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By treatment of chorioallantoic membranes from embryonated eggs with collagenase and hyaluronidase before the conventional application of trypsin cells could be grown in culture which supported growth of a large variety of myxoviruses, herpesviruses, avian reoviruses and the infectious bronchitis virus of chickens. The cultures could be used for sensitive plaque assays and neutralization tests.
Sera from 637 Athens residents of various age groups were examined by plaque reduction neutralization test for antibodies against Naples and Sicilian Phlebotomus fever viruses. A marked change in the prevalence of antibodies to both agents was observed in persons born after 1946, when residual insecticide spraying for malaria control was initiated in Greece. The prevalence of Naples and Sicilian neutralizing antibodies among residents greater than or equal to 30 years of age was 36% and 13%, respectively. In contrast, only 4% of persons less than or equal to 29 years of age had Naples antibodies and all were negative to Sicilian. These serologic data confirm previous clinical observations that sandfly fever becam uncommon in Athens after initiation of the insecticide spraying program. Presumedly the spraying program was effective in reducing the Phlebotomus population to levels where virus transmission was minimal. New information on the specificity and duration of Phlebotomus fever neutralizing antibodies is also presented.
Colostral globulins of sows immunized with an ethanol-saponin vaccine against Aujeszky's disease virus were fractionated through gel filtration by means of Sephadex-200. The virus-neutralizing activity of the globulin fractions identified through agar gel immunoelectrophoresis was evaluated by the virus-neutralizing test with cell cultures. The level of the antibodies found in class IgG substantially exceeded that of the IgA antibodies. It is believed that after muscular application of an ethanolsaponin vaccine against Aujeszky's disease the mammary gland of the vaccinated sows secretes virus-neutralizing antibodies mainly of the IgG class.
A chelonid herpesvirus was isolated from a group of tortoises in Italy with a history of increased mortality and upper digestive and respiratory tract disease. The isolated virus was inactivated with formalin and used to prepare a nonadjuvanted vaccine and a vaccine adjuvanted with aluminum hydroxide. 57 tortoises, 26 Testudo hermanni, 25 T. graeca, and 6 T. marginata, were included in the study. The animals were vaccinated 3 times at 45 day intervals. Blood was collected from the animals 14 days prior to the first vaccination, and on day 0, 25, 45, 90, 113 and 369 after the first vaccination. Plasma antibody titers to the homologous chelonid herpesvirus were determined using a virus neutralization test (VNT). No significant rise in antibody titer was noted in the vaccinated animals. Antibody titers measured dropped below the cutoff-level sporadically in all positive animals. Repeat serological testing may therefore be necessary in order to detect seropositive animals.
BACKGROUND: Lupus anticoagulant and anticardiolipin antibodies are antiphospholipid antibodies which have been independently associated with a high incidence of thrombotic diseases. However, the importance of their combined occurrence has not yet been examined. METHODS: We investigated 70 patients with systemic lupus erythematosus for the presence of anticardiolipin antibodies paying particular attention to a history of thrombosis and abortion. Lupus anticoagulant was detected using the kaolin clotting time, its mixing tests with normal plasma and the inosithin neutralization test. Anticardiolipin antibodies were detected using the ELISA technique. RESULTS: Lupus anticoagulant was detected in 11 patients (16%) and anticardiolipin antibodies in 13 (19%). Six patients were positive for both lupus anticoagulant and anticardiolipin antibodies. These patients had a higher incidence of thrombosis or recurrent abortion (5 of 6) compared to those with lupus anticoagulant (2 of 5) or anticardiolipin antibodies alone (1 of 7). The amount of inosithin required to neutralize lupus anticoagulant was greater [mean (SD) 27.5 (20.5) micrograms] in patients with both lupus anticoagulant and anticardiolipin antibodies than in patients with lupus anticoagulant alone [mean (SD) 4.0 (5.4) micrograms]. CONCLUSION: The presence of lupus anticoagulant is associated with thrombosis and recurrent abortion which are more frequent when both lupus anticoagulant and anticardiolipin antibodies are present and these patients probably have more severe disease as the amount of inosithin required to neutralize the lupus anticoagulant was greater.
