Cytoplasmic granule formation and translational inhibition of nodaviral RNAs in the absence of the double-stranded RNA binding protein B2.

Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3' end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.

Review of the regulation of veterinary drugs and residues in South Africa.

The food safety risk analysis framework of the FAO/WHO is used in the review of veterinary drug and residue regulation in South Africa to determine possible inefficiencies within this system. Results indicate that a variety of challenges relating to the processes of risk assessment, management, and communication do exist, although these occur within a fragmented system of legislation, functions, and structures. Addressing these challenges therefore requires a change to a more collaborative and integrated system. It is indicated that for such a change, the underlying challenges of inadequate horizontal communication, poor conceptualization, and awareness of functions of the system are required to be dealt with.

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies.

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.

Dual roles for an arginine-rich motif in specific genome recognition and localization of viral coat protein to RNA replication sites in flock house virus-infected cells.

Assembly of many RNA viruses entails the encapsidation of multiple genome segments into a single virion, and underlying mechanisms for this process are still poorly understood. In the case of the nodavirus Flock House virus (FHV), a bipartite positive-strand RNA genome consisting of RNA1 and RNA2 is copackaged into progeny virions. In this study, we investigated whether the specific packaging of FHV RNA is dependent on an arginine-rich motif (ARM) located in the N terminus of the coat protein. Our results demonstrate that the replacement of all arginine residues within this motif with alanines rendered the resultant coat protein unable to package RNA1, suggesting that the ARM represents an important determinant for the encapsidation of this genome segment. In contrast, replacement of all arginines with lysines had no effect on RNA1 packaging. Interestingly, confocal microscopic analysis demonstrated that the RNA1 packaging-deficient mutant did not localize to mitochondrial sites of FHV RNA replication as efficiently as wild-type coat protein. In addition, gain-of-function analyses showed that the ARM by itself was sufficient to target green fluorescent protein to RNA replication sites. These data suggest that the packaging of RNA1 is dependent on trafficking of coat protein to mitochondria, the presumed site of FHV assembly, and that this trafficking requires a high density of positive charge in the N terminus. Our results are compatible with a model in which recognition of RNA1 and RNA2 for encapsidation occurs sequentially and in distinct cellular microenvironments.

Microbial composition in bioaerosols of a high-throughput chicken-slaughtering facility.

The microbial composition of the air in various areas of a high-throughput chicken-slaughtering facility was investigated. Over a 4-mo period, 6 processing areas were sampled, and the influence of environmental factors was monitored. The highest counts of microorganisms were recorded in the initial stages of processing, comprising the receiving-killing and defeathering areas, whereas counts decreased toward the evisceration, air-chilling, packaging, and dispatch areas. Maximum microbial counts were as follows: coliforms, 4.9 x 10(3) cfu/m(3); Escherichia coli 3.4 x 10(3) cfu/m(3); Bacillus cereus, 5.0 x 10(4) cfu/m(3); Staphylococcus aureus, 1.6 x 10(4) cfu/m(3); Pseudomonas aeruginosa, 7.0 x 10(4) cfu/m(3); presumptive Salmonella spp., 1.5 x 10(4) cfu/m(3); Listeria monocytogenes, 1.6 x 10(4) cfu/m(3); and fungi, 1.4 x 10(4) cfu/m(3). Higher counts of airborne microorganisms found in the receiving-killing and defeathering areas indicate the importance of controlling microbial levels before processing to prevent the spread of organisms downstream. This should limit the risk of carrying over contaminants from areas known to generate high counts to areas where the final food product is exposed to air and surface contamination.

The influence of sanitizers on the lipopolysaccharide composition of Escherichia coli O111.

