[New cold-adapted donor strains for live influenza vaccine].

Cold-adapted (CA) strains A/Krasnodar/35 and B/Victoria/63 were isolated using passages of A/Krasnodar/101/59 and B/Victoria/2/87 wild type strains at low temperatures. The resulting CA strains possessed TS and CA phenotypes and had a reduced ability to reproduce in mouse lungs and nasal turbinates. They displayed a high protective efficacy in experiments on mice. The two CA strains reproduced well in chick embryos and MDCK cell line without change of TS and CA markers. The CA A/Krasnodar/35 strain during passages at low temperature acquired 13 mutations in the 6 internal genes, 8 of those mutations led to amino acid changes. The CA B/Victoria/63 strain acquired 8 mutations in the internal genes, 6 of which led to amino acid changes. The intranasal vaccination of mice with the CA A/Krasnodar/35 strain led to a transitory suppression of various lymphocyte subpopulations, as well as to an increase in the number of some other cell types. The CA strains in question may be used in the future as attenuation donors for live influenza vaccines.

[Investigation of antiviral activity of adamantan boron derivaties on pandemic influenza virus models].

Comparative investigation of the virus-inhibiting activity of some boron-containing compounds showed that products BG 12 and BG 4 had the highest inhibitory effect on pandemic viruses. The minimum inhibitory concentration (MIC) of the products was 0.1 mcg/ml. The use of liposomes loaded with BG 12 molecules in the optimal concentration (0.1 mcg/ml) resulted in inhibition of the avian plague virus growth in the MDCK cells. Possible design of efficient drugs for antiviral protection based on the complexes liposomes--boron-containing compounds is discussed.

[Enhancing the immunogenicity of inactivated polio vaccines with chitosan used as an adjuvant].

Addition of chitosan to inactivated trivalent polio vaccine or inactivated preparations of attenuated poliomyelitis viruses (Sabin strains) significantly increases immunogenicity of these inactivated poliomyelitis virus preparations. High neutralizing antibody titers are detected after two immunizations of mice and a single immunization of rats, as well as when the antigen dose was reduced by 4 times. Addition of chitosan as an adjuvant significantly induces cellular immunity.

[Comparative study of avian influenza virus propagation in the cell culture and chick embryos].

Comparative reproduction studies of 7 avian influenza virus strains (H5N1, H5N2, H3N2, H4N6, H7N7) in Vero and MDCK cell lines have indicated that the MDCK cell line is an optimal substrate for all study strains. The maximum viral output depends on trypsin concentrations and infection doses, which can differ for individual viral strains. The use of the optimal parameters of avian influenza virus replication in the MDCK cell lines yields virus titers comparable with virus reproduction in the chick embryos. The reproductive studies of the same avian influenza virus strains in chick embryos have shown that the maximum virus multiplication is seen when observing the optimum incubation time for infected embryos, which may be dissimilar in different strains. A considerable increase in hemagglutinin output can be achieved on adding trypsin to the infected chick embryos.

[Chitosan as an adjuvant for inactivated vaccines against avian influenza viruses].

AIM: To study chitozan as an adjuvant for inactivated vaccines against A/H5 influenza viruses. MATERIALS AND METHODS: Avian A/H5 influenza viruses were grown on chicken embryos or on MDCK cell line; viruses-containing fluid was inactivated with formalin. Mice were vaccinated intramuscularly with inactivated avian influenza virus mixed with chitozan and then levels of hemagglutination-inhibiting and neutralizing antibodies as well as protective efficacy against both homologous and drifted strains of avian influenza viruses A/H5 were measured. RESULTS: Addition of chitozan to inactivated preparations of A/H5 avian influenza viruses for immunization of mice significantly increased levels of hemagglutination-inhibiting and neutralizing antibodies to both homologous and drifted variants of A/H5 influenza viruses, including those containing neuraminidase from other subtype as well as strains isolated 10 - 20 years earlier than virus used for vaccination. Chitozan significantly improved protective efficacy of inactivated avian influenza vaccines against infection with both homologous and drifted variant of the virus. Vaccination with inactivated avian influenza viruses A/H5 and chitozan induced high levels of antibodies even after single immunization as well as after administration of 8-fold reduced dose of preparation. CONCLUSION: Chitozan is a perspective adjuvant for inactivated vaccines against avian influenza viruses, which could significantly improve immune response and protective efficacy against both homologous and drifted variants of avian influenza viruses A/H5.

