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At least 19 recordsLinked to original sources
PubMed1979

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.

Aedes↗
PubMed2026

Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings.

Fluorescence microscopy offers a highly sensitive and versatile approach for investigating alphavirus infection at the cellular level. By combining fluorescently labeled viruses with quantitative image analysis, this method enables detailed spatial and temporal characterization of infection dynamics, including the detection of subtle differences in replication kinetics and cell-to-cell spread. A central aim of this protocol is its application in antiviral screening assays. Image-based quantification of fluorescence intensity provides a robust and reproducible means to assess the efficacy of antiviral compounds, allowing early and sensitive detection of inhibitory effects in infected cells. This facilitates the identification of promising antiviral hits and supports the evaluation of dose-dependent responses. The approach is also well-suited for comparative studies of different alphavirus strains or mutants, as variations in replication behavior and dissemination patterns become readily apparent. Its flexibility, compatibility with multiple cell lines, and straightforward integration into automated imaging platforms makes the method scalable and suitable for high-throughput screening campaigns. Overall, this protocol advances the discovery and evaluation of antiviral strategies. Given that several alphaviruses cause significant human and veterinary diseases, lack approved antiviral therapies, and continue to expand geographically with emerging outbreaks, the identification of novel antivirals remains an urgent priority. Therefore, this fluorescence-based workflow represents a valuable and timely contribution to modern alphavirus research.

Antiviral Agents↗
PubMed1977

Evaluation of vascular clearance as a marker for virulence of alphaviruses: disassociation of rapid clearance with low virulence of venezuelan encephalitis virus strains in guinea pigs.

The concept that relates low virulence of certain alphaviruses to low viremia and efficient vascular clearance of virus was tested in guinea pigs. Previously published studies with hamsters suggested that virulent strains maintain high viremias primarily because they are cleared inefficiently from the blood. In the present study, with guinea pigs, six of six virulent strains of Venezuelan encephalitis virus were cleared inefficiently, whereas three of six nonlethal or benign virus strains were cleared rapidly. However, three other guinea pig-benign Venezuelan encephalitis virus strains cleared slowly, to produce a high viremia was correlated with inefficient growth in primary viral replication sites. Thus, the potential of some alphaviruses to produce destructive lesions may be restricted by efficient clearance of virus from the blood, whereas the growth of other benign alphavirus strains may be restricted after the virus is presented to target cells.

Animals↗
PubMed1977

Chromatographic separations of alphavirus strains by hydroxylapatite.

Hydroxylapatite column chromatography methods were developed to characterize selected alphavirus populations. Different conditions of pH and phosphate molarity were required to obtain satisfactory elution profiles and separations for Western equine encephalomyelitis virus strains, compared with Eastern equine encephalomyelitis virus and Semliki Forest virus strains. Raising the pH of the buffers effected earlier elutions of all viruses. Selection of phosphate gradients with more gentle slopes and adjustment to the proper pH effected better separations of virus subpopulations. Elution profiles were not affected by 0.85% NaCl, 10% fetal calf serum, or 1% bovine serum albumin, which are common constituents of virus-stabilizing diluents. Passage of Western equine encephalomyelitis and Semliki Forest viruses in BHK-21, Vero, or duck embryo cell cultures or in suckling mouse brains did not usually affect elution profiles, unless passage also resulted in a shift in the plaque size marker. Essentially all infectious virus applied to the column was recoverable in appropriate fractions. This permitted accurate determinations of heterogeneity within alphavirus populations. For Western equine encephalomyelitis large-plaque (LP) and small-plaque (SP) virus populations, it was possible to detect ratios of 1 LP in a population of 10(6) SP, and 1 SP in 10(3) LP by using linear phosphate gradients. When stepwise elution procedures were used, it was possible to detect ratios of 1 SP in a population of 10(5) LP. Hydroxylapatite column chromatography therefore appears to be a useful tool for characterizing alphaviruses and for isolating minority subpopulations of viruses of biological or epidemiological importance from apparently homogeneous virus stocks.

