3N2) recombinants subsets was variable. Subsequent study indicated that mutants in each of the three subsets shared one ts lesion and two of the subsets shared an additional ts lesion. The mechanism whereby viruses of the three subsets which share one or two ts lesions, and nevertheless undergo apparent complementation-recombination on occasion is not understood.

Temperature-sensitive mutants of influenza: VIII. Genetic and biological characterization of ts mutants of influenza virus A (H3N2) and their assignment to complementation groups

Abstract Fifteen ts mutants of influenza A virus were characterized genetically and assigned to at least seven complementation groups. Six of the mutants were recombinant viruses while nine were clones derived directly from mutagenized H3N2 virus. The genetic analysis used a complementation-recombination assay carried out directly on the tissue culture monolayer. The nine clones of virus derived directly from mutagenized H3N2 virus were characterized in vivo in hamsters with respect to their ability to replicate in the lungs and nasal turbinates and with respect to the appearance of phenotypically wild-type virus in those organs of the hamster.

Cold adapted variants of influenza A

SummaryThe genetic and biological properties of 13 recombinant influenza A clones derived at 25°C from the A/AA/6/60-cold variant (by crosses with 4 different wild type strains) were compared with a set of 5-FU inducedts-mutants. The 5-FU mutants had previously been placed into 7 complementation-recombination groups; the A/AA/6/60-cold parent (PI-7) and the 12 cold recombinant clones which werets were shown to share a lesion with only one of these groups. The parental strain and 5 recombinant clones were evaluated for replication in the lungs and nasal turbinates of hamsters. Each virus appeared to be attenuated; genetic stability correlated with the level of viral replication in the hamster lung, i.e., viruses which grew best showed a tendency to revert to thets+ phenotype. Characterization of thets+ revertants for the presence of the cold adaptation property revealed that these viruses exhibited a spectrum of cold adaptation properties. Two viruses, PI-7 (the parental cold variant) and the CR6 recombinant (A/Queensland/6/72) did not revert in either the lungs or nasal turbinates of hamsters.

Use of temperature-sensitive mutants of influenza A virus as live virus vaccine strains. Evaluation in laboratory animals, adults and children.

Temperature-sensitive (ts) recombinants of influenza A virus were evaluated for use in a live virus vaccine. Evidence from several sources suggested that the ts lesions were responsible for attenuation of these mutants. Specification of attenuation by defined genetic lesions which can be assayed for in the laboratory offers an advantage to the use of ts viruses for vaccination. This means that ts recombinants can be assessed for genetic stability during vaccine development, production and later during usage in man. One ts virus, influenza A/Hong Kong/68-ts-1[E], with a 38°C shut-off temperature, had the following properties desirable for a live virus vaccine: (1) satisfactory infectivity for seronegative (serum HI antibody titre ≤ 1:8) adults; (2) satisfactory attenuation for adults; (3) capacity to stimulate local and serum anti-haemagglutinin and anti-neuraminidase antibodies in seronegative volunteers; (4) stimulation of resistance to virulent, wild type virus; (5) relative genetic stability in vivo; (6) lack of communicability in man; (7) replication to high titre in avian leucosis virus-free eggs; and (8) localization of ts lesions to genes that do not code for the haemagglutinin and neuraminidase. The ts lesions of influenza A/Hong Kong/68-ts-1[E] virus were transferred to more current viruses within the H3N2 subtype (influenza A/Udorn/307/72 and influenza A/Georgia/101/74). These recombinant Udorn/72 and Georgia/74 ts viruses, which possessed the same shut-off temperature and the same ts lesions as the influenza A/Hong Kong/68-ts-1[E] parent virus, exhibited a pattern of infection and attenuation in hamsters and man similar to their ts parent. These data suggest that ts mutants which are sufficiently attenuated for man, could serve as donors of ts lesions for the rapid production of an attenuated vaccine when new antigenic variants arise. When the influenza A/Hong Kong/68-ts-1[E] virus was administered to children who lacked both anti-haemagglutinin and anti-neuraminidase antibody the virus replicated for a longer period than in adults and mild fever developed in some of the young vaccinees. A minority of children shed wild type revertant virus. The emergence of wild type virus in children, but not in adults, probably reflected the more extensive replication of the virus in the doubly seronegative children. The implications of these findings to the development and testing of live influenza A virus vaccines were discussed.

Temperature-sensitive mutants of influenza virus XV. The genetic and biological characterization of a recombinant influenza virus containing two is lesions produced by mating two complementing, single lesion is mutants

Abstract The influenza A/Hong Kong/68-ts-1[E] virus and its recombinants (38° shutoff temperature for plaque formation) were satisfactorily attenuated for individuals with neuraminidase immunity who lacked serum antibody for hemagglutinin antigen but not for persons who lacked immunity to both viral surface antigens, i.e., doubly seronegative individuals. It was necessary to produce a virus more defective than the Hong Kong/68-ts-1[E] virus to meet the need for an attenuated virus suitable for use in doubly seronegative persons. We produced a new recombinant which contained a ts mutation(s) on the genes believed to code for the Pl and P3 viral proteins, both of which are involved in cRNA synthesis. This recombinant was produced by mating two complementing ts mutants, one with a mutation affecting the Pl protein and the other with a mutation affecting the P3 protein. The new double ts recombinant, the Udorn/72-ts-1A2 virus, was 700-fold more restricted in plaque formation at 37° than either of its ts parents. In addition, it was defective in complementation-recombination when tested against prototypes of seven complementation groups. The Udorn/72-ts-1A2 recombinant failed to replicate in the hamsters lungs (37°) and was 100-fold restricted in its growth in the nasal turbinates (32°). Each of the isolates from Udorn/72-ts-1A2 infected hamsters was ts. Despite restricted replication in vivo, infection of hamsters with this virus induced significant resistance to wild-type virus challenge. Thus, the Udorn/72-ts-1A2 recombinant was (1) more restricted in replication in vitro at 37° and in vivo in the hamster and (2) more stable genetically in vivo than the HK/68-ts-1[E] virus.

