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In an effort to enhance genetic interactions by eliminating spatial or physical barriers between variants of Sindbis virus, MgCl2 was used to aggregate infecting viral particles. Mixing viral samples in a 1:1 ratio with 0.5m MgCl2 produced maximal reduction in plaque forming units (PFU) with minimal cell damage due to MgCl2. Aggregate size was determined to be about 7 PFU. Samples taken at 3, 5 and 10 hours after infection with mixed aggregates composed of large and small plaque forming virus indicated that only one type of genome was represented among the progeny particles. In addition, aggregation enhanced complementation and the progeny were stable after several cycles of sonication and passage.

A method for packing soil columns to investigate viral transport phenomena is described. The columns were 10 cm in diameter and ranged from 33 to 100 cm in length. Field conditions of the soil, including bulk density and profile, were reproduced in columns. Ionic gradients resulting from sequential applications of wastewater and distilled water affected the movement of poliovirus I (Chat) through soil. Compared with 33-cm- and 66-cm-length columns, lower concentrations of infectious virions were observed in the percolates from 100-cm soil columns. These results may be attributed to the greater pore volume in the longer columns (the greater volume of soil contained in these columns), whereas the volume of liquid applied was constant for all columns.

To ascertain the health risks that may be posed by the land application of sewage sludges, a scheme was devised to determine the types and numbers of pathogenic and potentially pathogenic bacteria present in sludges. A processing treatment was adapted to sludge to give a homogenate which yielded the greatest numbers of viable bacteria. Conventional methods were successful in enumerating Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used indicator organisms. Modifications of conventional methods improved the enumeration of Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., and Clostridium perfringens. However, Shigella methodology yielded only one isolate. Utilizing the proposed scheme, the population densities of these organisms were estimated in three domestic wastewater sludges. In light of these results, the potential impact of land application of sewage sludges is discussed.

In chicken cells infected by Sindbis virus and exposed to a variety of membrane-active compounds, virus release was inhibited. In infected cells exposed to antiserum directed against the virion glycoproteins E1 or E2, retinol, cortisone, Pb++ or insulin, the processing of two Sindbis virus precursor polypeptides which lead to the formation of virion polypeptides was inhibited. The B-protein, which is the precursor to both envelope proteins, accumulated in cells treated by these compounds. This precursor is generally not detected in chicken cells, presumably because it is processed rapidly. The cleavage of the precursor PE2 to the envelope glycoprotein E2 was also inhibited. E2 was also absent in cells exposed to menadione or to antiserum prepared against uninfected chicken cells. Since each of the compounds tested interfered with Sindbis virus polypeptide cleavage, despite their diverse mechanisms of action, it suggests that perturbation of normal membrane fluidity can interfere with Sindbis virus budding.

Laboratory models and field sewage samples were used to study viral association with particulates in suspension. Poliovirus and coliphages T2, T7 and f2 were tested for their adsorption to and elution from both organic and inorganic suspended solids at a variety of pH values and in both the presence and absence of a divalent cation. The results indicate that no one of the viruses tested can be used to describe a general pattern of adsorption. T2 and f2 readily associated with the clays used in the presence of divalent cation, while T7 and poliovirus adsorbed equally well to both organic and inorganic particulates. All viruses varied in the degree to which they associated with naturally-occurring suspended solids in primary and final sewage effluents; maximal association occurred at the pH extremes of 4.0 and 10.0 for all systems. Regardless of the conditions used to effect adsorption, all viruses tested, with the exception of f2, were infective by plaque assay in their adsorbed form. These data suggest that no one coliphage system can acceptably model the behavior of poliovirus in laboratory or field conditions. Furthermore, the results reaffirm and broaden the findings that viruses associated with suspended particulates are infective. Therefore, any system used to monitor virus levels in the environment must adequately account not only for free virus, but also for those which are solids-associated.

The viral polypeptides synthesized in cells coinfected with group C and group D or E Sindbis virus mutants were studied. Cleavage of the ts2 protein occurs in cells coinfected with ts2 and ts20. Since the ts2 protein fails to chase in cells infected with ts2 alone, the activity effecting this cleavage must be, at least in part, virus specified.

