Negative-sense virion RNA of segment 8 (NS) of influenza a virus is able to translate in vitro a new viral protein.

It was shown that full-length virion RNA of segment 8 of influenza virus A/Aichi/2/68 (H3N2) can initiate the synthesis of two major polypeptides with molecular weights of 23 and 13 kD and a minor polypeptide with a molecular weight of 19 kDa, which specifically reacted with the antibodies to the 30-membered peptide of the central part of the NSP protein of influenza A virus. Thus, the genomic-polarity RNA of segment 8 of influenza virus A has a translational template function. These data provide further confirmation of the concept of the bipolar (ambisens) strategy of functioning of the influenza A virus genome.

Miniature PCR based portable bioaerosol monitor development.

AIMS: A portable bioaerosol monitor is greatly demanded technology in many areas including air quality control, occupational exposure assessment and health risk evaluation, environmental studies and, especially, in defence and bio-terrorism applications. Our recent groundwork allowed us to formulate the concept of a portable bioaerosol monitor, which needs to be light, user friendly, reliable and capable of detecting airborne pathogens within 1-1·5 h on the spot. METHODS AND RESULTS: Conceptually, the event of a bioaerosol concentration burst is determined by triggers to commence the representative air sampling with sequential real-time polymerase chain reaction (PCR) confirmation of the targeted micro-organism present in the air. To minimize reagent consumption and idle running of the technology, an event of a bioaerosol burst is confirmed by three parameters: aerosol particle size, concentration and composition. Only particle sizes above 200 nm attract interest in the bioaerosol. Only an elevated aerosol concentration above the threshold (background aerosol concentration) is a signal to commence the analytical procedure. The combination of our previously developed personal bioaerosol sampler, aerosol particle counter based trigger and portable real-time PCR device formed the basis of the bioaerosol monitoring technology. The portable real-time PCR device was advanced to provide internally controlled detection, significantly reducing false-positive alarms. CONCLUSIONS: The technique is capable of detecting selected airborne micro-organisms on the spot within 30-80 min, depending on the genome organization of the particular strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to recent outbreaks of infectious airborne diseases and the continuing threat of intentionally released bioaerosol attacks, investigations into the possibility of the early and reliable detection of pathogenic micro-organisms in the air is becoming increasingly important. The proposed technology consisting of a bioaerosol sampler, technology trigger and PCR device is capable of detecting selected airborne micro-organisms on the spot within a short time period.

[Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin].

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.

Surface plasmon resonance-based bacterial aerosol detection.

AIMS: In the area of bioaerosol research, rapid methods for precise detection attracted much interest over last decades. One of such technologies operating in nearly real-time mode without any specific labelling is known as surface plasmon resonance (SPR). Recently, we validated a SPR protocol in conjunction with our earlier developed personal bioaerosol sampler for rapid detection of airborne viruses. Considering that the biological interaction between targeted micro-organism and corresponding antibody is strongly related to sizes of targeted micro-organisms, this research is vital validating suitability of SPR technique for bacterial aerosol detection, as characteristic size of bacteria is 2-3 orders of magnitude larger than sizes of common viruses. The combination of SPR with portable air sampling instrumentation could lead to the development of portable bioaerosol monitor. METHODS AND RESULTS: This study is focussed on the SPR technology application for direct detection of common environmental bacterial strain-Escherichia coli. The detection limit of developed SPR techniques based on utilization of a planar gold sensor chip functionalized with polyclonal antibody via NeutrAvidin junction for sensing of bacterial cells was found to be 1·5 × 10(3)  CFU ml(-1) , which corresponds to the limit of detection in the air to be 2·19 × 10(4)  CFU l(-1) for 1 min of sampling time. CONCLUSIONS: The technology was found fully suitable for rapid and reliable detection of large size bacterial aerosols. Low magnitude of the limit of detection looks very promising for sensitive detection of highly pathogenic airborne bacteria in the ambient air. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested technology based on a simple model organism is one of the first attempts to develop a real-time monitor for reliable detection of airborne bacteria. The outcomes would be of strong interest of professionals involved in monitoring and/or control of pathogenic airborne bacteria, including Legionella, Mycobacterium tuberculosis and Bacillus anthracis.

