Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein.

INTRODUCTION: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. OBJECTIVE: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. MATERIALS AND METHODS: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. RESULTS: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. CONCLUSION: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

[The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses].

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

[Development of entrepreneurship in the field of medical care based on the franchising model].

The article deals with the issues of functioning of Russian medical centers on the terms of franchising, relevant for the development of entrepreneurship in the field of medical care. Over the past few years, the number of private medical organizations in the Russian Federation has been growing, and the number of business representatives who want to participate in the development of entrepreneurship and conduct socially significant activities in the field of medical care has been increasing. The search for the most effective ways to establish and develop a business in the medical industry contributes to the growing interest in the franchise model, which allows you to expand the scale of your business. In the medical services market, the franchising model is currently much less common than in such areas as retail, hotel services and catering, however, it is in the medical industry that there is a great growth potential for the development of entrepreneurship in the field of medical services based on the franchising model. Thanks to the use of the franchising model, it is possible to predict an increase in the service culture in the business environment and the output of domestic companies to international markets. Infrastructure support plays an important role in the development of medical franchising: Internet resources, training technologies, platforms for exchanging information, experience and tools for promoting the network of franchising enterprises. Despite the fact that in modern conditions, commodity and productive franchising are predominant, and the practice of operating Russian medical centers on the terms of franchising is poorly developed, there are prospects for development and social value for the Russian market of medical services in the field of medical services.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

[Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus].

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.

[Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus].

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4-5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70-5.80 to 5.25-5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

[Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin].

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.

Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

[Testing of apathogenic influenza virus H5N3 as a poultry live vaccine].

Four H5N2 experimental vaccine strains and the apathogenic wild duck H5N3 influenza virus A/duck/ Moscow/4182/2010 (dk/4182) were tested as a live poultry vaccine. Experimental strains had the hemagglutinin of the A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site or the hemagglutinin from attenuated virus (Ku/ at) that was derived from the highly pathogenic influenza virus A/chicken/Kurgan/3/2005 (H5N1). The hemagglutinin of the Ku-at has the amino acid substitutions Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2, while maintaining the polybasic HA cleavage site at an invariable level. The other genes of these experimental strains were from the H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). A single immunization of mice with all tested strains elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal dose of A/chicken/Kurgan/3/05. The pathogenicity indexes of the Ku-at and the other strains for chicken were virtually zero, whereas the index of the parent H5N1 virus A/chicken/Kurgan/3/2005 was 2.98. Intravenous, intranasal, and aerosol routes of vaccination were compared. It was shown that the strain dk/4182 was totally apathogenic for one-day-old chicken and provided complete protection against the highly pathogenic H5N1 virus.

[New genotype of the sacbrood virus of the honeybee Apis mellifera].

The sacbrood virus (SBV) leads to death of honeybee larvae. Until now, there were known three SBV genotypes: European, Asian, and South African (E. Grabensteiner et al., 2001; S. E. Choe et al., 2012). Serologic assay, sequencing and phylogenetic analysis of the SBV RNA-polymerase gene fragments resulted in discovery of two genotypes of SBV circulating among the honeybee Apis mellifera in the European region of Russian Federation (RF). One of them forms a new distinct genetic lineage of SBV (genotype 4), which has not been described before and has an intermediate position between the Asian and the South African genotypes on a phylogenetic tree. The viruses belonging to this group were isolated from honeybee in Kaluga region in 1986, and in 2006 from honeybee introduced earlier to Moscow from Uzbekistan. The sequence homology inside this group is 94.2%, whereas this one between different groups is not higher then 80.6-83.5%. Another group represents the European genotype of SBV circulating in Krasnodar Territory, Adyghe Republic, and Moscow region. Two SBV genotypes from Russian Federation can be differentiated using PCR and radial immunodiffusion test (RID). As for RID, both genotypes react with antiserum #6(1), but only the fourth genotype reacts with antiserum #58.

[The generation and characteristics of reassortant influenza A virus with H5 hemagglutinin and other genes from the apathogenic virus H6N2].

