[Expression of hepatitis A virus procapsids in the insect cells infected by recombinant baculovirus]. / Ekspressia prokapsidov virusa gepatita A v infitsirovannykh rekombinantnym bakulovirusom kletkakh nasekomykh.

The recombinant baculocvirus containing genome P1-2A-P3 of hepatitis A virus (HAV) was constructed and used for infecting the Sf9 insect cells. It was demonstrated that the deletion of 2BC from HAV polyprotein and the insertion of a new 3C protease cleavage site between P1-2A and P3 did not interfere with the processing of polyprotein or with forming the 70S-procapsids. The identity of the protein contents as well as of morphological and antigen characteristics, obtained in Sf9-cells, to HAV empty capsids, which take shape in the infected mammal cells, proves that it is possible to use them in making the vaccine and diagnostic preparations.

[Synthesis and immunochemical properties of oligopeptides--fragments of capsid proteins of the hepatitis A virus]. / Sintez i immunokhimicheskie svoistva oligopeptidov--fragmentov kapsidnykh belkov virusa gepatita A.

The hepatitis A virus (HAV) capsid protein VP1, VP2 and VP3 are exposed at the virion surface and should therefore contain antigenic determinants. Algorithms for hydrophilicity, antigenicity and flexibility were used to predict probable antigenic sites. Synthesis of 7- to 23-membered overlapping peptides from seven sites, viz., 1-11, 1-17, 2-33, 11-25, 73-82, 76-86, 98-109, 98-112, 102-107, 102-108, 108-127, 113-123, 118-140, 276-298 from VP1, 42-62 from VP2, 76-85 from VP3, and 1-23 from VP4, was performed by various solid-phase methods. Free peptides and their conjugates with different carriers were used for immunization and study of antigenicity. The peptides did not interact with antibodies to the hepatitis A virus, whereas their conjugates did not induce the formation of anti-HAV-antibodies.

[A comparative study of the sensitivity and specific activity of an immunoenzyme test system for determining class-M antibodies to the hepatitis A virus]. / Sravnitel'noe izuchenie chuvstvitel'nosti i spetsificheskoi aktivnosti immunofermentnoi test-sistemy dlia opredeleniia antitel klassa M k virusu gepatita A.

The diagnostic value of the first experimental production batches of assay kit "DIAGN-A-HEP", produced at the Institute of Poliomyelitis and Viral Encephalitides (USSR Acad. Med. Sci.) and intended for the determination of IgM to hepatitis A virus (HAV) in the enzyme immunoassay (EIA), has been studied in comparison with that of the internationally known and widely approved commercial EIA system "HAVAB-MEIA" for the determination of antibodies to HAV (the product of Abbott, USA). The study has revealed that the EIA kit "DIAGN-A-HEP" is highly sensitive and specific, and the diagnostic value of this kit is not inferior to that of the commercial assay system "HAVAB-MEIA". On the basis of this study the use of the EIA kits "DIAGN-A-HEP" in medical practice has been allowed by the decree of the Ministry of Health of the USSR.

[The antigenic properties of hepatitis A virus particles possessing different physicochemical characteristics]. / Antigennye svoistva chastits virusa gepatita A, obladaiushchikh razlichnymi fiziko-khimicheskimi kharakteristikami.

A modified enzyme immunoassay based on adsorption of antihepatitis A virus (HAV) IgG-HRPO conjugate and monoclonal antibodies to HAV were used to investigate antigenic differences between mature HAV virions and subviral particles with different buoyant densities in CsCl produced in HAV-infected cells. The mature virions (1.34 g/cm3) appeared to have common antigenic determinants with subviral particles (1.20, 1.27, and 1.30 g/cm3) and possess some additional determinants. Nevertheless, both subviral particles and mature virions induced antibodies capable of neutralizing HAV infectivity in tissue culture.

[The immunogenicity of a cultured inactivated hepatitis A vaccine]. / Immunogennost' kul'tural'noi inaktivirovannoi vaktsiny gepatita A.

A trial of inactivated hepatitis A viral vaccine of heteroploid cell culture origin is described. The vaccine preparation was tested in guinea pigs and tamarins. The animals were immunized intramuscularly four or three times, respectively. The efficacy was judged by induction of anti-HAV antibody persisting for at least 12 months in guinea pigs, and development of immunity to subsequent virus challenge (monkeys only). The challenge dose of HAV was unable to produce any signs of HAV infection in the vaccinated tamarins, although the booster effect was observed in some animals. The study demonstrated that the tested batches of the vaccine were highly immunogenic.

[The synthetic peptide 62-75 VP3 of the hepatitis A virus induces virus-binding antibodies]. / Sinteticheskii peptid 62-75 VP3 virusa gepatitia A indutsiruet virussviazyvaiushchie antitela.

