[Growth characteristics of single gene mutants of A/Leningrad/134/57(H2N2) influenza virus and cold-adapted mutant A/Leningrad/134/17/57(H2N2)].

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

[Genetic and phenotypic analysis of heterogeneous population of a cold-adapted donor of the A/Leningrad/134/17/57 (H2N2) attenuation and of the donor-based reassortant influenza vaccine strains].

Cold-adapted (CA) temperature sensitive and attenuated virus A/Leningrad/134/17/57 (H2N2) (Len/17) has been recently used in Russia as a donor of internal genes in the preparation of reassortant vaccine strains of CA live influenza vaccine (LIV) for all age groups. The Len/17 population was found to be heterogeneous and to be made up of clones, which differ by combinations of mutations in internal genes. Around 50% of the Len/17 population had clones with all 8 coding mutations in internal genes. The others were made up of clones with mutation combinations, which were different from the original Len/17. The PCR restriction method was used to analyze 5 clones of Len/17 and 8 LIV vaccine strains. There were no Ala-86-Thr mutation in the M2 protein in 4 clones and 3 vaccine strains. The PB-1 gene of 4 clones and 3 vaccine strains had a mutation encoding Met-317-IIe more typical of a more attenuated virus A/Leningrad/134/47/57 (H2N2) (Len/47). The NP protein of a clone had a mutation Leu-341-IIe also typical of Len/47. However, neither the absence of mutation in the M2 gene nor an extra mutation in the PB1 gene affected the attenuation extent of reassortant CALIV.

[The temperature sensitivity of epidemic influenza A viruses as a marker of immunogenicity of reassortant vaccinal strains]. / Temperaturochuvstvitel'nost' épidemicheskikh virusov grippa A kak vozmozhnyi marker immunogennosti reassortantnykh vaktsinnykh shtammov.

The influence of ts-phenotype of epidemic viruses and of cold-adapted (CA) reassortant vaccines' strains, appropriately prepared, produced on the human immunogenicity was under investigation. A widespread variability of epidemic viruses' thermal sensitivity sign was established. It was shown that the CA reassortant vaccine strains, obtained through crossbreeding of attenuation donors and of thermally resistant epidemic viruses, are described by a higher immunogenicity. Therefore, the immunogenicity of live influenza vaccines (LIV) can be defined by the ts-phenotype of epidemic parent viruses, which must be sampled for the reassortant vaccine strains not only through searching for samples of antigenically actual viruses but also through search for non-ts-phenotype viruses.

[Criteria of primary screening of attenuated strains for live vaccine against Influenza A]. / Kriterii pervichnogo skrininga attenuirovannykh shtammov dlia zhivo'i vaktsiny protiv grippa tipa A.

Reassortant strains for live influenza vaccine (LIV) were selected using two additional markers: intensity of cytopathic effect (CPE) at 40 degrees C in MDCK cells and toxicity for mice (induction of acute hemorrhagic pulmonary edema after intranasal challenge with undiluted virus). All wild-type viruses induced a high CPE in MDCK cells, while the reassortants differed by this sign. Only vaccine strains and attenuation donors were characterized by a low CPE. Modern epidemic viruses are highly toxic for mice, causing the death of 60-100% animals from hemorrhagic pulmonary edema on days 3-4 after intranasal infection. Attenuation donors and vaccine strains were not toxic for mice, the level of toxic effect correlating with CPE in MDCK culture. Evaluation of CPE in MDCK culture and toxicity for mice can be used for primary screening of candidates for LIV.

[Antitumor activity of cold-adapted strains of influenza virus in a model transformed murine tumor cells]. / Protivoopukholevaia aktivnost' kholodoadaptirovannykh shtammov virusa grippa na modeli perevivaemykh linii opukholevykh kletok myshei.

