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.     

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.     

Growth of Venezuelan Equine Encephalomyelitis Virus in Human Diploid Cell Strain WI-38

We demonstrated that Venezuelan equine encephalomyelitis (VEE) virus replicated in and adapted rapidly to human diploid cell strain WI-38. Peak titers of approximately 10(9.8) mouse intracerebral 50% lethal doses were obtained at low passage levels in Eagles basal medium supplemented with calf serum. VEE virus replicated poorly in serum-free medium. Propagation of VEE virus was accompanied by the production of hemagglutinin and cytopathogenic effects.     

The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAV_UK. Various EIAV_UK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAV_UK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.     

Replication of Western Equine Encephalomyelitis Virus: I. Some Chemical and Physical Characteristics of Viral Ribonucleic Acid 1

The ribonucleic acid (RNA) from Western equine encephalomyelitis (WEE) virions sedimented through sucrose gradients with a sedimentation coefficient of 40S. Another viral RNA which was always associated with infected cells possessed a sedimentation coefficient of 26S. Both 40S and 26S RNA had identical base compositions and densities. The 40S RNA displayed a hyperchromic effect when heated with a T(m) of 57.5 C. When 40S RNA was heated at 90 C and cooled rapidly, it sedimented with a coefficient of 26S. Dialysis of 40S RNA against distilled water changed its sedimentation coefficient to 26S. The presence of 8 m urea or 50% dimethyl sulfoxide in the gradients also altered the sedimentation rate of 40S RNA to 26S. In the latter case, the 26S RNA retained 10% of the infectivity originally added as 40S RNA. Dialysis of 26S RNA against 0.5 m NaCl or 0.05 m acetate buffer at pH 4.0 altered it so that about 50% of the radioactivity sedimented with a coefficient of 40S. Chromatography on methylated albumin-kieselguhr columns failed to separate 40S RNA from 26S RNA. Viral RNA either exists in two conformations which sediment differently in sucrose or contains an extremely labile portion near the center and is easily broken into two equal pieces.     

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.     

Replication of Western Equine Encephalomyelitis Virus II. Cytoplasmic Structure Involved in the Synthesis and Development of the Virions

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with ³H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to ³H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of ¹⁴C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with ³H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.     

Formalin-inactivated Venezuelan Equine Encephalomyelitis (Trinidad Strain) Vaccine Produced in Rolling-Bottle Cultures of Chicken Embryo Cells

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr.     

Specific Role of Each Human Leukocyte Type in Viral Infections: II. Phytohemagglutinin-treated Lymphocytes as Host Cells for Vesicular Stomatitis Virus Replication In Vitro

The mitogenic agent, phytohemagglutinin (PHA), added to human mixed leukocyte cultures and to lymphocyte cultures converted small lymphocytes into lymphoblasts and increased lymphocyte susceptibility to vesicular stomatitis virus (VSV). Maximum virus yields were 30- to 1,000-fold higher in PHA-treated than in control cultures. VSV replicated to peak titers before lymphocytes were morphologically transformed by PHA, and virus titers fell as lymphoblast destruction began. PHA neither induced significant VSV replication in polymorphonuclear leukocyte cultures, nor increased the large virus yields in monocyte cultures. The treatment of PHA with heat, digestive enzymes, rabbit anti-PHA serum and serial dilutions failed to dissociate that portion of the PHA extract responsible for the conversion of lymphocytes into virus-susceptible cells from those components responsible for leukoagglutination or lymphocyte transformation.     

Immunofluorescence of Japanese Eneephalitis Virus Infection In Vitro: Localization of Viral Antigen in Canine Cerebellar Tissue Cultures

Propagation of Japanese encephalitis (JE) virus in cells of dog cerebellar tissue cultures was investigated by means of fluorescent antibody (FA) technique. The fluorescent globulin conjugate was made from the serum of a dog inoculated with partially purified JE virus, treated by Sephadex G-25 and DEAE cellulose column chromatography and then adsorbed with dog liver powder. This preparation was found to be appropriate for the present work. Fluorescence was demonstrable in virus-infected cultures of three different types of cells, fibroblast-like cells, nerve cells and some of the glial type cells. Fluorescence could first be demonstrated about 20 hours after virus inoculation and appeared to increase in intensity in proportion to the increase of infective virus present in the cultures. The specificity of the reaction was supported by the non-reactivity of control (non-infected) cultures and by the results of blocking tests. The infected nerve cells and glial type cells also exhibited morphological changes clearly detectable by the FA techniques, corresponding to the changes shown in Bodian-stained preparations. The localization of FA antigen in the fibers of these cells suggests a possible mode of spread of JE virus in the nervous tissues. In any of the cell types studied thus far, the nuclei remained FA-unstained even during the advanced stage of infection.     

