Genetic stability of cold-adapted A/Leningrad/134/47/57 (H2N2) influenza virus: sequence analysis of live cold-adapted reassortant vaccine strains before and after replication in children.

We previously reported that the A/Leningrad/134/47/57 (H2N2) cold-adapted virus (A/Len/47) used in preparing reassortant live attenuated vaccines for children acquired 14 (11 coding) mutations in genes coding for proteins other than haemagglutinin and neuraminidase during cold-adaptation. Preservation of these mutations in genomes of viruses isolated from children on the second, fifth, or eighth day after vaccination was examined by sequence analysis. The sequence data demonstrated that all nine coding mutations selected for examination were conserved in the genomes of all 11 strains investigated, indicating that the mutations accompanying cold-adaptation and attenuation of the A/Len/47 master vaccine are highly stable.

[ts-Mutations in the genomes of cold-adapted variants of influenza A/Hong Kong/1/68(H3N2) and A/Victoria/35/72(H3N2) viruses]. / ts-Mutatsii v genome kholodadaptirovannykh variantov virusov grippa A/Honkong/1/68(H3N2) i A/Viktoriia/35/72(H3N2).

The phenotype and localization of ts mutations in genomes of the influenza A/Victoria/30-ir (A/Vic/30-ir) and A/Hong Kong/17-ir (A/HK/17-ir) cold-adapted (ca) viruses were studied. Using the recombination analysis in chick embryo fibroblasts (CEF) we determined that influenza A/HK/17-ir ca virus carries ts mutations in three "internal" genes, i.e., PB1, NP and M, and influenza A/Vic/30-ir ca virus carries ones in four genes, i.e., PA, NP, M and NS. We have revealed ts mutations for NA gene in none of these viruses. Prior to the analysis of ts mutations in HA gene of influenza A/HK/17-ir and A/Vic/30-ir ca viruses, three cloning steps were performed in chick embryos (CE) by the method of limiting dilutions at 34 degrees C followed by selection of some strains with the most prominent ts phenotype. The cloned strains with such phenotypes were shown to repeat stable results within the recombination analysis in CE, i.e., none from the cloned strains of A/HK/17-ir ca virus recombined in CE at 40 degrees C with the 46 ts mutant, while recombination of this mutant with the cloned A/Vic/30-ir ca strains led to formation of the ts progeny. Thereafter our data result in conclusion that ts mutations in the PA gene must lead to some insignificant contribution for the expression of general ts phenotype among the ca strains as far as this sign is clearly displayed by both viruses, although only one of them, i.e., A/HK/17-ir carries ts mutation in the HA gene.

[Role of individual genes in expression of phenotypic properties of the cold-adapted strain A/Leningrad/134/47/57 (H2N2)-a donor of attenuation of live influenza vaccine]. / Rol' otdel'nykh genov v proiavlenii fenotipicheskikh svoistv kholodoadaptirovannogo shtamma A/Leningrad/134/47/57 (H2N2)-- donora attenuatsii zhivykh grippoznykh vaktsin.

The influenza A/Leningrad/134/47/57 (H2N2) (A/Len/47) cold-adapted virus expresses the ability to reproduce at 25 degrees C (the ca phenotype) and inability to reproduce at 40 degrees C (the ts phenotype). It was attenuated for mice. Reassortants of this donor virus with the genes coding for the surface glycoproteins from the epidemic viruses, i.e. hemagglutinin (HA) and neuraminidase (NA), have been shown to be attenuated, immunogenic and genetically stable. We made attempts to reveal the influence of individual genes from the A/Len/47 ca virus on the expression of some phenotypic properties. Different "single-gene" reassortants were created and investigated using the influenza A/PR/8/34 (H1N1) virus as another parent with the opposite phenotypic properties, i.e. lack of ca+ and ts+ phenotypes and high virulence for mice. We managed to obtain "single-gene" reassortants with PB1, NA and NS genes at this stage of the work. None of them (probably including the M gene as well) could determine the ca or ts phenotypes, nor could the presence of the NA and NS genes from the strain A/Len/47 influence these properties. However, our findings show that the combined influence (synergism) of the PB1 and NS genes from the ca donors results in the ts phenotype of the reassortants. Significant role of the NS gene for attenuation of influenza viruses in mice has been revealed.

[The evaluation of the degree of attenuation of cold-adapted influenza A virus strains in CBA-strain mouse and Syrian hamster models]. / Otsenka stepeni attenuatsii kholodo-adaptirovannykh shtammov virusa grippa A na modeliakh myshei linii CBA i siriiskikh khomiachkov.