Influenza virus suppressed tumor growth after injection to tumor zone in a dose of 7-8 lg EID50, as was shown for two continuous mouse tumor cell strains, Ehrlich's carcinoma and L-1210 lymphoma. Influenza virus strains differed by their antitumor activity which correlated with their interferonogenic activity. Antitumor activity of influenza virus depended on the site of application, dose of the virus, and interferonogenic activity. Virus therapy of tumors stimulated specific cytotoxic activity towards tumor antigens.

[Preliminary results of the Russia (St.Petersburg)/WHO program for the evaluation of the effectiveness of breast self-examination]. / Promezhutochnye rezul'taty programmy Rocciia (Sankt-Peterburg)/VOZ po otsenke éffektivnosti samoobsledovaniia molochnykh zhelez.

Although an absolute difference of 10% (65,4 vs. 54,9%) in 5- and 9-year survival in breast cancer patients was recorded between the self-examination and control groups a large-scale randomized population-controlled study of 122,471 females has failed to provide significant differences (Log-rank - 0,774, p > 0.05). No significant decrease in mortality was observed in the self-examination group as compared with the untrained controls. As a result of providing more information to the population on risk factors. twice as many of the trained females consulted oncologists. Also, the number of early detection of breast tumor (T1-2NOMO) in both groups was 1,5-2,5 times that recorded elsewhere. Since 3,55 per 1,000 patients with breast tumors per year, aged 50-59, died of cardio-vascular disease, i.e. 3,1 times the expected 1,16 per 1,000, more attention should be focused on timely diagnosis and treatment of concomitant cardio-vascular pathology.

[The NS gene--a possible determinant of apathogenicity of a cold-adapted donor of attenuation A /Leningrad/134/47/57 and its reassortants]. / Gen NS--veroiatnaia determinant apatogennosti kholodoadaptirovannogo donora attenuatsii A /Leningrad/134/47/57 i ego reassortantov.

Phenotypical properties of single-gene reassortants of attenuated cold-adapted strain A/Leningrad/135/47/57 (H2N2) and strain A/PR8/34 virulent for laboratory animals were studied. Only the group of reassortants inheriting NS gene from cold-adapted virus was fully attenuated for various animals species, similarly as reassortants with 6/2 genomic formula containing all the 6 internal protein genes from strain A/Leningrad/134/47/57. Reassortant 25A-1 single-gene for NS was temperature-sensitive (ts) on mammalian cells but formed plaques at 40 degrees C on chicken kidney cells. Reassortants with genomic formula 6/2 were temperature-sensitive in all types of cells used. Reassortant 25A-1 could synthesize normal amounts of polypeptides in MDCK cells at 39 degrees C, whereas protein synthesis of reassortants with 6/2 genomic formula was noticeably reduced at this temperature. Hence, a similar level of attenuation of both reassortant groups appears to be due to various molecular mechanisms. Possible role of NS2 gene mutation in attenuation of strain A/Leningrad/134/47/57 and its reassortants is discussed.

[Immunogenicity of attenuated reassortants of influenza A virus /Leningrad/134/47/57 (H2N2) for mice, depending on their genome]. / Immunogennost' dlia myshei attenuirovannykh reassortantov virusa grippa A /Leningrad/134/47/57 (H2N2) v zavisimosti ot sostava ikh genoma.

Reassortants of cold-adapted strain A/Leningrad/134/47/57 (H2N2) and virulent strain A/PR/8/34 (H1N1), modeling genomic composition of vaccinal strains, were tried in mouse experiments. Reassortants' genomes included the major part (5, 6, or 7) from cold-adapted strain and 1 to 3 genes of virulent strain. All the tested reassortants did not differ by temperature sensitivity or cold adaptation phenotypes, did not cause the death of mice, but differed by the level of reproduction in murine lungs and by immunogenicity. Strains with genomic formulas 6/2 (HA and NA of a virulent strain) and 5/3 (M) (HA, NA, and M of a virulent strain) were characterized by the highest immunogenicity. Reassortants including, besides HA and NA, PA gene 5/3 (PA) or inheriting only HA gene from strain A/pr/8/34 (H1N1) were hyperattenuated and low immunogenic.