Surface plasmon resonance-based real-time bioaerosol detection.

AIMS: Rapid and precise bioaerosol detection in different environments has become an important research and technological issue over last decades. Previously, we employed a real-time PCR protocol in conjunction with personal bioaerosol sampler for rapid detection of airborne viruses. The approach has been proved to be specific and sensitive. However, a period of time required for entire procedure was in manner of hours. Some new developments are required to decrease the detection time down to real-time protocols. METHODS AND RESULTS: Presently, a surface plasmon resonance (SPR)-based immunosensor that coupled with a specific antigen-antibody reaction could offer sensitive, specific, rapid and label-free detection. This study describes the possibility of combining the personal sampler with SPR technology for qualitative and extremely rapid detection of airborne micro-organisms. Common viral surrogate MS2 bacteriophage, frequently used in bioaerosol studies, was employed as a model organism. The results of the sensor functionalizing procedure with monoclonal anti-MS2 antibody and optimization of the chip performance are presented. The SPR-based detection of the airborne virus was found to be very fast; the viral presence was detected in less than 2 min, and the entire procedure (sampling and analysis) was undertaken in 6 min, which could be considered as real-time detection for this type of measurements. CONCLUSIONS: The combination of SPR with the personal sampler targeted towards bioaerosol detection was proven to be feasible. The SPR sensor was found to be highly stable and suitable for multiple utilizations without significant decrease in response. The suggested approach opens new possibilities for the development of portable and rapid (almost real time) bioaerosol monitors. SIGNIFICANCE AND IMPACT OF THE STUDY: This technology is the first in the world real-time bioaerosol monitor. This outcome would be of strong interest to individuals representing public health, biosecurity, defence forces, environmental sciences and many others.

[Molecular genetic characteristics of the newcastle disease virus velogenic strains isolated in Russia, Ukraine, Kazakhstan, and Kirghizia].

The F gene fragment of 79 Newcastle disease virus (NDV) strains isolated from domestic and synanthropic birds in Kazakhstan, Kirghizia, Ukraine, and Russia in 1993 to 2007 was comparatively analyzed. Phylogenetic analysis of test isolates and reference NDV strains obtained from the GenBank was carried out by polymerase chain reaction with subsequent sequencing and comparative analysis of 154-bp nucleotide sequences in the main functional region of the F gene. All newly characterized isolates belong to three NDV genotype VII subgroups: VIIa, VIIb, VIId. The results show it necessary to monitor of NDV strains isolated in the CIS countries since the spread of NDV among migratory and synanthropic birds (pigeons, crows, and jackdaws) poses a serious threat to commercial poultry industry.

[Spread of new pandemic influenza A(H1N1)v virus in Russia].

The paper presents the results of the investigations of the development of a influenza A(H1N1)v pandemic, conducted by the D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences, and collaborating laboratories in the European part of Russia, in the Urals, Siberia, and in the Far East. In the prepandemic period (April 27 - June 11, 2009) its first diagnosis was established on May 21, 2009; the first strain was isolated on May 24, 2009; the data on complete genome sequencing were sent to the GenBank; the sensitivity of the strain to commercial antiviral commercial agents was studied. In the early pandemic period (June 11 - August 15), 73 patients who had come from 14 countries of Europe, America, and Asia were identified; 19 virus strains (partially or completely sequenced) were isolated. The pandemic period (August 15 - December 1) was marked by absolute dominance of pandemic influenza virus virtually in the absence of seasonal influenza; the first death caused by pandemic influenza was detected in late August; 3053 subjects were infected with the pandemic strain, as shown by polymerase chain reaction diagnosis; 202 strains were identified.