We have demonstrated that the small plaque (SP) variant of Sindbis virus is more extensively neutralized by complete antibody and serum IgG fractions than is the large plaque (LP) variant. The Fc portion failed to demonstrate any neutralizing potential and did not block subsequent neutralization by IgG or Fab fragments. The LP and SP variants of Sindbis gave identical patterns upon isoelectric focusing. Treatment of the virus with intact serum IgG or isolated Fab fragments was found not to have an irreversible effect on the net charge of the virus population. The selective processes involved in the maturation of the nucleocapsid implicate the incorporation of charged host determinants which are then reflected in the charge of the virion. It is suggested that the virus core has a preferential affinity for those components of the plasma membranes which have an isoelectric point at pH 3.6.

The peptide composition of variants of Sindbis virus which differ in plaque-type, elution pattern from calcium phosphate columns and virulence in mice was analyzed. Differences in peptide number or composition were not detected in these variants. Since the Sindbis virion has an outer lipoprotein envelope which is derived at least in part from the plasma membrane of the host cell, peptide composition of purified chicken embryo membranes was analyzed. In contrast to the virions, the number of peptide units in the host cell membrane were too numerous to give resolution.

Rejection of viruses by commercial grade asymmetrical cellulose acetate membranes commonly used in the ultrafiltration and reverse osmosis processes was evaluated. Various concentrations of coliphage T2 and poliovirus were innoculated in the feed water of a bench scale ultrafiltration-reverse osmosis unit and virus concentrations in the produce were determined. The insoluble polyelectrolyte technique for concentrating extremely low levels of virus was modified and standardized for use in the study. Membrane penetration studies indicated that limited numbers of virus passed the membranes. The penetration of viruses may be attributable to the presence of random areas of imperfect crosslinkage of the cellulose acetate in the dense layer of the membrane. Despite limited virus penetration, all of the cellulose acetate membranes used in this study rejected an extremely high percentage of the viruses and provided a product water of excellent quality.

A method for concentrating and purifying enveloped viruses is described. Its salient feature is the use of an antihost immunoadsorbent column. Purified virus preparations were obtained in which viral-specific proteins per/mg total protein were enriched 800-fold, while host-specific complement fixing material became undetectable.

Sindbis virus, a group A arbovirus, is composed of a nucleocapsid and a glycolipoprotein membrane (Pfefferkorn & Hunter, 1963a; Strauss et al. 1968; Schelle & Pfefferkorn, 1969). The membrane protein is specified by the virus genome (Pfefferkorn & Clifford, 1964; Strauss et al. 1968; Strauss, Burge & Darnell, 1969) whereas the phospholipids and carbohydrate are derived from the host of origin (Pfefferkorn & Hunter, 1963b; Burge & Strauss, 1969; Strauss, Burge & Darnell, 1970) during maturation by budding through the membranes of cytopathic vacuoles (CPV) or the plasma membranes of the infected cell (Grimley, Berezesky & Friedman, 1968; Hackett et al. 1968; Holmes, Wark & Warburton, 1969; Nakai, Shand & Howatson, 1969). We have studied the maturation of Sindbis virus with the use of ferritin-conjugated antibody. After exhaustive adsorption with normal chick embryo cells, the globulin fraction precipitated from rabbit anti-Sindbis serum (Keckwick, 1940; Strauss et al. 1960) was conjugated with ferritin (Singer, 1959; Hsu, Rifkind & Zabriski, 1963; Rifkind, Hsu & Morgan, 1964).

The insoluble polyelectrolyte technique for concentrating virus is extended to extremely low virus levels. The effectiveness of this method employing a coliphage T2 model is a constant 20% over a range of virus levels from 10 3 to 10 −4 plaque-forming units/ml. The efficiency of the method is dependent upon p H control during the concentration phase. Although the study was initiated to develop a method for quantitating the effectiveness of water and wastewater treatment methods for the removal of viruses from waters at low concentrations, the potential of the technique for efficient monitoring of natural waters is apparent.