[The immunogenic properties and prophylactic efficacy of a live polyvalent influenza vaccine in children 5 to 14 years old]. / Izuchenie immunogennykh svoistv i profilakticheskoi éffektivnosti zhivoi grippoznoi polivalentnoi vaktsiny u detei 5--14 let.

Production lots of a live influenza vaccine made of strains A/47/T (N1H1), A/47/6/2 (H3N2), and B/60/32 were used for vaccination of 3663 children aged from 5 to 14 years inoculated twice with monovaccines, a trivaccine made of the above strains, or placebo. Both mono- and polyvaccine were practically areactogenic. An average per cent of subjects with a significant rise in antibody titres to the respective three antigens was 60%. The efficacy of the vaccination was 31.0-42.8% for monopreparations and 36.3% for the trivaccine. The studies showed the possibility and expedience of using for children the live influenza vaccine in the form of a polyvalent preparation including current influenza type A and B viruses.

[Interference and its overcoming during the reproduction of 2 influenza A virus strains in an experiment]. / Interferentsiia i ee preodolenie pri reproduktsii dvukh shtammov virusa grippa A v éksperimente.

The interference between two influenza A virus strains was investigated in vivo. In mixed infection of chick embryos or mice two results were observed: maximal reproduction of both strains or interference which resulted in a reduction of reproduction activity, antibody production, or virulence of one of the viruses. No interference was observed upon inoculation of two strains apathogenic for the study host combined with an equal concentration of an infectious virus.

[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.

[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 inoculation properties of live recombinant influenza vaccine types A and B used separately and jointly in children 3 to 14]. / Privivochnye svoistva zhivoi grippoznoi rekombinantnoi vaktsiny tipov A i B pri razdel'nom i sovmestnom primenenii detiam 3--14 let.

The reactogenicity and immunizing activity of vaccine influenza virus A (H1N1) and B strains used as mono- and bi-preparations in children of 3 to 14 years was studied. No increased reactogenicity after the use of bivaccine was observed in the children. Febrile reactions as well as 9 other clinical symptoms which could indicate the reactogenicity of the vaccines were identical for mono- and bivaccine and corresponded to the requirements of the technical documents for the vaccine. The optimal conditions for the evaluation of the immunogenicity of the B component by HI test were developed, and the necessity of using additionally the enzyme immunoassay for this purpose is substantiated. The above method demonstrated that the immunogenicity of the live influenza type A and B vaccine was high in children. No significant inhibition of immunological parameters was observed when the two viruses were combined in the bivaccine.

[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.

[Genetic basis for construction of the life influenza type A vaccine using temperature-sensitive mutants]. / Geneticheskie osnovy konstruirovaniia zhivoi grippoznoi vaktsiny tipa A na osnove temperaturochuvstvitel'nykh mutantov.

Published data and authors' original material on the use of temperaturesensitive influenza virus mutants (ts) as donors in attenuation process aimed at obtaining the recombinant live influenza vaccines (LIV) are reviewed. The so called cold adapted ts donors are shown to be superior for this aim. The data are presented in the review on the association of the mutations in the genome of cold adapted donors with attenuation, on the reactivity, immunogenicity and genetical stability of the recombinant LIVs constructed on the basis of the cold adapted donors.

[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.

[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.

[Variations in the temperature range for reproduction of virulent and attenuated cold-adapted influenza A viruses]. / Razlichiia v temperaturnom diapazone reproduktsii virulentnykh i attenuirovannykh kholodoadaptirovannykh virusov grippa A.

The informativity of RCT40, RCT37.5, and RCT25 markers for differentiation of wild type strains from ca-recombinants obtained on the basis of ca donors of attenuation was studied. The RCT25 marker and express RCT37.5 marker (determined within 24 hours) were found to be universal characteristics differentiating ca-recombinants from virulent viruses.