The pattern of the infectious process induced by the epidemic A/Leningrad/134/57 (H2N2) virus and its cold-adapted (CA) variants in CBA mice and Syrian hamsters was studied. The strains under study inoculated into the animals under a mild ether anesthesia differed by virulence, reproductive capacity in the nasopharynx, trachea and lungs, as well as by the isolation rate from extrarespiratory organs of both mice and hamsters. Upon intranasal inoculation of mice without anesthesia, the CA strains were found to be incapable of dissemination into the lower parts of the respiratory tract with distinguished these viruses from the original epidemic strain A/Leningrad/134/57 as well as from the mouse-adapted strain A/PR/8/34 (H1N1) used as control. The experimental results show that both models are suitable for laboratory evaluation of the attenuation degree of human influenza viruses.

[The selection of cold-adapted variants of the influenza viruses H1N1 and H3N2 and their antigenic and genetic characteristics]. / Selektsiia kholdoadaptirovannykh variantov virusov grippa H1N1 i H3N2, ikh antigennaia i geneticheskaia kharakteristika.

As a result of serial passages (42 passages) at low temperatures (26 degrees--28 degrees C) of two influenza H1N1 and H3N2 virus strains stable cold-adapted (ca) variants were produced. Investigations of them showed the ca A/USSR/03/84 (H1N1) variant to have ts-mutations in genes 1, 2, 4, 5, 7, and 8 and the ca A/USSR/215/79 (H3N2) to have ts-mutations in genes 1, 3, 4, 5, 7 and 8. These ca-variants may be recommended as attenuation donors to be used in recombination experiments with epidemic influenza viruses in order to obtain attenuated reassortant candidate vaccine strains.

[Mutations in the genes coding for hemagglutinin and neuraminidase in cold-adapted variants of the influenza virus A/Leningrad/134/57 (H2N2)]. / Mutatsii v genakh, kodiruiushchikh gemaggliutinin i neiraminidazu, u kholodoadaptirovannykh variantov virusa grippa A/Leningrad/134/157 (H2N2).

An analysis of ts-mutations in the genomes of native and cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus based on the use of fowl plague virus ts mutants was carried out. The recombination test was done by the conventional method in chick embryo fibroblast culture (genes PB2, PB1, PA, NP, NA, M and NS) or cell systems permissive for reproduction of human influenza virus (gene HA). The cold-adapted strain A/Len/17 used for preparation of live influenza vaccine (LIV) for adults was shown to have 4 ts mutations: three in "internal" genes (PB2, NP, and M) and one in gene 4 coding for hemagglutinin (HA). The more attenuated cold-adapted donor A/Len/47 for preparation of similar LIV for children acquired three additional ts mutations: two (PB1 and NS) in "internal" genes and one in gene 6 coding for neuraminidase (NA). The accumulation of ts mutations in the genome of cold-adapter strains was found to be accompanied by a decrease in their pneumotropicity for mice as well as their detectability in different organs of these animals.

[The ts phenotype of reisolates from children inoculated with live cold-adapted influenza vaccine type A]. / ts-fenotip reizoliatov ot detei, privitykh zhivoi kholodoadaptirovannoi grippoznoi vaktsinoi tipa A.

Using mutants of fowl plague virus (FRV) which have single temperature-sensitive (ts) mutations in some genes, an analysis was carried out on reisolates from children of 3-6 years, vaccinated with a monovaccine from recombinant strains of influenza type A virus. The recombinants were obtained by crossing of current epidemic strains of subtypes A (HINI) and a (H3N2) with the cold-adapted (XA) ts-donor of attenuation A/Leningrad/134/47/57 (H2N2) from which they, as a rule, inherited 5 ts-mutations in genes 1 (PB2), 2 (PB1), 5 (NP), 7 (M), and 8 (NS). All the reisolates were shown to retain the ts-phenotype. However, in the recombination test some reisolates (most frequently those isolated at late periods of vaccination infection) no ts-mutations could be found in 1-3 genes coding for proteins of the polymerase complex, less frequently for NP and NS proteins but not for M proteins.

Evaluation in children of cold-adapted influenza B live attenuated intranasal vaccine prepared by reassortment between wild-type B/Ann Arbor/1/86 and cold-adapted B/Leningrad/14/55 viruses.

A reassortant cold-adapted (ca) influenza B experimental live attenuated intranasal vaccine was evaluated for safety and immunogenicity in children by means of a blind, placebo controlled study. The vaccine contained the haemagglutinin and neuraminidase genes, and the gene for its non-structural proteins from wild-type (wt) B/Ann Arbor/1/86 virus, the contemporary strain at the time of the study. Other genes were derived from ca B/Leningrad/14/55 virus. No increase in illness rates was seen in the children from ages 3-15 years given vaccine at maximum potency (a one in two dilution of infectious allantoic fluid, having a titre of 10(7.0) EID50) compared to children given placebo. About 60% of seronegative children, ages 3-7 years, exhibited a detectible antibody response following one dose of intranasal vaccine, with the seroresponse rate rising to greater than 70% after two doses of vaccine. Immunogenicity was lowest in seropositive children age 8-15 years, reaching a maximum of 36% after two doses. Results indicated that the vaccine was highly attenuated, and probably of adequate immunogenicity for kindergarten age children. The lower immunogenicity in older children suggests the vaccine might be overly attenuated for use in school-age children who are more likely to have a history of prior natural infection with influenza B virus. Further clinical and epidemiological studies of protection are needed to fully assess this.

