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Five influenza pandemics emerged in the 20th and 21st centuries. Data regarding possibility of infection of swine with human influenza viruses-and persistent circulation of these strains among swine with subsequent infection of humans with these viruses were obtained in the recent years. A possibility of prediction of influenza pandemics by constant observation and study of influenza viruses circulating among swine is discussed in the paper.

A ts+ ca- (non-temperature-sensitive, non-cold-adapted) revertant of the A/Leningrad/134/47/57 ca strain influenza virus [A/Leningrad/134/47/ts+18/1957(H2N2)], obtained in our previous study, lost phenotypic manifestation of ts mutations by the PB2, NP and NS genes, although, according to sequencing data, it acquired only two true reversions of a mutation in the PB2 and PB1 genes. Direct sequencing showed the appearance of 27 additional mutations (13 coding) in the genes encoding the PB2, PB1, PA, NP, M and NS proteins of the revertant, along with the above-mentioned two true reversions. We conjecture that some of these mutations suppressed phenotypic manifestation of ts mutations in the NS and NP genes.

Optimal conditions are determined for growing cold-adapted reassortant strains of a live influenza vaccine in MDCK cell line cultivated in a fermenter with a serum-free medium and microcarriers. The studied MDCK cell line meet all national and WHO requirements for the finite cell lines used for the production of biological preparations. CA reassortant vaccine strains grown in such conditions which fully preserve its mutations and the mutations lead to amino acid substitution in all genome segments of the studied CA reassortants. Under optimal cultivation conditions, the output of a monovalent live CA influenza vaccine in a 10-l fermenter may reach 100,000 doses.

Eighteen influenza A (H3N2) viruses were isolated during a single outbreak in a closed community of Carmelite nuns. Serological analysis of the virus haemagglutinin (HAs), using a panel of monoclonal antibodies, demonstrated antigenic microheterogeneity. In contrast, no significant biochemical differences were detected in viral genes by RNA:RNA hybridisation or in structural or nonstructural polypeptides analysed by high-resolution polyacrylamide gel electrophoresis (PAGE) or by limited proteolysis followed by PAGE. Influenza A (H3N2) viruses isolated in the vicinity of the convent were distinguishable from each other and from the epidemic viruses isolated in the convent both antigenically and biochemically.

Genetic analysis of influenza B virus strains isolated during concurrent epidemics (February-March 1982) in three physically separated schools was carried out using the RNA:RNA hybridization technique. The data supports the hypothesis that the influenza B virus epidemics in each school were initiated by different viruses and the variability of the strains in a closed community was found to be minor though some variants were detected.

Some biological properties of recombinants obtained by crossing of fowl plague and human influenza viruses were studied. The capacity of the recombinants to reproduce in chick embryo fibroblast cultures was in reverse correlation to the number of genes coding for P proteins derived from the human influenza virus. The genome composition was of importance for the expression of ts-phenotype of the recombinants in different systems. Substitution of at least one gene in the fowl plague virus genome by a corresponding human influenza virus gene resulted in the decrease of virulence for 1-day-old chickens. The presence of three P genes from human influenza virus genome in the genome of the recombinant proved to be insufficient for the capability of the recombinant to reproduce in organ cultures of human origin.

Comparative studies have been undertaken on the H3N2 influenza virus strains isolated in Leningrad in May-June, 1982 and those isolated in the subsequent winter epidemic in February, 1983. Analysis of the isolates with ferret antisera against standard influenza viruses of the H3N2 subtype and with monoclonal antibodies against A/Bangkok/1/79 virus revealed a considerable but similar degree of heterogeneity in the HA antigenic specificity of the strains isolated in the spring-summer, 1982, as in the winter, 1983, periods and also a close resemblance between the antigenic specificities of the strains of these two epidemics. However comparative genome analysis of the strains using cRNA:vRNA hybridization technique revealed that in terms of the gene homology, the influenza virus strains which circulated in the spring-summer period of 1982 resembled the strains responsible for the previous rather than subsequent epidemic.

