Enzyme immunoassay for detection of antibodies against herpes simplex virus with the use of different viral antigens.

Enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to herpes simplex virus type 1 (HSV-1) have been performed by using different immunosorbents prepared by passive adsorption of four HSV-1 antigen preparations to the wells of polystyrene microtitre plates in order to compare the the sensitivity, specificity and reproducibility of the tests. The following antigen preparations have been used: virus-infected native Vero cells and their lysates, membrane glycoproteins and virus nucleocapsid proteins. The optimal conditions have been established for each assay system: the concentrations of adsorbed antigen and the "threshold" values of the optical density. For each antigen tested except of the nucleocapsid (NC) proteins, comparable antibody titres were found in the sera of 6 patients with different forms of herpes infections and in 86 healthy subjects. In the sera of 6 herpetic patients the antibody titres were higher against NC antigen than against other antigen preparations.

Standardization of ELISA for detection of antibodies to orthopoxviruses.

The experience with ELISA technique utilized at serological screening for orthopoxviruses in the Republic of Ivory Coast revealed factors reducing the sensitivity and the specificity of the test. It was found out that routine controls such as "positive" and "negative" sera as well as the accepted reading of the results by two-fold or higher increase of OD values as compared to the "negative" control may be not sufficient. A significantly enhanced sensitivity and specificity of reaction was achieved by simultaneous examination of each serum under study with a control antigen. Selection of optimal dilutions of each test component followed by spectrophotometric assay and calculation of results according to the given formula contributed to the same aim. As a result of these improvements the rate of antibody detection among revaccinees was enhanced from 19 to 78.8% and the titres of ELISA and virus neutralization tests correlated in 88% of cases.

Monoclonal antibodies to monkey pox virus: preparation and application.

Two fusion experiments with NSO myeloma cells yielded 30 hybrid cell lines which continuously and actively secreted monoclonal antibodies (MoAb) to monkey pox virus. Eight lines appeared to produce antibodies to specific antigenic determinants. The isotype and serologic reactivity of MoAb to monkey pox virus was characterized and the karyotype of several hybridomas determined. An enzyme labelled conjugate has been prepared from MoAb selectively reacting with monkey pox virus. This conjugate allowed the identification of monkey pox virus in the materials from patients. It also helped to determine that an orthopoxvirus isolated from wild squirrel in the Republic of Zaire was monkey pox virus.

Monkey pox in humans: current results.

The presented studies were performed at the WHO Collaboration Center for Smallpox in Moscow in the framework of the WHO monkey pox project. The authors recommend improved methods for rapid detection of orthopoxvirus antigen, namely passive haemagglutination (PHA) using dried stable red blood cells and ELISA in order to provide more rapid and efficient laboratory diagnosis under field conditions. Independent serologic diagnosis of monkey pox by ELISA-adsorption (ELISA-A) was proved of value for epidemiological studies and for detection of inapparent infections. The application range of the latter technique and its limitations were also determined.

[Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies]. / Obratnaia transkriptaza virusa immunodefitsita cheloveka: klonirovanie, ékspressiia v Escherichia coli, ochistka fermenta i poluchenie monoklonal'nykh antitel.

To express HIV-1 reverse transcriptase in E. coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained. The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced. The purification of reverse transcriptase was carried out. The substantial proteolysis of reverse transcriptase during purification was shown. The purified preparation is predominantly, an active protein with Mr 57 kDa. Some properties of this protein differed from the reverse transcriptase isolated from HIV.

[HIV-1 chemokine receptors and their role in the pathogenesis of AIDS]. / Khemokinovye retseptory VICH-1 i ikh rol' v patogeneze SPIDa.

Data on cellular receptors playing an important role in penetration of HIV into the cell and determining further development of HIV infection are reviewed. Special attention is paid to chemokine receptors CCR5 and CXCR4. The resistance of some humans to HIV-1 infection, probably due to defective CCR5 gene allele, is discussed.

[A nonisotopic method of quantitative PCR analysis in diagnosing HIV infection]. / Neizotopnyi metod kolichestvennogo PTsR-analiza v diagnostike VICh-infektsii.

A nonisotopic method for the detection of HIV DNA has been developed, based on the use of avidin-biotin complex to identify the products of polymerase chain reaction (PCR). Biotin label integrated in deoxyuridine triphosphate (bio-11-dUTP) was incorporated in the structure of amplified fragments in the course of PCR. The resultant products were identified in hybridization reaction with specific HIV DNA adsorbed on polystyrene plates. The suggested method was not inferior to the traditional radioisotope method in sensitivity and specificity and helped quantitatively assess the results of experiments using an objective criterion--optic density value.

[Quantitative determination of the infective activity of the human immunodeficiency virus by plaque formation using monolayer cultures]. / Kolichestvennoe opredelenie infektsionnoi aktivnosti virusa immunodefitsita cheloveka metodom bliashkoobrazovaniia s ispol'zovaniem monosloinykh kul'tur.

