A tissue culture color test for the titration of neutralizing antibodies against sendai virus (parainfluenza 1)

Summary A color test for the detection and titration of neutralizing antibodies against Sendai virus is described. Non-specific inhibitors should be removed by treatment with cholera filtrate R.D.E. In contrast to both the haemagglutination inhibition test and the complement fixation test, which do not allow a differentiation between mumps and Sendai virus infection because of cross reactions, the color test is assumed to be sufficiently specific for Sendai virus infection.     

Intraepidemic heterogeneity of influenza A (H3N2) viruses in 1985: antigenic analysis and sensitivity to non-specific inhibitors

During the influenza outbreak of 1984-85 22 strains of H3N2 viruses were isolated in Finland. An intra-epidemic heterogeneity was demonstrated in an antigenic analysis by haemagglutination inhibition test with antisera produced in rats. The strains could be classified into three groups which corresponded to the following reference strains: group I: A/Hong Kong/1/84, A/Hong Kong/3/84; group II: A/Philippines/2/82; group III: A/Caen/1/84. Seven of the isolates were entirely insensitive to gamma-inhibitors of guinea-pig sera, which is in contrast to the small number of these viruses found among H3N2 strains isolated in the 1970s. The insensitive strains could not be isolated until the second or third passage through the eggs, whereas about half of the sensitive and intermediate strains were already isolated during the first passage. Conversions in reactivity with gamma-inhibitors could be detected only from an intermediate or an insensitive virus to a sensitive virus when several strains were passed serially in ovo and in MDCK cultures. The findings suggest that the gamma-inhibitor-insensitive strains corresponded well to the viruses of the human host or arose from dimorphic virus populations under an arbitrary selection of terminal dilution conditions prevailing during isolation in eggs. The insensitive strains did not differ substantially from the sensitive viruses in their ability to agglutinate erythrocytes of different laboratory animals or in their disagglutination patterns. On the other hand, propagation of viruses in MDCK cultures had an effect on these properties. The results are discussed with respect to Q phase variants and receptor binding properties.     

Studies on the antigenic behaviour of egg- and egg-mouse-egg-lines of strains of influenza virus,with the aid of the haemagglutination-inhibition test

Summary Egg- and egg-mouse-egg-lines of strains of influenza virus were compared with the aid of the haemagglutination inhibition test. In most experiments it was found that the crossing of an antiserum of an egg-mouse-egg-line against the homologous egg-line or against a heterologous egg-line of a strain belonging to the same subgroup yields a low or very low antititre. We are indebted to DrG. K. Hirst (New York) and DrJ. Y. Sugg (New York) for sending us the strains of influenza virus Ala and Cam. Financial support was provided by the Institute for Preventive Medicine, Leyden; N. V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Cura?ao Fund for Preventive Medicine.     

Antibody response against strains of influenza-A virus in ferrets with basic immunity

Summary In Holland ferrets are offered for sale with basic antibodies against one or more sub-groups of strains of influenza-A virus. With the haemagglutination inhibition test low basic antibody titers can only be detected by neutralising the non-specific inhibitor in the ferret sera. When such a ferret is infected with a strain of a heterologous sub-group, a considerable rise in antibodies is obtained against the homologous and heterologous sub-groups. It is very probable that the same holds for human beings. Some examples are given of haemagglutination-inhibition tests with serum pairs from adults who suffered from influenza in the epidemic of the winter of 1949 in Holland from which the non-specific inhibitor had been eliminated.Sub-unit of the Influenza Research Team of the Institute of Preventive Medicine, Leyden. With the financial support of the Institute of Preventive Medicine, the Department of Medical Research Philips Roxane (Weesp), the State Department of Science, and the Jan Dekker Fund.     

Macrophage-tropic and T-cell line-adapted chimeric strains of human immunodeficiency virus type 1 differ in their susceptibilities to neutralization by soluble CD4 at different temperatures.

Molecular clones of three macrophage-tropic and three T-cell line-adapted strains of human immunodeficiency virus type 1 (HIV-1) were used to explore the mechanism of HIV-1 resistance to neutralization by soluble CD4 (sCD4). The three macrophage-tropic viruses, each possessing the V3 and flanking regions of JR-FL, were all resistant to sCD4 neutralization under the standard conditions of a short preincubation of the virus and sCD4 at 37 degrees C prior to inoculation of peripheral blood mononuclear cells. In contrast, the three T-cell line-adapted viruses, NL4-3 and two chimeras possessing the V3 and flanking regions of NL4-3 in the envelope background of JR-FL, were all sCD4 sensitive under these conditions. Sensitivity to sCD4 neutralization at 37 degrees C corresponded with rapid, sCD4-induced gp120 shedding from the viruses. However, when the incubation temperature of the sCD4 and virus was reduced to 4 degrees C, the three macrophage-tropic viruses shed gp120 and became more sensitive to sCD4 neutralization. In contrast, the rates of sCD4-induced gp120 shedding and virus neutralization were reduced for the three T-cell line-adapted viruses at 4 degrees C. Thus, HIV resistance to sCD4 is a conditional phenomenon; macrophage-tropic and T-cell line-adapted strains can be distinguished by the temperature dependencies of their neutralization by sCD4. The average density of gp120 molecules on the macrophage-tropic viruses exceeded by about fourfold that on the T-cell line-adapted viruses, suggesting that HIV growth in T-cell lines may select for a destabilized envelope glycoprotein complex. Further studies of early events in HIV-1 infection should focus on primary virus strains.     

