Neurotropic influenza type A viruses

Influenza A/turkey/England/63 is neurotropic for mice. Substitution of the hemagglutinin gene of this virus by the corresponding gene of A/FPV/ Rostok /34 virus results in the loss of the neurotropic properties of the original virus. Examination of recombinants produced by hybridization of parental strains nonpathogenic (or weakly pathogenic) for newborn mice revealed recombinants highly virulent for this host. A correlation between constellation of genes and neurovirulence for mice was established. After intranasal administration neurovirulent viruses were shown to be able to penetrate into the brain of the infected animal along the trigeminal nerve escaping the blood stream.     

Detection of human influenza A viruses by loop-mediated isothermal amplification

Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.     

Detection of respiratory viruses and subtype identification of influenza A viruses by GreeneChipResp oligonucleotide microarray

Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.     

Mixed infections with influenza A and B viruses

In the course of an influenza A and B epidemic, influenza virus was isolated from 46 patients. Seven of these patients showed seroconversion against influenza virus plus another respiratory virus. In 9 patients, simultaneous antibody increase against both influenza A and B viruses was demonstrated, but only a single virus type was isolated. In one case the isolated virus population could be separated into type A and B viruses.     

Rapid detection of influenza A and B viruses in clinical specimens by Light Cycler real time RT-PCR.

BACKGROUND: the influenza viruses cause morbidity and mortality annually among children and elderly. Surveillance and rapid diagnosis is imperative in the reference laboratory, as clinical symptoms are insufficient for proper diagnosis. OBJECTIVES: this study involved the design of a rapid detection method for influenza A and B viruses using real time RT-PCR from clinical specimens. Methods were specifically designed for use on the Light Cycler. The sensitivity and specificity were also to be determined. STUDY DESIGN: the identification and discrimination of influenza A and B viruses employs two dual probe systems based on fluorescence resonance energy transfer (FRET) technology. Following submission by physicians participating in the Florida sentinel influenza network, 58 specimens were chosen for testing using both tissue culture and Light Cycler methods. RESULTS: of the 35 identified positive for influenza virus via tissue culture isolation, the Light Cycler results matched identification and typing with 100% agreement. However, the Light Cycler recognized 16 additional specimens that were positive for the presence of the virus. RT-PCR and nucleotide sequencing confirmed the presence of influenza A virus in these specimens. Using tenfold serial dilutions, the sensitivity of the Light Cycler method was determined to be 0.01 TCID50. The lower limit of RNA detection was determined as 1.6 x 10(-7) microg for influenza A virus, and 1.2 x 10(-7) microg for influenza B virus. Specificity of the Light Cycler method was determined by testing specimens containing adenovirus, parainfluenza virus and echovirus, all of which yielded negative results with no discernible background. CONCLUSIONS: overall, this newly developed method of simultaneous detection and typing of influenza types A and B using the Light Cycler proves to be more sensitive than tissue culture isolation, with corresponding specificity. This technique may be valuable for surveillance and rapid identification of influenza for early diagnosis.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

Specific and nonspecific indicators of activation of influenza viruses before an epidemic caused by influenza viruses subtype A/H3N2 and type B in Czechoslovakia in 1986

The authors submit an aetiological and epidemiological analysis of the influenza epidemic which occurred in the CSR between the 4th and 14th week of 1986 and was caused by the influenza virus subtype A/H3N2/ and type B. The epidemic affected a total of 27.1% of the population, in the age group of 0-5 years 63.7%, in the age group 6-14 years 52.7% and in the age group above 15 years 17.1%. In the course of the epidemic 77,458 cases of pneumonia and bronchitis were reported and 1,412 deaths with the diagnosis influenza, bronchitis, pneumonia and chronic affection of the lungs. The authors analyze also specific indicators of the activation of influenza viruses and reach the conclusion that serological evidence of the circulation of influenza viruses in the population was detected already in the third quarter of 1985, the first isolations were made six weeks before the influenza epidemic. Activation of the influenza viruses is indicated already during the pre-epidemic period by some non-specific indicators which include the rising number of patients with acute respiratory affections in surgeries and the rising number of children absent from nurseries and nursery schools on account of these diseases. The most sensitive non-specific indicator is the rising number of patients with respiratory diseases in surgeries of the First aid medical service.     