The experimental reassortant vaccine strain VN-gull (H5N2) containing H5 hemagglutinin (HA) with a removed polybasic site in the connecting peptide and other genes from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (gull/M) was obtained using a two-step protocol. At Step 1, the reassortant with HA of A/Vietnam/1203/04-PR8/ CDC-RG and other genes from cold-adapted A/Leningrad/17/47 (VN-Len) viruses was generated due to selection with antibody to H2N2 at 26 degrees C. At Step 2, the reassortant VN-gull was obtained by replacing all genes from Len with those from gull/M due to selection with antibody to H6N2 at 39 degrees C. The reassortant VN-Len was apathogenic and the reassortant VN-gull was weakly virulent in mice. Both gave rise to specific antibodies and 4 weeks after single inoculation they provided complete protection against further challenge with highly pathogenic HSN1 virus A/chicken/Kurgan/3/05 (H5N1) (Ku-Len). The chickens infected with live VN-gull virus showed neither clinical symptoms, nor fecal virus excretion; nevertheless, they gave rise to antibodies and were protected from the further challenge with A/chicken/Kurgan/3/2005. The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

[A/H13 and A/H16 influenza viruses: different lines of one precursors].

Analysis of the data of annual (1980-2005) monitorings of influenza A viruses in the North Caspian Sea basin and the Volga river delta, as well as the primary hemagglutinin structure of isolates of different years revealed the circulation of A/H13 and A/H16 viruses among gulls since 1976. Phylogenetic analysis revealed 3 significantly different evolutionary lines: an American line, a European line, and a line comprising the isolates from America and Eurasia. The H13N6 and H16N3 viruses isolated in Russia replicated in the respiratory and intestinal tracts of ducks and induced the production of antibodies; the H16N3 viruses induced the antibodies neutralizing viruses of subtype H16 only. The use of glycoconjugate polymers showed that the receptor phenotype of the study H16 viruses differed from that of the H13 viruses in its capacity to bind to 3'SL with a higher affinity than alphaNANA. The comparative phylogenetic analysis suggests the existence of the common precursor of H13 and H16 viruses and their further evolution in relation to environmental conditions, including their adaptation to a new host.

[The study of the nonpathogenic influenza virus A/gull/Moscow/3100/2006 (H6N2) isolated in Moscow].

The influenza virus A/gull/Moscow/3100/2006 (H6N2) was isolated from gull feces within the precincts of Moscow in autumn 2006. The nucleotide sequence of the complete genome (GenBank, EU152234-EU152241) and genotype (K, G, D, 6B, F, 2D, F, 1E) for this virus were determined. Phylogenetic analysis suggests that the H6N2 virus derived by numerous reassortment between viruses that have been circulating among different birds in Europe since 1999 and in South-East Asia (NA gene) for last years. Migratory birds probably introduced some of these viruses from South-East Asia earlier. The strain A/gull/Moscow/3100/2006 is nonpathogenic for chicken embryos and mice and induces specific antibody production in mice. Similar to all avian influenza viruses A/gull/Moscow/3100/ 2006 it binds to Neu5Ac(2-3Gal receptors, but reveals higher affinity for fucosylated sialosugars (SLex) in contrast to the duck viruses, as was shown in receptor specificity assay and clarified due to modeling the accommodation of SLex into receptor binding site of duck and gull influenza virus hemagglutinin.

[Infection of pigs with influenza A/H4 and A/H5 viruses isolated from wild birds on the territory of Russia].