Cold-Adapted Variants of Influenza Virus A I. Comparison of the Genetic Properties of ts Mutants and Five Cold-Adapted Variants of Influenza Virus A

Abstract The genetic properties of seven cold-adapted variants of influenza virus A were compared with those of nine 5-fluorouracil (5-FU)-induced ts mutants. The 5-FU mutants had previously been placed into seven complementation-recombination groups; five of the seven cold-adapted variants also had the ts phenotype, and all five were shown to share the group 1 lesion. Three of the cold variants also had additional ts lesions.

Comparative Studies of Wild-Type and ‘Cold-Mutant’ (Temperature Sensitive) Influenza Viruses: Genealogy of the Matrix (M) and Non-structural (NS) Proteins in Recombinant Cold-Adapted H3N2 Viruses

The matrix (M) protein of the H2N2 virus A/Ann Arbor/6/60 may be distinguished from M protein of several H3N2 viruses and A/New Jersey/76 (HSWINI) by SDS acrylamide gel electrophoresis using a discontinuous buffer system. The smallest RNA (RNA 8) of the A/Ann Arbor/6/60 virus may be distinguished from RNA 8 of several H3N2 viruses by acrylamide gel electrophoresis in 3% or 3-6% gels in the absence of urea, if electrophoresis is done at 30 to 36 degrees C or 20 degrees C respectively. Ten clones of conditionally-lethal temperature-sensitive (ts) mutants were studied, which derived their cold-adaption and ts genes from mutant A/Ann Arbor/6/60, and their haemagglutinin from the H3N2 virus A/Scotland/840/74. Each clone was found to derive its M protein from A/Ann Arbor/6/60 mutant, and its RNA 8 from A/Scotland/840/74. The only assignment of genes 7 and 8 consistent with these findings for the recombinants is that in each parent virus (and in the recombinants) gene 7 codes for M protein, and gene 8 for NS protein. Furthermore, it may be concluded from the results that the biologically important ts lesions in the A/Ann Arbor/6/60 mutant parent are not present in the NS gene. In addition to the recombinants of A/Ann Arbor/6/60 and A/Scotland/840/74, five independent ts/cold-adapted recombinants of A/Ann Arbor/6/60 mutant with H3N2 and HSWINI wild-type viruses were examined, and all were found to contain the M protein of the A/Ann Arbor/6/60 mutant parent. This is suggestive that M protein may be at least partially responsible for the cold-adaptation and/or ts properties of the A/Ann Arbor/6/60 mutant and the recombinants.

Temperature-sensitive mutants of influenza virus: VII. Transfer of the ts-1[E] lesions to a wild-type influenza A virus with the HON1 surface antigens

Abstract The nature of the genetic defect(s) present in the influenza A/Hong Kong/1968-ts-1[E] (H3N2) candidate vaccine virus, was analyzed by transferring them by genetic recombination to a 1943 HON1 wild-type virus. Three classes of ts viruses bearing the HO hemagglutinin were identified. One class consisted of two clones of virus, R1 and R8, which underwent complementation-recombination with each other but not with the Hong Kong/1968 (H3N2)-ts-1[E] parent virus. The second class consisted of clones of virus, such as R4 and R11, that failed to undergo complementation-recombination with R1, with R8, or with the ts-1[E] parent. These data indicated that the parent Hong Kong ts-1[E] virus possessed two ts lesions that segregated independently of each other and were presumably on different segments of the influenza A virus genome. The third class of ts mutants consisted of a spontaneously occurring ts mutant, clone R9; this clone underwent complementation-recombination with the ts-1[E] parent and other HO-ts recombinants and was shown to belong to a complementation group different than clones R1 and R8. The ts defects present in the Hong Kong-ts-1[E] donor virus segregated independently of the genes that coded for the epidemiologically important surface antigens, i.e., the hemagglutinin and the neuraminidase glycoproteins. The implications of these findings for the development of a live attenuated influenza virus vaccine are discussed.

Localization of the TS defect in two TS mutants of influenza A virus: Evidence for the occurrence of intracistronic complementation between TS mutants of influenza A virus coding for the neuraminidase and nucleoprotein polypeptides

Abstract Temperature-sensitive mutants of influenza A/HK/68 belonging to seven complementation-recombination groups were isolated previously and analyzed genetically. Two groups, numbers 1 and 2, were characterized as defective in the P3 and NP polypeptides, respectively. The defects in HK group 3 (prototype ts -2C) and group 4 (prototype ts -304) were studied by preparing ts recombinants of the HK mutants and wild-type influenza A/WSN virus. An analysis of the genotype of ts recombinants by polyacrylamide gel electrophoresis of virion RNA indicated that the locus of the ts mutation in group 3 was on the RNA segment coding for the nucleoprotein, while the group 4 ts mutation was located on the gene coding for the neuraminidase. The group 3 and 4 HK prototypes each underwent genetic interaction with WSN ts mutants bearing a ts lesion on the corresponding RNA segment, suggesting that intracistronic complementation can occur between mutants with defects in the multimeric neuraminidase or the multifunctional nucleoprotein.

Enzymatic cleavage of a glycoprotein of respiratory syncytial virus

SummaryThe 75K glycoprotein of the A2 strain of respiratory syncytial virus was cleaved by digestion with trypsin orStaphylococcus aureus protease V8. The fragments resulting from trypsin digestion were 40K and 29K; those from the Staphylococcal protease were 49K and 37K.