Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA.

The integration of DNA of highly oncogenic simian adenovirus type 7 (SA7) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonuclease SalI (SA7) and BglII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome. Additionally integrated fragments were stable and preserved during numerous passages of cells lines, although they were no expressed, at least in the case of the Ad6-transformed cell line. The integration of the fragments of SA7 DNA was accompanied by loss of 25-50% of the mass of each fragment. Adding the linear form of the pBR322 plasmid to the preparation of transforming Ad6 DNA also contributed to its cointegration into the genome of the transformed cell. This technique of cell cotransformation with any foreign DNAs together with the viral oncogens may be used as an equivalent of an integration vector for eukaryotic cells.

A stable antibody-sensitized erythrocyte diagnostic preparation for detection of rhinovirus type 17 and its antibody.

A diagnostic preparation was obtained by sensitization of formalinized sheep erythrocytes with antibody to rhinovirus type 17. This preparation was agglutinated in the presence of relatively high concentrations of type 17 but not other types of rhinovirus. It could also be used for detection of antibody to rhinovirus type 17 in the antigen neutralization test, the antibody levels determined in this way being similar to those found by conventional virus neutralization tests in cell cultures.

Diagnosis of rhinovirus-infections by virological and immunofluorescent methods.

In January--May, 1971, detection of rhinoviurs infections among ambulatory patients with respiratory diseases in Moscow was carried out in parallel by virological and immunofluorescent methods. Rhinovirus infections were virologically confirmed in 13% out of 238 patients. By the indirect immunofluorescence technique, rhinovirus antigens of serotype 17 were detected in 2.2% out of 220 patients, of type 1B in 4.3% out of 183 patients, of type 48 in 5.6% out of 123 patients and of type 7 in 4.6% out of 86 patients. The use of immunofluorescence supplemented the results of virological examinations and increase the effectiveness of aetiological diagnosis of rhinovirus infections.

[Viral sequences in the genomes of rat and hamster cells transformed by simian adenovirus SA7(C8) and its DNA]. / Virusnye posledovatel'nosti v genome kletok krys i khomiakov, transformirovannykh adenovirusom obez'ian SA7(C8) i ego DNK.

Four cell lines transformed by simian adenovirus SA7 and its DNA were analysed by means of molecular hybridization. Content of viral DNA sequences in different lines varied from 10% (the left end) of the molecule to the entire genomes. Transcription of the D BglII fragment (coordinates 1.8--10) was observed in all examined lines. The integration type of highly oncogenic simian adenovirus SA7 DNA differed from the pattern of integration of the highly oncogenic human adenovirus type 12 and was similar to that of the non-oncogenic adenoviruses in adenovirus-transformed cells.

[Chronic infection of HeLa cells with respiratory syncytial virus]. / Khronicheskaia infektsiia kletok HeLa respiratorno-sintsitial'nym virusom.

Chronic infection of HeLa cells with respiratory syncytial virus (HeLa-RS) was produced at a multiplicity of infection of 0.00005 TCD50/cell. During 144 days, 21 passages of HeLa-RS culture were done. The chronically infected cells did not differ from the control culture in the growth pattern and their proliferative activity. The infectious virus was found in the culture fluid with cells at 7 and 14 days after inoculation of the culture. Subsequent attempts at isolation of the infectious virus from the culture fluid and homogenates of the infected cells were fruitless. However, the specific antigen was detected by the immunofluorescence procedure in 17.9-26.0% of cells (6-7 days after cell plating). The number of the infected cells increased after treatment of the culture with 5-iodo-2-deoxyuridine. Passage of HeLa-RS cells in the medium containing specific antibody had no effect on the number of cells containing RS-virus antigen. HeLa-RS cells were moderately resistant to superinfection with RS virus and retained their sensitivity to adenovirus type 3. Out of 9 clones examined 8 were infected. No interferon was found.

[Study of the possibilities of inactivating adeno-associated virus type 4]. / Izuchenie vozmozhnostei inaktivatsii adenoassotsiirovannogo virusa tipa 4

Adeno-associated type 4 virus (AAV-4) is inactivated by formalin dilutions 1 : 400, 1 : 1000 and 1 : 2000 for 24 hours at 37 degrees C. Under these conditions hemogglutinins (HA) and complement-fixing antigen (CFA) are retained. The residual formalin must be neutralized with sodium bisulphite. AAV-4 can also be inactivated by 3% hydrogen peroxide and HA and CFA are retained too. Hydrogen peroxide may be neutralized with catalase. No inactivation of AAV-4 could be achieved with lowere concentrations of hydrogen peroxide, even in the presence of copper sulphate.

