The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector.

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed.

The yellow fever 17D vaccine virus: molecular basis of viral attennuation and it use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed.

Measles antibody prevalence after mass immunization campaign in Niterói, state of Rio de Janeiro, Brazil.

Three months after a mass vaccination campaign (coverage: 100%) against measles a random seroepidemiological survey was carried out in students aged 1 to 19 years old in the Municipality of Niterói, State of Rio de Janeiro. Blood samples were tested for measles antibodies by enzyme immunosorbent assay (EIA) and negative cases were tested again using hemagglutination inhibition (HI) and plaque reduction neutralization (PRN). Of the 798 samples tested by EIA, 718 (90.2%) were positive for measles antibodies. PRN test was more sensitive than EIA and HI in detecting measles specific antibodies. The total antibody prevalence increased from 90.2% to 93.2% when HI was employed in EIA negative specimens and to 98.9% when PRN was used. After the mass vaccination campaign a marked decrease in measles incidence was observed in the municipality studied, showing the effectiveness of the strategy used for measles control in developing countries.

Biological characterization of clones derived from the Edmonston strain of measles virus in comparison with Schwarz and CAM-70 vaccine strains.

Four virus clones were derived from the Edmonston strain of measles virus by repeated plaque purification. These clones were compared with the vaccine strains Schwarz and CAM-70 in terms of biological activities including plaque formation, hemagglutination, hemolysis and replication in Vero cells and chick embryo fibroblasts (CEF). Two clones of intermediate plaque yielded mixed plaque populations on subcultivation whereas the other two, showing small and large plaque sizes, showed stable plaque phenotypes. The vaccine strains showed consistent homogeneous plaque populations. All the Edmonston clones showed agglutination of monkey erythrocytes in isotonic solution while both vaccine strains hemagglutinated only in the presence of high salt concentrations. Variation in the hemolytic activity was observed among the four clones but no hemolytic activity was detected for the vaccine virus strains. Vaccine strains replicated efficiently both in Vero cells and CEF. All four clones showed efficient replication in Vero cells but different replication profiles in CEF. Two of them replicated efficiently, one was of intermediate efficiency and the other showed no replication in CEF. Two of the clones showed characteristics similar to vaccine strains. One in terms of size and homogeneity of plaques, the other for a low hemolytic activity and both for the efficiency of propagation in CEF.

Loss of maternal measles antibody during infancy in an African city.

Measles epidemics with 20% of the cases under nine months of age continue to occur in Brazzaville, Congo, even though measles vaccination coverage was 77% in the 12- to 23-month age group in 1986. In order to estimate the duration of passive immunity against measles, we conducted a serologic survey of infants aged 2 to 9 months. Measles antibody was measured from capillary blood with the plaque inhibition test. An antibody titre of 40 milli-International Reference Units per ml of serum (mIRU/ml) or less was defined as seronegative. Among the 252 infants studied, the proportion with detectable antibody dropped from 95.8% at 2 months of age to 48.5% at 4 months of age, and to 8.2% in the 7-8 month age group. A simple logistic model with age as the only variable provided an excellent fit to the observed values. Between the ages of 8 and 28 weeks, there was an almost steady decline of approximately 4.7% per week in the proportion of infants who were seropositive. These findings suggest that loss of maternal measles antibody during infancy might be faster than reported in other African populations. Giving measles vaccine to infants before the age of nine months currently recommended by the Expanded Programme on Immunization may be useful in some populations. Further studies of seroconversion and impact on measles are needed.

Effect of gamma irradiation on the human immunodeficiency virus and human coagulation proteins.

The effect of gamma irradiation on HIV and plasma coagulation factors F VIII:C, F VIII:vWF and FIX was studied. Donor plasma was harvested from single donations, frozen and irradiated in the frozen state at target doses from 0 to 40 kGy (0-4 mRad). HIV was inoculated into human plasma and irradiated in a similar manner. A range of other viruses, not suspended in plasma, were also irradiated to establish viral inactivation. An inactivation rate of 0.164 TCID50 dose/ml/kGy was demonstrated for HIV compared to rates of 0.00173, 0.00526 and 0.00286 log10 units/ml/kGy for F VIII:C,F VIII:vWF and FIX respectively. The use of gamma irradiation to inactivate infectious agents present in human plasma may eliminate the need for any post-production viral inactivation methods and provide a means of assuring the safety of as yet untreated products such as cryoprecipitate and fresh frozen plasma.

Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: specificity of subgenomic probes in quantitative slot blot and in situ hybridisation.