A method for assessment of the infective activity of HIV using plaque formation method with human diploid cells (strains L-65, L-68, L-72) has been developed. This method is not inferior to plaque formation with poly-L-lysine in sensitivity, but superior to it in reproducibility of results and standardization of the test conditions. The method is fit for titration of HIV and virus-neutralizing antibodies, as well as for assessment of the activity of anti-HIV preparations.

[Immunoenzyme test systems for detecting the HIV antigen and a trial of their use in examining HIV-infected subjects]. / Immunofermentnye test-sistemy dlia vyiavleniia antigena VICh i popytka ikh ispol'zovaniia pri obsledovanii VICh-infitsirovannykh lits.

The results of the development and trials of two variants of EIA test system for the detection of HIV-1 antigen using biotinated HIV-antibody and streptavidin-peroxidase or biotin-beta-lactamase enzyme complexes are presented. These diagnostic preparations proved to be highly sensitive and specific allowing the detection of the antigen in the blood of HIV-infected subjects.

[Stability of the main components of an EIA test system for the serodiagnosis of HIV infection]. / O stabil'nosti osnovnykh komponentov test-sistemy IFA dlia serodiagnostiki VICh-infektsii.

The stability of two most important components of the EIA test system for diagnosis of HIV infection, an immunosorbent (HIV antigen adsorbed on polystyrene plates) and antispecies peroxidase conjugate, was studied. The possibility to prolong the shelf life of the test system for at least up to 6 months was demonstrated. At the same time, it is proposed to supplement the existing methods of production control with the conjugate thermostability test developed by the authors who believe that this will allow to exclude conjugates with insufficient stability from the test system kit.

[Increase in the specificity of immunoenzyme analysis by using a monoclonal antibody-based conjugate]. / Povyshenie spetsifichnosti immunofermentnogo analiza za schet ispol'zovaniia kon"iugata na osnove monoklonal'nykh antitel.

Examinations by EIA of laboratory cultures of various orthopoxviruses and specimens from human-monkey pox cases demonstrated the advantages of a monoclonal preparation over the peroxidase polyclonal conjugate prepared from hyperimmune vaccinia antiserum.

[Detection and quantitative determination of antigen to the human immunodeficiency virus during development of a test-system for the serodiagnosis of AIDS]. / Vyiavlenie i kolichestvennoe opredelenie antigena virusa immunodefitsita cheloveka v protsesse polucheniia test-sistemy dlia serodiagnostiki SPID.

One of the variants of IFA using a conjugate on the basis of anti-HIV-IgG was used for control of the content of human immunodeficiency virus (HIV) antigen at different stages of production of a test system for serodiagnosis of AIDS. This method permits HIV antigen quantitation in virus lysates, its nonpurified concentrates, and in native culture fluid in titres from 2000 to 60,000 and from less than 27 to greater than 729, respectively. In most cases, high titres of antigen in the liquid fraction of the culture corresponded to high values of the antigen-containing cells on the basis of immunofluorescence data and signs of intensive HIV production in electron microscopic culture control. The authors developed a method allowing to determine the antigen titre in the study material using only one dilution (1:9) of it in IFA technique.

[Primers for the nef gene in the diagnosis of HIV infection using the polymerase chain reaction]. / Praimery k genu nef v diagnostike VICh-infektsii s pomoshch'iu polimeraznoi tsepnoi reaktsii.

A new system of primers and internal probe identifying the nef gene of HIV-1 was selected and tested for the laboratory diagnosis of HIV infection by means of PCR. The proposed primers and probe by their characteristics approached the optimal ones for PCR diagnosis. The diagnostic value of the above system was confirmed by the PCR analysis of blood samples from 29 patients including subjects with a doubtful diagnosis of HIV infection. The results of the PCR analysis using primers nef1/nef2 correlated with the results obtained by employing the widely used primers for the gag gene SK38/SK39 and attested to the high specificity and diagnostic efficiency of the proposed system.

[Development and practical use of new experimental models of the various forms of herpetic infection]. / Razrabotka i prakticheskoe ispol'zovanie novykh éksperimental'nykh modelei raznykh form gerpeticheskoi infektsii.

New experimental models of neurological herpes in cotton rats and genital herpes in male guinea pigs have been developed which are more adequate to the corresponding human diseases, and models of ophthalmic herpes in rabbits and guinea pigs have been improved. These models may be used for screening and evaluation of the effectiveness of drugs for herpes. A high activity against herpes of bromovinyldeoxyuridine and acyclovir has been verified, a marked therapeutic effect of Soviet monophosphates ara-A, ara-C, and original silur preparation in some forms of herpes infection has been demonstrated.

[Variability of the HIV-1 nef regulatory gene and its association with different HIV stages]. / Variabel'nost' reguliatornogo gena nef VICH-1, sviazannaia s razlichnymi stadiiami VICH-infektsii.

