Application of Laser Correlation Spectroscopy for Measuring Virus Size.

Dynamic light scattering method or laser correlation spectroscopy was applied to evaluation of the size of viruses. We measured correlation functions of the light scattered by human immunodeficiency viruses (HIV) and hepatitis A viruses (HAV) and found that size of HIV-1 (subtype A and B) and HAV virions were 104 nm and 28 nm, respectively. Comparison of these findings with electron microscopy data for fixed samples of the same viruses showed good agreement of the results.

Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity.

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~ 25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell.

[Comparative study of carbon nanotubes and polymer composites with silver as sorbents of the influenza A and B viruses].

The comparative examination of the interaction of the influenza A and B viruses and fragments of DNA with the carbon nanotubes--composites of polyaniline (PANI) nanotubes and granules containing Ag and without Ag was performed. The increased absorption of the allantois viruses and DNA was demonstrated in composites with Ag. The influence of temperature in the range of 4-36 degrees C was not found to be essential. The intensive absorption took place within the first 15 min of the contact with the sorbents. In total, the properties of the composites of PANI nanotubes + Ag 30% are the most promising for the influenza viruses and DNA absorption in water solutions.

Abnormal Morphological Vesicles in Influenza A Virus Exposed to Acid pH.

Vesicles on the virion surface, which continued the lipoprotein membrane but had no spikes of virus glycoproteins hemagglutinin (HA) and neuraminidase (NA), were detected. These vesicles and virus particles were 18±7 and 103±12 nm in diameter, respectively, and, as a rule, one vesicle was found per virion. The locus of the external protrusion in the virion presumably corresponded to the site of virus budding during assembly in infected cell free from HA and NA spikes outside and M1 matrix protein inside, but enriched with ionic channel protein M2. Particles with vesicles constituted ~3-10% of the virus population produced in MDCK-H culture and containing uncleaved HA0 hemagglutinin. The content of vesicular virions increased slightly after trypsin cleavage HA0→HA1+HA2 and reached 10-15%. Exposure of the virus in acid medium (pH 4.3) led to a drastic increase of vesicular virions - to 60-80% for HA0 and HA1+HA2 virus. This was paralleled by changes in contrast permeability (for phosphotungstic acid). HA0 virions remained contrast-impermeable, while HA1+HA2 particles let the contrast in through the vesicles detected on the surface and were rapidly destroyed after incubation in acid medium. Hence, cleavage of the surface glycoprotein HA0 into HA1 and HA2 stimulated the acid-dependent permeability of the lipid membrane and led to attenuation of the ribonucleoprotein and protein matrix M1 contacts inside the virion.

[pH-dependent rearrangements in the influenza A virus].

The Influenza virus possesses two modules: internal ribonucleoprotein (RNP) containing the viral genome RNA and external lipid envelope with transmembrane ionic channel protein M2 and embedded glycoproteins hemagglutinin (HA) and neuraminidase (NA) forming surface spike ends. These modules are combined in a whole virion by the matrix protein M1. The effect of the acidic pH 4,2-4,5 on the influenza virus grown in MDCK-H cells was tested. The A/Aichi/68 (H3N2) virus synthesized in MDCK-H cells was shown to contain uncleaved HA0 (m.w. 78 kD) and provide low infectivity. This virus was resistant to acidic medium and non-permeable to the phosphotungsten acid (PTA) used in electron microscopy as a contrast stain, and did not reduce infectious potential after acidic treatment. The trypsin-activated virus containing cleaved HA1 (56 kD)+HA2 (22 kD) was sensitive to acidic exposition resulting in the appearance of permeability to PTA, reduction of infectivity, enhancement of the M1-RNP interlink. These data indicate that the structural form of the cleaved HA1 +HA2 surface hemagglutinin coordinates a transmembrane interaction between surface and internal virus components.

[An ultrastructural study of the cervix epitelium infected with the human papillomavirus types 16 and 18 before and after treatment with contrasting thermo-laser therapy].

The results of the ultrastructural study of the epithelium of the patient cervix infected by the human papillomavirus (HPV) types 16 and 18 before and after treatment by contrasting thermo-laser therapy (CTLT) are presented. It was shown in this work that 1.5 and 6 months after treatment HPV DNA was not detected in the biopsy and the smear of the cervix using the polymerase chain reaction (PCR). In the ultrathin sections, the structure of the epithelial cells from the biopsy after treatment corresponded to norm. There was effective elimination of HPV types 16 and 18 as Induces by CTLT method.

[Interaction of influenza A and B viruses with nanodiamond-based sorbents].

The paper presents data on the sorption of influenza A(H1N1), A(H1N1)v, A(H3N2) viruses, cDNA of A(H1N1)v and B viruses on nanodiamonds and furnace charge. The sorption of viruses occurred in different solutions at 4-37 degrees C during 10-20 min. The rate of sorption varied with the concentration of a sorbent in the solution and its structure, but did not with the antigenic formula of viruses or temperature. The sorption capacity of furnace charge towards influenza A and B viruses was higher than that of nanodiamonds. Nonviral proteins (bovine serum albumin and influenza virus antibodies) were found to be bound by both sorbents. Viral desorption did not take place in physiological solution at 4 and 22 degrees C for 48 hours.

[Viral sorption on polyaniline, carbon nanotubes and their based nanocomposites].

The paper gives data on the sorption of influenza virus pandemic strain A/IIV-Moscow/01/2009 (H1N1)swl, avian influenza viruses with A/H5 and A/H7 hemagglutinin, poliomyelitis virus, and T4-D bacteriophage on polyaniline sorbents, carbon nanotubes, and their based nanocomposites. The sorption of viruses occurred in different solutions at 4-37 degrees C during 15 min or more. The rate of viral sorption depended on the structure of sorbents.