[Virus inactivation by hydrogen peroxide]. / Inaktivatsiia virusov perekis'iu vodoroda.

The effect of H2O2 on adenovirus types 3 and 6, adenoassociated virus type 4, rhinoviruses 1A, 1B, and type 7, myxoviruses, influenza A and B, respiratory syncytial virus, strain Long, and coronavirus strain 229E was studied in vitro, using different H2O2 concentration and timec of exposure. H2O2 in a 3 percent concentration inactivated all the viruses under study within 1--30 min. Coronavirus and influenza viruses were found to be most sensitive. Reoviruses, adenoviruses and adenoassociated virus were relatively stable. H2O2 is a convenient means for virus inactivation.

[Development of a method for preparing adeno-associated virus type 4 antigen]. / Razrabotka metoda polucheniia antigena adenoassotsirovannogo virusa tipa 4

A method for preparation of adeno-associated type 4 virus (AAV-4) purified from group-specific adenovirus antigen by adsorption on formalinized sheep erythrocytes and elution into hypertonic NaCl solution was developed. In 1 M naCl solution the purified AAV-4 retained its infectivity and the complement-fixing and hemagglutinating activities. Separation of AAV-4 and adenovirus group-specific complement-fixing antigen was based on differences in conditions of their adsorption and elution. AAV-4 was inactivated by treatment with both formalin and hydrogen peroxide but retained its complement-fixing antigen and hemagglutinating properties. The purified antigen or virus is recommended for serologic tests and other purposes.

[Formation and action of interferon in experimental respiratory syncytial infection]. / Obrazovanie i deistvie interferona pri éksperimental'noi respiratorno-sintsitial'noi infektsii

Respiratory syncytial virus multiplying activity in the lungs of newborn and 6-day-old cotton rats, induced production of considerable amounts of interferon. Interferon production correlated with virus multiplication. Lower interferon titers were observed in the lungs of newborn cotton rats than in the lungs of 6-day-old animals. RS virus also induced production of serum interferon upon intraperitoneal inoculation, its titers being 2 times as low as after inoculation of equal doses of Newcastle disease virus. Poly I: poly C was shown to be a quite active interferon inducer in cotton rats and to inhibit RS virus reproduction in their lungs.

[Pathogenesis of rhinovirus infection]. / O patogeneze rinovirusnoi infektsii.

Rhinoviruses (RV) are the etiological factors of not only acute respiratory diseases of the common cold type but also of acute and chronic diseases of otorhinolaryngological organs. Virological and bacteriological examinations of patients with chronic pneumonia in the stages of exacerbation and incomplete remission revealed a high degree of the infection activity. Virus-bacterial associations were frequently found. RV were isolated from the lower respiratory tract (bronchopulmonary secrete, biopsy materials). The association of RV with the involvement of the lower respiratory tract in patients with chronic pneumonia was demonstrated. Rhinoviremia was detected in one child dying with acute respiratory disease. The results of studies on some factors of specific and nonspecific immunity in patients with chronic pneumonia are presented.

[Isoenzyme characteristics of glucose-6-phosphage dehydrogenase and lactate dehydrogenase of continuous cells from the collection of the Sverdlovsk Research Institute of Virus Infections]. / Izofermentnaia kharakteristika gliukozo-6-fosfatdegidrogenazy i laktatdegidrogenazy perevivaemykh kletok kollektsii Sverdlovskogo nauchno-issledovatel'skogo instituta virusnykh infektsii

An analysis of isoenzymes of glucose-6-phosphage dehydrogenase (G-6-P-DH) and lactage dehydrogenase (LDH) of continuous cells from the collection of Sverdlovsk Research Institute of Virus Infections was carried out. Seventeen continous human and animal cell lines were examined by electrophoresis in a vertical block of 7% polyacrylamide gel followed by histochemical detection of the enzymes. The HEp-2, HEp-2 clone No. 23, KB, RH, and FL cells were shown to have the electrophoretic motility of G-6-PDG characteristic of HeLa cell line. LEP and SOC cell lines were contaminated with mouse cells. A culture of rat fibroblasts had isoenzymes of G-6-PDG and LDG characteristic of HeLa cell line. The remaining cell lines had the isoenzymatic characteristics corresponding to their species appurtenance.