Crossing of an attenuated influenza B virus strain (B/Leningrad/14/17/55) passaged at a low temperature with a virulent influenza B virus strain (B/England/2608/76) yielded recombinants similar in the antigenic specificity of their haemagglutinin (HA) and neuraminidase (NA) to B/England/2608/76 strain, but possessing an RCT37.5 marker alike to the attenuated donor. Analysis of the genome composition of 2 recombinants has shown that they inherited genes coding for P (1, 2, 3) and M (7) proteins from the attenuated parent, but genes coding for HA (4), NA (6), NP (5) and NS (8) proteins from the virulent parent. All recombinants proved to be areactogenic for adult volunteers with no pre-existing antibody to the corresponding HA (less than or equal to 8); however, they had a reduced immunogenicity as compared to parent viruses.

On interaction of ts mutants of fowl plague virus (FPV) belonging to different complementation groups and human influenza A viruses under conditions of abortive infection for both partners, complementation was marked and recombinants occurred with a high frequency. The level and degree of complementation as well as the frequency of recombinants depended on the stage at which the reproduction of the human influenza A viruses involved was blocked. On simultaneous inoculation of chicks with two apathogenic ts mutants of FPV belonging to different complementation groups, pathogenic ts+ recombinants appeared. On interaction of influenza B virus with ts mutants of FPV or ultraviolet-irradiated FPV, neither complementation nor recombination was observed. There was no complementation between influenza B virus and ts mutants of FPV even at the level of the formation of virus-specific substructures.

Direct biochemical evidence for the existence of mutations in five of the RNA segments of the A/Leningrad/134/57 cold-adapted 47th passage mutant as compared with its wild-type progenitor has been obtained using two techniques. T1 oligonucleotide mapping of total viral RNA as well as of individual RNA segments revealed changes in RNAs 4, 5 and 6. Analysis of S1 nuclease-treated RNA-RNA hybrids on polyacrylamide gels revealed changes in at least one of the polymerase genes as well as in RNAs 4, 5, 6 and 7. These findings provide a direct demonstration for the existence of multiple mutations in the cold-adapted mutant vaccine strain.

The ability of the fowl plague virus (FPV) M protein to form a complex with FPV RNP and to inhibit the RNP transcriptase activity in vitro depended on NaCl concentration and did not depend on the concentration of nonionic detergents. The results obtained indicate that the M protein-RNP links formed were of an electrostatic rather than a hydrophobic nature. As demonstrated using individual RNP components, vRNA and RNA-free protein structures, M protein formed complexes only with vRNA, and the complex formation was salt-dependent. Analysis of products formed in the in vitro system containing RNP of FPV in the presence of the M protein showed impairment in the transcription of all RNA segments. The degree of inhibition correlated with the size of a segment, transcription of high molecular weight RNA segments being inhibited significantly more than that of low molecular weight RNA segments.

Organ cultures of human nasal polyps were shown to support the replication of five out of seven human influenza A viruses and three out of six avian strains with varying degrees of efficiency. The ability to replicate was independent of the antigenic formula of the virus. The structure of nasal polyps closely resembled that of normal nasal mucosa and infection with influenza A virus resulted in histological changes analogous to those seen in natural infections. This system provides an in vitro method for more detailed studies of influenza A virus and possibly other respiratory virus infections of man.

The effect of proteins soluble in acidic chloroform-methanol (ACMS proteins) on the transcriptase activity of virus ribonucleoproteins (RNPs) in vitro has been studied. Experiments with ACMS membrane (M) proteins from type A and B orthomyxoviruses, as well as from vesicular stomatitis virus, showed that inhibition of the viral RNP transcriptase activity occurred when they interacted with M proteins isolated from viruses of a different serotype, or even of a different family. The presence of ACMS proteins capable of inhibiting the transcriptase activity of orthomyxovirus RNP in vitro was also detected in human blood plasma and among proteins produced by human leukocytes. Determination of the minimum concentration of M protein inhibiting the RNP transcriptase activity, and analysis of the fowl plague virus M protein-RNP complex formed in the in vitro system, showed that the M protein was capable of inhibiting RNP transcriptase activity at a M:RNP ratio of 0.1 to 0.2:1.