[Molecular mechanisms of reversions to the ts+ -phenotype of cold-adapted influenza virus A strains--attenuation donors for live influenza reassortant vaccines].

A ts+ revertant of cold-adapted (ca) strain A/Leningrad/134/47/57--the attenuation donor for live influenza reassortant vaccines--was obtained by passages of the ca strain in chick embryos at nonpermissive temperatures. The ts+ revertant acquired the ability to grow in chick embryos at 40 degrees C and lost the capacity to reproduce there at 25 degrees C. A complementation-recombination test using the fowl plague virus (FPV0 ts-mutants showed the loss of the ts-phenotype in the RNA-segments of ts+ revertants' genome coding for PB2, NP, and NS (NS2) proteins. However, PCR-restriction analysis revealed a true reversion in RNA-segment coding for PB2 protein only. All the investigated mutations in the ts+ revertant genome were preserved. This phenomenon could be explained by the appearance of intragenic and extragenic suppression mutations in the ts+ revertant genome. The data of the complementation-recombination test suggest that reversion of ts-phenotype occurs more frequently due to extra- or intragenic suppression rather than as a result of a true mutation loss. Estimation of the genetic stability of vaccine ca strains of influenza virus should be based on the combined use of PCR-restriction and complementation tests.

Development of cell culture (MDCK) live cold-adapted (CA) attenuated influenza vaccine.

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.

[Further development (MDCK) of live cold-adapted influenza vaccine: cultivation of vaccine strains in production fermenters].

Optimal conditions were developed for cultivating the cold-adapted reassortant live influenza vaccine (CARLIV) in MDCK cells, which were in their turn cultivated in fermenters with serum-free medium and microcarrier. The use of MDCK cells meets all national and WHO requirements to continuous cells used in the production of biological preparations. CARLIV cultivated under such conditions well preserve their ts-mutations and mutation, which entail substitutions of amino acids, in all CARLIV genome segments. Provided the cultivation conditions are optimal, the output of multivalent CARLIV in a 101 fermenter can reach 100000 doses.

[Genetic characterisation of wild measles strains circulating in European part of Russia in 1998-2002]. / Geneticheskaia kharakteristika dikikh shtammov virusa kori, tsirkuliruiushchikh na evropeiskoi territorii Rossii v period 1998-2002 gg.

The primary structure of the N-gene COOH terminus of measles virus isolated, 1998-2000, in Russia's European part was investigated. The general analysis as well as an analysis of the primary gene structure showed the two group's isolates as belonging to the D4 genotype. A subsequent analysis of the primary structure of the N-gene COOH-terminus of Moscow/2002/61 isolated during the 2002 measles outbreak in Moscow also showed it as belonging to the D4 genotype. The obtained data are indicative of that the wild measles strains belonging to the D4 genotype have been recently circulating in Russia's territory.

[Features of primary structure of genes and proteins from passaged variations of measles virus]. / Osobennosti pervichnoi struktury genov i belkov passazhnykh variantov virusa kori.

Primary structure and proteins of measles virus variants passaged in tissue culture were studied. The findings suggest that genetic determinants responsible for measles virus attenuation are not linked with the genes coding for envelope proteins and nucleoprotein of this virus. However the detected nucleotide substitutions can be considered as the main prerequisites for the appearance of mutations in other regions of viral genome, leading to decrease of virulence for humans.

[Experimental measles infection in rodents]. / Eksperimental'naia korevaia infektsiia u gryzunov.

The sensitivity of newborn hamsters to inoculation with the vaccine L-16 strain of measles virus and the Lec strain isolated from a patient with subacute sclerosing panencephalitis as well as the possibility of persistence of these viruses in the animals were studied. Intracerebral inoculation of the L-15 strain was shown to produce in hamsters acute meningoencephalitis leading to death in 85%-100% of cases. Over 30 days after inoculation, the infectious virus, the virus-specific antigen and virus genome were found in the brain. In the brains of the sick animals, all the structural proteins of measles virus with the exception of hemagglutinin were expressed. After inoculation with the Lec strain, the clinical signs of the disease were less manifest, and mortality was 40%. The infectious virus could be detected in the brain up to 20 days postinoculation, the genome, up to 31 days. All the structural proteins of measles virus were expressed in the brains of the inoculated animals. No persistence of L-16 and Lec strains of measles virus could be demonstrated at langer intervals after inoculation (90-180 days) in the brains of hamsters.