[The 24 May, 2009 isolation of the first A/IIV-Moscow/01/2009 (H1N1)swl strain similar to swine A(H1N1) influenza virus from the first Moscow case detected on May 21, 2009, and its deposit in the state collection of viruses (SCV No. 2452 dated May 24, 2009)].

The paper presents the results of the first isolation of the new influenza virus in Moscow and the Russian Federation, which was similar to the swine A/IIV-Moscow/01/2009(H1N1)swl strain isolated on May 24, 2009 from a Russian arrived in Moscow from the USA on May 19, 2009. The antigenic, biological, and molecular genetic properties of this virus were studied. The virus was isolated on MDCK and chick embryos, the hemagglutination titers being 1:8-1:16 AE; the infectious titers being 6.51g of the tissue cytopathogenic infective dose (TCID50) and 7.01g of the common infective dose (CID50). The virus was sensitive to arbidol, ribavirin, oseltamivir, and resistant to rimantadine. The complete virus genome was sequenced; the data were accepted to the Gen Bank on May 28, 2009 under GQ219584-GQ219590 and GQ202724. The significant gene substitution of neuraminidase Asp for Gly in position 451, which has been undetectable in any other strain published in the Gen Bank by the present time is unique only to A/IIV-Moscow/01/2009 (H1N1)swl. The virus has been deposited in the State Collection of Viruses, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, under No. 2452 dated May 24, 2009.

[Isolation of influenza virus A (Orthomyxoviridae, Influenza A virus), Dhori virus (Orthomyxoviridae, Thogotovirus), and Newcastle's disease virus (Paromyxoviridae, Avulavirus) on the Malyi Zhemchuzhnyi Island in the north-western area of the Caspian Sea].

The paper presents the results of the 2003 and 2006 environmental virological monitoring surveys on the Malyi Zhemchuzhnyi Island where a large breeding colony of sea gull (Laridae) is located. In the past several years, expansion of cormorants (Phalacrocorax carbo) has enhanced the intensity of populational interactions. The investigators isolated 13 strains of influenza A virus (Orthomyxoviridae, Influenza A virus) subtype H13N1 (from sea gulls (n = 4), cormorants (n = 9) 1 strain of Dhori virus (Orthomyxoviridae, Thogotovirus) from a cormorantwith clinical symptoms of the disease, 3 strains of Newcastle disease virus (Paramyxoviridae, Avulavirus) from cormorants. RT-PCR revealed influenza A virus subtype H5 in 3.1% of the cloacal lavages from cormorants. Neutralization test indicated that sera from cormorants contained specific antibodies against West Nile (Flaviviridae, Flavivirus) (15.0%), Sindbis (Togaviridae, Alphavirus) (5.0%), Dhori (10.0%), and Tahini (Bunyaviridae, Orthobunyavirus) (5.0%); sera from herring gulls had antibodies against Dhori virus (16.7%); there were no specific antibodies to Inco (Bunyaviridae, Orthobunyavirus) and mountain hare (Lepus timidus) (Bunyaviridae, Orthobunyavirus) virus.

[Molecular genetic characteristics of the strain A/chicken/Moscow/2/2007 (H5N1) strain from a epizootic focus of highly pathogenic influenza A among agricultural birds in the neear-Moscow region (February 2007)].

Among agricultural birds in the near-Moscow Region (February 2007), local epizootics caused by the highly pathogenic avian influenza A/H5N1 virus seem to be of unintended manual origin. Such a situation may be considered to be model when the source of inoculation is elucidated in cases of potentially possible acts of bioterrorism. Molecular genetic analysis of isolated A/chicken/Moscow/2/2007 strain established its genetic similarity with the highly pathogenic strains detected in the Black-and-Caspian Sea region in 2006. At the same time, comparison of nucleotide sequences of the strain A/chicken/Moscow/2/2007 with the strains of Qinghai-Siberian genotype (CSG) for which the sequences of full-sized genomes are known in the international databases revealed a significant distinction of the near-Moscow strain from the earlier known analogues. The uniqueness of the primary structure of the PB1 gene is shown. The paper discusses the functional value of amino acid substitutions in the proteins of the strain A/chicken/Moscow/2/2007 and in other variants of CSG of the subtype H5N1.