Molecular characterization of the HA gene of influenza type B viruses

Nucleotide sequences of the HA1 subunit of influenza B viruses isolated in Portugal between 1994 and 2003 influenza winter seasons were analyzed by the Neighbor-Joining algorithm and rates of HA1 evolution estimated by linear regression. From 1994 to 2002, all influenza B viruses studied were of the Yamagata lineage. Strains isolated from 1994 to 1996, 1996 to 1999, and 1999 to 2002 revealed a high similarity with B/Beijing/184/93, B/Yamanashi/166/98, and B/Sichuan/379/99, respectively, and strains isolated during 1994-1995, 1996-1997, and 1998-1999 clustered in more than one branch of the phylogenetic tree. Victoria-related strains reappeared during 2002/2003 and formed only one branch in the phylogenetic tree revealing a closer relationship to B/Shandong/7/97. Evolutionary rates for strains from the Yamagata lineage were estimated as 3.82x10(-3) nucleotides/site/year and 2.62x10(-3) nucleotides/site/year for Victoria-related strains. In order to identify putative influenza B HA1 codons under selective pressure, a codon-substitution model for heterogeneous selective pressure at amino acid sites was used. A percentage of 97.3% of codons under negative selective pressure and 2.7% of codons under positive selective pressure (omega=dN/dS=2.65) were estimated, with posterior probability higher than 0.90. Amino acid sites 75, 197, and 199 were found more likely to be under positive selective pressure.     

The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance

Detection of human immunodeficiency virus (HIV) infections has been enhanced by incorporating p24 antigen detection with current HIV antibody detection using enzyme immunoassays (EIAs). However, screening for HIV antibodies has increased through the use of rapid, lateral-flow HIV antibody detection assays that currently do not have the capability to detect HIV p24 antigen. In this report, a lateral-flow based assay using super-paramagnetic particles as the detection marker was developed for the detection of HIV-1 p24 antigen. This magnetic immuno-chromatographic test (MICT) uses an inexpensive, low-maintenance instrument that detects the magnetic moment of the super-paramagnetic particles in a magnetic field. MICT is simple to perform, provides a numerical output for easier determination of reactive results and can be completed in 40 min. The lower limit of detection for HIV-1 p24 spiked into assay sample buffer and 50% plasma was 30 pg/ml for both. Detection of HIV-1 p24 antigen at 50 pg/ml was reproducible in both inter-run and intra-run assays with coefficients of variation of <13%. Furthermore, the MICT p24 assay was able to detect intact virus spiked into 50% plasma (lower detection limit of 250,000 viral RNA copies/ml). MICT detection of increasing HIV-1 p24 levels in commercially available seroconversion panels by MICT was only slightly later than that detected by much more complex EIAs. MICT could provide a simple, low-cost, and portable method for rapid HIV-1 p24 detection in a variety of testing environments.     

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.     

流感/禽流感病毒及其致病力鉴别的基因芯片技术研究

目的 建立流感/禽流感病毒及其致病力鉴别的基因芯片检测技术.方法 以血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)基冈作为靶片段,设计病毒检测和致病力特异性鉴别探针,建立基因芯片鉴别检测技术,采用单引物扩增法(SPA)处理样本核酸,分别对此芯片进行特异性、敏感性和符合率评价.结果 此芯片能够特异性的检测H1N1、H3N2、B型流感病毒及H5N1、H9N2禽流感病毒,敏感性分别为8HAU、16HAU、32HAU及8HAU、8HAU.致病力鉴别探针敏感性为32HAU.同RT-PCR方法比较,检测灵敏度为83.9%.结论 建立的常见流感病毒检测基因芯片特异性高、敏感性高、灵敏度高,更能够对致病力进行有效甄别,可作为临床诊断、传染病防控等方面的有益补充.     

Reassortment and evolution of current human influenza A and B viruses

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.     