Biological properties of HIV-1 laboratory strains and isolates were studies and compared with research results of the nef gene fragment obtained from their proviral DNA and RNA as well as from RNA and HIV-1 DNA isolated immediately from the blood of patients at early HIV stages. The electrophoresis pattern as well as the results of determination of nucleotide sequences showed that the HVI-1 RNA nef gene of rapid/high laboratory strains of HIV isolates and RNA obtained from patients at late infection stages had a 135-nucleotide inner deletion. The phenomenon can be regarded is proof of that nef gene structure, if violated, causes an enhanced virulence of and an intensified multiplication of the virus (according to laboratory markers) observed at late HIV stages, which triggers, at least in a number of cases, the infection aggravation.

[Use of an immune blotting method (western) for studying viral antigens and for detecting antibodies to viral proteins]. / Primenenie metoda immunnogo ("vestern") blotinga dlia izucheniia virusnykh antigenov i vyiavleniia antitel k virusnym belkam.

The technique of the procedure and the results of the use of immune ("western") blotting method for studies on viral antigens and detection of antibodies to individual proteins of viral particles are described. The possibility of detection and study of individual viral antigens in the whole plasma of patients or carriers is demonstrated by the example of HBs antigen of human hepatitis B virus. The method of immune blotting was used for screening of human sera for the detection of antibodies to the AIDS virus proteins. The sera under study positive for antibodies to AIDS virus by preliminary solid-phase enzyme-immunoassay were shown to contain actually the antibodies to different proteins of AIDS virus. The antibody levels to individual AIDS virus proteins varied in different sera. Some sera positive for antibodies to AIDS virus by the solid-phase enzyme-immunoassay contained no antibody to AIDS virus proteins but reacted with cellular proteins present in the antigen. The immune blotting method was also used for determinations of the spectrum of antibodies in animal sera produced by immunization with different viral antigens.

[Results of examining wild monkeys for the presence of smallpox antibodies and smallpox group viruses]. / Rezul'taty obsledovaniia dikozhivushchikh obez'ian na nalichie protivoospennykh antitel i virusov ospennoi gruppy

The results of examinations of sera, blood and organs of different species of monkeys from some Asian and African countries for the presence of antibody to smallpox and viruses of the smallpox group. Significant titers of smallpox antibodies (antihemagglutinins virus-neutralizing and, in some cases, precipitating antibody) were found in a considerable number of monkeys shot near foci with human cases (Equatorial province of Zair Republic). In the same monkeys kidney tissues yielded 3 isolates of smallpox virus group two of which were indistinguishable in the laboratory tests from variola virus. On the basis of these data it is concluded that smallpox viruses circulate among wildlife monkeys in some areas of Equatorial Africa. Further studies along these lines are necessary.

[The strain features of the HIV-1 circulating in the USSR based on data from a study of its properties in cell cultures]. / O shtammovykh osobennostiakh tsirkuliruiushchego v SSSR VICh-1 po dannym izucheniia ego svoistv v kletochnykh kul'turakh.

Both variants of HIV-1 reported in the literature: slow/low and rapid/high types, were detected among the strains isolated from the subjects examined in 4 foci of HIV-1 infection in the south of the RSFSR and Byelorussia. All the 17 strains isolated in the southern RSFSR foci belonged to the slow/low type and had a low and unstable replication potential in donor peripheral blood mononuclear cells and in MT-4 cell line. All of them were isolated from subjects with asymptomatic infection and from children with initial clinical manifestations of the disease. Only one strain isolated in Byelorussia belonged to the rapid/high type. Its replicative activity was very similar to that of the classical HIV-1--HTLV-IIIB strain. Long-term (up to 7 months) propagation of slow/low strains did not result in any increase of their replicative activity. The capacity to form syncytia was found not only in the rapid/high type strains but also in the majority of slow/low strains under study.

[Experimental study of cellular immunity after administration of inactivated and live virus vaccine]. / Z Eksperimental'noe izuchenie kletochnogo immuniteta pri vvedenii inaktivirovannogo i zhivogo virusa vaktsiny.

Experiments were conducted on guinea pigs with the use of cell migration inhibition test of the peritoneal exudate; stimulation of a definite level of cell immunity in response to the administration of both live and of inactivated vaccine virus was shown. The results obtained are used for the interpretation of the action mechanism of the inactivated preparation in two-stage smallpox vaccination.

[Use of the passive hemaglutination reactions for the determination of anti-smallpox antibodies in primary vaccination and revaccination against smallpox]. / Primenenie reaktsii passivnoi gemaggliutinatsii dlia vyiavleniia protivoospennykh antitel pri pervichnoi vaktsinatsii i revaktsinatsii protiv ospy

The authors present the results of studying the PHAT sensitivity in comparison with the neutralization test and the hemagglutination inhibition test in examination of 254 sera of revaccinated and 95 sera of primarily vaccinated children. It appeared that PHAT was characterized by a sufficiently high sensitivity, and reproducibility, in case the same batch of erythrocytic diagnostic agent was used; at the same time the test was simple, accessible, economic, and gave rapid results. This test can be used for assessment of the immunological efficacy of smallpox revaccination.