Development and Pre-Clinical Evaluation of Two LAIV Strains against Potentially Pandemic H2N2 Influenza Virus

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.     

Clinical infection of Cantabrian chamois (Rupicapra pyrenaica parva) by louping ill virus: new concern for mountain ungulate conservation?

Louping ill virus (LIV) was recently involved in an outbreak of encephalitis in domestic goats from Asturias region, northwestern Spain. Since livestock and wildlife in Asturias are frequently in close contact, we designed a retrospective survey for LIV antibody prevalence in wild ungulates by testing sera from 51 red deer (Cervus elaphus), 19 Cantabrian chamois (Rupicapra pyrenaica parva) and 8 roe deer (Capreolus capreolus) by the haemagglutination inhibition (HI) test. Only two Cantabrian chamois out of the 78 tested (2.6?±?3.5 %) gave positive results. Seroprevalence in chamois was 10.5?±?13.8 %. One of these chamois was found dead after falling down a cliff and the other one was found alive but with neurological signs. Histological examination of brain samples revealed that both animals showed severe inflammatory lesions compatible with a viral encephalitis caused by LIV, but LIV antigen was not detectable by specific immunohistochemistry. Real time RT-PCR was performed on formalin-fixed paraffin embedded sections of brain but was unable to confirm the presence of LIV RNA due to poor sample quality. By testing one of two HI positive sera from chamois by virus neutralization test and plaque reduction neutralization test against West Nile virus, Bagaza virus, Usutu virus, LIV and tick-borne encephalitis virus, we confirmed the presence of high antibody titres (1:10240) against LIV in the absence of antibodies to another Flavivirus. This work describes the first association between LIV and clinical encephalitis in chamois, which suggests that special attention should be paid to the impact on chamois conservation and management in Asturias, and perhaps in other European regions.     

Effect of polymerized orosomucoid on some strains of influenza virus

1. Polymers of orosomucoid were produced in two molecular shapes, filamentous (;chain') and spherical (;ball'), by heating the sodium salt of the monomer in either water or high concentrations of sodium chloride. An ;intermediate' state containing both shapes in various proportions was found in preparations obtained by polymerizing orosomucoid in intermediate concentrations of sodium chloride. 2. The filamentous form of polymer was found to inhibit strongly the haemagglutination of some (;sensitive') strains but not of other (;insensitive') strains of influenza virus; the ;intermediate' form feebly inhibited haemagglutination by ;sensitive' strains. 3. The filamentous form agglutinated both ;sensitive' and ;insensitive' strains of virus; the other forms of polymer did not. It also inhibited multiplication of both ;sensitive' and ;insensitive' strains when inoculated into embryonated and de-embryonated eggs. 4. The ;intermediate' and spherical forms of the polymer had no effect on the virus multiplication. 5. Polymers of orosomucoid from which neuraminic acid had been split off had no detectable effect on influenza viruses.     

Studies of the adaptation of influenza viruses to lowered temperatures of replication. II. Studies in vivo

Swiss white mice were given intranasally suspension of influenza A virus (H3N2) isolated at different period of time and replicated in lowered temperatures in 11 days old chicken embryos. The presence of antigen in lung of animals was detected by IF. They were given the virus replicated at 30 degrees C at different rate depending on strain tested. No distinct differences were observed in haemagglutination inhibition antibody level. On the other hand the level of neuraminidase activity inhibiting antibodies level was significantly higher after giving virus replicated at 30 degrees C than after giving the virus replicated at 37 degrees C. In the case of epidemic strains 4-5 fold fold increase of immunogenicity of neuraminidase component was observed and in the remaining strains immunogenicity of neuraminidase increased 1-5-fold only.     

Preparation and properties of Vibrio cholerae antifimbrial antibody

The presence of fimbriae on the Vibrio cholerae strains used was assessed by pellicle formation, haemagglutination activity and electron microscopy. Fimbrial suspensions were prepared by shearing them off the organisms, then separating them from other components by absorbing them on to rabbit red blood cells. Rabbits were then immunized with the fimbrial-red cell suspensions and the antibodies evoked were titrated by haemagglutination inhibition, agglutination, vibriocidal and immobilization techniques.     

Preparation and properties of Vibrio cholerae antifimbrial antibody

The presence of fimbriae on the Vibrio cholerae strains used was assessed by pellicle formation, haemagglutination activity and electron microscopy. Fimbrial suspensions were prepared by shearing them off the organisms, then separating them from other components by absorbing them on to rabbit red blood cells. Rabbits were then immunized with the fimbrial-red cell suspensions and the antibodies evoked were titrated by haemagglutination inhibition, agglutination, vibriocidal and immobilization techniques.     

