[Use of an immunoperoxidase method in situ for detecting recombinant clones of vaccinia virus containing an expressed gene of influenza viral hemagglutinins]. / Ispol'zovanie immunoperoksidaznogo metoda in situ dlia vyiavleniia rekombinantnykh klonov virusa ospovaktsiny, soderzhashchikh ékspressiruemyi gen gemaggliutinina virusa grippa.

The vaccine L-IVP strain of vaccinia virus (VV) was used to construct the recombinant viral clones containing the influenza A hemagglutinin gene. The recombinant T plasmid was obtained with HA gene inserted in the vector pGS-20 (B. Moss) under the 7.5 K promoter of VV. A homologous recombination technique was used to insert the gene with the flanking TK sequences into vaccinia virus genome. The recombinant clones were selected by dot-hybridization with [32P]-labeled HA-probe. These recombinants were analysed for HA gene expression by the indirect immunoperoxidase method in situ using the peroxidase conjugate of the staphylococcal A-protein. This technique allows to obtain stable stained preparation and analyse the protein behavior at the ultrastructural level.

[Changes in the amino acid sequence of hemagglutinin during sequential adaptation of human influenza virus A to replication in mice]. / Izmeneniia aminokislotnoi posledovatel'nosti gemaggliutinina pri posledovatel'nykh étapakh adaptatsii virusa grippa A cheloveka k razmnozheniiu v organizme myshei.

The primary structure of hemagglutinin (HA) gene of Influenza virus A/USSR/90/77 (H1N1) variants after 3 and 11 passages has been determined. In the HA1 coding region of mice-adapted virus (11 passages) there are two amino acid substitutions: Thr 89----Ala and Asn 127----Asp. At the first stage of adaptation (3-rd passage) only a single mutation was detected: Asn 127----Asp. The adaptation is accompanied by the loss of specific carbohydrate attachment sites adjacent to the receptor-binding site located at HA1 subunit with a concomitant variation in antigenicity.

[Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens]. / Metody polucheniia i selektsii rekombinantov virusov ospovaktsiny, ékspressiruiushchikh chuzherodnye virusnye antigeny.

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.

[Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin]. / Konstruirovanie rekombinantnogo shtamma virusa ospovaktsiny, soderzhashchego ékspressiruemyi gen gemaggliutinina virusa grippa A.

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.

[Primary structure of fragment 7 of the influenza virus A/USSR/90/77(H1N1) RNA]. / Pervichnaia struktura segmenta 7 RNK virusa grippa A/USSR/90/77(H1N1).

The double-stranded DNA copy of the matrix protein (M) gene of the influenza virus A/USSR/90/77 (H1N1) has been inserted in PstI site of plasmid pBR322 and cloned in E. coli. The full-length DNA copy of the M gene has been sequenced using Maxam-Gilbert method. Analysis of nucleotide sequence of segment 7 RNA of influenza virus A/USSR/90/77 and nucleotide substitutions, as compared with known primary structures of segment 7 RNA for other strains, is presented. A hypothetical model of secondary structure of segment 7 RNA of influenza virus and repeating sequences at nucleotide and amino acid levels, revealed in the central region of M1 protein, are discussed.

[Polyadenylation of influenza virion RNA in an in vitro system]. / Poliadenilirovanie virionnoi RNK grippa v sisteme in vitro.

A method for isolation of terminal polyriboadenylate transferase from E. coli cells (MPE 600) is presented. The specific activity of the enzyme yield at the terminal stage was 933 units per 1 mg of protein. Analysis of polyadenylated in vitro virion RNA of influenza virus A/USSR/90/17 strain in polyacrylamideagarose gel (2.2%-0.6%) in the presence of 6 M urea showed all the 8 fragments of genome RNA to be adenylated, their sizes being retained with regard to the distribution in gel of the initial RNA fragments. In vitro polyadenylated virion RNA was an effective matrix in reverse transcription reaction with RNA-dependent DNA-polymerase using oligo (dT) as a primer. Complementary DNAs obtained in this way may be the starting material for synthesis of double-stranded DNAs and subsequent construction of recombinant DNAs containing influenza virus genetic information.

[Isolation of interferon messenger RNA and the synthesis of the double-stranded DNA product]. / Vydelenie informatsiomnoi RNK interferona i sintez dvunitevogo DNK-produkta.

The data on preparation of the fraction of "enriched" interferon messenger RNA of chick fibroblasts by fractionation of biologically active RNA molecules by molecular weight are presented. The conditions of enzymatic synthesis and characteristics of the single- and double-stranded DNA product are described. The ways of producing more highly specific messenger RNA and DNA products are discussed.