Categorization of nucleoproteins and matrix proteins from type A influenza viruses by peptide mapping

Relationships between the nucleoprotein (NP) and matrix protein (M) of 16 human and 3 lower vertebrate strains of type A influenza virus were investigated by two-dimensional mapping of [³⁵S]methionine peptides. By regarding certain common groups of peptides as taxonomic criteria, all NP proteins could be classified as belonging to one of two basic patterns. Differences existed between maps of either category but these have not been investigated in detail. The chymotryptic-tryptic peptide maps of the M proteins were too similar overall to warrant dividing them into subgroups but differences between individual strains were clearly apparent. Both patterns of NP protein occurred within a subtype. The change from one NP pattern to another, and back again, within a subtype is consistent with the hypothesis that genetic assortment of NP genes occurs during the reign of a particular subtype. This and other implications are discussed.     

Induction and evasion of type I interferon responses by influenza viruses

Influenza A and B viruses are a major cause of respiratory disease in humans. In addition, influenza A viruses continuously re-emerge from animal reservoirs into humans causing human pandemics every 10-50 years of unpredictable severity. Among the first lines of defense against influenza virus infection, the type I interferon (IFN) response plays a major role. In the last 10 years, there have been major advances in understanding how cells recognize being infected by influenza viruses, leading to secretion of type I IFN, and on the effector mechanisms by how IFN exerts its antiviral activity. In addition, we also now know that influenza virus uses multiple mechanisms to attenuate the type I IFN response, allowing for successful infection of their hosts. This review highlights some of these findings and illustrates future research avenues that might lead to new vaccines and antivirals based on the further understanding of the mechanisms of induction and evasion of type I IFN responses by influenza viruses.     

Influence of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses

Pteridine derivatives neopterin and 7,8-dihydroneopterin are produced by human macrophages and dendritic cells upon stimulation with interferon-γ (IFN-γ) and therefore become detectable in increased amounts in humans during cell-mediated (Th1-type) immune response. Compounds produced upon influence of cytokine IFN-γ often exert antiproliferative and antiviral activity. The aim of this study was to investigate the effect of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses. The changes in the replication of these viruses were evaluated by the degree of cytopathic effect and their ability to form plaques in Coxsackie B5-infected human larynx carcinoma epithelial (Hep-2) cells and in influenza A-infected canine kidney epithelial cells (MDCK). Potential toxicity of neopterin and 7,8-dihydroneopterin was estimated by the incorporation of ³H-thymidine and ³H-uridine into Hep-2 and MDCK cells. Whereas 30 nmol/l neopterin delayed the development of the cytopathic effect of Coxsackie B5 virus in Hep-2 cells (P < 0.01), 7,8-dihydroneopterin did not have any essential influence at any of the concentrations tested between 10 nmol/l and 1,000 μmol/l. However, 100–1,500 μmol/l 7,8-dihydroneopterin significantly suppressed the propagation of influenza A virus. Neopterin and 7,8-dihydroneopterin were practically nontoxic for Hep-2 and MDCK cells even at high μM concentration. Results suggest that the increased production of neopterin derivatives by activated macrophages and dendritic cells may represent part of the antiviral armature induced by IFN-γ. The mechanisms of the inhibitory effects of neopterin and 7,8-dihydroneopterin on virus replication apparently are different.     

Interaction of influenza A and B viruses with a carbon-containing sorbent

The investigation demonstrated that influenza A and B viruses actively interacted with a sorbent obtained from modified oxygen-containing graphite via hydrothermal treatment irrespective of the antigenic structure of surface proteins. Virionic sorption occurred in a wide range of temperatures from 8 to 34 degrees C for 15 min or more. After interaction with the sorbent, the titer of a virus decreased 4- to 256-fold. The immobilized viruses were able to interact with homologous antibodies and immune sera. Desorption of viruses with the sorbent was extremely slight. In addition to viruses, the proteins of nonviral nature--those of allantoic hen embryo liquid, immune serum, and 1% bovine serum albumin--could be immobilized to the sorbent.