Laboratory properties of cold-adapted influenza B live vaccine strains developed in the US and USSR, and their B/Ann Arbor/1/86 cold-adapted reassortant vaccine candidates.

The adaptation of two influenza B strains (B/Leningrad/14/55 and B/Ann Arbor/1/66) to replication at 25 degrees C is described. Comparison of the two viruses indicates that both also exhibit temperature sensitive phenotypes, although that of the virus B/Leningrad/14/55 is less pronounced. When inoculated into ferrets both viruses replicate well in the trachea, but only the B/Leningrad/14/55 cold-adapted virus replicates in the lungs. This virus exhibited a moderate level of attenuation in the animals, in contrast to the B/Ann Arbor/1/66 cold-adapted virus, which was fully attenuated. Reassortant viruses deriving the surface antigens of the contemporary wild type virus B/Ann Arbor/1/86 and most or all of their other genes, from one or other cold-adapted parent, were virtually indistinguishable from their respective cold-adapted parents. The B/Leningrad/14/55 reassortant was slightly more attenuated than its cold-adapted parent in ferrets. These studies extend knowledge of the properties of viruses used to prepare experimental live influenza B human vaccines.

[Heterogeneity of current epidemic influenza A viruses with respect to reproduction activity in a tissue culture]. / Geterogennost' sovremennykh épidemicheskikh virusov grippa A po priznaku reproduktsionnoi aktivnosti v tkanevoi kul'ture.

The reproduction activity in human embryo kidney cell culture (HEK) of epidemic influenza A viruses (H3N2 serosubtype) isolated in different years and with a certain level of virulence for man was studied. The cells were inoculated with the viruses at a multiplicity of infection of 0.001 EID50/cell, and the reproductive activity was judged by the levels of infectious and hemagglutinating activity during 96 hours of observation. There was a clear-cut correlation between the virus virulence level and the reproductive activity in HEK cell culture. Epidemic influenza A (H1N1) viruses of the A/Chile/1/83 variant were found to be heterogeneous by the marker of their reproductive activity in HEK cells. It is presumed that the observed differences in the infectious activity of the viruses of this group are also associated with different levels of their virulence for man.

[Attenuated recombinant influenza type B virus obtained during crossing of virus B/Ann Arbor/2/86 with the cold-adapted strain B/Leningrad/14/17/55]. / Attenuirovannyi rekombinant virusa grippa tipa B, poluchennyi pri skreshchivanii virusa B/énn arbor/2/86 s kholodoadaptirovannym shtammom B/Leningrad/14/17/55.

Crossing the cold-adapted B/Leningrad/14/17/55 strain with the temperature-sensitive virulent B/Ann Arbor/2/86 strain yielded a recombinant B/14/5/1 which, by the antigenic specificity of hemagglutinin and neuraminidase, corresponded to the B/Ann Arbor/2/86 strain but, like the attenuated donor, had the cold-adapter characteristics. The B/14/5/1 recombinant inherited the genes coding for proteins PB2, PB1, PA, NP, and M from the attenuated master strain and the genes coding for hemagglutinin, neuraminidase, and proteins NS from the virulent master strain. This strain was nonreactive for adults and for children with the initial anti-hemagglutinin antibody titre less than or equal to 1:20 (the reactogenic index being 1 and 0.9% respectively) and was moderately antigenic inducing a 4-fold or more rise of anti-hemagglutinins in the blood of 48.8% of seronegative adults and in 46.6% of seronegative children of 3 to 15 years of age. The highest indices of seroconversions (60%) were recorded in a group of preschool children.

[The effect of amplifying reproduction of influenza virus in mouse lungs during simultaneous infection with two cold-adapted strains]. / Effekt usileniia reproduktsii virusa grippa v legkikh myshei pri odnobremennom infitsirovanii dvumia kholodoadaptirovannymi shtammami.

Reproduction of cold-adapted (ca) strains of influenza virus in the lungs of white mice after separate and combined inoculation and the properties of isolates derived from the infected animals were studied. It was shown that after combined inoculation with ca and ts strains A/Leningrad/134/17/57 (H2N2) and A/PR/8/59/1 (H1N1) ca recombinants could develop loosing some ts mutations and possessing (unlike the master strains) pneumo-virulence for mice. All the pneumo-virulent reassortants inherited hemagglutinin from the ca A/PR/8/59/1 strain and PB1 protein from the ca A/Leningrad/134/17/57 strain. The results indicate that it is unsafe to construct live recombinant divaccines by combining the recombinants produced from different donors of attenuation.