A recombinant (R3/3) has been obtained which inherited the gene coding for the haemagglutinin from human influenza virus A/Hong Kong/1/68 (H3N2), and seven other genes from influenza virus A/duck/Ukraine/1/63 (H3N8). The recombinant R3/3 had properties, a ts+ phenotype and a capability of reproducing in chick embryos, similar to those of the duck influenza virus, but, in contrast to this parent, had lost its ability to reproduce in organ cultures of colon of ducks and chickens as well as in monolayer cultures of chick embryo fibroblasts.

Two fowl plague virus temperature-sensitive (ts) mutants belonging to different complementation groups were studied. Both were defective in the syntheses of unpolyadenylated complementary RNA [A(-)cRNA] and virus RNA (vRNA) at non-permissive temperature whereas primary transcription was unaffected. In addition, ts 29, in which the ts mutation is in gene 1 coding for polypeptide P3, has a defect in 'secondary' synthesis of mRNA at non-permissive temperature whereas inhibition of mRNA synthesis by ts 131, in which the ts mutation is in gene 2 coding for polypeptide P1, appeared to result from a defect in vRNA synthesis. These results indicate, therefore, that different enzymes are responsible for the syntheses of virus mRNAs and A(-)cRNAs, which is consistent with the apparent differences in initiation and termination of transcription in the two reactions. The patterns of synthesis of the various types of virus RNA during infection are discussed.

Comparative studies on the electrophoretic mobility of double-stranded RNA segments constructed of the genome of isolates under study and of reference influenza virus strains of respective serotypes are suggestive for the evolution of majority of the genes of both A and B influenza viruses, including their genes coding for non-glycosylated proteins. Among influenza A and B virus strains which were circulating during the same epidemic, there were variants differing from each other in a number of genes including those coding for virion internal proteins. A recombinant (reassortant) detected among the influenza virus strains, which circulated in Moscow in 1979, contained the genes 4, 6, 7 and 8 identical with those of the reference H1N1 strain, and the genes 1, 2, 3 and 5 identical with those of the reference H3N2 strain. Comparative analysis of reference influenza virus strains, both the H3N2 serotype (being isolated from 1968 to 1979) and the H1N1 serotype (being isolated from 1977 to 1980) differing in the antigenic specificity of their haemagglutinin (HA) suggested the increasing variability of either genes coding for glycoproteins as well as for non-glycosylated proteins.

The biological and molecular properties of both virulent and vaccine types of influenza virus strains are reviewed, including their analysis in laboratory animals and in organ tissue cultures, as well as studies of their genome peculiarities. Methods are discussed for obtaining attenuated donor strains of influenza virus, and the properties of these strains are analysed. The methods of studying the genome composition and the genes carrying mutation lesions in recombinant vaccine strains obtained by recombination of an attenuated donor strain with current epidemic influenza virus variants are also reviewed.

A fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 degrees C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.

Thirteen strains of human influenza virus producing in chick embryo cell (CEC) cultures either virions with low infectivity or no virions were studied. In CEC, most of the strains induced synthesis of viral RNA, polypeptides, and ribonucleoprotein and produced functionaly active haemagglutinin, neuraminidase and virions lower infectivity. The low infectivity of virions produced by strains of this functional group was due to disturbed cleavage of a polypeptide, haemagglutinin precursor, formed in CEC, into a heavy a light haemagglutinin chain. Two strains belonging to another functional group induced no synthesis of virus-specific macromolecules in CEC, but were able to adsorb onto the cells. With one of these viruses, no transcription of parental RNA could be detected in CEC. There was no relationship between the grouping of the studied strains into a certain functional group with their antigenic characteristics, the year of isolation, the passage history in chick embryos and human pathogenicity.

The genetic characteristics of 24 representative influenza B viruses isolated in widely different geographical areas of the world between 1940 and 1980 were analysed using either RNA:RNA hybridisation or oligonucleotide mapping. Additional biochemical characterisation included electrophoretic analysis of virus-induced polypeptides and virion RNAs. A panel of monoclonal antibodies to virus HA was used to investigate serological relationships between the viruses. The influenza B viruses examined constituted a genetically and serologically related group but mutational changes were detected in all eight genes of the viruses isolated in different eras and also in genes of viruses isolated in the same epidemic year. Regardless of the overall and dominating similarities, at a higher level of discrimination it was clear that certain genetic and serological relationships were more complex than expected and, for example, some recently circulating field viruses were apparently more closely related antigenically and genetically to viruses isolated five to twelve years previously than to other viruses isolated concurrently. No evidence of recombination with hitherto undescribed influenza B viruses and with genes coding for internal proteins was detected.