Peptide (P6) representing amino acids 62-75 of HAV capsid protein VP3 was synthesized. Pure P6, octamer form of P6, and P6 conjugated to KLH were injected into rabbits. The sera against these preparations reacted with denaturized VP3 in immunoblotting tests; however, only anti-P6-KLH serum was capable of binding the native HAV.

[Mapping of the structural proteins of the hepatitis A virus]. / Kartirovanie strukturnykh belkov virusa gepatita A.

The Western blot procedure with highly specific antipeptide antibody was applied to identify the electrophoretic mobility of hepatitis A virus capsid proteins. Polypeptides with molecular weights of 33, 29 and 27 kDa proved to be VP1, VP2, and VP3 proteins as they reacted with sera generated to VP1 recombinant protein and to synthetic oligopeptides 42-62 VP2 and 62-75 VP3, respectively.

[The results of serological research to determine hepatitis A and B markers in the blood sera of the population of the Republic of Guinea]. / Rezul'taty serologicheskikh issledovanii po opredeleniiu markerov gepatitov A i B v syvorotakh krovi naseleniia Gvineiskoi Respubliki.

The results of examinations of sera from apparently normal urban and rural residents of the Guinea Republic (GR) for markers of viral hepatitis A (anti-HAV) and B (HBsAg) are presented. The number of HBsAg-positive subjects was 16 +/- 1% (1199 serum specimens were examined by direct enzyme immunoassay, EIA, and HI test), the rate of HBsAg findings in sera from children (less than 16 years) and adults (greater than or equal to 16) did not differ significantly (p less than 0.05). The rate of a HBsAg carrier state did not depend on sex and residence of the subjects under study (p less than 0.05). The detection rate of total (IgM + IgG) anti-HAV antibody was 67 +/- 2% (812 serum specimens were examined by a variant of EIA block). The detection rate and titres of anti-HAV in children were higher than in adults, and in urban residents higher than in rural subjects (p less than 0.05). The results of detection of HBsAg and total anti-HAV antibody in the sera of GR residents are close to those obtained in examinations of sera from the populations of countries bordering GR in Western Africa (Senegal, Mali, Liberia) and are typical of Africa as a whole.

[A trial of a cultured inactivated vaccine against hepatitis A on Saguinus mystax tamarins]. / Ispytanie na tamarinakh Saguinus mystax kul'tural'noi inaktivirovannoi vaktsiny protiv gepatita A.

An experimental batch of inactivated hepatitis A vaccine was prepared using hepatitis A virus (HAV), HAS-15 strain, adapted to cell culture and purified by ultracentrifugation. The vaccine was tested in tamarins immunized intramuscularly three times one month apart. Three tamarins received a vaccine preparation containing 10 ng of immunogen each, three--100 ng each, and three animals were used as controls. The efficacy was judged by the anti-HAV antibody response in the vaccinated animals and development of immunity to subsequent virus challenge two months after the last immunization. The criteria of infection were: elevation of serum alanine aminotransferase, fecal excretion of HAV and production of specific antibody of IgM class. The immune response to 10 ng of the immunogen was lower than to 100 ng, however, both doses produced complete resistance to infection. The booster effect was observed in animals receiving 10 ng of the immunogen. The vaccine batch under study in the indicated doses was shown to have a good immunogenic potency and protective activity for tamarins.

[A highly sensitive method of detecting the structural proteins of hepatitis A virus]. / Vysokochuvstvitel'nyi sposob vyiavleniia strukturnykh belkov virusa gepatita A.

A modified technique of protein staining with silver nitrate was employed in electrophoretic analysis of hepatitis A virus structural proteins. The modifications of the original technique, i.e. thorough washing of the gel, increased formaldehyde concentration and a more intensive lighting of the gel, have elevated the method sensitivity 10-fold permitting the detection of up to 0.1 ng protein. The modification has allowed detection of VP1, VP2, and VP3 structural proteins with molecular masses 33, 29, and 27 kD, respectively, in virus preparations of low concentrations.

[Detection of hepatitis A virus RNA by molecular hybridization]. / Obnaruzhenie RNK virusa gepatita A metodom molekuliarnoi gibridizatsii.

Hepatitis A virus (HAV) was propagated in continuous rhesus monkey embryo kidney cells (FRhK-4 line). HAV RNA extracted with phenol from purified HAV pretreated with proteinase K was used for molecular cloning experiments. Two radioactive plasmids, pHAV-23 and pHAV-5p, containing HAV cDNA fragments complementary to structural and nonstructural parts of HAV genome, respectively, were found to be useful for discrimination between mature virions and subviral structures. HAV cDNA-RNA hybridization seems to be 10 times more sensitive than the enzyme immunoassay (ELISA).

[Trial of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (an anti-HAV IgM test system)]. / Ispytanie otechestvennogo diagnosticheskogo komplekta dlia opredeleniia immunoglobulinov klassa M k virusu gepatita A immunofermentnym analizom (test-sistema na anti-VGA IgM).