A distilled water elution-bentonite concentration technique was developed and used to monitor indigenous viruses present in liquid sludges undergoing land application at six field sites.

Complement-fixing antigens were detected in the nucleocapsid and envelope of Sindbis virus. Neutralizing antibodies are directed against antigens in the viral envelope.

Movement of poliovirus I (Chat) through nonsterile core samples of a sandy forest soil was monitored, using several regimens of loading with either dechlorinated final effluent from an operating activated sludge treatment plant or distilled water. Stimulated cycles of rainfall and effluent applications, resulting in ionic gradients, were shown to affect virus movement. Such studies indicate that poliovirus applied in effluents may move considerable distances through this soil after rainfall. Survival of poliovirus in the soil at 4 and 20 C has been monitored for 84 days. During this period, the capacity of the virus to migrate is unchanged.

Poliovirus I, a human enterovirus, was studied in field-scale model wastewater holding ponds. Viruses were distributed between the sediment and overlying water in the ponds. Virions were inactivated more rapidly in the water than in the sediment. The rate of inactivation was higher during the warmer months of the year for virions in both water and sediment. Primary wastewater effluent in ponds was more conducive to prolonged virus survival than was final effluent. The metabolic activity of algae elevated the pH of the pond waters, correlating with a more rapid inactivation of poliovirus.

Encephalomyocarditis viruses adsorb to introduced organic and inorganic solids in water over a wide range of pH and with various concentrations and species of metal cations. Visible flocculation of solids was not a prerequisite for significant virus association. Virus adsorption to natural solids in various types of natural waters was significant but variable. Clay-adsorbed virus retained its infectivity in tissue culture monolayers. These solids-associated viruses also retained infectivity in mice.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.

SummaryHeLa cells and chick embryo fibroblasts were grown on glass and harvested under various conditions. Trypsinization and scraping were each compared with alcoholether denaturation. Trypsinization was found to be less efficient as a means of obtaining material for chemical analysis although less damaging to cell viability than scraping. Alcohol-ether denaturation in situ was the preferred means of harvesting cells for chemical analysis.

The development of resistance of outbred white mice to two plaque size variants of Sindbis virus was studied as a function of host age. A sudden drop in mortality occurred over a 3-day period. As little as 1 plaque-forming unit (PFU) per mouse was lethal in 1-day-old animals, whereas inocula of 10 9 PFU per mouse failed to produce high mortality in weanlings. A comparison of viral growth kinetics shows that early replication was similar in newborns and weanlings but that after 6 to 8 hr the production rate and, therefore, the maximal viral titer were suppressed in weanlings. The data demonstrate a lethal viral threshold. Titers in excess of this level lead to mortality, whereas those below it allow survival.

The mechanism of penetration and uncoating of enveloped animal viruses has recently been the subject of renewed controversy. Electron microscopic studies have led a number of investigators to conclude that penetration takes place by cellular engulfment (viropexis) of intact virus particles (Dales & Choppin, 1962; Mussgay & Weibel, 1962; Dales, 1963; Simpson, Hauser & Dales, 1969). Other investigators have concluded that enveloped animal viruses penetrate by fusion of the virus envelope with the host cell plasma membrane (Morgan & Howe, 1968; Morgan, Rose & Mednis, 1968a, b; Heine & Schnaitman, 1969). We now report on the role of the Sindbis virus envelope in the attachment of virus to host cells. Friedman & Berezesky (1967) and Sreevalsan & Allen (1968) noted that nucleocapsid components obtained from cytoplasmic extracts of arbovirus-infected cells or by detergent treatment of intact virus particles were non-infective for chicken embryo cells. However, the RNA released from arboviruses by detergent treatment is infective when assayed appropriately (Richter & Wecker, 1963; Nakamura & Eguchi, 1968).

A mathematical analysis of virus production with accompanying heat inactivation, from which the rate of virus release and total virus production are readily calculated, is presented. Applications of this analysis for Sindbis and Chikungunya viruses are discussed.