Buffers↗
PubMed1978

Radioimmunoassay for quantitation of antibodies to alphaviruses with staphylococcal protein A.

A radioimmunoassay (RIA) procedure is described for measuring antibodies to alphaviruses in human and other mammalian sera. The test employed protein Abearing Staphylococcus aureus as a solid-phase immunoadsorbent for (3)H-labeled viruses complexed with immunoglobulin G. Using antibodies produced in humans and guinea pigs, the RIA procedure clearly differentiated among antibodies to Venezuelan, western, and eastern equine encephalomyelitis viruses. Sensitivity of the RIA depended on the concentrations of labeled viruses employed. The dilution of serum that effected binding of 50% of the (3)H-labeled virus (determined by probit analysis) was consistently higher than the neutralizing antibody titer determined by a conventional plaque reduction neutralization test using 80% plaque reduction end points. In addition, sera from 73 individuals were screened for seroconversion following live attenuated Venezuelan equine encephalomyelitis virus vaccine (strain TC-83) inoculation, by RIA using a single serum dilution (1:80); results were identical with seroconversions identified by plaque reduction neutralization test. Hyperimmune Venezuelan equine encephalomyelitis virus sera from a number of mammalian species were successfully titrated by RIA; the species tested were human, guinea pig, white rat, rabbit, burro, dog, monkey, sheep, and cotton rat. The protein A-mediated RIA is a rapid, sensitive, specific, and precise serological tool for measuring antibodies to surface antigens of alphaviruses, and should allow the subsequent development of a competitive binding RIA to measure antigenic potency of inactivated alphavirus vaccines.

Animals↗
PubMed2025

Proximity interactome of alphavirus replicase component nsP3 includes proviral host factors eIF4G and AHNAK.

All positive-strand RNA viruses replicate their genomes in association with modified intracellular membranes, inducing either membrane invaginations termed spherules, or double-membrane vesicles. Alphaviruses encode four non-structural proteins nsP1-nsP4, all of which are essential for RNA replication and spherule formation. To understand the host factors associated with the replication complex, we fused the efficient biotin ligase miniTurbo with Semliki Forest virus (SFV) nsP3, which is located on the cytoplasmic surface of the spherules. We characterized the proximal proteome of nsP3 in three cell lines, including cells unable to form stress granules, and identified >300 host proteins constituting the microenvironment of nsP3. These included all the nsPs, as well as several previously characterized nsP3 binding proteins. However, the majority of the identified interactors had no previously identified roles in alphavirus replication, including 39 of the top 50 interacting proteins. The most prominent biological processes involving the proximal proteins were nucleic acid metabolism, translational regulation, cytoskeletal rearrangement and membrane remodeling. siRNA silencing confirmed six novel proviral factors, USP10, AHNAK, eIF4G1, SH3GL1, XAB2 and ANKRD17, which are associated with distinct cellular functions. All of these except SH3GL1 were also important for the replication of chikungunya virus. We discovered that the small molecule 4E1RCat, which inhibits the interaction between the canonical translation initiation factors eIF4G and eIF4E, exhibits antiviral activity against SFV. Since the same molecule was previously found to inhibit coronaviruses, this suggest the possibility that translation initiation factors could be considered as targets for broadly acting antivirals.

Viral Nonstructural Proteins↗
PubMed1976

Antigenic relationships of alphaviruses by a simple micro-culture cross-neutralization method.

A cross-neutralization study of 22 alphaviruses disclosed two major antigenic complexes and two viruses that were distinct from all the others. Cross-reactions were common but were restricted within the complexes. Sub-groupings within a complex were also shown, and some viruses proved to be indistinguishable by neutralization testing. The results generally paralleled previously reported data. Investigations were carried out, using BHK 2I cells in a micro tissue-culture system, on all available alphaviruses.