[Preparation and properties of monoclonal antibodies to influenza virus A/Krasnodar/101/59 (H2N2)]. / Poluchenie i svoistva monoklonal'nykh antitel k virusu grippa A/krasnodar/101/59 (H2N2).

Somatic hybridization produced a set of 6 mouse hybridomas producing monoclonal antibodies of G isotype to influenza A/Krasnodar/101/59 (H2N2) virus. MCA were characterized by solid phase enzyme-immunoassay, hemagglutination-inhibition test, and indirect immunofluorescence technique. According to the results of radioimmunoprecipitation, all 6 hybridomas produced MCA to hemagglutinin of influenza A/Krasnodar/101/59 (H2N2) virus.

[Analysis of the possible mechanisms for the persistence of the vaccinal strain of the measles virus in a human cell culture]. / Analiz vozmozhnykh mekhanizmov persistentsii vaktsinnogo shtamma virusa kori v kul'ture kletok cheloveka.

The mechanisms (factors) of the measles virus vaccine L-16 strain persistence in HEp-2 cell culture were analysed. Among the known mechanisms, most likely is the reduction of the cell-destroying properties of the persisting virus due to mutations in nucleoprotein gene manifested by changes of the isoelectric point of NP protein and temperature sensitivity of its synthesis.

[Electron microscopy study of the nucleocapsid structure of the measles virus isolated from cell cultures with primary and chronic infections]. / Elektronno-mikroskopicheskoe izuchenie struktury nukleokapsidov virusa kori, vydelennykh iz kul'tur kletok pri pervichnoi i khronicheskoi infektsii.

Electron microscopic examination of isolated intracellular measles virus nucleocapsids (NC) revealed a relationship between their structure, cell system, and the type of infection. Acute virus infection of Vero or Japanese quail embryo cells gave rise to the formation of linear NC strands with regularly and tightly stacked turns. Acutely infected L-41 or HEp-2 cells contained heteromorphous viral NC populations which included both typical and loosely packed NC. Persistently infected L-41 and Hep-2 cells predominantly contained NC of the latter type with the appearance of a "strings of beads".

[Characteristics of the ribonucleoprotein of the measles virus persisting in a human cell culture]. / Kharakteristika ribonukleoproteida virusa kori, persistiruiushchego v kul'ture kletok cheloveka.

The properties of virus-specific RNPs recovered from human HEp-2 and L-41 cells chronically infected with measles virus were studied in comparison with those of RNPs formed in acute infection of L-41 cells. The persisting RNP was shown to contain nucleoprotein not differing in the electrophoretic mobility from the same protein of measles virus virions, and RNA in the persisting RNP was found to be insensitive to the action of RN-ase. RNP from chronically infected cells had a changed ultrastructure and conformation as compared with RNP of the original virus and, unlike the latter had no infectivity upon transfection of the sensitive cells by calcium-phosphate precipitation. No differences in relationships of RNP with the cytoskeleton of the infected cells in the acute and chronic infection were observed.

[Effect of elevated temperature on the persistence of measles virus vaccinal strain L-16 in a human cell culture]. / Vliianie povyshennoi temperatury na persistentsiiu vaktsinnogo shtamma L-16 virusa kory v kul'ture kletok cheloveka.

The influence of high temperature (40 degrees C) on the virus carrier state and synthesis of virus-specific macromolecules in the HEp-2 cells--measles virus system was studied. The fluorescent antibody technique showed that cultivation at this high temperature led to changes in virus antigen morphology, a decrease in the number and then complete disappearance of the cells producing virus-specific antigen. Simultaneously, gradual cessation of synthesis of all kinds of virus-specific RNA was observed in chronically infected cells. The method of radioimmunoprecipitation showed the incubation at 40 degrees C to affect first of all the synthesis of virus nucleocapsid protein. The temperature-sensitive nature of synthesis of virus macromolecules may be explained by the existence of its mutations in the genome of the persisting virus.