Changes in the Receptor-Binding Properties of H3N2 Viruses during Long-Term Circulation in Humans.

It was previously shown that hemagglutinin residues Thr155, Glu158, and Ser228 are crucial for the recognition of Neu5Gc. In this study, we demonstrated that the ability to bind the Neu5Gc-terminated receptor is related to the amino acid 145: viruses of years 1972-1999 with Lys145 bind to the receptor, whereas viruses with Asn145 do not. Sporadic appearance and disappearance of the ability to bind Neu5Gc oligosaccharides and the absence of Neu5Gc in the composition of human glycoconjugates indicate the non-adaptive nature of this ability. It was previously shown that unlike H1N1 viruses, H3N2 viruses of years 1968-1989 did not distinguish between Neu5Acα2-6Galß1-4Glc (6'SL) and Neu5Acα2-6Galß1-4GlcNAc (6'SLN). H3N2 viruses isolated after 1993 have acquired the ability to distinguish between 6'SL and 6'SLN, similarly to H1N1 viruses. We found that the affinity for 6'SLN has gradually increased from 1992 to 2003. After 2003, the viruses lost the ability to bind a number of sialosides, including 6'SL, that were good receptors for earlier H3N2 viruses, and retained high affinity for 6'SLN only, which correlated with the acquisition of new glycosylation sites at positions 122, 133, and 144, as well as Glu190Asp and Gly225Asp substitutions, in hemagglutinin. These substitutions are also responsible for the receptor-binding phenotype of human H1N1 viruses. We conclude that the convergent evolution of the receptor specificity of the H1N1 and H3N2 viruses indicates that 6'SLN is the optimal natural human receptor for influenza viruses.

Detecting 2009 pandemic influenza A (H1N1) virus infection: availability of diagnostic testing led to rapid pandemic response.

Diagnostic tests for detecting emerging influenza virus strains with pandemic potential are critical for directing global influenza prevention and control activities. In 2008, the Centers for Disease Control and Prevention received US Food and Drug Administration approval for a highly sensitive influenza polymerase chain reaction (PCR) assay. Devices were deployed to public health laboratories in the United States and globally. Within 2 weeks of the first recognition of 2009 pandemic influenza H1N1, the Centers for Disease Control and Prevention developed and began distributing a new approved pandemic influenza H1N1 PCR assay, which used the previously deployed device platform to meet a >8-fold increase in specimen submissions. Rapid antigen tests were widely used by clinicians at the point of care; however, test sensitivity was low (40%-69%). Many clinical laboratories developed their own pandemic influenza H1N1 PCR assays to meet clinician demand. Future planning efforts should identify ways to improve availability of reliable testing to manage patient care and approaches for optimal use of molecular testing for detecting and controlling emerging influenza virus strains.

Influenza epidemiology and characterization of influenza viruses in patients seeking treatment for acute fever in Cambodia.

The epidemiology, symptomology, and viral aetiology of endemic influenza remain largely uncharacterized in Cambodia. In December 2006, we established passive hospital-based surveillance to identify the causes of acute undifferentiated fever in patients seeking healthcare. Fever was defined as tympanic membrane temperature >38 degrees C. From December 2006 to December 2008, 4233 patients were screened for influenza virus by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). Of these patients, 1151 (27.2%) were positive for influenza. Cough (68.8% vs. 50.5%, P < 0.0001) and sore throat (55.0% vs. 41.9%, P < 0.0001) were more often associated with laboratory-confirmed influenza-infected patients compared to influenza-negative enrollees. A clear influenza season was evident between July and December with a peak during the rainy season. Influenza A and B viruses were identified in 768 (66.3%) and 388 (33.7%) of the influenza-positive population (n = 1153), respectively. In December 2008, passive surveillance identified infection of the avian influenza virus H5N1 in a 19-year-old farmer from Kandal province who subsequently recovered. From a subset of diagnostic samples submitted in 2007, 15 A(H1N1), seven A(H3N2) and seven B viruses were isolated. The predominant subtype tested was influenza A(H1N1), with the majority antigenically related to the A/Solomon Island/03/2006 vaccine strain. The influenza A(H3N2) isolates and influenza B viruses analysed were closely related to A/Brisbane/10/2007 or B/Ohio/01/2005 (B/Victoria/2/87-lineage) vaccine strains, respectively. Phylogenetic analysis of the HA1 region of the HA gene of influenza A(H1N1) viruses demonstrated that the Cambodian isolates belonged to clade 2C along with representative H1N1 viruses circulating in SE Asia at the time. These viruses remained sensitive to oseltamivir. In total, our data suggest that viral influenza infections contribute to nearly one-fifth of acute febrile illnesses and demonstrate the importance of influenza surveillance in Cambodia.

[Study of biological properties of cold-adapted reassortant strain of influenza virus subtype H7N3].