Antigens, Viral↗
PubMed1979

Cross-reactive cytotoxic T cells to alphavirus infection.

Secondary Tc cells immune to alphaviruses (BEB, SIN and SFV) cross-react between serologically defined subgroups at the level of target lysis and at the level of induction of response. Despite this apparently complete Tc cell cross-reactivity between BEB, SIN and SFV, antisera raised against BEB and SIN showed virus specificity in their ability to block Tc cell-mediated lysis of alphavirus-infected targets. This result suggests that Tc cells recognize the same viral antigen molecule as antibodies, but with less specificity. Other possible interpretations are discussed.

Animals↗
PubMed1975

Comparative aspects of the replication of alphaviruses and poliovirus.

The replication of both alpha- and picornaviruses results in the production of plus-stranded viral progeny. Each of these two classes of virus devotes about the same amount of viral genetic information to structural proteins but the alphaviruses use more genetic information for non-virion proteins. This additional information may be required because the alphaviruses have a much broader host range. Alternatively, the additional information may be required for interrupted transcription that yields a subgenomic mRNA that encodes all of the alphaviral structural proteins. This subgenomic mRNA allows preferential synthesis of a precursor af all virion proteins. Cleavage of this precursor has been observed during in vitro protein synthesis and can be prevented by an appropriate temperature-sensitive mutation.

Arboviruses↗
PubMedDate not supplied

Investigations on the presence of antibodies to alphaviruses, flaviviruses, Bunyavirus and Kemerovo virus in humans and some domestic animals.

The presence of antibodies to some alphaviruses, flaviviruses, bunyaviruses and orbiviruses was studied in the sera from humans and domestic animals of biotopes "C" and "T" in the south-east of Romania. Specific antibodies were found in humans to 3 alphaviruses (eastern equine encephalitis, western equine encephalitis and Sindbis), in a very low proportion, and to 3 flaviviruses (tickborne encephalitis, West Nile and Ntaya), at a much higher percentage. Very low percentages of antibodies to Bunyavirus and Kemerovo virus were also detected. The proportion of antibodies to flaviruses (tick-borne encephalitis, West Nile and Ntaya) detected in domestic animals (sheep, cattle and swine) is much lower than that found in humans of the same biotope ("T").

Animals↗
PubMed1979

Persistence in humans of antibody after immunization with four alphavirus vaccines.

Immunization with attenuated VEE virus vaccine resulted in persistence of neutralizing antibody for 12 years. Immunization with inactivated WEE vaccine converted 83% of the subjects, killed EEE vaccine converted 27% and killed Chikungunya vaccine induced no significant titers. Antibody formed as a result of immunization with inactivated vaccines was of short duration, i.e. less than 1 year. Attenuated VEE was responsible for some heterologous antibody rises to the other three alphaviruses. Among the inactivated vaccines WEE and Chikungunya vaccines produced one heterologous rise each to EEE virus.

Alphavirus↗
PubMed1977

Isolation of Sindbis (alphavirus) and Leanyer viruses from mosquitoes collected in the Northern Territory of Australia, 1974.

Mosquitoes collected near Darwin, Northern Territory, in 1974 yielded two virus strains. One was identified as Sindbis virus, not previously isolated from the Northern Territory. The other is antigenically distinct from viruses previously isolated from arthropods in Australia, and the name "Leanyer" is proposed for it. Its properties suggest that it may be a togavirus serologically unrelated to available alphaviruses and flaviviruses.

Animals↗
PubMed1978

Use of Semliki forest virus to identify lipid-mediated antiviral activity and anti-alphavirus immunoglobulin A in human milk.