The authors have studied the effectiveness of the first Soviet test system for the diagnosis of hepatitis A by means of the enzyme immunoassay (Diagn-A-Hep), developed at the Institute of Poliomyelitis and Viral Encephalitides, Moscow, under the conditions of different epidemic situations. In the process of this trial the high specificity and sensitivity of this test system, established earlier in the certification and commission trials, have been confirmed. Diagn-A-Hep has proved to be highly effective in the diagnosis of acute forms of hepatitis A and permitted its detection in patients during the incubation period, as well as in patients with anicteric and asymptomatic subclinical forms. Besides clinical diagnosis, the kit Diagn-A-Hep may be used in large-scale seroepidemiological surveys of the immune structure of the population, as well as in detection of HAV in different material under test.

[Development of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (a test system for anti-HAV IgM)]. / Razrabotka otechestvennogo diagnosticheskogo komplekta dlia opredeleniia immunoglobulinov klassa M k virusu gepatita A immunofermentnym analizom (test-sistema na anti-VGA IgM).

A new test system Diagn-A-Hep for the laboratory diagnosis of hepatitis A (HA) by means of the enzyme immunoassay has been developed at the Institute of Poliomyelitis and Viral Encephalitides (Moscow). The sensitivity and specificity of the newly developed test system have proved to be similar to those of the well-known commercial diagnostic system HAVAB manufactured by Abbott Laboratories (USA). Diagn-A-Hep permits the diagnosis of HA with 96-100% effectiveness both in patients with the acute form of the disease and in patients with its anicteric or inapparent forms. This system is simple and convenient, it may be employed in inadequately equipped laboratories or even under field conditions. The rules for the selection of immunobiological preparations to be included in the test system have been worked out.

[Hepatitis A in Macaca fascicularis and M. arctoides infected by the Java monkey-55 strain of hepatitis A virus]. / Gepatit A u iavanskikh i burykh makak pri ikh zarazhenii shtammom virusa gepatita A IaM-55.

The results are presented dealing with experimental inoculation of M. fascicularis and M. arctoides with a strain of hepatitis A virus (HAV), YaM-55, isolated from a M. fascicularis with spontaneous hepatitis A, and parallel experiments on inoculation of these monkey species with HAV preparations (strain HAS-15) obtained as a result of the strain propagation in FRhK-4 cell culture and with specimens from human hepatitis A patients containing HAV particles. The YaM-55 strain of HAV was found to be capable of producing an infectious process quite similar to HA in the inoculated seronegative M. fascicularis and M. arctoides. Two different isolates of HAV derived from humans and the HAS-15 strain of HAV propagated for a long time in FRhK-4 cell culture failed to induce a disease in these monkey species. The classification of the YaM-55 strain with HAV was verified by specific serological studies and by molecular hybridization with cloned cDNA of HAV.

[Accumulation of the infectious virus and the viral antigen during the multiplication of the hepatitis A virus in an embryonic kidney cell culture of the rhesus macaque (FRhK-4)]. / Nakoplenie infektsionnogo virusa i virusnogo antigena pri razmnozhenii virusa gepatita A v kul'ture kletok pochki émbriona makaki rezusa (FRhK-4).

Growth characteristics of the HAS-15 strain of human hepatitis A virus (HAV) in rhesus monkey foetal kidney cell line (FRhK-4) are described. The conditions optimal for the accumulation of infectious HAV and viral antigen (HAAg) in the infected cells and tissue culture fluids were studied. The production of infectious HAV occurred in the first stage while in the second stage predominantly HAAg was accumulating intracellularly. Serological properties of the cultivated HAV proved to be very similar to those of the HAV occurring in hepatitis A patients.

[Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture]. / Adaptatsiia shtamma virusa gepatita A (IaM-55), iskhodno patogennogo dlia obez'ian, k kletochnoi kul'ture.

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.

[Properties of hepatitis A virus particles produced during infection in vitro]. / Svoistva chastits virusa gepatita A, formiruiushchikhsia pri vosproizvedenii infektsii in vitro.

Four types of virus-specific particles with different sedimentation coefficients and buoyant densities in CsCl were shown to be accumulated in hepatitis A virus (strain HAS-15) infected fetal rhesus monkey kidney cells (FRhK-4 line). Unlike the mature virions (155S, 1.34 g/cm3), cell-associated isosedimenting 92 S-particles (buoyant densities of 1.30 and 1.20 g/cm3) proved to be sensitive to lipase action. Particles of all four types were shown to contain similar sets of polypeptides, and, with the exception of "empty" 1.30 g/cm3-particles, appeared to be "full" under the immune electron microscopic examination. The viral RNA was unequivocally identified by the molecular hydridization test only in the mature virions.

[Dynamics of the accumulation of virus particles with different physico-chemical properties in FRhK-4 cells infected with hepatitis A virus]. / Dinamika nakopleniia virusnykh chastits s razlichnymi fiziko-khimicheskimi svoistvami v infitsirovannykh virusom gepatita A v kletkakh FRhK-4.

The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.