[Biological characteristics of epidemic and interepidemic strains of the influenza A (H3N2) virus]. / Biologicheskie osobennosti épidemicheskikh i mezhépidemicheskikh shtammov virusa grippa A (H3N2).

Comparative studies of biological properties of influenza A (H3N2) viruses isolated in the epidemics of 1980 and 1983 and in the interepidemic period of 1982 showed a sharp reduction of the biological activity of the interepidemic viruses as compared with that of the epidemic ones. This was manifested by low isolation rate of virus in the interepidemic period, reduced hemagglutinating, interfering, and immunogenic activity of these viruses, poor reproduction in the lung tissue of white mice and the lack of virus-specific syntheses in splenocytes of infected Syrian hamsters. Unlike epidemic strains, the interepidemic strains had ts mutation in gene 6 coding for neuraminidase. There are reasons to associate the observed defect in gene 6 with frequent development of asymptomatic forms of influenza infection in the interepidemic period.

Characterization of the reproduction of influenza A epidemic viruses in cell cultures.

Two influenza A epidemic viruses with different indices of virulence for humans have been compared with respect to their reproduction in human embryo kidney (HEK), human embryo lung (HEL), and chick embryo kidney (CEK) cell cultures. It has been shown that the highly virulent for humans A/Victoria/35/72 (H3N2) strain reproduced intensively in HEK and HEL cells irrespective of the inoculated dose (multiplicity of infection = 1 EID50 per cell and of 0.001 EID50 per cell, respectively). Efficient infection of a moderately virulent virus A/Bangkok/1/79 (H3N2) was registered in these cell cultures only after addition of trypsin to the maintenance medium. The viruses tested exhibited essentially no difference as to the intensity of their reproduction in CEK cell culture whose sensitivity remained unchanged after addition of trypsin to the maintenance medium.

[Persistent influenza virus infection. Molecular genetic characteristics of ts mutants selected during persistence]. / Persistentnaia infektsiia virusa grippa. Molekuliarno-geneticheskaia kharakteristika ts-mutantov, selektsioniruiushchikhsia v protsesse persistentsii.

Properties of ts mutants isolated from systems of persistent influenza infection formed in MDCK cell culture by A/Victoria/35/72 virus were studied. The ts mutants isolated at later intervals of persistent infection (158 days) were characterized by thermolability of hemagglutinin and neuraminidase, changes in the EP mobility of HA2 polypeptide, decrease in the molecular weight of this polypeptide, appearance of multiple ts mutations in genes 3, 5, 6, 7, and 8, coding for P2, NP, NA, M, and NS proteins, respectively.

[Changes in the biological properties of influenza virus (HON1) strains virulent for mice when exposed to homologous antibodies]. / Izmeneniia biologicheskikh svoistv virulentnykh dlia myshei shtammov virusa grippa (HON1) pod vozdeistviem gomologichnykh antitel.

A decrease in the virulent properties of two influenza virus strains (A/PR/8/34 and A/Mel/35 (HON1) pathogenic for mice was demonstrated in multiple passages in chick embryos in the presence of homologous antibody. The artificially developed avirulent A/PR/8/240 and A/Mel/375 variants had low immunogenic activity and a marked temperature sensitivity to 40 degrees C. Common ts mutations in the genomes of these avirulent variants located in genes 1 and 7 coding for P3 and M proteins were demonstrated.

Recombinant cold-adapted attenuated influenza A vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants.

A previously described cold-adapted attenuated virus, A/Leningrad/134/17/57 (H2N2), was further modified by 30 additional passages in chicken embryos at 25 degrees C. This virus had a distinct temperature-sensitive (ts) phenotype, grew well in chicken embryos at 25 degrees C, and failed to recombine with reference ts mutants of fowl plague virus containing ts lesions in five genes coding for non-glycosylated proteins (genes 1, 2, 5, 7, and 8). Recombination of A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79 (H1N1) and A/Bangkok/1/79(H3N2) yielded ts recombinants 47/25/1(H1N1) and 47/7/2 (H3N2). These recombinants inherited their ts phenotype and ability to reproduce in chicken embryos at 25 degrees C from the cold-adapted parent. Analysis of the genome composition of the recombinants obtained by recombination of the cold-adapted donor with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) showed that recombinants 47/25/1(H1N1) and 47/7/2 (H3N2) inherited five and six genes, respectively, from the cold-adapted parent, and hemagglutinin and neuraminidase genes from the wild-type strains.