In a simple and reliable assay system, Semliki forest virus (SFV) was used to detect the activity of antiviral factors in human milk. Fractionation of the milk showed that a heat-stable, lipid-associated activity and an immunoglobulin-associated activity were present, either singly or together, in 85% of the human milk samples tested. Cow and synthetic milk showed neither activity. Extraction of the neutral milk lipids allowed the antiviral to be located with the monoglyceride and free fatty acid fractions. The milk low in antiviral lipids and high in triglycerides also lacked a strong lipase activity. The immunoglobulin anti-SFV activity was shown to be due to immunoglobulin A, the major milk immunoglobulin, and appears to be directed against an alphavirus closely related to SFV, possibly Ross River virus.

Antibodies, Viral↗
PubMed1979

Adoptive transfer of cross-protection among alphaviruses in mice requires allogeneic stimulation.

Cell-mediated (T-effector cell) immunity is proposed as playing the major role in cross-protection between Sindbis and Semliki Forest viruses, which are alphaviruses that do not elicit cross-neutralizing antibodies. In adoptive transfer experiments, T-cells from spleens of Sindbis virus-immunized mice were found to confer specific cross-protection to Semliki Forest virus upon recipient mice. This cross-protection was observed in the outbred ICR strain of mice and when transfers were made between several combinations of inbred and hybrid strains. Cross-protection was substantially reduced if syngeneic rather than allogeneic cell transfers of one spleen equivalent per mouse were made. The results suggest that allogeneic stimulation (mixed lymphocyte reaction in vivo) is necessary to increase the number of effector cells (donor) in the recipient. This was supported by the observation that blastogenic stimulation of donor cells in vitro by concanavalin A induces cross-protection in syngeneic animals. Conversion of recipient cells to specific effector cells also appears to play a role in protecting mice against Semliki Forest virus. This was concluded from the experiments described above, a time course study, and the results of experiments that involved serial passages of transferred cells across histocompatibility barriers. Thus, we propose that both donor and recipient cells are active in protecting recipient mice against challenge with Semliki Forest virus after adoptive transfer.

Animals↗
PubMed1979

Restricted replication of two alphaviruses in ricin-resistant mouse L cells with altered glycosyltransferase activities.

Two mouse L cell variant lines (CL 3 and CL 6) selected for resistance to the toxic plant lectin ricin were restricted in their ability to replicate the two alphaviruses Sindbis virus and Semliki Forest virus. CL 3 cells have been shown to exhibit increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, whereas CL 6 cells have been shown to contain decreased UDPgalactose:glycoprotein galactosyltransferase and UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activities. The adsorption of Sindbis virus to CL 6 cells was considerably reduced, suggesting that the loss or inaccessibility of the receptors for Sindbis virus accounted for a major defect in virus production in these cells. In contrast, CL 3 synthesized Sindbis viral RNA and proteins but were unable to convert the precursor glycoprotein PE2 to the structural protein E2. The cleavage of PE2 to E2 was also blocked in both CL 3 and CL 6 cells infected with Semliki Forest virus.

Galactosyltransferases↗
PubMedDate not supplied

[Kyzylagach virus (family Togaviridae, genus alphaviruses), a new arbovirus isolated from Culex modestus mosquitoes trapped in the Azerbaijani SSR].

A strain of LEIV 65A virus was isolated in 1969 from Culex modestus mosquitoes collected in the Kyzylagach preserve, the Azerbaijan SSR, in a colony of Ciconiiformes birds. Investigations of the antigenic properties of this virus by the complement fixation and neutralization tests as well as by electron microscopy showed it to belong to alphaviruses of the Togaviridae family. Kyzylagach virus had one-way relations with Sindbis virus. Some biological and biophysical-biochemical properties of the virus were studied.

Animals↗
PubMed1977

Plaque formation by alphavirus (Getah) in Culex Mosquito cells.

Plaques were formed by Getah virus (an Alphavirus) which had been grown in BHK cells and passaged once in Culex cells before inoculation of Culex cell cultures. Plaques were not produced either by Getah or Japanese encephalitis (JE) viruses which had been passaged only in BHK cells. Plaque counts in Culex cell monolayers were almost the same as in BHK cells.

Arboviruses↗