Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Factors Influencing Virulence and Plaque Properties of Attenuated Venezuelan Equine Encephalomyelitis Virus Populations

A minority of stable large-plaque virus increased proportionally in stored unstable attenuated (9t) Venezuelan equine encephalomyelitis virus populations. L-cell-grown progeny (9t2) of stored 9t showed large amounts of large-plaque virus and increased virulence. Small-plaque virus inhibited large-plaque virus but not the reverse. Serial passage of small-plaque virus from 9t2 yielded a strain (20t) that was more attenuated than 9t.     

Properties of Venezuelan Equine Encephalomyelitis Virus Grown In Vivo

One intracerebral passage of either the parent egg seed (PES) or an attenuated variant (10t) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus in young adult mice produced progeny that were no longer differentiated unequivocally on the basis of plaque size. Plaques averaging about 2 mm in diameter, which was somewhat smaller than those formed by the PES virus and larger than those of the 10t strain, were formed by both strains. Seven serial passages of the PES virus in mouse brain failed to alter its virulence appreciably. In contrast, passage in mouse brain progressively changed the properties of the attenuated 10t strain. A substrain was isolated that possessed virulence similar to that of the PES virus and formed small plaques similar to those of the 10t strain. These findings showed a unique dissociation between the plaque size and virulence of the 10t strain. The new substrain differed from the PES virus and the 10t strain in its capacity for growth in mouse tissues after intraperitoneal inoculation. The substrain multiplied poorly in splenic tissue, which supports growth of the PES and 10t strains, but grew to high titers in the brain, which does not support appreciable growth of the 10t strain.     

Virulence of Venezuelan Equine Encephalomyelitis Virus Subtypes for Various Laboratory Hosts

Mice, guinea pigs, and duck embryo cell cultures were inoculated with known subtypes of Venezuelan equine encephalomyelitis (VEE) virus and the attenuated (TC-83) strain of VEE. With the exception of TC-83, all strains were highly pathogenic for suckling mice by either intracranial or intraperitoneal routes of inoculation used. Virulence for older mice and guinea pigs provided a means to distinguish strains. In addition, virulence or lack of virulence for adult mice or guinea pigs provides a rapid method for separating epizootic subtype IB from TC-83 VEE virus isolates.     

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.     

The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]

Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.     

Melatonin treatment enhances the efficiency of mice immunization with Venezuelan equine encephalomyelitis virus TC-83

To determine whether treatment with melatonin (MLT) improves the efficiency of immunization against Venezuelan equine encephalomyelitis (VEE) virus, mice were vaccinated with TC-83 VEE virus and treated daily with MLT (1 or 5 mg/kg) starting 3 days before immunization, until 10 days after. IgM antibody titers were determined at days 7, 14, and 21 post-immunization. IL-10 levels were assayed at day 14 postvaccination. Treatment with MLT increased antibody titers 14 days after the immunization. IL-10 levels also increased with MLT treatment (1 and 5 mg/kg). Mice were challenged with live VEE virus at day 21 postimmunization, and viral titers were plaque assayed in chicken embryo fibroblasts 4 days after the infection. Following this challenge brain virus levels were significantly reduced. The results suggest that MLT treatment enhances the efficiency of mice immunization against VEE virus.     

Development of a new live attenuated mumps virus vaccine in human diploid cells

A new live attenuated mumps vaccine was developed in human diploid cells. The S-12 virus was isolated from a 10-year-old girl showing typical symptoms of mumps infection, the diagnosis was confirmed by a pediatrician. The virus was isolated in green monkey kidney cells, without passage in chick embryo cavity or chick embryo fibroblasts. Attenuation of the wild virus was performed by serial passages in human diploid cells (MRC-5). The attenuated virus was characterized by identity tests, as well as by a reduction in plaque size, as marker tests. The virus was free from adventitious agents and safe for laboratory animals as well as for monkeys. The reactogenicity and immunogenicity of the S-12 virus for man was investigated by administration of a monovalent vaccine to 20 seronegative adult male volunteers and 30 children aged 1 to 5 years without history of mumps infection or vaccination. Seroconversion was obtained in 95% of the vaccinees. The new vaccine has the advantage of not requiring specific pathogen-free eggs, and being free from avian proteins and therefore can be used in sensitized patients.     