For the development of live attenuated influenza vaccine (LAIV) against influenza virus strains with pandemic potential, method of classic genetic reassortment of donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic influenza viruses of different subtypes was used. Strain with genome formula 6:2, which contains HA and NA genes from avian apathogenic virus A/wild duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] exhibited ts- and ca- phenotype specific for cold-adapted strains. Reassortant was identical on antigenic profile to parent avian virus H7N3-wt. Like cold-adapted donor strain Len/17, Len17/H7 was attenuated for chickens, whereas wild-type parent strain was lethal in 60% of birds after its intravenous challenge. Reassortant strain Len17/H7 was attenuated during intranasal inoculation of 6 EID50 to white mice, which was confirmed by absence of its isolation from the lungs, actively reproduced on nasal mucosa and stimulated specific systemic and local antibody response.

[The inoculative properties of cold-adapted reassortant A(H5N2) influenza strain during intranasal administration to mice].

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).

[PCR restriction analysis of the genome composition of reassortant cold-adapted influenza B virus strains].

A simple and sensitive method has been developed to determine the genome composition of the reassortant on the basis of B/USSR/60/69 by the restrictase analysis of DNA copies of RNA sites containing the nucleotide replacements typical of B/USSR/60/69.

[Growth characteristics of single gene mutants of A/Leningrad/134/57(H2N2) influenza virus and cold-adapted mutant A/Leningrad/134/17/57(H2N2)].

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

[Life expectency and fertility of postembrio stage drosophila after the pulse-periodic X-ray irradiation].

The effect of 13 Hz repetition rate X-ray pulses with 3 x 10(-6)-1.5 x 10(-4) Gr per pulse dose during 5 minute on drosophila's larvae and on pupae vas investigated. It was shown that the effect depends on drosophila's age as well as on X-ray dose and manifests itself in variation of life expectancy and fertility.

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential.

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.

[Different receptor specificity of influence viruses from ducks and chickens and its reflection in the composition of sialosides on host cells and mucins].

The ability of influenza viruses from different hosts to bind to the intestinal epithelium of various birds (Anseriformes (Anatidae), Galliformes, Charadriiformes (sandpipers and sea gulls), Ciconiiformes (storks), Podicipediformes (grebes), and Gruiformes was studied. The composition of sialo-containing receptors on the epithelia was examined, by using lectins. Intestinal epitheliocytes of the Anatidae (Anseriformes) family was shown to have a low content of receptors binding both Sambucus nigra agglutinin (SNA) lectin specific to Siaalpha-6Gal, and Maackia amurensis agglutinin (MAA) lection specific to Siaalpha2-2Gal. Nevertheless, these cells well bound duck influenza viruses. The intestinal epithelium of Ciconiiformes, Podicipediformes, and Gruiformes well bound MMA lection, but avian influenza viruses weakly bound the latter. The intestinal cells of Gallinaceae bound both MMA and SNA lectins and avian and human influenza viruses. Thus, the composition of natural sialosides is different in various avian species whereas the receptor specificity of influenza viruses from various hosts reflects these differences. This can be accounted for by the differences in the ability of influenza viruses from different birds to break through the interspecies barrier, infecting mammals and human beings in particular.

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005.

Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003-04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.

[Assessment of some metabolic parameters of white rats' liver after exposure to repetitive x-ray or microwave pulses].

Effects of repetitive X-ray and microwave pulses on the rat liver functions were investigated. The action of repetitive nanosecond X-ray is characterized by the metabolic dysfunction of the liver. In particular, it results in a considerable reduction in the ALT activity, augmentation of the AST/ALT ratio and decrease of the total protein content. The most considerable effect is observed at 16 Hz. Microwave pulses render a less significant effect on metabolic functions of the rat liver as compared to X-rays. The effect depends on the frequency of pulses.

[Effect of impulse-intermittent ultrahigh frequency irradiation on synthesis of nucleic acids in tumor cells].

In this work is shown that the repetitive high power microwaves is able to exert an inhibitory influence on the process of DNA and RNA syntheses in tumor cells of P-815 mastocytoma. This effect depends on pulse repetition rate. High power microwave pulses inhibit the process of transcription in tumor cells. No activation of DNA reparation system due to the irradiation of non-proliferating mononuclear blood cells was found. This indicates that the repetitive high power microwaves are not able to initiate single-filament rupture in DNA of tumor cells. The conformation of transcription enzymes is assumed to be changed under the influence of the microwave irradiation that makes for significant inhibition of RNA synthesis.

[Genetic and phenotypic analysis of heterogeneous population of a cold-adapted donor of the A/Leningrad/134/17/57 (H2N2) attenuation and of the donor-based reassortant influenza vaccine strains].

Cold-adapted (CA) temperature sensitive and attenuated virus A/Leningrad/134/17/57 (H2N2) (Len/17) has been recently used in Russia as a donor of internal genes in the preparation of reassortant vaccine strains of CA live influenza vaccine (LIV) for all age groups. The Len/17 population was found to be heterogeneous and to be made up of clones, which differ by combinations of mutations in internal genes. Around 50% of the Len/17 population had clones with all 8 coding mutations in internal genes. The others were made up of clones with mutation combinations, which were different from the original Len/17. The PCR restriction method was used to analyze 5 clones of Len/17 and 8 LIV vaccine strains. There were no Ala-86-Thr mutation in the M2 protein in 4 clones and 3 vaccine strains. The PB-1 gene of 4 clones and 3 vaccine strains had a mutation encoding Met-317-IIe more typical of a more attenuated virus A/Leningrad/134/47/57 (H2N2) (Len/47). The NP protein of a clone had a mutation Leu-341-IIe also typical of Len/47. However, neither the absence of mutation in the M2 gene nor an extra mutation in the PB1 gene affected the attenuation extent of reassortant CALIV.