Sensitive, reliable, and rapid methods of virus culture are essential for wild poliovirus isolation and identification from stool specimens collected from cases of acute flaccid paralysis. Recently, recombinant murine cell lines expressing human poliovirus receptor (CD155) on the cell surface have become available. These cells are sensitive selectively to poliovirus because the poliovirus receptor is not used by other enteroviruses. In early field studies non-polio enteroviruses were not isolated from stool samples of cases of acute flaccid paralysis using L20B cells. For the past 3 years, L20B cells were used extensively in our laboratory for virus culture. The objective of the present study was to identify non-polio enteroviruses causing cytopathic changes in L20B cells. Stool specimens collected from 1,153 cases of acute flaccid paralysis and 2,670 apparently healthy children were tested for enteroviruses using RD and L20B cell lines. A small number of viruses other than poliovirus causing cytopathic effect in L20B cells were isolated. Such isolates detected in other polio network laboratories in India were also included. The virus isolates were typed using partial VP1 nucleotide sequence analysis and virus neutralization tests. Of the 111 viruses studied, 8 were non-enteroviruses. Among the 103 non-polio enteroviruses, 73 were identified as group A coxsackieviruses. Of the 30 isolates that could not be characterized, 1 remained unidentified even by sequence analysis and 29 did not reach high titers in L20B as well as RD cells. In conclusion, coxsackie A viruses multiply in L20B cells causing cytopathic effect. Coxsackievirus A8 and coxsackievirus A10 were predominant among the eight coxsackie A virus types so far identified.
Circulating antibody appeared in the convalescing NDV-infected chicken concomitantly with the disappearance of virus from the tissues. The antigenic response to the CG 179 and B strains was demonstrated to be approximately equal. The neutralization test in the embryo and the hemagglutination inhibition technique yielded parallel results in the measurement of antibody early in convalescence, but late in convalescence the hemagglutination inhibition titers were relatively lower. This disparity indicates the possible duality of the antibodies. There was a wide ratio between the neutralizing antibody titers found in the brain and in the serum after an asymptomatic infection with NDV. The antibody level in the brain appeared to be related to the extent of virus growth and damage in the central nervous system. It appeared likely that a major factor in determining the virulence of the CG 179 strain was the more rapid attainment in the central nervous system of high virus concentration which outstripped the defense mechanisms of the host.
Although antibodies to viruses in both the epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) sero-groups have been reported from white-tailed deer (Odocoileus virginianus) in Texas (USA), there are few reports of hemorrhagic disease (HD) in these populations. To understand the extent and diversity of exposure to the North American EHDV and BTV serotypes in these deer populations, we serologically tested 685 white-tailed deer collected from November 1991 through March 1992 throughout their range in Texas. Overall, 574 (84%) of deer had antibodies to EHDV or BTV. Prevalence estimates varied according to ecological region, from 57% in the Gulf Prairies to 100% in the northwest Edwards Plateau. Based on serum neutralization tests, the deer had evidence of previous exposures to multiple EHDV and BTV serotypes, with evidence of exposure to two to five serotypes detected in each ecological region. The apparent lack of HD in relation to this high antibody prevalence cannot be explained, but may be related to enzootic stability in which a near perfect host-virus relationship exists.
Between 2001 and 2002, samples from 1,413 animals in 21 Spanish small ruminant flocks, most of them with animals showing clinical signs compatible with Border disease (BD), were screened for the presence of Pestivirus antigen and antibodies by an indirect peroxidase monolayer assay (IPMA) and the virus neutralization test (VNT), respectively. Although all flocks harboured seropositive animals, virus could only be isolated from animals in five of the flocks. Between 4 and 11 months later all animals older than 6 months in three of the flocks were resampled. At this time, 51-83% of them had neutralizing antibodies. The prevalence of persistently infected (PI) animals within two of the flocks was 0.3 and 0.6%, respectively. The third flock presumably had eliminated all the PI animals. Fourteen virus isolates were obtained. The 5' untranslated region (5'UTR) was amplified by RT-PCR and directly sequenced. Phylogenetic analyses classified them as a group of Border disease viruses (BDV), separated from BDV-1, but showing a relatively low bootstrap value. Three of the 14 isolates were in the same subgroup as a set of formerly characterised Spanish isolates from the Basque Country, which were allocated to subgroup BDV-C. In addition, they were in the group with an isolate from chamois, which is currently allocated in group BDV-4. Because of its close relation to the chamois isolate, these isolates were tentatively reallocated in a subgroup BDV-4a. The remaining isolates generated a new subgroup, related but not in the same cluster as the chamois isolate, and was therefore tentatively assigned to a new subgroup BDV-4b. Our results show that classification and nomenclature of BDV needs to be harmonised.
Research on HIV vaccines, as well as studies on HIV pathogenesis in human and SIV in the macaque model, require the availability of simple and standardized assays for quantification of neutralizing antibodies to primary virus isolates. We have recently developed and standardized assays using human cell lines engineered to express CD4 and co-receptors for HIV and SIV entry. One cell line originated from a glioma (U87) and the other from an osteosarcoma (HOS). Both cell lines and their derivatives form monolayer cultures, a prerequisite for counting plaques. HIV-infected U87.CD4-CCR5 or -CXCR4 cells form syncytia, that is, plaques that can be stained with hematoxylin and enumerated by light microscopy. In addition to CD4 and co-receptors (most often used CCR5 and CXCR6 by SIV), GHOST(3) cells have been engineered to express the green fluorescent protein following virus infection. Infected cells show green fluorescence and can be enumerated by fluorescence microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. Both assays are run in microtiter format and neutralization is evaluated after 3 d. Intra-assay variation has been used for estimation of the cutoff for neutralization. Testing 15 serum-virus combinations in the U87.CD4 assay and four serum-virus combinations in the GHOST(3) assay revealed that standard deviation of differences ranged from 9.1% to 9.9% in the two assays. This allowed the use of a cutoff >3 SD; that is, 30% neutralization. Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The assays have high specificity and reproducibility, and are simple and sensitive high-throughput assays.