[Complex environmental and virological monitoring in the Primorye Territory in 2003-2006].

The paper presents the results of monitoring of viruses of Western Nile (WN), Japanese encephalitis (JE), tick-borne encephalitis (TBE), Geta, Influenza A, as well as avian paramicroviruses type I (virus of Newcastle disease (ND)) and type 6 (APMV-6) in the Primorye Territory in 2003-2006. Totally throughout the period, specific antibodies to the viruses were detected by neutralization test in wild birds (7.3%, WN; 8.0%, Geta; 0.7% Batai; 2.8%, Alpine hare (Lepus timidus); by hemagglutination-inhibition test in cattle (11.4% WN; 5.9%, JE; j 3.0%, TBE; 11.6%, Geta), horses (6.1, 6.8, 0, and 25.3%, respectively), and pigs (5.4, 1.5, 0, and 5.9%, respectively) by enzyme immunoassay (IgG) in human beings (0.8, 0.5, 6.8, and 3.2%, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was used to reveal RNA of the NP segment of influenza A virus in 57.9 and 65% of the cloacal swabs from wild and domestic birds, respectively; and the HA-segment of subtype HH was not detected in 2005. HA/H5 RNA was recorded in 5.5 and 6.7% of the swabs from wild and domestic birds, respectively; 6% of the specimens from domestic birds were M-segment positive in 2006. RNA of influenza A virus NA/H7 and RNA was not detected throughout the years. In 2004, the cloacal swabs 8 isolated influenza A strains: two H3N8 and two H4N8 strains from European teals (Anas crecca), two (H3N8 and H6N2) strains from Baikal teals (A. formosa), one (H10N4) strain from shovelers (A. clypeata), and one (H4N8) from garganeys (A. querquedula). In 2004, one ND virus strain was isolated from the cloacal swabs from European teals (A. crecca). RT-PCR revealed RNA of this virus in some 8 more cloacal swabs from black ducks (A. poecilorhyncha) (3 positive specimens), pheasants (Phasianus colchicus) (n = 2), garganeys (A. querquedula) (n = 1), gadwalls (A. strepera) (n = 1), and geese (Anser anser domesticus) (n = 1). Sequencing of the 374-member fragment of the ND virus F gene, which included a proteolytic cleavage site, could assign two samples to the weakly pathogenetic variants of genotype 1, one sample to highly pathogenic variants of genotype 3a, five to highly pathogenic ones of genotype 5b. Isolation of APMV-6 (2003) from common egrets (Egretta alba) and geese (Ans. anser domesticus) is first described.

[Molecular virological monitoring of Newcastle disease virus strains (Paramyxoviridae, Avulavirus) in the populations of wild birds in the Volga estuary (the 2001 data)].

The paper analyzes the results of isolation of Newcastle disease virus (NDV) strains from 336 swaps of 31 wild bird species collected in the 2001 summer in the Volga estuary (Astrakhan Region). Twenty-seven NDV strains were isolated from little terns (Sterna albifrons) (n=11; infection rate, 24.4%), great cormorants (Phalacrocorax carbo) (n=6; 11.1%), coots (Fulica atra) (n=8; 6.5%), sandwich terns (Sterna sandvicensis) (n=1; 100%), and common redshanks (Tringa totanus) (n=1; 50.0%). Four strains were sequenced by the 374 n. a. residue fragment from the beginning of the F gene, one of them was by the full F gene, and another (Stemal/Astrakhan/2755/2001) was by the full genome. Nucleotide sequences have allowed the authors to classify corresponding NDV strains as 5b genotype and the analysis of the amino acid sequence of the F-protein cleavage site has shown them to belong to a non-pathogenic group.

