Selection of Domestic Cell Lines for the Creation of Diagnostic and Preventive Preparations against Enterovirus 71.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID50/ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

[Further studies of continuous human and animal cell lines for the manufacture of viral vaccines and diagnostic kits].

Long-term experiments have provided conditions for the optimal conditions for reproduction of vaccine strains of poliomyelitis, measles, tick-borne and Japan encephalitis on the continuous cell lines. This makes it possible to solve one of the most urgent problems of modern biotechnology, namely to refuse to use primary cell cultures in vaccinology and to apply a more accessible, safe, and reference biological substrate that are stable cell lines.

[Combined (associated) vaccination against quarantinable infections]. / Kompleksnaia (assotsiirovannaia) vaktsinatsiia protiv karantinnykh infektsii.

The presence of several active foci of infection of different etiology is an indication for complex (combined) immunization against these diseases. The scheme of complex (combined) immunization against plague, cholera and yellow fever has been experimentally substantiated and successfully tested on volunteers.

Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines.

Cell substrate DNA was shown to be an abundant contaminant in the clarified preparations of the Sabin type 1, 2 and 3 poliovaccines produced on a continuous cell line (4647). The size of the DNA, as evaluated for the Sabin type 1 poliovaccine, was highly heterogeneous, ranging from 100 to 20,000 base pairs. In view of potential oncogenicity of this DNA a simple and efficient procedure for its elimination is proposed. The method is based on use of protamine sulphate which at the concentration of 2.0 mg ml-1 precipitated cell DNA almost completely without affecting the virus titres.

[Creation of an experimental cell culture vaccine against distemper using cell line 4647]. / Sozdanie éksperimental'noi kul'tural'noi vaktsiny protiv chumy plotoiadnykh s ispol'zovaniem kletok 4647.

Experiment in susceptible animals demonstrated the effectiveness and safety of several samples of a distemper vaccine prepared in cell line 4647. Preparations of the vaccine virus variants generated in line 4647 after 10 passages at 34 degrees C, 37 degrees C and 40 degrees C and 20 passages of rapidly multiplying virus (34 degrees C) possessed the protective activity when used for immunization in a dose of 1000 PFU/animal. The results obtained in observations of the animals and examinations of sera for antibodies did not differ from those of control tests of "Vakchum".

[Changes in cellular microfilaments in viral infection]. / Izmeneniia kletochnykh mikrofilamentov pri virusnoi infektsii.

The effect of disorders in actin microfilament polymerization by cytochalasin B (CB) on the development of cytopathic changes was studied in two models. CB in concentrations of 0.1-1.0 microgram/ml enhanced 3-5-fold the formation of symplasts in a line of kidney cells from green monkey 4647 infected with carnivore distemper virus and inhibited this process in higher concentrations (2.5-5.0 micrograms/ml). The addition of CB in concentrations 5-10 micrograms/ml into the medium of a line of Chinese striped hamster cells infected with Sindbis virus led to disorders in the permeability of plasmolemma and destruction of cells although virus reproduction and production of virus antigens remained uninvolved. The above results not only demonstrate the role of actin microfilaments in the development of cytopathic lesions caused by paramyxo- and alphaviruses but also allow the assessment of separate stages of these processes.

[RAMT cell line as a substrate for the propagation of vaccinal viral strains]. / Liniia kletok RAMT kak substrat dlia razmnozheniia vaktsinnykh shtammov virusov.

Properties of the PAMT cell line which are deficient in hypoxanthine guanyl phosphoribosiltransferase were studied. The PAMT cell line derived from African green monkey kidney tissue may be identified by selective nutrient media, contains no contaminants, is not tumorigenic, is susceptible to a wide range of viruses. Titres of poliomyelitis, tick-borne encephalitis, and carnivore plague viruses were similar in cell cultures grown in the medium with 10% serum and in those adapted to growth in the medium containing 1% serum. The properties of PAMT cell line allow it to be used in manufacture of killed virus vaccines.

[Cytolar microcarriers for the cultivation of surface-dependent cells]. / "Tsitolar"--mikronositeli dlia kul'tivirovaniia povekhnostno--zavisimykh kletok.

The results of trials of one of the microcarriers (MC) developed by the authors, Cytolar, synthesized from denaturated collagen are presented. Both primary chick embryo cell and green monkey kidney cell cultures and diploid fibroblast-like human embryo cells were successfully cultivated on these MC. The properties of the developed MC were not inferior to those of the best known analogues.

[Comparison of methods for the large-scale culture of cells and viruses]. / Sravnenie metodov krupnomasshtabnogo kul'tivirovaniia kletok i virsuov.

The paper presents the results of experiments on propagation of primary, secondary, and continuous diploid and heteroploid human and animal cells in 2 different systems for large-scale propagation: in perfusion tank with Rashig rings and in tanks with microcarriers. Both methods of large-scale cultivation produce higher cell yields than the traditional cultivation methods. A yield of tick-borne encephalitis virus per 1 cell in the perfusion cultivator was 12 times as high as in roller cultures. Poliomyelitis virus titres were practically equal with both methods of large-scale cultivation and with the conventional method (7.73 lg PFU/ml in GMKC on microcarriers). The method of cultivation on microcarriers is more acceptable and advantageous as it gives higher yields of cells necessary for growth of poliomyelitis virus.

[Fragmentation of the DNA of simian adenovirus type 38 by using restriction endonucleases]. / Fragmentatsiia DNK adenovirusa obez'ian tipa 38 s pomoshch'iu restriktiruiushchikh éndonukleaz.

The effect of restricting endonucleases Eco R I, BgI II and Sal I on simian adenovirus type 38 (SV-38) DNA was studied. Bgl II restrictase cleaves the virus DNA into 4 fragments, A, B, G, and D, with molecular weights 9.3 x 10(6), 3.3 x 10(6), and 2.9 x 10(6) daltons, respectively. After restriction with Eco R I and Sal I SV-38 DNA cleaves into 5 and 6 fragments, respectively. The molecular weights of Eco R I fragments are A--8.2 x 10(6), B--6.5 x 10(6), C--4.0 x 10(6), D--1.27 x 10(6), and of Sal I fragments: A--6.5 x 10(6), B--5.4 x 10(6), C--4.2 x 10(6), D--2.8 x 10(6), E--2.5 x 10(6), and F--0.25 x 10(6). By restriction of DNA-protein compex by means of partial DNA hydrolysis, combined Bgl II and Eco R I hydrolysis and secondary restriction of a fragment eluted from the agar gel, the alternation of Eco R I and Bgl II fragments in SV-38 genome was determined.