Pigs were intranasally infected with avian influenza A/H5 (H5N1, H5N3) and A/H4 (H4N6, H4N8) viruses in mono- and coinfection. Infection with both apathogenic and pathogenic strains caused no clinical manifestations. A virus and/or fragments of its genome retained in nasopharyngeal fluid as long as 6-8 days after infection. During monoinfection, the structure of the hemagglutinin (HA) receptor site of isolates from the pigs infected with A/H5N1 strains (A/chicken/Kurgan/3/2005, A/duck/Russia/5354-vac/2005) and A/H5N3 (A/duck/Primorje/2633/01) remained unchanged during 6-7 days. When two animals infected with avirulent A/H5N3 viruses (A/duck/Primorje/2633/01, A/duck/Altai/1285/91) that differed in immunogenic properties were kept together, the A/duck/Altai/1285/91 virus that induced a later IgG generation was prevalent in the nasopharyngeal fluid of both animals. Moreover, 4 significant nucleotide replacements were detected in the HA gene on days 7-8. Infection of pigs with avian influenza A/H4 viruses yielded the similar results. The joint keeping of animals infected with Algarganey teal/Astrakhan/309/102 (H4N8) strain and A/ musk beaver/Buryatia/944/00 (H4N6) isolated from musk beaver exhibited fragments of "a variant" of the identical structure in the nasal swabs of both animals on days 7-8. A nucleotide sequence from 37 nucleotide replacements differing from both baseline sequences was revealed in the HA 364-1045 gene region. The amino acid sequence of the variant was similar to Algarganey teal/Astrakhan/3091/02, other than one position 264 < Lys > (numeration by H3), which coincided with the A/ musk beaver/Buryatia/1944/00 strain. The latter induced the antibody generation on day 5 after infection while the A/garganey teal/Astrakhan/3091/02 strain did only on day 14. It is possible that under co-circulation of two different influenza A viruses, the virus causing a slower development of an immune response showed a higher probability of transiting to another host (specimen) and changing the HA receptor site in the organism of a new host.

[Ecology and evolution of influenza viruses in Russia (1979-2002)]. / Ekologiia i évoliutsiia virusov grippa v Rossii (1979-2002 gg.).

The research results on ecology and evolution of influenza A viruses, which has been conducted by the Center of Ecology and Evolution of influenza Viruses of Ivanovsky's Institute of Virology, Russian Academy of Medical Sciences, for more than 30 years, are summarized in the paper. A gene pool of influenza A viruses circulating in Russia's territory was defined. Foci of influenza A viruses were detected in natural biocenosis. Issues conditioned by the population interrelations of influenza viruses, i.e. between the populations of wild and home animals and the populations of people, are also under discussion.

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

[Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22]. / Izuchenie molekuliarnykh osnov izmeneniia nekotorykh biologicheskikh svoistv virusa iashchura podtipa A22.

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.

[Nucleotide sequence of the RNA polymerase gene attenuated by a variant of foot-and-mouth disease virus and its comparison with the virulence of related variants of subtype A22]. / Nukleotidnaia posledovatel'nost' gena RNK-polimerazy attenuirovannogo varianta virusa iashchura i ee sravnenie s virulentnymi rodstvennymi variantami podtipa A22.

The nucleotide sequence of RNA-polymerase gene and 3'-terminal untranslated genome region of attenuated foot-and-mouth disease virus (FMDV) strain A(22)645 has been determined. RNA-polymerase gene and predicted amino acid sequences of attenuated FMDV strain A(22)645 were compared with those of the original virulent FMDV strain A(22)550. The examined genome region of strain A(22)645 differed from that of strain A(22)550 by 22 nucleotides and 3 amino acids. Three mutations occurred within nucleotide residues 883 to 1026, which encode an extremely conserved amino acid domain corresponding to RNA polymerization and nucleoside triphosphate binding functions. Mutation in 920n caused substitution of conserved amino acid Asn for Ser in the core motif for nucleotide binding Gxxx-TxxxN(S/T). It is possible that change in functional motif caused the alteration of phenotypic virus and loss of pathogenicity for cattle. This may be finally confirmed only after comparative analysis of complete genomes of the examined virus strains.

[Cloning fragments of the RNA polymerase gene of an attenuated variant of the foot-and-mouth disease virus A22]. / Klonirovanie fragmentov gena RNK-polimerazy attenuirovannogo varianta virusa iashchura A22.

The cDNA fragments complementary to RNA-polymerase gene and 3'-untranslated genome region of attenuated foot-and-mouth disease virus strain A(22)645 have been synthesized and cloned into a plasmid vector pUC19 in E. coli JM109. The cloned cDNA fragments were characterized as to their size, orientation towards the plasmid, and localization in the virus genome. Restriction maps for complete gene and two cDNA clones were constructed.