A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.

Freon 113 (Freon) is an essential component used in some viral purification methods to separate virus from infected cell debris. With its environmental and toxic hazards, Freon's availability is limited and more tightly regulated. Several organic solvent substitutes were selected to identify a suitable Freon replacement for the purification of both cultivable reovirus and fastidious calicivirus. Reovirus was extracted from tissue cultured cells with each solvent tested and purified in cesium chloride gradients by standard techniques. Purified virions were analyzed for conservation of physical and biological properties by morphological examination and infectivity studies. The purification of calicivirus nucleic acid from stool samples using selected solvents was also examined. Solvent-extracted calicivirus RNA was reverse transcribed and quantified by polymerase chain reaction amplification of a standard diagnostic 117 bp amplicon. These studies indicated that Vertrel XF (a newly developed environmentally friendly Freon substitute) and a 7:3 mixture of isopentane/1-chlorobutane are suitable replacements. Considerations of flammability and ease of use suggest that Vertrel XF is the preferred choice as a Freon substitute for the purification of these non-enveloped viruses.

The reovirus mutant tsA279 has temperature-sensitive lesions in the M2 and L2 genes: the M2 gene is associated with decreased viral protein production and blockade in transmembrane transport.

Temperature-sensitive mutants provide an ideal means for dissecting viral assembly pathways. The morphological variants produced by and biological characteristics of tsA279, a previously uncharacterized mutant from the Fields' panel of temperature-sensitive mutants of reovirus, were determined under restrictive growth conditions. The mutant showed a distinctive pattern of increased temperature sensitivity as the temperature was raised from 39 degrees to 40 degrees. Wild-type reovirus type 1 Lang and the mutant were crossed to generate reassortants. Efficiency of plating analyses of the reassortants showed that tsA279 has temperature-sensitive lesions in two genes, a mildly temperature-sensitive one in L2, which encodes core spike protein lambda 2, and a stronger, dominant lesion in M2, which encodes major outer capsid protein mu 1. Electron microscopic examination of thin-sectioned tsA279-infected cells showed three ways in which the mutant phenotypes were expressed. The mutant appeared to be blocked in transmembrane transport of virions, a phenotype that mapped to the M2 gene; the mutant produced significantly reduced amounts of identifiable particles; and those particles that were produced appeared to be morphological variants. Immunofluorescent microscopy and immunoprecipitations of tsA279- and various T1L x tsA279 reassortant-infected cells suggested that the reduction in observed progeny was caused by a decreased production of viral proteins at the nonpermissive temperature. This phenotype also mapped to the mutant M2 gene.

The reovirus mutant tsA279 L2 gene is associated with generation of a spikeless core particle: implications for capsid assembly.

Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified two ts lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46-58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the lambda2 protein, is associated with the accumulation of a core-like particle defective for the lambda2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein lambda2, and the minor core protein micro2. Core particles with the lambda2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins lambda1, lambda3, and sigma2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein mu2, either preceding or collateral to the condensation of the lambda2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.

Comparison of direct and indirect enzyme immunoassays with direct ultracentrifugation before electron microscopy for detection of rotaviruses.

A direct and an indirect enzyme immunoassay (EIA) were evaluated against a standard of electron microscopy after direct ultracentrifugation of the specimen for their performances in detecting rotaviruses. The indirect EIA had variable background activity which influenced test specificity. The indirect EIA control (test system without the detector antibody) plus a regression line (which reflected background noise) improved test specificity. However, the results of direct EIA (Rotazyme; Abbott Laboratories, North Chicago, Ill.) sensitivity (86%) and specificity (96%) were better than those of the indirect EIA in tests on 73 rotavirus-positive and 78 rotavirus-negative specimens. Endpoint titrations of purified SA-11 rotavirus showed greater sensitivity of the direct EIA test. Electron microscopy, performed after direct ultracentrifugation, and direct EIA were approximately 2 log10 more sensitive in the detection of purified SA-11 rotavirus than was electron microscopy with standard methods of unconcentrated specimen preparation. Direct EIA test are potentially sensitive, specific, and practical for the rapid detection of rotaviruses from human clinical specimens. Further studies are needed before EIA methods for detection of human rotaviruses can be equated with the level of reliability of results obtainable with sensitive electron microscopy techniques.

Human rotavirus detection by counterimmunoelectrophoresis versus enzyme immunoassay and electron microscopy after direct ultracentrifugation.

The sensitivity of the counterimmunoelectrophoresis test with NCDV, Wa, and SA-11 rotavirus antisera was 60, 60, and 67%, respectively. The counterimmunoelectrophoresis specificity was greater than 99%, but the low sensitivity is a limiting feature of this test as a first-line immunodiagnostic test for rotavirus detection.

Improved detection of viruses by electron microscopy after direct ultracentrifuge preparation of specimens.

We have adapted the Beckman Airfuge air turbine ultracentrifuge and the new EM-90 particle-counting rotor to improve detection by electron microscopy of viruses in clinical specimens. Samples were clarified by centrifugation, pelleted in the EM-90 rotor directly to Formvar-coated copper grids, and strained with 1.5% sodium phosphotungstate. Virus counts and endpoint titrations of serial dilutions of partially purified preparations of poliovirus, SA11 rotavirus, herpes simplex virus, and vaccinia virus showed an increase of ca. 1.5 log10 to 3.0 log10 over the virus titers of unconcentrated preparations of the same material. An increased yield of 14% more positive specimens for rotavirus was obtained after preparation of clinical samples by direct ultracentrifugation versus a method without virus concentration (82 versus 72). A prospective study showed that detection of adenoviruses, herpesviruses, and enteroviruses increased when specimens were prepared by direct ultracentrifugation. Direct ultracentrifugation with the EM-90 rotor in the Airfuge ultracentrifuge is a rapid concentration method which enhances the rate and yield of virus detection from clinical specimens by electron microscopy and is easily adaptable to a diagnostic virology laboratory.

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.