[Newcastle disease virus in the populations of wild birds in the south of the Primorye Territory in the period of autumn migrations in 2001-2004].

The paper presents the results of molecular virological monitoring of Newcastle disease virus (NDV) by reverse-polymerase chain reaction (followed by sequence of F-gene fragment 374 p.n.) and chick embryo isolation of samples from the avian cloacal swabs collected in the south of the Primorye Territory in September-October 2001-2004. It shows that before 2004, there were only slightly pathogenic variants of NDV of genotype 1 in this region and in 2004 they were added by highly pathogenic variants of subtypes 3a and 5b. The impact of landscaping features of the south of the Primorye Territory on the environment of NDV is discussed.

[Highly pathogenic influenza A/H5N1 virus-caused epizooty among mute swans (Cygnus olor) in the lower estuary of the Volga River (November 2005)].

Molecular virological studies of the field material collected in the epicenter of epizooty with high mortality among mute swans (Cygnus olor) in the area of the lower estuary of the Volga River (November 2005) could establish the etiological role of highly pathogenic influenza A (HPAI) virus of the subtype H5N1. Ten HPAI/H5N1 strains deposited at the State Collection of Viruses of the Russian Federation with the priority dated December 1, 2005 were isolated from the cloacal/tracheal swabs and viscera of sick and freshly died mute swans. Complete nucleotide sequences of all fragments of the genome of 6 strains have been deposited in the Gene Bank. The paper discusses the molecular genetic characteristics of isolated strains.

[Molecular genetic characteristics of the Newcastle Sterna/Astrakhan/Z275/2001 virus isolated in Russia].

The genome of the NDV strain Sterna/Astrakhan/2755/2001 isolated from a wild bird of the Volga River delta in 2001 was completely sequenced. The phylogenetic analysis of the strain investigated and other NDV strains clearly demonstrated that Sterna/Astrakhan/2755/ 2001 belonged to the lineage 5b. Comparative analysis of molecular genetic markers of pathogenicity of strain Sterna/Astrakhan/2755/ 2001 allows its velogenic character to be suggested.

[Isolation of highly pathogenic avian influenza (HPAI) A/5H5N1 strains from wild birds in the epizootic outbreak on the Ubsu-Nur Lake (June 2006) and their incorporation to the Russian Federation State Collection of viruses (July 3, 2006)].

Virological and molecular genetic studies of the field material collected in the epicenter of epizooty with high mortality rates among the wild birds on the coast of the Ubsu-Nur lake (Republic of Tyva, 51 degrees NL, 93 degrees EL, June 2006) revealed the etiological role of highly pathogenic avian influenza (HPAI) H5N1. Seven HPAI/H5N1 strains were isolated from the tracheal/cloacal swabs of clinically healthy, ill and recently dead great-crested grebes (Podiceps cristatus), cormorants (Phalacrocorax carbo), balt-coots (Fulica atra), and common terns (Sterna hirundo) collected on June 24, 2006, and incorporated to the RF State Collection of Viruses (with the July 3, 2006 priority). Full-length genome nucleotide sequences were incorporated to the GenBank (with the July 23, 2006 priority) (DQ852600-DQ852607). Comparative analysis of molecular genetic characteristics showed their belonging to the Qinghai-Siberian genotype. The strains were sensitive to rimantadine.

[Analysis of new variants of West Nile fever virus]. / Analiz novykh variantov virusa likhoradki Zapadnogo Nila.

The complete nucleotide sequences for 6 strains of the West Nile fever virus were determined. For the first time the complete nucleotide sequences of the Indian isolate and Krsn190 strain, that is the most far phylogenetically from all isolates known at present time were established. The scheme for separation of virus variants into 4 groups and criteria for determination the group to which the isolate belongs are suggested.