This study focused on the influence of typical sanitizers on the composition of the lipopolysaccharides (LPS) produced by the verocytotoxin-producing (VTEC) Escherichia coli O111. We also aimed to cast light on the applicability of O-antigen-based serotyping and endotoxin based Limulus Amebocyte Lysate (LAL) assays applied in the food industry for the identification and quantification of Gram-negative bacteria. E. coli O111 was propagated in the presence of three typical commercially applied sanitizing solutions that included a Clean in Place (CIP) chlorinated sanitizer (bacteriocidal), heavy-duty alkaline sanitizer (bacteriocidal) and a phenolic hand wash solution (bacteriostatic). After the required growth phase was reached the LPS from both the intact cells and debris was extracted and methanolysed followed by trifluoroacetylation. Subsequently GC-MS analysis and the chromogenic LAL assay were applied to assess both the ultra-structure and the toxicity of the extracted LPS. The viability and debris formation during growth was also evaluated to verify the bacteriocidial and static effect of the applied sanitizers as well as to assess its relationship with LPS formation. The total LPS produced was quantified at 1.3 x 10(6) [KDO] x OD(620 nm)(-1) for the control samples, 6.5 x 10(3) [KDO] x OD(620 nm)(-1) for E. coli grown in the presence of CIP chlorinated sanitizer and 2.1 x 10(5) and 2.85 x 10(6) [KDO] x OD(620 nm)(-1) for the organisms grown in the presence of heavy-duty alkaline sanitizer and phenolic hand wash solution respectively (KDO = 2-keto-3-deoxy-octulosonic acid). A negative correlation (gamma(2)= -0.880) between the [KDO] and Delta viability was evident and indicated that E. coli O111 responds to factors that hinder viability by producing more LPS in its outer membrane. Subsequent assessment of the LPS ultra-structure revealed a definite change in both the total assessed saccharide and lipid fractions. The cumulative change of the LPS in response to the sanitizers further appeared to influence the toxicity of the LPS as the latter change could not be related to an individual compound within any of the assessed fractions. This emphasised the fact that the quantity of LPS obtained from E. coli O111 in this study, did not seem to determine the toxicity of the organism. From the results we further propose a coefficient that could be applied to describe the response of E. coli O111 LPS to sanitizers and caution against the application of serotyping (based on the O-antigen) and the LAL assay to quantify and identify E. coli O111 obtained from food strata where the possibility of sanitizer contamination exists.

Influence of commercial sanitizers on lipopolysaccharide production by Salmonella Enteritidis ATCC 13076.

The effect of typical sanitizers on the composition and toxicity of lipopolysaccharides (LPSs) produced by Salmonella Enteritidis ATCC 13076 was analyzed. Salmonella Enteritidis was propagated up to the late exponential phase in the presence of commercial sanitizing solutions. LPS was extracted and derivatized with trifluoroacetylation, and gas chromatography-mass spectrometry analysis and the chromogenic Limulus amoebocyte lysate assay were used to assess the ultrastructure and toxicity of the LPS. The viability and debris formation during growth were evaluated to verify the bactericidal and bacteriostatic effects of the sanitizers and to assess sanitizer effects on LPS formation. The LPSs produced were quantified at 1.7 x 10(4), 1.2 x 10(4), 3.6 x 10(3), and 9.6 x 10(4) [KDO] x OD(620nm)(-1) for the controls and the organisms grown in the presence of a chlorinated sanitizer, a heavy-duty alkaline cleaner, and a phenolic hand wash solution, respectively. In response to these treatments, the short-chain polysaccharide fractions of the LPSs in the Salmonella Enteritidis cells increased. This finding suggests that this organism increases the low-molecular-weight fraction of the LPS in relation to the high-molecular-weight fraction to survive these unfavorable conditions. The cumulative change in the LPS in response to the sanitizers influenced the toxicity of the LPS; however, this change could not be related to an individual compound within any of the assessed fractions.

The distribution of Staphylococci in bioaerosols from red-meat abattoirs.

The quality and shelf-life of perishable foodstuffs can be reduced by high concentrations in the processing environment of bioaerosols consisting of spoilage microbiota. A lack of documented literature on the distribution of such bioaerosols has, however, led to the underestimation of their impact. In the study reported here, the deboning rooms of selected South African red-meat abattoirs were investigated for airborne concentrations of staphylococci; the authors studied the distribution of Staphylococcus species in general, as well as the coagulase types of Staphylococcus aureus in particular. Average staphylococci bioaerosol concentrations varied considerably among the abattoirs investigated, with Abattoir B having the highest counts (3 x 10(2) CFUs/m3) and Abattoir A having the lowest (7.6 CFUs/m3). There was a significant link between bioaerosols and microbial loads from red meat in the same environment. The recorded levels were, however, well below the recommended maximum limits for bioaerosols suggested by various international and governmental authorities. Staphylococcus xylosus and S. saprophyticus were found to be the most abundant species in the air of the deboning rooms, while among S. aureus coagulase types, Type III and Type VIII were predominant. On the basis of the ecology of the bacterial groups, the authors suggest probable sources of staphylococcal bioaerosols and propose strategies that could be developed for red-meat abattoirs to reduce the levels of airborne pathogens.

Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

Biological dynamics and distribution of 3-hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy. Evidence for a putative regulatory role in the sexual reproductive cycle.

Dipodascopsis uninucleata has been recently shown to produce 3-hydroxy polyenoic fatty acids from several exogenous polyenoic fatty acids. In order to examine whether endogenous 3-hydroxy fatty acids (3-OH-FA) may be implicated in the developmental biology of this yeast, we mapped by immunofluorescence microscopy their occurrence in fixed cells with or without cell walls using an antibody raised against 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE), the biotransformation product from arachidonic acid (AA). This antibody turned out to cross-react with other 3-OH-FA. 3-OH-FA were detected in situ in gametangia, asci, as well as between released ascospores, and proved to be associated with the sexual reproductive stage of the life cycle of the yeast. Acetylsalicylic acid (1 mM), which is known to suppress the formation of 3-OH-FA from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as the sexual phase of the life cycle suggesting a prominent regulatory role of 3-OH-FA for the latter.

Hysterosalpingo-radionuclide scintigraphy (HERS).

A radionuclide procedure, hysterosalpingo-radionuclide scintigraphy (HERS), was designed to evaluate the migration of a particulate radioactive tracer from the vagina to the peritoneal cavity and ovaries as well as to image and functionally outline the patency of the pathways between these two extremes of the female reproductive system. Technetium-99m human albumin microspheres (99mTc-HAM) were deposited in the posterior fornices of patients who were divided into two specific groups. Group I consisted of patients who were to undergo different elective gynecologic operations, in which besides obtaining sequential images, radioactivity levels were measured in the removed organs and tissues. Group II consisted of patients referred by the Infertility Clinic for evaluation of their reproductive system pathways patency. In this latter group, HERS was compared with contrast hysterosalpingography (HSG) and peritoneoscopy (PCP). The results obtained from measurements of radioactivity levels on the removed surgical specimens and comparison with other conventional gynecologic diagnostic procedures provide accurate evidence of the migration of 99mTc-HAM from the vagina, through the uterus and tubes, to the peritoneal cavity and ovaries, and show that HERS is a simple noninvasive method for functionally imaging and assessing the patency of the female reproductive system pathways.

The Martin-Bell syndrome in South Africa.

A national screening programme was introduced in 1980 when the first cases with the Martin-Bell syndrome were diagnosed in South Africa. This survey includes patients from all the major population groups in South Africa. One thousand patients, who include 354 relatives of 21 index cases, were investigated cytogenetically. About 75% of the 354 relatives were either affected males or obligate or possible carriers. The segregation pattern of the fragile site was investigated in 271 offspring of 58 carrier women. At least 30% of the carriers were mildly mentally retarded with most expressing the fragile site. Various other investigations, such as measurements of testes, speech, verbal and IQ evaluations and hormone studies were done on several affected males. No fragile site could be demonstrated in 57 unselected autistic children. The results of this programme show that this syndrome is a common cause of mental retardation and that prevention of mental retardation is possible if all the involved families could be identified.

Acrocallosal syndrome in two African brothers born to consanguineous parents.

We describe two mentally retarded brothers with craniofacial anomalies, polydactyly, and other clinical manifestations compatible with the acrocallosal syndrome (ACS). These are the first black patients from Africa with this diagnosis. They are also the fourth set of sibs described with ACS, and together with the parental consanguinity documented in this family, confirm autosomal recessive inheritance of this syndrome. The clinical manifestations in our patients confirm the intrafamilial variability of the syndrome. Postnatal onset of growth retardation is proposed as an additional manifestation of ACS.

Folate status, homocysteine metabolism, and methylene tetrahydrofolate reductase genotype in rural South African blacks with a history of pregnancy complicated by neural tube defects.

The birth incidence of neural tube defects (NTDs) in South Africa is threefold to sixfold higher in rural compared with urban blacks. We investigated whether folate deficiency and aberrant homocysteine metabolism could explain the high NTD incidence in rural black populations. Plasma folate and total homocyst(e)ine (tHcy) concentrations were determined in apparently healthy rural black women (n = 107), rural black women with a history of pregnancy complicated by NTDs (n = 54), and urban blacks (n = 101). Methionine load tests were performed on the 54 women with a history of NTD-affected pregnancy and 54 controls matched for age and body mass. The presence of the 677C --> T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene was investigated in both groups by a polymerase chain reaction (PCR) of genomic DNA and HinfI digestion of the PCR product. Apparently healthy urban black women (n = 101) had a lower (P < .001) plasma folate concentration compared with rural black women (n = 107). Women with a history of NTD-affected pregnancy did not differ significantly from controls with respect to plasma folate, fasting homocyst(e)ine, methionine, and the post-methionine load increase in plasma homocyst(e)ine. More than 50% of both of the latter groups had a post-methionine load increase in plasma tHcy less than the fifth percentile as observed in a healthy white control group. No homozygotes for the 677C --> T mutation in the MTHFR gene were found in black mothers with NTD-affected offspring or controls. It is concluded that black urbanization is characterized by a diminished folate status that is paradoxically associated with a lower NTD birth incidence. Homozygosity for the 677C --> T mutation in the gene coding for MTHFR does not constitute a genetic risk factor for NTDs in blacks. No aberrant homocysteine metabolism could be demonstrated in black women with NTD-affected pregnancies.

Giant neurofibromas of the labia.

Two cases of giant neurofibroma of the vulva are described. The finding of a solitary neurofibroma in a patient who shows no other stigmata of multiple neurofibromatosis is unusual. Also unusual was the exceptional size of these tumors.

Extraction methods for lipopolysaccharides from Escherichia coli ATCC 25922 for quantitative analysis by capillary electrophoresis.

Lipopolysaccharides (LPS) are major constituents of the cell wall of Gram-negative bacteria. Capillary zone electrophoresis (CZE) was applied to distinguish between lipopolysaccharides extracted from Escherichia coli ATCC 25922 with various techniques. Extraction methods proposed by Westphal and Jann [Methods Carbohydr. Chem. 5 (1965) 83], Galanos et al. [Eur. J. Biochem. 9 (1969) 245], Ni Eidhin and Mouton [FEMS Microbiol. Lett. 110 (1993) 133] and Nichols [Infect. Immun. 62 (1994) 3753] for LPS preparation were evaluated. Electrophoresis buffers with varying pHs were applied to assess the structure stability of the extracted LPS samples. Variations in structural breakdown were apparent demonstrating that different extraction methods removed different LPS molecules. Furthermore, the results obtained proved the CZE useful as an analytical technique for LPS evaluation. The LPS removed with the Nichols extraction procedure presented a unique electrophorogram that could in future be applied in the rapid identification of Gram-negative foodborne pathogens.

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata.

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.

Bacterial populations associated with meat from the deboning room of a high throughput red meat abattoir.

Developing countries are faced with high incidences of food poisoning outbreaks, with obvious economic consequences. In highly perishable foodstuffs such as fresh red meat the threat of food poisoning is particularly intense. In this study, red meat samples were collected from a deboning room of a high throughput abattoir. The samples were analysed for the presence of Bacillus cereus., Staphylococcus aureus., Pseudomonas spp., Listeria monocytogenes., Escherichia coli and Salmonella spp. The aerobic plate counts as well as Enterobacteriaceae were also enumerated. Almost without exception the counts exceeded the microbiological guidelines for raw meat as proposed by the South African Department of Health. The average B. cereus count over the sampling period was 8.32 × 10(3) cfu, g (-1), for S. aureus and Pseudomonas spp. 1.72 × 10(5) and 1.7 × 10(5) cfu g(-1) respectively and for E. coli 3.4 × 10(5) cfu g(-1). Sixty percent of the samples were positive for presumptive Salmonella spp. while 52% of the samples tested positive for the presence of L. monocytogenes. The aerobic plate and Enterobacteriaceae counts were 1.7 × 10(7) and 4.6 × 10(6) cfu g(-1), respectively. The data highlighted the need for a more systematic approach to ensuring safe food through implementing quality control methods to prevent the entry and proliferation of pathogens in meat and meat products, especially during processes with a high degree of handling, such as deboning.

Quantification of bioaerosols in automated chicken egg production plants.

The quantity and composition of bioaerosols in a typical automated chicken egg layer management system (LMS) with a controlled internal climate (B) and without (A) were compared. The LMS-A used a fecal matter disposal system featuring a central opening in the floor through which the matter automatically dropped to an open-air lower level; the LMS-B used a conveyer belt below each hen battery set, which removed the fecal matter frequently. Bioaerosols were collected by impaction on agar. Humidity, wind velocity, temperature, and dust particle concentration were also analyzed at several locations in the LMS. The average bioaerosol concentrations (total viable aerobic bacteria) associated with the inside of LMS-A reached X = 1.1 x 10(5) cfu/m3 with counts in LMS-B being X = 9.2 x 10(4) cfu/m3. In both systems, the bacterial counts were significantly higher on the inside of the LMS than the outside. The LMS-A showed yeast counts of X = 6.7 x 10(1) cfu/m3 with none detectable in LMS-B. Total culturable mold counts were X = 7.0 x 10(2) cfu/m3, with significantly higher presumptive Salmonella spp. counts (X = 6.6 x 10(1) cfu/m3) inside both LMS when compared with the outside. Escherichia coli and total culturable gram-negative counts were significantly higher in LMS-B at concentrations of X = 3.6 x 10(1) cfu/m3. These counts were significantly higher compared with the outside environment. We concluded that the live birds were the major source of bioaerosols in both LMS, with the fecal matter disposal systems attributing to the difference in bioaerosol composition. Modifications to the operation protocols of both LMS to limit the contamination of eggs by bioaerosols are suggested.

Differential segregation of nodaviral coat protein and RNA into progeny virions during mixed infection with FHV and NoV.

Nodaviruses are icosahedral viruses with a bipartite, positive-sense RNA genome. The two RNAs are packaged into a single virion by a poorly understood mechanism. We chose two distantly related nodaviruses, Flock House virus and Nodamura virus, to explore formation of viral reassortants as a means to further understand genome recognition and encapsidation. In mixed infections, the viruses were incompatible at the level of RNA replication and their coat proteins segregated into separate populations of progeny particles. RNA packaging, on the other hand, was indiscriminate as all four viral RNAs were detectable in each progeny population. Consistent with the trans-encapsidation phenotype, fluorescence in situ hybridization of viral RNA revealed that the genomes of the two viruses co-localized throughout the cytoplasm. Our results imply that nodaviral RNAs lack rigorously defined packaging signals and that co-encapsidation of the viral RNAs does not require a pair of cognate RNA1 and RNA2.