Subgenomic cDNA clones representing defined regions of the genome of Coxsackie B3 virus were used as hybridisation probes to detect RNA of various enteroviruses in cell culture and mouse model systems. Radiolabelled probes were used in slot blots to quantitate the RNA in samples; biotinylated probes were used to localise virus RNA at the cellular level by in situ hybridisation with monolayers of infected cells or thin sections of tissue samples. Probes derived from the 5' or 3' terminal regions of Coxsackie virus RNA, which are highly conserved in the genus Enterovirus, detected RNA of various serotypes in infected cell cultures, but failed to hybridise with hepatitis A virus (HAV) RNA. Although HAV is clearly a Picornavirus, our data support the view that its taxonomic position within the enteroviruses should be reconsidered. The biotinylated probes were also used to detect in situ virus RNA in paraffin-embedded tissue samples from experimental mouse models of Coxsackie B3 virus-induced myocarditis or Coxsackie B1 virus-induced myositis. Since the integrity of the tissues was preserved during the process, and viral RNA was localised in the affected muscle fibres, this has enabled us unequivocally to relate the infecting virus to the underlying tissue injury.

Chronic enterovirus infection in patients with postviral fatigue syndrome.

76 patients with the postviral fatigue syndrome (PVFS) and 30 matched controls were investigated. Virus isolation was attempted from concentrated faecal samples by direct culture and after acid dissociation of virus from antibody. Positive cultures of enteroviruses were obtained from 17 (22%) patients and 2 (7%) controls. An enterovirus-group-specific monoclonal antibody, 5-D8/1, directed against the VP1 polypeptide, was used to detect enteroviral antigen in the circulation, either free or complexed with antibody. VP1 antigen was detected in the serum of 44 (51%) of a further group of 87 PVFS patients. The number of patients positive for VP1 antigen was greater (42/44) when IgM complexes were detectable than when they were not (2/23). 1 year later, the 17 patients of the first group of 76 with positive cultures were again studied. The same virus was again isolated from 5 (29%), 13 (76%) had detectable IgM responses to enteroviruses, and 9 (53%) were positive for VP1 antigen in the serum. These results show that chronic infection with enteroviruses occurs in many PVFS patients and that detection of enterovirus antigen in the serum is a sensitive and satisfactory method for investigating infection in these patients.

Clinical and research application of an enterovirus group-reactive monoclonal antibody.

Diagnostic methods employed in enterovirus laboratories are generally laborious, slow and expensive. This is largely because type-specific neutralization tests still play the major role in identification and diagnostic serology. In the companion paper we describe the derivation of monoclonal antibodies against epitopes of the VPI peptide which are shared by all of the enteroviruses tested to date, with the exception of hepatitis A virus. This study describes the application of one of these monoclonal antibodies in several research and diagnostic procedures, illustrating a special utility in a wide variety of assay systems. This monoclonal antibody has proved particularly useful in the detection of enterovirus antigens in circulating immune complexes, and in identifying field isolates of this group of viruses. Immunohistochemistry, previously almost impossible in enterovirus diagnosis and research due to the large number of serotypes, is now shown to be practical and informative when this monoclonal antibody is used.

A semi-automated system for the assessment of toxicity to cultured mammalian cells based on detection of changes in staining properties.

We have established a semi-automated microtiter-based system for the quantification of dye binding to cultured eukaryotic cells. This system has been applied to the quantitation of toxic activities that disrupt cell monolayers and their neutralization. We have used this background as a basis for developing a detection and characterization system for activities that do not cause such gross toxicity. A prototype system has been established based on three staining procedures which in broad terms assess cellular dehydrogenase activity, and protein, DNA, and RNA content. The activity of several agents affecting cyclic nucleotide metabolism, including cholera toxin, on the staining properties of exposed monolayers has been assessed. Several new categories of cellular response are readily discernible in this latter system indicating that biological activities may be identified on the basis of the pattern of such responses. Since microtiter based systems show considerable potential for automation, it is suggested that the further development of this approach could offer a realistic prospect for numerous forms of toxicity testing on an industrial scale.

Immunisation of 4-6 month old Gambian infants with Edmonston-Zagreb measles vaccine.

Five different vaccination schedules were used to immunise Gambian infants aged 4-6 months against measles with the attenuated Edmonston-Zagreb strain of virus, which has a history of passage in human diploid cells. Vaccine aerosol given either by mask in a dose of 3500 or 7000 plaque-forming units (PFU) or from a plastic bag at a dose of 7000 PFU raised haemagglutinin-inhibiting or plaque-inhibiting measles antibody 16-24 weeks after vaccination to a titre of 1 in 8 or greater in all but 3 of the 51 children so vaccinated. All 21 infants given 11 400 PFU of vaccine intradermally in two divided doses and the 21 given 39 000 PFU of the virus subcutaneously also had satisfactory levels of measles antibody 16 weeks after vaccination. None of the vaccinated children had clinical evidence of measles in the 12 to 17 months after vaccination. The Edmonston-Zagreb vaccine, given subcutaneously or by other routes at 4-6 months, may be useful in preventing measles in infants in African cities, where 15-30% of children have measles before they are 9 months old, which is the recommended age for immunisation with the chick-cell-adapted strains of measles virus.