[Analysis of mutations in the genome of cold-adapted strains of influenza A virus using extended modification of polymerase chain restriction method]. / Analiz mutatsii v genome kholodoadaptirovannykh shtammov virusa grippa A s ispol'zovaniem rasshirennoi modifikatsii PTSP-restriktsionnogo metoda.

Cold-adapted influenza viruses A/Leningrad/13 4/17/5 7 (H2N2) (Len/17) and A/Leningrad/I 34/4 7/57 (H2N2) (Len/47) are used in Russia to prepare live reassortant cold-adapted influenza vaccines (LIV) for adults and children, respectively. Comparison between the nucleotide sequences of the Len/17 strain and the initial wild-type strain A/Leningrad/13 4/5 7 (H2N2) revealed ten nucleotide substitutions (eight of them encoding). Four additional substitutions (three encoding) were found in the genome of the Len/47 virus. Gene segment restriction site (PCR-restriction) analysis was used for identification of the genotype of reassortant influenza viruses. Conventional methods of PCR-restriction analysis detect only five encoding nucleotides substitutions in the internal genes of the Len/17 and seven substitutions in the internal genes of the Len/47 virus. An extended modification of the PCR-restriction method detect all encoding mutations in the internal genes of the Len/17 and Len/47 viruses (eight and eleven encoding substitutions, respectively). This method is advantageous for genome composition analysis of reassortant influenza vaccine strains and for investigating the genetic stability of LIV during replication in vaccines.

[Criteria of primary screening of attenuated strains for live vaccine against Influenza A]. / Kriterii pervichnogo skrininga attenuirovannykh shtammov dlia zhivo'i vaktsiny protiv grippa tipa A.

Reassortant strains for live influenza vaccine (LIV) were selected using two additional markers: intensity of cytopathic effect (CPE) at 40 degrees C in MDCK cells and toxicity for mice (induction of acute hemorrhagic pulmonary edema after intranasal challenge with undiluted virus). All wild-type viruses induced a high CPE in MDCK cells, while the reassortants differed by this sign. Only vaccine strains and attenuation donors were characterized by a low CPE. Modern epidemic viruses are highly toxic for mice, causing the death of 60-100% animals from hemorrhagic pulmonary edema on days 3-4 after intranasal infection. Attenuation donors and vaccine strains were not toxic for mice, the level of toxic effect correlating with CPE in MDCK culture. Evaluation of CPE in MDCK culture and toxicity for mice can be used for primary screening of candidates for LIV.

Immunogenicity and efficacy of Russian live attenuated and US inactivated influenza vaccines used alone and in combination in nursing home residents.

The immunogenicity and efficacy of Russian live attenuated and US inactivated trivalent influenza vaccines administered alone or in three different combinations were evaluated in a randomized, placebo-controlled, double-blinded study of 614 elderly or chronically ill nursing home residents in St. Petersburg, Russia during the 1996-97 influenza season. Postvaccination serum antibody responses were more frequent among individuals administered the combination vaccines than among those vaccinated with live or inactivated vaccine alone. Only individuals who received live vaccine, alone or in combination with inactivated vaccine, achieved significant postvaccination increases in virus-specific nasal IgA. Efficacy in preventing laboratory-confirmed influenza in vaccinated versus nonvaccinated individuals was 67% (95%CI, 36-81%) for recipients of a combination of the vaccines compared with 51% (95%CI, -17-79%) for recipients of live vaccine alone and 50% (95%CI, -26-80%) for recipients of inactivated vaccine alone. These results suggest that administration of a combination of influenza vaccines may provide a strategy for improved influenza vaccination of elderly people.

Low incidence of rimantadine resistance in field isolates of influenza A viruses.

The spread of drug-resistant influenza viruses type A to close contacts in families, schools, and nursing homes has been well documented. To investigate whether drug-resistant influenza viruses circulate in the general population, 2017 isolates collected in 43 countries and territories during a 4-year period were tested for drug susceptibility in a bioassay. Drug resistance was confirmed by detection of specific mutations on the M2 gene that have been shown to confer resistance to amantadine or rimantadine. Sixteen viruses (0.8%) were found to be drug-resistant. Only 2 of these resistant viruses were isolated from individuals who received amantadine or rimantadine treatment at the time the specimens were collected. For 12 individuals use of amantadine or rimantadine could be excluded, and from the remaining 2 patients information about medication was unavailable. These results indicate that the circulation of drug-resistant influenza viruses is a rare event, but surveillance for drug resistance should be continued.