Studies on the elimination of non-specific inhibitors in sera against influenza viruses with the aid of filtrates ofVibrio cholerae

Summary In the sera of humans and various animals there are two different non-specific inhibitors (α-and β-inhibitors) which may be specifically abolished by two substances (α-and β-“enzymes”) both present in filtrates ofV. cholerae. This neutralization is necessary for the rapid classifying of influenza virus strains with the aid of the haemagglutination inhibition test. The egg line strains of the A- and A′-groups are only sensitive to β-inhibitor, while the mouse line strains of the same groups are only sensitive to the α-inhibitor. This does not apply to strains of the B group, where mouse as well as egg lines are sensitive to both inhibitors. With the aid of isolated β-inhibitor (easily to be prepared from cattle serum), it is possible to decide in a quick manner whether or not a strain from the A- or A′-group has previously been adapted to the mouse. With the technical assistance of Miss I.de Nooyer and with the financial support provided by the Institute for Preventive Medicine, Leyden; N.V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Curacao Fund for Preventive Medicine.     

Prevalence in England of Antibody to Human Polyomavirus (B.K.)

Over 500 human sera were tested by complement fixation and haemagglutination inhibition tests for antibody to the human polyomavirus (B.K.). Both tests showed that antibody to this virus was very common in the population and began to be acquired after the age of 1 year. No clinical illness has so far been associated with the development of this antibody in a series of paired sera from children.     

Virucidal effect of sodium p-chloromercuribenzoate on influenza viruses attributable to inhibition of virus particle-associated RNA-dependent RNA polymerase.

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 mug/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H2N2) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 mug/ml and that of Lee strain of type B influenza virus at 8.5 mug/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

Two viruses isolated from rodents (Clethrionomys gapperi and Microtus pennsvlvanicus) trapped in St. Lawrence County, New York

Four strains of C. gapperi virus were isolated from 3 Clethrionomys gapperi and 47 strains of Microtus virus from 15 Microtus pennsylvanicus and 1 Mus musculus. One of the Microtus strains was isolated from a pool of 20 mites while the others were from rodent tissues. These agehts were insensitive to ether and sodium desoxycholate, withstood freezing at -70 C for 3 years and lyophilization without loss of titer, and were not killed when heated at 60 C for 1 hour. Their size as determined by filtration was less than 50 mg and greater than 20-35 mmicro. The strains within each group appear to be similar. The illness induced in suckling mice by the C. gapperi agents had a 5-day incubation period followed by prostration and death with a histologic picture of extensive encephalomalacia. The incubation period in mice for the Microtus agents was 9 to 11 days followed by convulsions and death. Histopathology showed meningeal infiltration and necrosis of the molecular layer. No antigenic similarity was detected between the C. gapperi and Microtus viruses by cross complement-fixation test.     

Evaluation of Some Serological Techniques for The Identification of Mycoplasma Hyorhinis and Mycoplasma Suipneumoniae

Eighteen strains of Mycoplasma hyorhinis and a strain of Mycoplasma suipneumoniae were tested in 4 serological tests, i. e., disc growth inhibition, metabolic inhibition, indirect haemagglutination and indirect epi-immunofluorescence. Only with immunofluorescence could all tested strains of M. hyorhinis be shown; no cross-reactions between M. hyorhinis and M. suipneumoniae could be detected. The other tests failed in many cases to identify strains of the same species, and they gave cross-reactions between M. hyorhinis and M. suipneumoniae.     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

Abstract It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysacharides also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Eliminating bias in the estimation of the geometric mean of HI titres.

The standard test for anti-haemagglutinin antibody titration is the haemagglutination inhibition (HI) test. The HI titre is defined as the dilution factor of the highest dilution that still completely inhibits haemagglutination. If the highest dilution tested (1:2560) still completely inhibits haemagglutination, an HI titre value of 2560 is assigned. Logarithmically transformed HI titres tend to be normally distributed. But because dilutions less than 1:2560 are not tested, the distribution may be truncated and the assumption of normality may not hold. As a consequence, the geometric mean titre (GMT) will be underestimated. Using data from 10 clinical studies, it is shown here that the GMT may be underestimated by 5-13%. An unbiased estimate of the GMT can be obtained by a statistical method that originates from the analysis of survival data: maximum likelihood estimation for censored observations. The maximum likelihood estimate of the GMT of truncated HI titres can be readily obtained using the statistical software package SAS.     

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.     

Interpretation of the results of identification of measles virus antibodies in the serum and cerebrospinal fluid from patients with suspected subacute sclerosing panencephalitis (SSPE)]

Clinical diagnosis of subacute sclerosing panencephalitis+ (SSPE) requires laboratory confirmation relying on an evaluation of immune response in serum and in cerebrospinal fluid (CSF) to measle virus. In our study a comparison of antibody level for this virus by ELISA and haemagglutination inhibition test was made in materials derived from 1396 patients with SSPE, sclerosis multiplex, acute measles++ neuroinfections and other diseases of central nervous system (CNS). Statistical analysis permitted to settle criteria for differentiation of SSPE from remaining diseases of CNS and usefulness for diagnosis of particular methods as well as analysis of results obtained with single sample of serum or CNS. An advantage of ELISA for diagnostic purposes and CSF samples were confirmed.