PIP: 5 different vaccination schedules were used to immunize Gambian infants aged 4-6 months against measles with the attenuated Edmonston-Zagreb strain of virus, which has a history of passage in human diploid cells. Vaccine aerosol given either by mask in a dose of 3500 or 7000 plaque-forming units (PDUs) or from a plastic bag at a dose of 7000 PFU raised hemagglutinin-inhibiting or plaque-inhibiting measles antibody 16-24 weeks after vaccination to a titer of 1 in 8 greater in all but 3 of 51 children so vaccinated. All 21 infants given 11,400 PFU of vaccine intradermally in 2 divided doses and the 21 given 39,000 PFU of the virus subcutaneously also had satisfactory levels of measles antibody 16 weeks after vaccination. None of the vaccinated children had clinical evidence of measles in the 12-17 months after vaccination. The Edmonston-Zagreb vaccine, given subcutaneously or by other routes at 4-6 months, may be useful in preventing measles in infants in African cities,where 15-30% of children have measles before 9 months of age, which is the recommended age for immunization with the chick-cell-adapted strains of measles virus.

Stability of further-attenuated measles vaccines.

Accelerated stability tests on lyophilized measles vaccines show two distinct mechanisms of virus inactivation. A rapid initial loss of infectivity occurs only on exposure to temperatures above the ambient temperature. This loss is temperature related and may be attributable to the movement of residual moisture from the virus pellet into the void space of the vial. Subsequent inactivation of virus occurs at all temperatures as a first-order reaction that follows Arrhenius kinetics. Integration of values for these two components allows precise prediction of vaccine stability at any temperature. Analysis of the results obtained for greater than 30 vaccines shows that those which are stable for one week at 37 C have a predicted life of more than one year at 8 C. This simple test is now being applied to the identification of unstable products. The rate of this reaction is closely, if conservatively, matched by a time-temperature color indicator, which may be useful for monitoring vaccine quality.

Replication of influenza A and B viruses in human diploid cells.

Under optimal conditions, of high multiplicities of infection and with trypsin included in the medium throughout the incubation period, high yields of infectious influenza A and B viruses (10(6 . 5) p.f.u./ml) and of antigenically active haemagglutinin (HA)(1 microgram/HA/10(6) cells) were produced in human diploid MRC-5 cells. Budding virus particles were seen as spherical or short rod-like protrusions on the surface of the infected cells, and also on cell filopodia. Virus-induced cytoplasmic and nuclear inclusions were present in infected cells. This virus-human cell system may be suitable for studies of influenza virus persistence and for production of immunologically active HA antigen.

Detection of HBsAg in a clone derived from the PLC/PRF/5 human hepatoma cell line.

A total of 28 clones were established from the PLC/PRF/5 hepatoma cell line by a plating procedure. All clones were found to secrete HBsAg into the supernatant culture fluids. Of these, one clone (No. 23) free of detectable mycoplasma contamination and showing smooth epithelial morphology was selected for further study. Maximum accumulation of HBsAg occurred 9 days after sub-culture and intracellular antigen was detected by indirect immunofluorescence both in the cytoplasm and at the plasma membrane. Granules and perinuclear staining reactions were also observed in clone 23 cells and these findings are compared to the previously published properties of the parental PLC/PRF/5 cell line.

Nondetection of infectious hepatitis B virus in a human hepatoma cell line producing hepatitis B surface antigen.

The PLC/PRF/5 human hepatoma cell line producing hepatitis B surface antigen (HBsAg) was studied to determine whether infectious hepatitis B virus (HBV) was also being produced. 2 chimpanzees with no previous exposure to HBV and no serologic markers of past or active HBV infection were inoculated intravenously with 50 ml of either tissue culture supernatant fluid (357 ng/ml HBsAg) or a suspension of cells disrupted by repeated freeze-thaw cycles (57 ng/ml HBsAg). No evidence of HBV infection was detected in either chimpanzee during 6 months of evaluation. This study suggests that the expression of a portion of the HBV genome, when a portion or all of that genome has been incorporated into a host cell, can result in the production of HBsAg without infectious HBV. If it becomes possible to produce a similar expression of this portion of the genome by itself in nonmalignant cells, HBsAg without HBV may be produced in vitro for use in hepatitis B vaccines.

Hepatitis B surface antigen production as a growth cycle-related terminal event in PLC/PRF/5 hepatoma cells.

The dynamics of hepatitis B surface antigen production of PLC/PRF/5 hepatoma cells were studied using a quantitative radioimmunoassay method. Maximum rates of antigen production were found in nutrient-depleted, non-dividing cultures and were temporally related to cytological changes preceding cell death. These results indicate that antigen production may be cell-cycle related and represent a terminal event.