During 1972 an apparently new enterovirus with characteristics of the Coxsackievirus group A was isolated from 11 residents of New York State, 10 of whom had central nervous system disease. The representative strain S/Albany 1/72 has the physical and chemical properties of an enterovirus and causes the pathology typical for Coxsackievirus group A in 1-day-old mice. Cross-neutralization tests indicated that the virus is distinct from currently recognized enteric viruses of man. Antibody levels to the representative strain were moderate to high in patients and were low to moderate in 26% of healthy individuals tested.
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Investigated were 11 phages isolated from lysogenic strains of Pseudomonas aeruginosa. Based on the results of the neutralization test the phages were divided into 3 clearly distinguished groups. The first group embraced phages P28 and C18, the second one--phages 5118, 05, 14, and 141, and the third one--phages 159 and 32. Bacteriophages 10, 12, and 149 produced slightly manifested (23 to 43 per cent) neutralization with P28 and 5118 sera. High-titer and specific antiphage sera were obtained.
The liposomal technology for preparing the immunostimulating complex (ISCOM) helped obtain a complex measles preparation whose antigens are represented by the structural proteins of measles virus in the bilayer phosphate-lipid membrane. Immunization of mice with the resultant preparation induced antimeasles antibodies with the maximum titer of antihemagglutinins 1:640 and of antihemolysins 1:1280. The biological activity of antibodies was confirmed in the neutralization test.
The capsid protein genes of five feline calicivirus (FCV) isolates associated with different disease manifestations were cloned and sequenced. The viruses represented two recent isolates from cats with chronic stomatitis, one recent isolate from a cat with acute stomatitis, one recent isolate each from a cat with acute respiratory symptoms and the classical limping syndrome strain FCV-2280. The amino acid sequences were compared with eight other published sequences and analyzed for their relationships. Phylogenetic analysis of the complete capsid protein sequences or of known antigenic regions of that protein (hypervariable regions A and E) did not group the isolates of different disease manifestations in distinct subclusters. Monoclonal antibodies (MAbs) generated against either a chronic stomatitis isolate or a recent isolate associated with respiratory symptoms were tested against a panel of 11 recent isolates and four "classical' FCV strains, covering all known disease associations. With those MAbs no obvious clustering with respect to disease manifestation could be seen. Four specific sera prepared in rabbits against our prototype isolates also failed to cluster those isolates according to the disease manifestations when examined in neutralization tests. From these antigenic and genetic analyses of the capsid protein the hypothesis of the existence of biotypes of FCV responsible for distinct disease manifestations could not be confirmed.
This report describes the first isolation and partial characterization of a herpesvirus from the harbor seal (Phoca vitulina). The virus was isolated during a disease outbreak in a group of young seals nursed in a seal orphanage in The Netherlands. Almost half of the seals died with symptoms of acute pneumonia and focal hepatitis and the virus was isolated of organs of most of the dead animals. Seven out of ten seals of which paired serum samples were obtained showed seroconversion in a virus neutralization test during this outbreak. The virus was tentatively characterized as a herpesvirus (seal herpesvirus: SeHV or phocid herpesvirus 1) on the basis of its characteristic morphology in electron microscopy, buoyant density in sucrose, sensitivity to ether and heat treatment and its antigenic relationship with other probable members of the Alphaherpesvirinae subfamily. The virus caused cytopathic changes within 24 hours after inoculation in seal kidney cells, consisting of a focal rounding of cells and syncytium formation. No cytopathic changes were observed in the cells of nine other mammalian species tested.
Two group A rotaviruses (ISU-64 and ISU-65) with subgroup I antigen were isolated from pigs with diarrhea and were shown by two-way neutralization tests to be two new serotypes of porcine rotavirus.
Tick-borne encephalitis can be prevented by active immunization, for which different schedules and booster recommendations exist. However, recommended booster intervals are often exceeded. In the present study, 178 adults aged 18-81 years received the first booster dose 2-11 years after primary immunization (instead of 12-18 months as recommended) according to the rapid schedule (vaccination on days 0, 7, and 21). The booster dose was well tolerated. All subjects showed a typical anamnestic response with an 11-fold increase in the geometric mean titre as measured both by neutralization test and ELISA.