[Human rhinovíruses circulating in Moscow (author's transl)]. / Rinovirusy cheloveka, tsirkuliruiushchie v Moskve

In the period of 1971--1973 in Moscow, 85 strains of rhinoviruses (types IB, 14, 20, 27, 33, 48, 56, 60 and 69) were isolated from patients with respiratory diseases. In March-May 1971 in one administrative district of Moscow, from ambulatory patients with ARD rhinoviruses of types 48 (24 strains) and 27 (8 strains) were predominantly isolated. An outbreak of diseases due to these two types appeared to have occurred. From children rhinoviruses were isolated not only from nasopharyngeal washings. From 3 children they were isolated from the lower respiratory tract (from the blood collected before his death due to staphylococcal sepsis. Surveys for virus-neutralizing antibody to 17 rhinovirus serotypes in 10 lots of gamma globulin prepared in different towns of the Soviet Union revealed wide spread of rhinovirus types IB, 13, 18, 31 and 32 limited spread of types 17 and 42 and moderate spread of types 2, 3, 7, 10, 14, 21, 22, 24, 48 and 53.

[Preparation of immune serums for the identification of rhinoviruses]. / Prigotovlenie immunnykh syvorotok dlia identifikatsii rinovirusov

Six schedules of immunization of rabbits, guinea pigs and rats for production of antirhinovirus immune sera were studied. Sera with virus-neutralizing antibody titers of 1:1000 to 1:50,000 were obtained in rabbits immunized into popliteal lymph nodes. The resulting sera had high titers of antibody to cell components and neutralized the cytopathic effect induced by heterologous strains. Sera from which cell components had been absorbed showed strict type-specificity in the neutralization and indirect immunofluorescent tests. A mixture of sera has been shown to be useful for identification of rhinoviruses by the immunofluorescent procedure.

[Viremia in respiratory syncytial virus infection]. / Virusemiia pri respiratorno-sintsitial'noi (RS) virusnoi infektsii

Viremia was demonstrated to occur in experimental respiratory syncytial (RS) virus infection in suckling cotton rats and in natural infection in children. RS virus was isolated from the whole blood of the animals in 3 out of 6 experiments at 2, 5, 6, 7 and 15 days after inoculation, the maximum infectious titer being more than 10(4) TCPD50/0.1 ml. RS virus was also isolated from the blood of 7 out of 15 examined children presenting the typical clinical picture of RS virus disease during the epidemic season of RS virus infection. In 6 patients RS virus was isolated from one blood specimen at 1, 6 and 7 days after the onset, in one patient from 3 blood specimens at 6, 9 and 22 days after the onset. The demonstrated long-term persistence of virus in the blood suggests the possibility of existence of chronic RS virus infection.

[Development of optimal conditions for isolation of rhinoviruses]. / Razrabotka optimal'nykh uslovii dlia vydeleniia rinovirusov

When 366 dispensary patients with respiratory diseases were examined in Moscow during the period December 1969--May 1971, 49 strains of rhinoviruses were isolated out of which 21 strains were identified as type 48. Using Eagle's medium with a single set of amino acids and vitamins for isolation rhinoviruses were isolated in 4.7% of cases, empdoying Eagle's medium with a double set of amino acids and vitamins in 16% of cases. Virus isolation experiments were carried out in HeLa-Bristol cell cultures and diploid strain of skin-muscle cells KM-13-70 in the presence of 30 mM MgCl2 and 2% aminopeptide. The isolation rate of rhinoviruses in both the cell cultures was approximately similar. The average duration of time for isolation of rhinoviruses in diploid cells was 13.4 days, in HeLa-Bristol cells 15 days. All rhinovirus strains were isolated within 3 passages. In addition to rhinoviruses, from the same group of patients 25 Coxsackie A-21 strains, 12 adenovirus strains and 2 herpesvirus strains were isolated using Eagle's medium with double set of amino acids and vitamins.

[Identification of a new antigenic variant of simian adenovirus (SV30N)]. / Identifikatsiia novogo antigennogo varianta adenovirusa obez'ian (SV30N).

A new variant of simian adenovirus SV30-N is described. The variant is antigenically close to SV30 virus in the neutralization test but has some antigenic relationship to SA7 virus. The properties of SV30-N virus were retained after 5 clonings by the plaque method, after passage and cloning of the virus treated with immune sera to SV30 or SA7. The heteroduplex analysis showed DNA of the virus under study to be by 80% and 40% homologous to SV30 DNA and SA7 DNA, respectively, whereas no heteroduplex molecules between SV30 and SA7 DNA were found. By the set of polypeptides the antigenic variant SV30-N is close to SV30 but differs from SV30 and, particularly from SA7. Unlike SV30 and SA7 viruses, the SV30-N virus showed no oncogenic activity for hamsters.

