Studies on the enhancement of virus titre of Japanese B encephalitis virus propagated in chick embryo cells by D2O and its possible mechanism.
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Deuterium↗
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Results for “ENCEPHALITIS VIRUSES”
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Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection.
A series of double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding the immunogenic proteins of Japanese encephalitis virus (JEV) [prM-E, prM-E-NS1, NS1-NS2A, 80%E (encodes the amino-terminal 80% part of E), and NS1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent JEV. The cassettes were introduced into both 5' [second subgenomic promoter of the vector precedes the SIN structural open reading frame (SP-ORF)] and 3' (the promoter follows the SP-ORF) dsSIN vectors. The longest cassette (prM-E-NS1) was 3.2 kb in length, which is remarkable for such a small vector virus as SIN (SIN genome is roughly 11.8 kb in length). The level of expression of JEV proteins appeared similar for both 5' and 3' recombinants. In general, the stability of the recombinants obtained was found to be low (expression was lost following one to five passages at low multiplicity of infection, depending on the recombinant). However, 5' recombinants containing longer cassettes (prM-E-NS1, prM-E, NS1-NS2A) were more stable than the corresponding 3' recombinants. Intraperitoneal inoculation of mice with 10(7) PFU of dsSIN-JEV recombinants induced antibodies against JEV proteins and low titers of JEV-neutralizing antibodies were produced by mice inoculated with recombinants expressing 80%E, prM-E, and prM-E-NS1. A single immunization of mice with the dsSIN-prM-E or dsSIN-prM-E-NS1 recombinants provided 40-65% protection against peripheral lethal challenge with 10(4) LD50 of neurovirulent JEV. The results demonstrate that genetically engineered togaviruses can be successfully used as vaccine vectors.
Animals↗
Simultaneous detection of three mosquito-borne encephalitis viruses (eastern equine, La Crosse, and St. Louis) with a single-tube multiplex reverse transcriptase polymerase chain reaction assay.
Three mosquito-borne human encephalitis viruses (eastern equine encephalitis virus [EEE], St. Louis encephalitis virus [SLE], and La Crosse encephalitis virus [LAC]) are sympatric in the southeastern United States. However, little is known concerning the temporal and spatial pattern of the distribution of these viruses in this area. As part of surveillance activities to detect the transmission of these 3 viruses in the Tennessee Valley area, we developed a single-tube multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay capable of detecting these 3 mosquito-borne viruses in a single reaction. Three viruses were differentiated by size of amplified products. Sensitivities of the multiplex RT-PCR assay for SLE, EEE, and LAC were 1-3 log median tissue culture infective doses per pool, roughly comparable to the reported sensitivity of PCR detection assays for the individual viruses, and 1 log more sensitive than antigen-capture assays for SLE and EEE. The sensitivity of the multiplex PCR was not changed significantly when carried out in the presence of extracts prepared from 50 uninfected mosquitoes. The cost of the assay is estimated at $2.98 per test, similar to the cost of other RT-PCR-based assays for viruses. However, adaptation of the RT-PCR to a multiplex format adds less than $0.01 to the per-unit cost of an RT-PCR assay targeting a single virus species. Analysis of these data suggests that the single-tube multiplex RT-PCR assay represents a sensitive, specific, cost-effective, and rapid method for monitoring activities of the 3 endemic mosquito-borne human encephalitis viruses in mosquito populations in the southeastern United States.
Animals↗
[Electrophoretic and sedimentation heterogeneity of the virion antigen of the tick-borne encephalitis virus].
Analysis of tick-borne encephalitis virus virion antigen by cross immunoelectrophoresis showed it to be separated into 2 subpopulations of virions differing by their mobility in the electric field. It was demonstrated that in immunoelectrophoresis the formation of precipitation bands typical of virion antigen occurred due to the movement of soluble immune complexes in the electric field. The immunoelectrophoretic characteristics of TBE virus virions are determined by the membrane E protein which was also found to be heterogeneous in its immunoelectrophoretic characteristics. The nucleocapsid of tick-borne encephalitis virus virions formed no precipitation bands in immunoelectrophoresis. The virion antigen not sedimenting in ultracentrifugation was represented by morphologically intact virions the structural protein composition of which differed from that of the structural proteins of virions sedimentable by ultracentrifugation. The possible causes of electrophoretic and sedimentation heterogeneity of tick-borne encephalitis virus virion antigen are discussed.
Antigens, Viral↗
ISOLATION FROM IXODES PERSULCATUS TICKS OF CYTOPATHIC AGENTS (KEMEROVO VIRUS) DIFFERING FROM TICK-BORNE ENCEPHALITIS VIRUS AND SOME OF THEIR PROPERTIES.
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Animals↗
Immunoglobulin response and viremia in dengue-vaccinated gibbons repeatedly challenged with Japanese encephalitis virus.