Temperature-sensitive (ts) mutations occurring in three genes, 1, 2, and 7, and 1, 5 and 7 were found in two cold-adapted (ts) attenuated influenza virus strains A/Leningrad/9/37/46 (H0N1) and A/Leningrad/134/17/57 (H2N2) respectively. The recombinants, obtained by crossing these cold-adapted strains with virulent influenza virus strains, had different genome structures and inherited from one to six genes from the cold-adapted parents. Tests of reactogenicity of recombinants in volunteers showed all the recombinants to be non-reactogenic irrespective of the number of genes inherited from the cold-adapted parent.

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.

The transcriptase activity of influenza A virus ribonucleoproteins was inhibited by 42 to 49% in vitro in the presence of membrane (M) protein. The addition of M protein to the system of ribonucleoprotein preparations isolated from rimantadine-sensitive or rimantadine-resistant influenza virus strains, as well as the addition of M protein isolated from a sensitive strain, in the presence of rimantadine further inhibited the transcriptase activity of such complexes by approximately 40%. In the system containing the same ribonucleoprotein preparations, but with M protein isolated from a rimantadine-resistant influenza virus strain, the transcriptase activity was not sensitive to rimantadine. The data show that M protein can influence the activity of influenza A virus virion transcriptase and that the susceptibility of influenza virus to rimantadine may be due to the peculiarities of M protein.

Genome composition and polypeptides of H1N1 influenza virus strains isolated in the U.S.S.R. during an epidemic in 1961-1962 have been studied as well as H2N2 influenza virus strains that dominated at that time. The H1N1 isolates did not contain genes of the simultaneously circulating H2N2 viruses. H1N1 influenza viruses have proved to be genetically similar to A/FM/1/47 (H1N1) influenza virus strain differing in only two genes, rather than to H1N1 influenza viruses isolated in 1977 differing in six genes.

The activities, the temperature and pH optima of in vitro functioning and stability upon heating of virion transcriptase of 10 human influenza virus A strains differing in reactogenicity and isolated in different epidemiological situations, and of fowl plague virus (FVP) were compared. As compared with virion transcriptase of human influenza virus strains studied, that of FPV had a higher pH optimum, was capable of functioning in vitro at a higher temperature and was more stable on heating. Freshly isolated and vaccine influenza virus strains on the one hand and strains isolated at the peak and in the end of an epidemic did not differ in the virion transcriptase properties. The virion transcriptase of a strain isolated from a local influenza outbreak was much less active than transcriptase of a highly epiedmic strain.

Temperature-sensitive (ts) mutants of fowl plague virus (FPV) were divided into six complementation groups. Experiments with ts mutants having defects of transcription showed that in FPV strain Weybridge, protein P1 coded by gene N2 takes part in primary transcription, and protein P3 coded for by gene N1 takes part in secondary transcription. Ts mutants of FPV with lower pathogenicity were present in all six complementation groups under study. Simultaneous inoculation of chickens with two pathogenic ts mutants of FPV caused death of the chickens and a pathogenic virus with ts+ phenotype was isolated from their organs. By recombination of ts multimutant FPV with human influenza virus a recombinant was obtained that contained genes coding for the haemagglutinin and neuraminidase of human influenza virus; all other genes were derived from FPV. In experiments on volunteers this recombinant appeared to be non-reactogenic but capable of inducing antibody formation.

Reproduction and synthesis of virus-specific macromolecules were studied in chick embryo fibroblast cultures co-infected with influenza viruses type A (FPV) and B (B/Japan/73). When a multiplicity of infection (MOI) of B/Japan/73 virus (10 EID50/cell and higher) was equal to, or exceeded that of FPV, formation of infectious FPV virions in coinfected cells was suppressed significantly. At equal MOI of FPV and B/Japan/73 synthesis of all proteins of one partner and some proteins of the other was observed. However, when a MOI of one virus was 10 times higher than that of the other, proteins of the virus used at a higher MOI were formed. Studies of the synthesis of virus-specific cRNAs formed in the presence of cycloheximide have shown that at equal MOI. cRNAs were detected that corresponded only to one of the partners involved in the reproduction. The data obtained suggest that intrinsic interference between A and B viruses occurs at a stage of primary transcription.