Relationship of Plaque Size and Virulence for Chickens of 14 Representative Newcastle Disease Virus Strains

Ability of 14 Newcastle disease virus strains to produce large plaques was related to virulence for chickens. Plaque-size comparisons were made under standard conditions in chick embryo cell monolayers. All plaque-producing strains showed a range of plaque sizes modified to a degree by the overlay medium used. An increase in size was found for most strains under methyl-cellulose overlay medium. Markedly larger plaques were found under this medium for both Calif-RO and Calif-CG strains. Heterogeneity in plaque size was most pronounced in velogenic (high virulence) strains. Only populations of small plaques were found in mesogenic (intermediate virulence) strains, and plaques were rarely found in lentogenic (low virulence) strains. Statistical analysis showed that the plaque size of velogenic strains differed significantly from mesogenic strains. None of the 11 plaque-producing strains had a normal distribution of plaque sizes, owing primarily to the presence of different genotypes within the plaquing population of a strain. This was demonstrated by derivation of clones from two of the strains. The populations of the large (Herts L) and small (Herts S) clear plaque clones derived from Eng-Herts were homogenous and distinct from one another on the basis of plaque size. Herts L was more virulent than Herts S. Although Herts L became more heterogenous in respect to plaque size upon repeated passage in embryonated eggs, no decrease in virulence of the strain was observed.     

<Emphasis Type="Italic">In Vitro</Emphasis>, Melatonin Treatment Decreases Nitric Oxide Levels in Murine Splenocytes Cultured with the Venezuelan Equine Encephalomyelitis Virus

The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 μg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.     

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.     

Characteristics of the factors that suppress and stimulate interferon production

A repressor of interferon production is contained in the hyaloplasm of the chick embryo fibroblast cells in the state of hyporeactivity. A factor stimulating interferon production (FSIP) was found in the hyaloplasm of the cells subjected to single or double induction with poly (pI) x poly (pC) followed by exposure to actinomycin D. Both preparations were of the protein nature and possessed high biological activity. They repressed or stimulatd interferon production at a dilution of 1:512-1:1024. The preparations had tissue and inductor specificity, did not affect the Venezuelan equine encephalomyelitis virus and interferon antiviral activity. Unlike the repressor, the FSIP proved to be thermolabile and pH-sensitive in acid media. The half lives of the repressor and stimulator were about 10 and 5 hours respectively.     

Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]

The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus.     

Heterogeneous RNA of the Prague strain of Rous sarcoma virus caused by undiluted passages.

The size of genomic RNA in PR-RSV A passaged in chick embryo fibroblasts (CEF) or quail embryo fibroblasts (QEF) was determined by gel electrophoresis. The results showed that 3 undiluted passages resulted in heterogeneity of RNA. The heterogeneity of the smaller incomplete RNAs in the virus stock was decreased by diluted passage or cloning, but RNA of the b subunit size and a subunit RNA of complete genome size were relatively stable. These heterogeneous RNAs were characterized by hybridization analysis. The RNAs from 4 peaks hybridized with both cDNAtotal and cDNAsrc to appreciable extents, indicating that they were derived from viral RNA and that at least some of them contained the src sequence. This finding and the failure to isolate a td mutant from the undiluted-passaged virus stock or from some subclones that had a and b subunits of RNA indicate that the td virus was only a minor constituent of the incomplete virus population caused by undiluted passages. Some viruses with incomplete RNA in the virus stock could produce foci with the aid of td B77 or RAV-60. The emergence of rd viruses by undiluted passages was indicated.     

Nonviable Venezuelan Equine Encephalomyelitis Hemagglutinin Prepared from Tissue Cultures by Gamma Radiation

Hemagglutinins of Venezuelan equine encephalomyelitis virus were produced in tissue cultures, and the infective virus was rendered nonviable by exposure to gamma radiation.     

Structural changes in influenza virus RNA during its attenuation

Changes in structures of only two genes of influenza virus--M and NS genes were found during virus attenuation by the method of oligonucleotide mapping. Such changes were observed in virulent and attenuated viruses (passages 10-23 in chick embryos) by comparing intermediate variants of the virus (passages 11-16 in chick embryos). These results allow us to conclude the important role of these genes in virus attenuation and in connection with virulence of the virus.     

Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.     

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.     

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.     

A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors.