Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature

The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies.     

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.     

Complex of fd gene 5 protein and double-stranded RNA

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.     

DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

Reliability in the genetic code.

Base sequences of φ × 174 and MS2 viruses genomes and of some mRNAs (Coat protein fd virus, Rabbit B. Globin, Rat Growth Hormone and Human Chorionic, Somatomammotropin) show a preferential use of some amino-acid codons. Based on this observation the reliability of three non-degenerate codes are analyzed. All of them display higher reliability than the standing genetic code and specially one formed by a set of non-directly related codons.The absence of these type of codes in Nature is discussed in terms of a balance between reliability and mutability of the genetic information, able to preserve species and maintain evolution     

The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

Summary Polyamines are able to affect Fe²⁺ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe²⁺ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe²⁺ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe²⁺ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe²⁺ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe²⁺ autoxidation, is also discussed.Abbreviations poly [A] polyadenylic acid (5) - poly [C] polycytidylic acid (5) - poly [1] polyinosinic acid (5) - poly [G] polyguanylic acid (5) - poly [A. U] polyadenylic-uridylic acid - poly [A] poly [U] polyadenylic-polyuridylic acid     

Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid.

Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.     

The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses.     

Folding of two large nucleotide chains

We have already described the FOLD-A code designed for folding mRNA's and single stranded DNA molecules (Nussinov & Pieczenik, 1984). In this paper we describe its application to two long polynucleotide chains: the A protein gene of the MS2 RNA and the whole genome of the phi X 174 phage. The folded form of the single stranded DNA of the phi X 174 is a six armed star with the origin of replication in its center.     

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.     

Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3

The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations. Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids. Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure [Thomas, G.J., Jr., & Wang, A.H.-J. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol. 2, Springer-Verlag, Berlin]. The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3. The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti. However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers. Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3 and ε3/ε4) were identified from dsDNA obtained by PCR from corresponding patients.     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Photochemical stability of aromatic amino acids under picosecond laser UV-irradiation

Two-step photochemical decomposition of aromatic amino acids under picosecond laser UV-irradiation was investigated. These results were compared with the photochemical stability of nucleic acid bases. Using the known ratio between the nucleic acid bases and aromatic amino acids in native bacteriophages lambda and phi X174 it was shown that picosecond laser UV-inactivation of viruses occurred due to the photodegradation of nucleic acid.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

白花蛇舌草醇提取物对乳腺癌T-47D细胞株生长的抑制作用

目的：观察白花蛇舌草醇提取物对乳腺癌细胞T-47D生长的影响。方法：采用平皿克隆形成实验、软琼脂集落形成实验和BrdUrd（溴脱氧尿苷）掺入实验,观察不同浓度的白花蛇舌草醇提取物（1.0μg/mL,3.0μg/mL,10μg/mL,30μg/mL,100μg/mL）对T-47D细胞锚定依赖性生长和软琼脂集落形成能力及核酸合成的抑制作用。结果：①随着白花蛇舌草醇提取物浓度的升高,对T-47D细胞的生长呈现明显抑制作用;②白花蛇舌草醇提取物可呈浓度依赖性抑制对数生长期T-47D细胞核酸合成。结论：白花蛇舌草醇提取物可能通过影响肿瘤细胞核酸合成而抑制T-47D细胞生长。     

A dendrogram of plant viruses.

To facilitate the recognition of plant viruses with similar characteristics a dendrogram of characterized viruses was constructed. The sequence of criteria included: type of nucleic acid; single or double stranded; presence or absence of lipid envelope; helical or nonhelical symmetry; and divided or single genome. Nonhelical RNA viruses with divided genomes were further divided into viruses with one or more than one capsid size. Those with one capsid size were subdivided into viruses with one or more than one sedimenting component. Nonhelical RNA viruses with a single genome were divided according to their RNA size, and their sensitivity to sodium dodecyl sulfate and ethylenediaminetetraacetic acid.     

Solar Disinfection of Viruses in Polyethylene Terephthalate Bottles

Solar disinfection (SODIS) of drinking water in polyethylene terephthalate (PET) bottles is a simple, efficient point-of-use technique for the inactivation of many bacterial pathogens. In contrast, the efficiency of SODIS against viruses is not well known. In this work, we studied the inactivation of bacteriophages (MS2 and ϕX174) and human viruses (echovirus 11 and adenovirus type 2) by SODIS. We conducted experiments in PET bottles exposed to (simulated) sunlight at different temperatures (15, 22, 26, and 40°C) and in water sources of diverse compositions and origins (India and Switzerland). Good inactivation of MS2 (>6-log inactivation after exposure to a total fluence of 1.34 kJ/cm²) was achieved in Swiss tap water at 22°C, while less-efficient inactivation was observed in Indian waters and for echovirus (1.5-log inactivation at the same fluence). The DNA viruses studied, ϕX174 and adenovirus, were resistant to SODIS, and the inactivation observed was equivalent to that occurring in the dark. High temperatures enhanced MS2 inactivation substantially; at 40°C, 3-log inactivation was achieved in Swiss tap water after exposure to a fluence of only 0.18 kJ/cm². Overall, our findings demonstrate that SODIS may reduce the load of single-stranded RNA (ssRNA) viruses, such as echoviruses, particularly at high temperatures and in photoreactive matrices. In contrast, complementary measures may be needed to ensure efficient inactivation during SODIS of DNA viruses resistant to oxidation.