Evaluation of human papillomavirus elimination from cervix uteri by infrared laser exposure.

Elimination of types 16 and 18 human papilloma virus from the surface of cervix uteri for secondary prevention of cervical cancer was evaluated. The method is protected by patent of invention of the Russian Federation. Infrared laser therapy of cervix uteri was carried out in patients with precancer diseases of cervix uteri at Department of Gynecology of Municipal Clinical Hospital No. 52 (Moscow). Papillomavirus infection was eliminated using a Russian diode laser (lambda=1.06 µ, radiation power 10 W) with a collimating headpiece using carbon die at a distance of 10-12 cm from the exposed surface. The treatment resulted in a high percentage of elimination of types 16 and 18 oncogenic virus 4-6 weeks and during delayed periods after exposure.

[Localization of human cytomegalovirus immediate-early protein IE-1 pp72 in the juxtanuclear inclusion at the late stage of infection].

Immediate-early protein IE-1 pp72 is one of the most abundant proteins at the early stage of human cytomegalovirus infection and has a number of intranuclear activities. This paper gives immunocytochemical and ultrastructural data on IE-1 pp72 accumulation in the juxtanuclear inclusion at the late stage of low-multiplicity infection. Detection of a new localization site infers that this protein may participate in the final steps of virus morphogenesis and play a functional role in the pathogenesis of cytomegalovirus infection.

[Use of atomic force microscopy to study the cytomorphological signs of papillomavirus infection].

The paper presents the results of studying genital squamous epitheliocytes from human papillomavirus-infected female patients by cytology and atomic force microscopy. The squamous epitheliocytes with and without cytomorphological signs of papillomavirus infection (koilocytosis, dyskeratosis, parakeratosis, hyperkeratosis) have been compared. Examining the surface of the squamous epithelium has yielded quantitative characteristics of infection-induced surface changes.

[Interaction of influenza A and B viruses with polyaniline nano complexes].

The paper gives data on the sorption of influenza A and B viruses diluted from different solutions on polyaniline sorbents synthesized with low and high molecular-weight polymer acids. Irrespective of the antigenic structure of surface proteins, the influenza A and B viruses isolated in the period of 1977 to 2008 were shown to be able to be immobilized on polyaniline sorbents in the temperature range of 4 to 37 degrees C. The immobilized viruses interacted with homologous immune sera.

[Application of confocal microscopy to the use of a study of the morphogenesis of human immunodeficiency virus type 1].

Confocal microscopy is widely used in different fields of biology. By using the reports on the study of the morphogenesis, diagnosis, drug therapy of HIV-1 as an example, the authors consider the procedures of confocal microscopy, which have found wide application in virology.

[Diversity and dynamics of bacteriophages in horse feces].

The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5-11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (10(3)-10(7) plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain.

[Internal epidemic influenza virus proteins: isolation and investigation].

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.

[Detection of hepatitis C virus RNA in the liver biopsy specimens from patients with chronic hepatitis C and hepatocellular carcinoma by means of in situ RT-PCR].

The in situ RT-CPR technique has been first adapted in Russia to detect hepatitis C virus (HCV) RNA in the samples from patients with chronic hepatitis C (CHC) and hepatocellular carcinoma (HC). A total of 18 patients with CHC and HC were examined. Fourteen (78%) samples were ascertained to be positive. A positive reaction was found in 2 of 3 patients with HC. A label was revealed in individual hepatocytes without any regularity in the distribution along the tissue section. A reaction was negative in all samples from control groups. The label was observed in both the cytoplasm and the hepatocytic nuclei. There was no correlation between the degree of hepatic lesion, estimated after Knodelle, the results of the classical RT-CPR and the in situ RT-CPR techniques.

[The use of bromelain in obtaining the subviral particles of influenza A and B viruses]. / Ispol'zovanie bromelaina dlia polucheniia subvirusnykh chastits virusov grippa A i B.

Subviral particles of modern strains of influenza A viruses, i.e. A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), reassortant X-31 (subtype H3N2) and B/Sichuan/379/99, were obtained by using two preparations of bromeline ("Sigma Co., Catalogues' Nos. B2252 and B5144). A selective ability of bromeline B5144 was detected to the proteolytic splitting of hemagglutinin of influenza A and B viruses. An influence of enzyme B5144 produced on influenza B viruses brought about an appearance of subviral particles. As for influenza A(H1N1) virus, the above enzyme did not have any impact on it under the similar experimental conditions. An incomplete effect was noted for the influenza A(H3N2) virus with particles (with intact external coatings) being found in the reaction mixture. Enzyme B2252 was found to be effective in respect to all viruses selected for testing, however, the highest effect was noted for influenza A(H1N1) and B viruses.

[Detection of DNA packing density in bacteriophages Phi KZ and T4 by atomic force microscopy]. / Opredelenie plotnosti upakovki DNK u bakteriofagov Phi KZ i T4 metodami atomnoi silovoi mikroskopii.

Comparative analysis of DNA packing density in Phi KZ and T4 bacteriophages was carried out by atomic force microscopy. Irrespective of the support (mica or highly ordered pyrrolytic graphite), Phi KZ bacteriophage was compressed stronger than T4. The most probable causes of this difference are analyzed.

Electron microscopic study of DNA packing in phi(kz) bacteriophage (Pseudomonas Aeruginosa) irradiated with laser in the presence of a dye.

DNA packing of bacteriophage phikz (Pseudomonas aeruginosa) was studied by electron microscopy after gentle destruction of the capsid with powerful laser impulse in aqueous virus suspension in the presence organic dye. Suspension of destroyed phage heads contained complexes of genomic DNA with endogenous capsid proteins characterized by pronounced regular structure. The type of these structures corresponds to one the main model of phage DNA packing, the so-called coil packing.