Antagonistic Effect of Luzindole in Mice Treated with Melatonin During the Infection with the Venezuelan Equine Encephalomyelitis Virus

The effect of Luzindole (LZ) in mice treated with melatonin (MEL) during the infection with the Venezuelan equine encephalomyelitis (VEE) virus was examined. Melatonin (500 μg/Kg b.w.) was administered daily 3 days before and 5 days after the infection. Luzindole (5 mg/Kg b.w.) was injected intraperitoneally 3 days before (pre-infection) or 5 days after (post-infection) the infection. Mortality rates in the infected mice treated both with MEL and LZ were higher than in those treated with MEL alone in which the lowest brain and serum viral titers were detected. On the third post-infection day, viral titers of the MEL + VEE + LZ (pre-infection) group were higher than those of the remainder groups. On the fifth day, viral titers in infected mice were similar to those of the MEL + VEE + LZ (pre-infection) group, but higher than those detected in the MEL + VEE + LZ (post-infection). In conclusion, the protective effect of MEL in mice infected with VEE virus was inhibited by LZ suggesting that this protection is mediated by MEL receptors.     

Assignment of the Multifunctional NS3 Protein of Bovine Viral Diarrhea Virus during RNA Replication: an In Vivo and In Vitro Study

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products-negative-strand intermediate and progeny positive-strand RNA-in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein-in particular the ATPase/RNA helicase-play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.     

Replication of Sendai Virus: II. Steps in Virus Assembly

Chick embryo fibroblast cultures infected with Sendai virus were incubated with (3)H-uridine in the presence of actinomycin D beginning at 18 hr after infection. The 35 and 18S virus-specific ribonucleic acid (RNA) components were found in a ribonuclease-sensitive form in the cell and appeared to be associated with polyribosomes. Newly synthesized 57S viral RNA was rapidly coated with protein to form intracellular viral nucleocapsid, and no 57S RNA was found "free" (ribonucleasesensitive) in the 2,000 x g supernatant fraction of disrupted cells. The nucleocapsid from detergent-disrupted Sendai virus and that from disrupted cells were indistinguishable in ultrastructure and buoyant density, and neither was found to be infectious or have hemagglutinating activity. Kinetic studies of nucleocapsid and virus formation indicated a relative block in conversion of viral nucleocapsid to complete enveloped virus in these cells, resulting in accumulation of large amounts of nucleocapsid in the cell cytoplasm.     

Specific Role of Each Human Leukocyte Type in Viral Infections I. Monocyte as Host Cell for Vesicular Stomatitis Virus Replication In Vitro

Each major leukocyte type of the peripheral blood of healthy donors was studied in vitro for its ability to support vesicular stomatitis virus (VSV) replication. Purified cultures of each white blood cell type were prepared by the selective adsorption and elution of cells from silicone-treated glass beads. It was found that monocytes and macrophages (derived from the rapid transformation of monocytes in vitro) were the principal host cells for VSV replication. Interferon added to mixed leukocyte cultures, prior to virus inoculation, reduced virus yields and prevented destruction of macrophages. Cultures of small lymphocytes, containing no detectable monocytes or macrophages, produced amounts of virus equivalent to 1% of that produced in leukocyte cultures which contained 7% monocytes. Small lymphocytes did not undergo demonstrable cytopathic alterations in virus-infected cultures. VSV neither replicated nor produced cytopathic effects in polymorphonuclear leukocytes.     

In Vitro Processing of Herpes Simplex Virus Type 1 DNA Replication Intermediates by the Viral Alkaline Nuclease, UL12

Herpes simplex virus type 1 (HSV-1) DNA replication intermediates exist in a complex nonlinear structure that does not migrate into a pulsed-field gel. Genetic evidence suggests that the product of the UL12 gene, termed alkaline nuclease, plays a role in processing replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, J. Virol. 70:2075–2085, 1996). In this study we have tested the hypothesis that alkaline nuclease acts as a structure-specific resolvase. Cruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of being resolved into linear duplexes as would be expected of a resolvase activity, the artificial cruciforms were degraded. DNA replication intermediates were isolated from the well of a pulsed-field gel (“well DNA”) and treated with purified HSV-1 alkaline nuclease. Although alkaline nuclease can degrade virion DNA to completion, digestion of well DNA results in a smaller-than-unit-length product that migrates as a heterogeneous smear; this product is resistant to further digestion by alkaline nuclease. The smaller-than-unit-length products are representative of the entire HSV genome, indicating that alkaline nuclease is not inhibited at specific sequences. To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate that replicating DNA apparently contains no accessible double-stranded ends but does contain nicks and gaps. Our data suggest that UL12 functions at nicks and gaps in replicating DNA to correctly repair or process the replicating genome into a form suitable for encapsidation.