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Animals↗
Comparative vector competence of Culex tarsalis and Culex quinquefasciatus from the coachella, imperial, and San Joaquin Valleys of California for St. Louis encephalitis virus.
St. Louis encephalitis (SLE) virus has continued to be active in the Colorado Desert region of Southern California (Coachella and Imperial Valleys) since 1973, while it has virtually disappeared from the Sacramento and San Joaquin Valleys of Central California. Consequently, comparative vector competence studies were undertaken from 1978 to 1981 to evaluate the susceptibility of populations of Culex quinquefasciatus and Culex tarsalis from the San Joaquin and Coachella/Imperial Valleys to oral infection with representative SLE viral strains from each geographical area. Culex quinquefasciatus females from both areas were equally susceptible to infection with both viral strains by the pledget and viremic chick feeding techniques. Although susceptibility profiles were similar by both feeding techniques, infection thresholds (i.e., ID50S) obtained with Cx. quinquefasciatus females were at least 10,000-fold lower when fed on viremic chicks than on virus soaked pledgets. Culex tarslis females from the two geographical areas were equally susceptible to infection with the indigenous viral strain by feeding on pledgets, and were uniformly more susceptible than sympatric populations of Cx. quinquefasciatus. Thus, differences observed in SLE viral activity in different geographical areas of California since 1973 cannot be explained by differences in the susceptibility of Cx. quinquefasciatus or Cx. tarsalis to oral infection with SLE virus.
Animals↗
Infections of congenitally athymic (nude) and normal mice with avirulent and virulent strains of Venezuelan encephalitis virus.
Two strains of Venezuelan encephalitis virus that are avirulent for normal BALB/c mice inoculated subcutaneously were also avirulent for infected congenitally athymic (nude) mice of the same strain. Viremias were of similar magnitudes and durations in normal and nude mice. Brain concentrations were higher in nude mice with the one avirulent strain tested, although the periods of detectable virus in brains were similar. No lesions were found in brains, spleens, or lymph nodes by ordinary histopathological examination. Viral neutralizing antibody titers in plasmas at 1 to 3 weeks after infection were lower and more transient in nude than in normal mice, and implantations of thymic tissues into nude mice partially restored their neutralizing antibody responses. Concentrations of spleen cells producing antibodies that lysed sheep erythrocytes 4 days after inoculation of erythrocytes and avirulent virus into nude mice were above the levels of uninfected nude mice. These concentrations were similar in infected and uninfected normal mice. In contrast, two mouse-virulent strains of Venezuelan encephalitis virus killed nude mice faster than normal mice after subcutaneous inoculation. Yet concentrations and durations of virus in bloods and brains were not consistently different between nude and normal mice. There were perivascular monocytes in brains and slight architectural alterations of spleens and lymph nodes. Concentrations of spleen cells producing antibodies hemolytic for sheep erythrocytes 4 days after inoculation with erythrocytes were depressed in nude and normal mice by infection with virulent strains.
Animals↗
Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine.
We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 micro g per 10(4) cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10(6) PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.
Animals↗
Detection of antibodies to alphaviruses and discrimination between antibodies to eastern and western equine encephalitis viruses in rabbit sera using a recombinant antigen and virus-specific monoclonal antibodies.
Three arthropod-borne alphaviruses, western equine encephalitis viruses (WEEV), eastern equine encephalitis viruses (EEEV) and Venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the New World. With regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus Alphavirus in the American continent. However, this has been hampered in the past by (a) the close antigenic relationship between alphaviruses in traditional serological assays, especially in the routinely used haemagglutination-inhibition, and (b) the need of biosafety level 3 facilities to grow the viral antigens. An epitope blocking assay using an EEEV glycoprotein E1-expressing recombinant Sindbis virus and virus-specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut-off value of 30% inhibition for antigenic complex-specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus-, EEEV- and WEEV-complex-specific serum antibodies. As this test is based on the inhibition of binding of virus-specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation.
Animals↗
The response of goat brain cells to infection by caprine arthritis-encephalitis virus.
Caprine arthritis-encephalitis virus is a lentivirus of goats causing encephalomyelopathy acutely in young goats and occasionally in late stages of chronically infected adult goats. To determine the specific brain cells infected by the virus and the response of other cell types, goat cerebrum explant cultures prepared from newborn goat kids were infected by virus application. Infection of brain cultures was also possible by coculture with virus-infected macrophages. Predominantly, microglia cells were specifically infected by the virus, resulting in death of more than 50% of the cells within 2 weeks of infection. Infected cells enlarged and formed multinucleated giant cells. Infection of oligodendrocytes was observed, but infrequently. Astrocytes were not infected. Proviral deoxyribonucleic acid (DNA) was detected in infected cultures by amplification of a 490 base-pair fragment of the viral gag gene by the polymerase chain reaction (PCR) technique. Virus budding from plasma membrane of infected microglia cells was demonstrated by transmission electron microscopy. The supernatant of infected brain cells induced cytopathic effect when applied to indicator cells. These observations suggest productive infection of brain cultures. Virus infection of neurons was not observed. Culture of neurons in virus-free supernatant of CAEV-infected microglia or mixed glia culture resulted in death of 50% of the neurons in 11 days, suggesting the production of neurotoxic substances by the virus-infected glial cells.
Animals↗
Pseudotyped viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus.
Virus envelope proteins are the primary targets of neutralizing antibody responses. The epitopes recognized differ sufficiently between virus subtypes and species to distinguish viruses and provide an important basis for disease diagnosis. Venezuelan equine encephalitis virus (VEEV) causes acute febrile illness in humans and has high mortality in equines. The most specific detection methods for serum antibodies use live virus in neutralization assays or in blocking enzyme linked immunosorbent assays. However, work with Venezuelan equine encephalitis virus requires biosafety level 3 containment and select agent security in the United States. We report two new assays for detection of Venezuelan equine encephalitis virus neutralizing antibody responses, based on virus pseudotypes. The first provides detection by marker gene expression after 20 hours and is particularly suited for high-throughput screening; the second uses a new, rapid virus entry assay to give readouts within 1 hour. Both assays are safe, sensitive, and in general recapitulate neutralizing antibody titers obtained by conventional plaque reduction assays. Each is suitable as a rapid primary screen for detection of neutralizing antibodies against Venezuelan equine encephalitis virus.
Analysis of Variance↗
[Karyological characteristics of L cells and the identification with them, of the antigen of persisting Japanese encephalitis virus].
The immunofluorescence procedure revealed the virus-specific Japanese encephalitis virus antigen in latently infected L cells as well as differences in the percentage of antigen-positive cells and in the localization of the virus-specific antigen under conditions of acute, chromic, and latent infection and in the period of activation of the latent state of the virus at 28 degrees C. Karyological studied demonstrated no marked differences between persistently infected and original lines.
Animals↗
Herpes simplex virus encephalitis: intrathecal synthesis of oligoclonal virus-specific IgG, IgA and IgM antibodies.
Paired specimens of serum and CSF from seven patients with acute herpes simplex virus encephalitis were examined during the acute illness or the convalescent stage or during both stages. Imprint immunofixation analyses of viral antibodies separated by agarose electrophoresis and by electrofocusing disclosed intrathecal production of herpes simplex virus IgG antibodies in all seven patients, and of IgA and IgM antibodies in six and three of six patients, respectively. Intrathecal production of herpes simplex virus-specific IgG and IgA was observed in two patients from whom samples were collected after 1 year, while intrathecal production of virus-specific IgM was not demonstrated later than 5 weeks after onset. The intrathecally synthesized IgG and IgM, and to a lesser extent IgA antibodies displayed oligoclonal characteristics. Oligoclonal bands of IgG were observed in the CSF of all patients. Evidence is presented to show that the bulk of the oligoclonal CSF IgG represents herpes simplex virus-specific antibodies. Intrathecally synthesized populations of herpes simplex virus antibodies cross-reacting with varicella-zoster virus were identified in three of the patients.
Adolescent↗
[The homogeneity of a population of production strain 205 of the tick-borne encephalitis virus].
Tick-borne encephalitis virus strain 205 was characterized by such sign as the presence of negative colonies. In the swine fibroblast cell line the population of the strain was represented mainly by large plaque (67.3%). Medium-sized plaques prevailed in chick embryo fibroblast cell culture. Protamine sulfate and magnesium chloride reduced the period of incubation, increased the resolving capacity of the plaque method and the proportion of medium- and small-sized plaques. Agarose B rendered the virus colonies less contrasting. The production strain and the clones of its population were found to have pronounced reproductive and protective properties. The S+ variants of the virus exhibited the highest reproductiveness.
Animals↗
Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination.
West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.
Dengue↗
Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus.
Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.
Animals↗
Protease treatment and chemical crosslinking of a flavivirus: tick borne encephalitis virus.
Tick-borne encephalitis virus was treated with pronase or thermolysin. The resulting particles were banded in sucrose gradients and analyzed for polypeptide composition. Both enzymes caused a reduction in particle density from 1.19 to 1.15--1.16 g/cm3. No loss of viral lipid or nucleic acid could be observed. SDS-polyacrylamidegel electrophoresis showed that only the core protein V2 was unchanged whereas the envelope proteins V3 and V1 had disappeared from their original positions in the PAGE profile. Instead a new peptide(s) with molecular weight of 4000--6000 was found in which hydrophobic amino-acids were enriched. Crosslinking by dimethyl-3.3'-dithiobispropionimidate (DTBP) made the virus resistent to solubilization of the envelope proteins by TX-100. This could be interpreted by the formation of a dense envelope protein network around the nucleocapsid preventing its liberation by TX-100. Some data however indicate that direct crosslinking of at least one of the envelope proteins with the core cannot be excluded.
Cross-Linking Reagents↗