[Isolation of adeno-associated virus type 4 from a culture of green monkey kidney cells]. / Vydelenie adenoassotsiirovannogo virusa tipa 4 iz kul'tury kletok pochek zelenykh martyshek.

In the process of biological control of uninfected green monkey kidney (GMK) cell cultures a thermostable hemagglutinating agent designated No. 5056 was isolated alongside with adenovirus-SV. By its antigenic properties the 5056 strain was identified as adeno-associated virus type 4 (AAV-4). In control of 574 specimens of GMK culture batches, 40 AAV-4 strains were isolated in the presence of a helper adenovirus. Some biological properties of the isolates and their resistance to certain physical and chemical treatments were studied. Two isolates of the satellite virus were examined in the electron microscope. A correlation between the rate of AAV-4 isolation from GMK cultures and the presence of complement-fixing antibody to AAV-4 in monkey sera was observed.

[Experimental infection of green monkeys with adenoassociated virus]. / Eksperimental'naia infektsiia zelenykh martyshek adenoassotsiirovannym virusom.

Primary infection and reinfection with adeno-associated virus type 4 (AAV-4) was reproduced in green monkeys experimentally infected with AAV-4 in mixture with adenovirus. Wide dissemination of the satellite virus in animals was observed. AAV-4 and its antigen were detectable 5 to 23 days after inoculation. In monkeys infected with a mixture of AAV-4 and adenovirus or with one of them the infection was accompanied by a marked fever persisting from the 5th to the 20th day after inoculation. The infected monkeys showed an intensive rise of homologous antibody titer most marked on the 10th-15th day after inoculation with AAV-4. AAV-4 and its antigen were detected in smears from conjunctival and tonsillar mucosa, rectal specimens in the time course of the infectious process, as well as from the trachea, lungs, liver, spleen, intestines and kidneys of the sacrificed monkeys. Besides, AAV-4 antigen was found in cells of the tonsils and blood leukocytes of the sacrificed monkeys. No virus or its antigen were found in the brain and heart tissues. Virions of adeno-associated virus were found by electron microscopic examinations of kidney cells of one of 3 monkeys infected with AAV-4.

[Isolation and translation of late mRNA coding for the structural proteins of simian adenovirus SA7]. / Vydelenie i transliatsiia pozdnikh mRNK, kodiruiushchikh strukturnye belki adenovirusa obez'ian SA7.

A total preparation of cytoplasmic RNA was isolated from late stage-infected African green monkey kidney cells using phenol extraction procedure. The poly (A)-containing mRNA fraction was selected on oligo(dT)-cellulose columns. The resulting mRNA preparations were heterogenous in size and contained about 20--60% of SA7-derived sequences. SA7 late mRNA was efficiently translated in rabbit reticulocyte cell-free system giving rise to a number of polypeptide products that were related by antigenicity to authentic SA7 virion proteins. The main translation product having a molecular weight of 114 kilodaltons was identified as intact SA7 hexon protein.

[Biological properties of large-plaque clone 230 of simian adenovirus SA7(C8)]. / Nekotorye biologicheskie svoistva krupnobliashechnogo klona 230 adenovirusa obez'ian SA7(C8).

The clone KB-230 of simian adenovirus SA7(C8) is described differing from the reference SA7(C8) strain and clones KB-2 and MB-1 by the presence of additional recognition sites when treated with different endonucleases. The KB-230 clone differs antigenically in the neutralization test from the MB-1 clone. Some biological and molecular-biological properties of the KB-230 clone were studied. All the simian cell cultures under study were highly sensitive to the cytopathic effect and replication of the KB-230 clone. The reproduction cycle of the KB-230 clone was 12 h, that of KB-2 clone 16 h. The maximum accumulation of virus in the cells was observed by 27-29 h for both clones. The KB-230 clone differed from the KB-2 clone by early development of the CPE (by 9 h of cultivation against 19 h for the latter). The oncogenic activity of the KB-230 clone was less marked than that of the MB-1 clone. The methods of heteroduplex and polypeptide analysis established the difference of the KB-230 clone from the reference SA7(C8) strain and KB-2 and MB-1 clones.