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.

Three different types of impairment in the synthesis of virion RNA (vRNA) were detected in three groups of temperature-sensitive (ts) mutants of fowl plague virus (FPV) having ts mutations in genes 1, 3 and 5 respectively. Normal synthesis of poly-(A+) cRNA, poly(A-) cRNA and vRNA was observed under non-permissive conditions early in infection in cells infected with the ts43 mutant having a ts mutation in gene 1 coding for the PB2 protein. However, 4 h after infection synthesis of vRNA ceased, synthesis of poly(A+) cRNA was reduced drastically, but the rate of poly(A-) cRNA synthesis was the same as that in cells infected with wild-type FPV. In cells infected with the ts 166 mutant having a ts mutation in gene 3, coding for the PA polypeptide, a drastic reduction was observed in poly(A+) cRNA synthesis under non-permissive conditions. Synthesis of poly(A-) cRNA was also reduced and synthesis of vRNA was not detected. The ts 60 mutant, having a ts mutation in gene 5 coding for the NP polypeptide, induced synthesis of all types of virus-specific RNA under non-permissive conditions, but the regulation of synthesis of vRNA and poly(A+) cRNA was affected, there being predominant syntheses of RNA segments 5 and 8 late in infection. In cells infected with mutants ts43 and ts 166 synthesis of virus-specific proteins was impaired, which reflected defects in the synthesis of virus-specific RNAs. The data obtained suggest that the PB2 protein may be contained in an enzyme complex responsible for synthesis of vRNA, that different enzyme complexes may be involved in the synthesis of poly(A-) cRNA and vRNA, and that the NP protein plays a significant role in the regulation of vRNA synthesis.

The cRNA:vRNA hybridization technique was used to analyse H3N2 influenza virus isolates obtained from influenza patients in the United Kingdom and the U.S.A. (Alaska) during the epidemic season of 1982-1983. The majority of isolates differed from reference H3N2 influenza virus strains A/Bangkok/1/79 and A/Philippines/2/82 as well as from one another in the homology of nearly all the genes. No identical strains were detected among the isolates including the ones isolated in the same town and at the same time.

Fowl plague virus (FPV) ribonucleoprotein (RNP) bands in sucrose density gradient in a heterogeneous peak with sedimentation coefficients from 45 to 70 S, whereas in cesium chloride gradient it has a homogeneous density of 1.33-1.34 g/cm3. FPV RNP contains 7.4-8% RNA. Upon inoculation of chick embryo cell cultures. FPV RNP shows no infectivity, does not induce virus-specific protein synthesis and does not participate in complementation or recombination interactions with ts mutants of FPV. The biological activity of FPV RNP demonstrable under certain experimental conditions is due to admixture of undestroyed virions and is completely eliminated by treatment of the preparation with gamma-globulin fraction of antiserum to FPV haemagglutinin, but not with antiserum to RNP proteins.

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.

Acetone added to the maintenance medium in a 1% concentration reduces the yield of infectious rabbitpox virus in L-cell monolayer cultures by 90 to 97%. This concentration of the inhibitor is not toxic for cells. It was established that there is an inhibitor-sensitive stage late in the infectious cycle. Acetone exerted no significant influence on production of early viral messenger ribonucleic acid, formation of polyribosomes, deoxyribonucleic acid (DNA) replication, or protein synthesis. In the presence of acetone, the assembly of so-called "acetone particles" occurred. These particles are similar to normal virions by morphological and sedimentation properties but are slightly different from them in buoyant density. The amount of virus-specific DNA and the optical density of the "acetone particles" are the same as those of normal virions despite a 10- to 25-fold difference in the infectivity of the preparations.

Ribonucleic acid (RNA) synthesis of chick embryo fibroblasts was inhibited by two members of the myxovirus group, Newcastle disease virus (NDV) and fowl plague virus. It was also found that cellular deoxyribonucleic acid-dependent RNA polymerase was inhibited by a cytoplasmic factor induced by NDV infection.